Conștiință și cunoaștere. Volumul I - Lucrări științifice [1 ed.] 9789737036179, 9789737036308

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Universitatea ,,Alexandru loan Cuza" din la§ii aniverseaza un secol §ii jumatate de la momentul fondarii sale, care este, in acela§ii timp, momen.tul inaugural al invajamantului superior romanesc, organizatpe baze modeme. Colecµa EXCELLENTIA-150, coordonata de prof. univ. dr. Gheorghe Iacob, ~i propune sa reuneasca lucrari reprezentative - stabilite de fiecare facultate -, dedicate personalitajilor vieµi universitare ie§iene.

Redactor: M. Pricop Coperta: Manuela Oboroceanu Tehnoredactor: Luminija Raducanu

ISBN general 978-973-703-617-9 ISBN vol. I 978-973-703-630-8

© Editura Universitclp.i ,,AJexandru loan Cuza", 2010

700109 - laf?i, str. Pinului, nr. lA, tel./fax: (0232) 314947 http:// www.editura.uaic.ro e-mail: [email protected]

Con§tiinta ~i cuno3.§tere volumulI Lucrari ~tiintifice

GR.T.POPA

Ediµe alcatuita ~i ingrijita, nota asupra ediµei, studiu introductiv, repere biobibliografice de Dana Baran

EDITURA UNJVERSITATII nALEXANDRU IOAN CUZA* IA$I

2 0 1 0

GRlGORE T. POPA (1892-1948)

CUPRINS

Nota asupra editiei / 9 Grigore T. Popa, un cruciat al cunoa~terii / 11 Repere biografice / 63 Bibliografie selectiva / 75

LUCRARI ~TIINTIFICE The vascular link between the pituitary and the hypothalamus ( Gregor Popa and Una Fielding) I 85 A portal circulation from the pituitary to the hypothalamic region (Gregor Popa and Una Fielding) I 89 Hypophysio-portal vessels and their colloid accompaniment (G.T. Popa and Una Fielding) I 93 Les conditions morphologiques de la circulation hypophysaire (Gr.T. Popa) I 101 Le pouvoir hemoclasique de l'hypophyse (Gr.T. Popa) I 122 Studies on Hypophysis and Its Relations (Gregor T. Popa and Una Fielding) I 138 Neurotropic Principles in the Sheep Hypophysis Including a ,,Cerebrostimuline" (Gr.T. Popa) I 192 La cerebrostimuline, telle qu'on la connait aujourd'hui (L'effet des injections intracerebrales avec le chlorure de potassium el avec la cerebrostimuline (Gr.T. Popa, E. Lucinesco, L. Wasserman, E. Gheller) I 224 Nouvelles contributions a !'analyse de la cerebrostimuline (Gr.T. Popa) I 264

A technique for operations on the hypothalamo-hypophysial region of the rabbit (G. W Harris and Gr.T. Popa) I 283 Les meninges, leurs dependances et leurs relations (Origine, developpement, anatomie et physiologie) (Gr. T. Popa, E. Lucinesco, Fiorica Popa, I. Frasin, E. Repciuc, L. Wasserman) I 292 The influence of the sympathetic on the pigeon's wing ( Gregor T. Popa and Fiorica Popa) I 453 Certains functions of the midbrain in pigeons (Gr.T. Popa and Fiorica Gr. Popa) I 458 A Lipo-Gel Reaction Exerted by Follicular Fluid Upon Spermatozoa and Its Significance (Lillie's Reaction) (Gregor T. Popa) I 463 The distribution of substances in the spermatozoon (Arbacia and Nereis) (Gregor T. Popa) I 475 The mechano-structure of the pericardium ( Gregor T. Popa and Eug. Lucinescu) I 491

NOT A ASUPRA EDITIEI ,

inchinate memoriei Profesorului Dr. Gr. T. Popa- ramas in Academia Romana, prin vicisitudinea vremurilor ~i indiferenta oamenilor - doar membru corespondent, cele doua volume de fata, :rara a fi exhaustive, aduna texte ilustrative din creatia sa ~tiintifica ~i eseistica: sunt, pe de o parte, scrieri care ii definesc gandirea, cercetarea ~i contributiile originale in plan bio-medical, iar, pe de alta parte, scrieri privind spiritul ~tiintific in Romania ~i, 1ndeosebi, in Moldova, statutul universitatii ~i al universitarului in propria tara, ora~ul Ia~i ~i institutiile sale de asistenta sociomedicala, portretul unor figuri reprezentative din istoria ~tiintei medicate romane~ti ~i universale. Seleqia s-a :racut astfel incat opere de referinta ale marelui savant sa fie repuse acum pentru mtaia oara la indemana tuturor celor interesati m a le cunoa~te nemijlocit. Datele bio-bibliografice ~i textele volumelor au avut la baza Memoriul de titluri ~i lucrari publicat de Prof. Dr. Grigore T. Popa, la Tipografia Brawo din Ia~i, in 1940, scrierile lui Gr.T. Popa din colectiile Bibliotecii Centrale Universitare ,,Mihai Eminescu" ~i ale Bibliotecii Centrale a Universitatii de Medicina ~i Farmacie din Ia~i, precum ~i volumele Grigore T. Popa, Reforma spiritului, aparut la Editura Viata Medicala Romaneasca din Bucure~ti, in 2002, ~i Ion T. Ro~culescu, Profesorul Grigore T. Popa, in amintirea contemporanilor sai (din presa vremii), aparut la Editura Sitech din Craiova, in 2010. Aducem multumiri tuturor celor care au sprijinit aceasta prima ampla reeditare a unor materiale semnate de Gr.T. Popa in sfera atat a medicinii experimentale romane~ti, care 1-a consacrat. dt ~: a public1sticii cu un evident caracter civic, educativ-militant, ce au deschi~ 0 dezbatere socio-culturala ampla ~i avizata asupra ~tiintei ~i politicii vremii. Aceasta constanta verticalitate a caracterului i-a dat, de altfel, Profesorului Popa, insu~i sensul dramatic al destinului.

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Volumul I include lucrarile ~tiintifice care 1-au consacrat pe Grigore T. Popa, referitoare la domeniile sale predilecte de cercetare: alcatuirea ~i functionarea sistemului port hipofizar, neurocrinia ~i producerea de cerebrostimulina hipofizara; ipoteza capacitatii hemoclazice a hipofizei; o tehnica de abord experimental al hipofizei; mecanica morfo-functionala a meningelui; structura mecanica a pericardului; dubla inervatie somatica ~i simpatica a fibrei musculare striate; biologia reproducerii ~i histochimia spermatozoidului. Semnate singur sau alaturi de colaboratori romani ~i straini, contributiile medicale de relevanta ale lui Gr.T. Popa au fost redactate, in principal, in limbile engleza ~i franceza, ceea ce le-a asigurat o circulatie nestanjenita in lumea ~tiintifica internationala. Volumul reproduce textele conform originalelor, aparute fie in jurnale de specialitate universal recunoscute, precum ,,Journal of Anatomy" ori ,,The Lancet" (Londra), fie in ,,Memoriile Sectiunii $tiintifice" a Academiei Romane. La lucrarile in limba franceza, s-au corectat unele acorduri, iar, unde a fost cazul, s-a introdus conjunctivul. S-a intervenit, in toate situatiile, asupra gre~elilor de tipar. D. B.

GRIGORE T. POPA, UN CRUCIAT AL CUNOASTERII

I. Grigore T. Popa: destin ~i vocatie universitara Puterea carfii

Grigore T. Popa a avut o existenta fulguranta, intre 1892, cand a vazut lumina zilei, ~i 1948, cand a incetat din viata, trasand o cale aparte in istoria medicinei ~i universitatii romane~ti. Dupa o ascensiune profesionala rapida, confirmat profesor universitar la numai 36 de ani, la 44 de ani era deja ales - lucru putin obi~nuit -, in unanimitate, membru corespondent al Academiei Romane, dupa stagii repetate de cercetator in Statele Unite ale Americii ~i in Marea Britanie ~i dupa ce la 37 de ani se consacrase pe plan international prin descrierea fara tagada a sistemului port hipofizar. Cadru didactic de talent ~i necontestata probitate, ilustrand Facultatile de Medicina din Ia~i ~i Bucure~ti, demonstrator Ia universitatile din Chicago ~i Cambridge, Gr.T. Popa a imbrati~at multiple domenii, de la Embriologia Generala, Anatomia Umana ~i Anatomia Comparata la Antropologie, de la literatura, istoria ~tiintei ~i epistemologie la arta oratorica, prin care ~i-a sustinut pana la sacrificiu crezul etic ~i deontologic, dorind sa modeleze con~tiinta publica. A crezut in rostul culturii, al cunoa~terii, indeosebi ~tiintifice, de a ,,desmargini" limitele fapturii, de a preschimba in bine societatea, de a-i indruma ,,devenirea intru fiinta" in mod con~tient. ,,Cand bagi de seama intaia oara ca asimilarea, pe care ai facut-o odata, nu-i definitiva ~i ca trebuie s-o reincepi, te cuprinde o adanca descurajare. Curajul iti revine de abea atunci cand distingi prin observatia intema ca folosul real al invataturii nu sta in capitalizarea cuno~tintelor in scoarta cerebrala ca in ni~te coloane de dictionar enciclopedic, ci sta mai ales in sporirea puterii de pricepere, in perfectionarea aparatului propriu de gandire cu care apoi vei prelucra lumea. Daca ajungi sa poti observa pe tine insuti augmentarea intelectului, sa-ti urmare~ti in taina perfectiunea, atunci nimic nu te mai poate sdruncina in lenta dar sigura ta evolutie" (Evolufia unui om $i evolufia unei $fiinfe).

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Astazi, cand dreptul la clarificarea destinului istoric ~i la lectura nestingherita, necenzurata, a operei vaste a lui Gr.T. Popa a fost ca~tigat, orizontul intelepciunii romane~ti are ~ansa extinderii prin reintegrarea · acestui tezaur de excelenta, prin reactualizarea unor teme ,,bio-socio-gnoseologice" ~i ,,bio-filosofice", prin polemici constructive, prin redimensionarea unor modele umane ~i repere axiologice, aproape complet ignorate anterior, deliberat inlaturate sau uitate. Toate merita acum centrul atentiei, spre reimpamantenirea tuturor fiilor ratacitori ai neamului ~i revitalizarea etemei Alma Mater nationale. Evolutia omului, ideilor ~i aspirapilor Jzul pamantului

fntrupand ,,izul pamantului" - pentru a prelua titlul unuia din eseurile sale literare -, Grigore T. Popa a rasfrant, prin intreaga sa personalitate, miresmele lumii care 1-a nascut, din care s-a inaltat, asemenea unui arbore purtator de rod imbel~ugat, in ogorul ~tiintei ~i con~tiintei neamului sau, proiectate pe coordonatele exigentelor ~i implinirilor mondiale. Personalitate de eruditie renascentista, a trudit asupra caqii ~i adevarului, reu~ind sa se afirme ca genial savant, riguros ~i justitiar ganditor social ~i politic, epistemolog militant pentru ~tiinta - factor axial de progres -, ca literat sensibil ~i universitar misionar, dedicat instruirii totale a studentilor sai, cautarii, revelarii ~i cultivarii valorii. Pentru Gr. T. Popa, arta, inclusiv arta medicala, trebuia sa traseze o tendinta, sa nu ramana o forma fiira fond ori arta pentru arta. Omul de ~tiinta care nu creeaza, nu cerceteaza ~i nu lasa unna trecerii sale prin taramul revelatiilor promise nu conteaza, este o fiinta sterila (Evolu/ia unui om $i evolu/ia unei $fiin/e). Si-a asumat conditia de ie~ean prin formatie, de moldovean prin na~tere, dar s-a ridicat intotdeauna deasupra ,,fiziologiei provincialului", in speta a ,,fiziologiei ie~eanului", evitand atat complacerea in gloria trecuta ~i stinsa a inainta~ilor, cat ~i lamentatia vana ~i orgolioasa a epigonilor (Starea trecutii $i actualii a la:jului). Fatalismului innascut, visarii molatece ~i lenevirii orientale ale moldoveanului le-a opus foqa criticii constructive, comandamentul afimarii ~i daruirii absolute in actiune, imparta~ind lumii prodigiul romanesc ~i deschizand tarii sale o perspectiva planetara. El insu~i a dat substanta propriilor concluzii: cu toate defectele lor de grup, ,,nu este etapa mai insemnata din viata poporului nostru, nu este merit mai insemnat al sau, la initierea ~i desavir~irea caruia sa nu fi participat Moldoveni" (Starea trecutii :Ji actualii a la:jului). Era unul dintre ace~tia. Repetatele calatorii de studiu intreprinse de Gr.T. Popa ca bursier al Fundatiei Rockefeller din New York, in Statele Unite ale Americii ~i in Anglia, s-au dovedit prolifice itinerare initiatice pentru cercetatorul in devenire, pentru savantul ce i~i cristaliza valentele multiple ale propriei individualitati, in relatie cu intregul corp social ~i profesional. in 1925-1926, la Chicago ~i Woods-Hole, a fost indrumat de profesorii Frank R. Lillie, pre~edinte al Academiei de Stiinte a SUA, Charles J.

GRJGORE T. POPA, UN CRUCIAT AL CUNOA$TERII

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Herrick, Carl R. Moore, iar, la Hampstead-Londra, intre 1926-1928, a beneficiat de ·indrumarea unor profesori precum sir Grafton Elliot-Smith, sir Archibald Vivian Hill, laureat Nobel in 1922, ~i sir Henry Hallett Dale, laureat Nobel in 1936, directorul Institutului de Cercetari Medicate. A revenit, pentru studii ~i ca demonstrator de anatomie la Universitatea din Chicago, in 1926, ~i la University College din Londra, cate cinci luni pe an, in 1929, 1930 ~i 1932, iar apoi, cate patru luni pe an, in 1935 ~i 1936, fiind demonstrator ~i research fellow la Institutul de Anatomie al Universitatii din Cambridge. in spatiul sacru al acestei batrane cetati universitare, a fost invitat ~i pentru o conferinta asupra cerebrostimulinei, eveniment petrecut in mai 1940, intr-o ~edinta prezidata de profesorul de anatomie Henry Albert Harris. Experienta luminilor Apusului ~i-a pus amprenta asupra mentalitatii tanarului profesor roman, a operat o stralucita transmutatie euristica. Gr.T. Popa, omul universal, a facut la randul sau cunoscut ~colilor vizitate peste Ocean ~i in matca Albionului aspiratiile, vointa ~i capacitatile semenilor sai din aria danubiano-pontica. De acum incolo, plecarea la studii in centrele cele mai vestite ~i contactul nemijlocit cu oameni ~i locuri consacrate erau sfaturi constante date celor mai merituo~i discipoli ai sai. Printre ultimii beneficiari ai recomandarilor lui Popa catre colegii occidentali anglo-americani s-a numarat George Emil Palade, fostul sau student, unicul roman ce avea sa primeasca, pana in ziua de azi, un premiu Nobel pentru medicina, in 1974. Confirma astfel, ,,cu asupra de masura", increderea ~i optiunile magistrului sau, dar ~i calitatile ~colii de morfologie din care venea. G.E. Palade - eel care, similar lui Gr.T. Popa, bursierul fundatiei Rockefeller, ~i-a inceput cariera americana la Institutul Rockefeller pentru Cercetare Medicala - admitea a-~i fi faurit nucleul gandirii bio-medicale la Bucure~ti, orientat de Francisc Rainer spre studiul unei anatomii funqionale ,,teleologice", de Andre Boivin, spre patrunderea biochimiei pana in intimitatea proteinelor ~i acizilor nucleici, ~i de Gr.T. Popa spre 1 destoinicia experimentului perseverent ~i meticulos • Paul Gore, medicul-scriitor

Ca medic-scriitor, Gr.T. Popa a semnat nuvele ~i schite, mereu sub misterul unui pseudonim, Paul Gore, care trimitea cu gandul nu doar la propriul prenume, ci ~i la numele de familie al mamei sale, Gorea. Randurile lui Gr.T. Popa ii atesta sensibilitatea sufletului, mereu receptiv la bine, adevar ~i frumos, indurerat la nedreptate, apasat de tragedia r;izboiului, de uratenia comportamentala a semenilor, pe care ii voia imbunatatiti prin cultura ~i educatie, intristandu-1, dar ~i mobilizandu-l. intre incercarile literare s-au inscris o serie de schite, printre care: lmpeifectiunile din nou, Aporii, Bobolocul, Cei plecap, 0 vizita medicala, La 1

Radu Iftimovici, George Emil Palade. Spovedania unui invingiitor, Editura Academiei Romane, Bucure~ti, 2007, pp. 24-25, 30.

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Bucurqti, in drum spre Oituz, La Tazlau, Parintele Ilarion, Mo:j Vranceanu, La boierul Tache, Nai/a Vanturescu,Un /aranist romantic, Laurii izbanzii, Ironia vie/ii, i n/elesul mor/ii, Decep/ii, Rataciri, Flamanzii, Dibuiri sentimentale, 0 vanatoare cu urmari, in cantec de /it era, Izul pamantului, Cercul durerii, Abisul, Spafiul vital !ji autarhie, Subt impresia focului, Amintiri din sat, Amintiri din ora!j. Parte din acestea erau fragmente ale proiectatului roman Ione! Nazuinfa. Cazul controversat al militantului comunist basarabean Timotei Marin, student la Facultatea de Litere ~i Filosofie a Universitatii ie~ene in anii 1920, pre~edinte al Cercului studentesc democrat, a fa.cut ~i el subiectul randurilor lui Gr.T. Popa. Majoritatea paginilor beletristice au dat consistenta ,,insemnarilor ie~ene", revista democratica, dorita echidistanta politic. Fondata de Sadoveanu, Toparceanu ~i Gr.T. Popa la 1 ianuarie 1936 ~i acoperind o perioada de framantari sociale ~i conflicte politice, a supraviejuit pana in 1940, cand a fost suprimata de legionari. 1 Gr.T. Popa a fost silit sa piece din Ia~i . Dupa moartea lui Toparceanu in 1937, locul sau a revenit sonetistului Mihai Codreanu. in aceasta initiativa cu totul laudabila, perseverand in traditia umanismului medical dintotdeauna ~i de pretutindeni, Gr.T. Popa s-a implicat cu daruire, cuget ~i trup, ca publicist, critic literar, eseist, prozator, popularizator al ~tiintei2. Parasita, in 1933, de ,,Viata romaneasca", publicatie exemplara a Ia~ului universitar ~i cultural, dupa mutarea redactiei la Bucure~ti, cetatea moldava a fost rede~teptata la exigentele spiritului critic ~i literar prin noul jumal ,,insemnari ie~ene". in paginile acestuia, eminentul anatomist diseca simtirea sa ~i gandul profund interferand cu natura, mai cu seama natura oamenilor ~i a evenimentelor. De altfel, ~edintele redactionale se tineau, prin 3 grija secretarului de redactie Gr.T. Popa, duminica dimineata , in cabinetul Profesorului. Dupa Armistitiul din 1918 ~i realizarea Marii Uniri, la propunerea profesorilor Rainer ~i Ibraileanu, aproape in fiecare seara se reuneau, la Institutul de Anatomie ie~ean, studenti ~i colegi basarabeni, spre a stapani mai bine limba romana, citind ~i comentand scriitorii romani, mai ales moldoveni. ,,Printre ace~tia, mai toti din grupul « Vietii romane~ti» au dat contributiile lor: Ibraileanu, Sadoveanu, 4 Toparceanu ~i I.I. Mironescu" - i~i amintea Gr.T. Popa • Comparabile, oarecum, serilor de luni ale lui Claude Bernard ~i colocviilor de miercuri ale lui Pavlov, doi Prometei ai fiziologiei de notabila deschidere culturala, Miercurile de la ,,insemnari ie:Jene ,,s reuneau oameni de litere ~i de ~tiinta, facand epoca. intrunirile saptamanale 1

Ion T. Ro~culescu, Profesorul Grigore T. Popa in amintirea contemporanilor (din presa vremii), Editura Sitech, Craiova, 2010, pp. 33-34, 60. 2 *** in memoria lui Grigore T. Popa. Studii, documente, marturii, volum publicat de Universitatea de Medicina ~i Farmacie ,,Gr.T. Popa", Editura Omnia, Ia~i, 1999, pp. 29-42, 92-97. 3 E. Lucinescu, ,,Topil.rceanu la «insemnari iel?ene»", in G. Topdrceanu in amintirile contemporanilor. Antologie de Silvia Popescu, edi1ia a II-a, p. 166, Princeps Edit, Ia~i, 2007, p. 185. 4 Gr.T. Popa, ,,Prietenie til.rzie", in G. Topdrceanu in amintirile contemporanilor. Antologie de Silvia Popescu, edi1ia a II-a, Princeps Edit, Ia~i, 2007, p. 185. 5 Traian Gheorghiu, Miercurile de la ..fnsemniiri ie:jene ", Editura Junimea, Ia~i, 1980.

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ale ,,Insemnar1lor ie~ene", in sala ,,Rockefeller" a Institutului de Anatomie, al caroi ·director era acela~i eficient redactor-strateg ~i neobosit medic-scriitor, derulate la un ceai ~i covrigi calzi, sau la o cafea ~i un corn, se colorau cu taifasuri pline de verva ~i duh, pe cand studentii pandeau ora sa zareasca siluetele celebre ale Ia~ului, care, dupa nume, le erau familiare sau indragite. ,,insemnari ie~ene" a debutat ca tribuna a asociatiei ,,Prietenii Universitatii", alt for strans legat de numele lui Gr.T. Popa ~i incercarea sa perseverenta de a combate ,,ambulantismul" magi~trilor. Straduindu-se sa ridice prestigiul Universitatii, Asociatia, pe care o fondase in 1935, se ocupa in egala masura de probleme culturale ~i ~tiintifice, asemenea Societatii Prietenii Ia~ului, pe care acela~i neobosit 1 Gr.T. Popa o intemeiase • Atingand curand o incontestabila recunoa~tere nationala, continuand sa promoveze, dupa pilda ,,Vietii romane~ti", specificul romanesc ~i talentul scriitoricesc autohton, analiza literara, ca ~i analiza sociala, ,,insernnari ie~ene" pleda pentru ratiune ~i emancipare intelectuala, impotrivindu-se extremismului ~i fanatismului, meschineriei politicianiste ~i imposturii. La ,,Insernnari ie~ene", Profesorol Popa mai era coleg de condei, pe langa Sadoveanu, Toparceanu ~i Codreanu, cu doctorii I.I. Mironescu ~i Eugen Lucinescu, dar ~i cu Paul Anghel, Gheorghe Tudoranu ~i Ion Balteanu, cu Otilia Cazimir, Lucia Mantu ~i Magda Isanos, Stefan Ciobotara~u ~i Traian N. Gheorghiu, Ionel ~i ALO. Teodoreanu, George Lesnea ~i Demostene Botez, Nicolae Tatomir ~i Felix Aderca, Alexandro Pogonat ~i Alexandro Voitin, Orest Tafrali, Emil Diaconescu ~i Ion Petrovici, Stefan Procopiu, Dan Badarau ~i P. P. Stanescu, George Calinescu ~i George Iva~cu, Alexandro Piru ~i Octav Botez, N.N. Tonitza, N.I. Popa ~i Radu Beligan, alaturi de decanul de varsta al ziari~tilor locali, Constantin Meissner. Prestigiul lui Gr.T. Popa a facut ca nume mari ale ~tiintei mondiale, ca profesorii Leriche ~i Grosser, sa apara in revista ,,insernnari ie~ene". Generatii, orgolii ~i unghiuri de vedere diferite slujeau un acela~i scop nobil. ,,Miss-(terul) Grete Popa" - cum i s-a spus la un moment dat printr-un joc de cuvinte - a fost, intr-adevar, vedeta in Ia~ul interbelic al Romaniei Mari - ceea ce remarca, sub titlul tocmai reprodus, ziarul ,,Lumea" din 20 martie 1935, la rubrica ,,Ochiul Lumei". In mod cu totul sernnificativ, in 2004, la aniversarea a 125 de ani de invatamant medical superior in cetatea celor ~apte coline, in memoria crezului jurnalistic al lui Gr.T. Popa, s-a dorit ~i s-a implinit reeditarea ,,insemnarilor ie~ene" - serie noua, prin implicarea nemijlocita a Universitatii de Medicina ,,Gr. T. Popa" din Ia~i.

1 I. Iancu, Profesontl Grigore T. Popa, jigura proeminenta a viefii universitare ie:jene, Institutul de Medicina ~i Farmacie Ia~i, 1879-1979, Centenar Sesiune ~tiintifica, 1-2 decembrie 1979, Ia~i, vol. I, p. 47.

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Concuren(a in campul civiliza(iei

in studiul intitulat Starea trecuta ~i actuala a Ja~ului, Profesorul Gr.T. Popa, ie~ean de onoare, explica raspicat: ,,Universitate de prim rang la Ia~i inseamna Ia~ul in fruntea culturii romiine~ti. Universitate decazuta la Ia~i inseamna ie~irea acestui ora~ din riindurile factorilor determinanti ai culturii nationale". Doua categorii de argumente convingatoare pentru fiinta romiineasca se impuneau. Una se referea la capacitatea universitatii de a educa identificarea complementara in alteritate, in diversitate, atiit de la indemiina ie~enilor, ce au convietuit peste veacuri cu popoare, culturi ~i civilizatii diverse: ,,Cu spirit larg considerata dezvoltarea unui popor, nu trebuie sa ne sparie adausurile civilizatiei altora. Bine utilizate, elementele de cultura nemtasca ~i rusasca pot spori enorm tezaurul nostru national, in care toate sa se contopeasca, pentru o forma noua de civilizatie. Dar tocmai de aceea e nevoe de un aparat central puternic, asimilator al tuturor nuantelor de cultura, ~i acela nu poate fi deciit universitatea" (Starea trecuta ~i actuala a Ia~ului). 0 a doua categorie comporta capacitatea universitatii de a deslu~i ideologiile in mod obiectiv, de a decripta mesajele politice ~i a disceme pericolul ori ocazia favorabila. in 1932, prin urmare, la un deceniu ~i jumatate de la Revolutia din Octombrie ~i instaurarea celui dintiii regim sovietic al lumii in coasta Romiiniei, Gr.T. Popa invita, cu deplina responsabilitate, la estimarea fenomenului in toate implicatiile sale multiple, la intelegerea transformarilor dramatice petrecute la rasarit de Nistru, pentru a minimiza posibilele riscuri viitoare, pentru a trage invatamintele cuvenite din vreme: ,,Cu idei nascute in universitatea din Ia~i se poate lupta mai cu succes impotriva bol~evismului rusesc deciit cu armata la Nistru. Dar pentru a-~i indeplini aceste rosturi universitatea din Ia~i trebue pusa pe primul plan de atentie, sa i se dea toate mijloacele de dezvoltare ~i de prosperare". Mult timp, doar prin centrele V echiului Regat - aprecia Popa - vom putea proba vitalitatea filonului autohton: ,,Numai prin Bucure~ti ~i Ia~i vom dovedi noi vreodata ca avem o cultura romiineasca, ca avem merite reale comparabile" cu cele ale marilor no~tri vecini ~i rivali (Starea trecuta ~i actuala a Ja~ului). Conform devizei sale, nu era suficient sa piara lumea, ca pret al dreptatii, ci lumea trebuia sa piara ca pret chiar ~i numai al adevarului. ,,Alta.data se spunea aforistic: ~i de s-ar pierde lumea totu~i sa se faca dreptate. Am mai putea adauga: chiar daca s-ar risipi universul, adevarul sa nu fie innabu~it. Caci asta inseamna ca dreptatea ~i adevarul trebue sa ciirmuiasca existenta ~i nu existenta sa ciirmuiasca 1 adevarul ~i dreptatea" . Acest principiu-ratiune de a fi, de altfel, 1-a condus spre finalul tragic al unui parcurs existential consecvent absolutului, spre experienta unui martiriu liber acceptat. Pentru ca: ,,Firea lucrurilor cere ca, daca purtam o flacara, s-o trecem altora, peste abisul mortii" - spunea Gr.T. Popa, incheindu-~i cursul magistral din 1942, de la Facultatea de Medicina din Bucure~ti, unde tocmai 1

Gr.T. Popa, ,,Substratul biologic al progresului", in Viafa Ji societate, FRLA, Bucure~ti, 1946, p. 293.

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17 1

se transferase, inlocuindu-1 pe Francisc Rainer, apreciatul sau magistru • ,,Traim •prin altii in spiritul carora urma noastra, daca am lasa-o, nu s-ar pierde". Vizionarismul sau s-a implinit. Pa~ii sai profetici ii descoperim, reconstituind anevoie calea sa de suferinµ ~i bucurie, de lumina ~i de nadejde ce a strabatut tenebrele mintii ~i simtirii omene~ti, la cumpana catastrofelor evolutive de la mijlocul veacului al XX-lea. Articolele din volumul Via/a '}i societate, majoritatea publicate in ,,Insemnari ie~ene", vin sa confirme eminenta savantului democrat Gr.T. Popa, eel caruia, a~a cum s-a spus incii in epoca sa, ,,Medicina ii pune la indemana mijloacele cu care tamiiduie~te corpul uman", ~i eel care ,,ca om politic ~i ca om de initiativa ( ... ) lupta ca sa prefaca corpul social". Acesta era ,,Miss-(terul) Grete Popa". Spiritul §tiinfific in Moldova

Patruns de rostul oamenilor ~i semnificatia evenimentelor derulate sub auspiciile unui generos genius loci, Gr. T. Popa a scris despre Ia~i ~i despre Moldova, despre zbaterea de fiecare zi a ~colii, a spitalului, a cetatii. Liceului National din Ia~i, pe care 1-a absolvit in 1911, institutie de traditie ~i perpetuat renume, i-a liimurit rolul ~i legaturile cu Academia Mihaileana, cu Societatea de Medici ~i Naturali~ti, cea dintai societate ~tiintifica din Principatele Dunarene, ~i cu prima universitate a Romaniei, fondata de Principele Cuza la 1860 in fosta capitala a Moldovei. Dovedind stapanirea datelor ~i familiaritatea cu istoria nationala, discursul doctorului Gr. T. Popa cauta, mereu, in evocarea retrospectiva a faptelor, desprinderea unei lectii pentru contemporaneitate, reaprinderea sacrei datorii a memoriei (Desvoltarea spiritului '}tiin/[fic fn Moldova .yi contribu/ia Academiei Mihiiilene la aceastii desvoltare). Destinul avea sail a~eze in locul ce i se cuvenea, spre a aplica spiritul critic ~i a-~i imparta~i efervescenta idealurilor: in 1930, ca pre~edinte al corpului didactic ie~ean, in 1932-1933 ~i 1939-1940, ca pre~edinte al Societatii Medico-Naturaliste, iar intre 1938-1940, ca decan al Facultatii de Medicina ~i prorector al Universitatii din Ia~i2. Institutiile medicale filantropice ie~ene, precum Azilul de biitrane ,,Eufrosina Balf', Eforia Spitalelor ,,Sfantul Spiridon" in ansamblul sau, i-au captat, deopotriva, interesul. Asistenta sanitara ~i sociala se gasea in situatia de a imbina vechiul deziderat moral al filantropiei ~i milei cre~tine, predicata de Biserica ~i transpusa in fapt prin a~ezamintele sale caritabile, cu exigentele moderne ale unei societati in care mentalitatea laica i~i cerea acceptarea ~i aplicarea. Odata cu punerea la punct a serviciilor de asigurari de sanatate de stat se preluau, in maniera nesatisfacatoare, din pacate, atributii socio-administrative importante, detinute anterior precumpanitor de autoritatile ecleziastice, de societatile filantropice, alaturi de influente persoane caritabile. Trecerea nu a fost intotdeauna un succes; din contra, a~a cum reliefeaza 1

2

Gr.T. Popa, Evolufia unui om $i evolufia unei $fiinfe, Tipografia Cultura, Bucure~ti, 1942, p. 37. Ion T. Ro~culescu, op. cit., pp. 32, 52, 56-59.

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Gr.T. Popa, el insu~i ales epitrop al Spiridoniei (1938-1940), surse de neajunsuri grave erau, pe de o parte, politizarea excesiva ~i instabilitatea conducerii, iar, pe de alta parte, abandonarea institutiei filantropice: ,,E de mare itnportanta sa nu fie sdruncinate sentimentele adanci de caritate ale marelui public, aratand ca vointa testatorilor nu conteaza ~i deci donatiile care eventual s-ar face, i~i vor schimba destinatia. Oamenii vor sa traiasca dincolo de existenta lor materiala, prin actele ~i vointa lor, care sa se perpetueze in timp. Trebuie sa le lasam aceasta credinta, aducatoare de noi donatii ~i sa nu le-o risipim prin acte nechibzuite, care se do1 vedesc pana la urma a fi profund daunatoare societatii" . Prin aceasta dragoste de aproapele ~i credinta in divinitate, prin ravna de a dobandi o ve~nica amintire, ,,( ... ) Spiridonia din Ia~i - conchideau Popa ~i colaboratorii sai, A.M. Frimu ~i Gr.Gr. Iamandi - este eel mai bun solidarizator de oameni, peste generatii" (Spiridonia din la§i, Raportul dintre Spiridonie §i Facultatea de Medicina, Azilul de batrane ,,Eufrosina Bal§"). Mai mult, Spiridonia opera unirea intre romanii suferinzi de pretutindeni: ,,Pana la exproprierea mo~iilor ea i~i indeplinea rolul atat de bine, incat actiunea ei intinsese faima pana peste vechile graniti ~i numero~i basarabeni ~i bucovineni cautau ingrijire in spitalele ei, iar unii dintr-in~ii ~i-au lasat apoi averile acestui a~ezamant, stabilind astfel, in raport cu suferinta umana, o timpurie unire romaneasca" (Raportul dintre Spiridonie §i Facultatea de Medicina). Odata devenit universitar, spitalul ,,Sfantul Spiridon" din Ia~i, ca ~i alte stabilimente spitalice~ti, a evoluat in stransa dependenta de Facultatea de Medicina, cu prioritatile ~i cu dificultatile sale. Gr.T. Popa a incercat sa evidentieze unele din aceste opreli~ti, generalizand cazurile particulare, individualizandu-le ca tip de problema universitara provinciala, in care ~i altii se puteau regasi ~i afla solutii. Ce ~i cat s-a schimbat fata de 1929? Iara ce spunea Gr.T. Popa mai ieri, parca vorbind despre zilele de azi: ,,Dar cand e~ti calic ~i o spui tuturora, cand nu ai spitale ~i te distrug epidemiile, cand populatia sufera de foame, cand nu ai laboratoare in Universitati ~i nici biblioteci, sa faci o catedrala de un miliard, sa ridici statui ~i arcuri de triumf, inseamna sa nu-ti dai seama ce faci. Acuma chiar, dupa ce am milogit imprumuturi in tara straina - pentruca am ajuns la limita rezistentei financiare - ne vine gust a doua zi dupa incheiere sa construim catedrala de un miliard ~i sa facem iar sarbari costisitoare ~i sa ridicam nu ~tiu cate statui" (Facultatea de Medicina din la§i ca tip de problema universitara provinciala). Blestemul ,,eternei reintoarceri", al zborului in cerc, al starii pe loc! Intransigent, Profesorul Popa a respins intotdeauna jumatatile de masura, verdictul sau vizand o decantare axiologica reala, o ierarhizare a mintilor izvoditoare, dupa modelul celor care au izbandit: ,,Scoala este in conceptia barbatilor no~tri de stat un accesor, nu ceva esential, cu toata protestarea lor oratorica". insa, a~a cum ~i frebuie: ,,Pentru

1

Gr.T. Popa, Spiridonia din la!ji, ,,Insemnari ie~ene", Anul III, No. 6, Institutul de Arte Grafice Brawo, Ia~i, 1938.

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Americani Universitatea este parghia de rezistenta ~i obiectul principalei lor atentii" (Facultatea de Medicina din la$i ca tip de problema universitara provinciala).

Imperativul categoric in mod incontestabil, ca om de ~tiinta, om de litere ~i om al cetatii, Profesorul Gr.T. Popa a trait sub semnul imperativului categoric al ideilor ~i al moralei absolute. A analizat, cumpanind in balanta gandului, viata universitara romaneasca din Ia~i, Bucure~ti ~i Cluj, a identificat problemele false ori reale ce ii stateau in fata, ~i-a sustinut mae~trii ~i ucenicii de exceptie, a comparat, a integrat realitatile romane~ti intr-un program-cadru european avant la lettre (un ,,program Cambridge", ar putea fi numit) (Din via/a universitara, Problema fnvafamiintului superior, Procesul fnva/amiintului, Drama universitara, Starea trecuta $i actuala a la$ului, Facultatea de Medicina din la$i ca tip de problema universitara provinciala... ). Ratiunea, bazata pe cunoa~tere ~i pe emotie, pe vointa ~i pe morala, era invocata sa indice solutii: ,,Creerul insa, care guverneaza intreg organismul, reu~e~te sa se sustraga mai mult tiraniei modelatoare a mediului, carui adeseori i~i impune initiativa sa, a~a cum se intampla in cazul omului. Mediul il influenteaza ~i pe el prin organele de simt, dar influenta aceasta este un stimul ~i nu o rigida impunere. A~adar, creerul este substratul biologic al progresului" (Substratul biologic al progresului). in desavar~irea acestui deziderat al propa~irii, arata Grigore T. Popa, institutia Universitatii trebuie privita ca Statul Major al o~tirii nationale, capabil sa elaboreze tactici ~i strategii educationale, ai carei generali sunt profesorii, deoarece orice ,,initiativa a fost totdeauna ~i va fi legata de Universitate, care este centrul ~i plamaditoarea de mi~cari intelectuale". $i ,,absurditatile patente cand sunt sociale au nevoie sa fie respinse, ~i pentru asta ele trebue sa fie cercetate ... Unde sa se cerceteze atent incercarile de organizare ale altei civilizatii, decat in campul ideilor, in lumina criticii nepartinitoare. $i asta in universitate ar trebui s-o gasim" (Starea trecuta $i actuala a la$ului). Entuziasmat de lectia occidentala, indeosebi americana, a insufletit la Ia~i 1 societatea ,,Prietenii Universitatii" ~i a luat parte la infiintarea Universitatii Libere ori a Universitatii Muncitore~ti ( 1939-1940)2. Pentru el, cartea de vizita ~i destinul Ia~ului erau insu~i destinul Universitatii. Universitatea ie~eana trebuia sa fie perceputa, onorata, ca un Cambridge ori Oxford al romanilor, trebuia sa ajute la faurirea, la desavar~irea sperantelor nationale insele: universitatile, ,,ca ~i parlamentul, oriunde ar functiona, reprezinta poporul, pentru ca in ele se manifesta suma calitatilor unui neam, ~i totalitatea sfoqarilor spre gasit orientari noi" (Facultatea de Medicina din la$i ca tip de problema universitara provinciala). in acest sens, Universitatea ie~eana, in opinia lui Gr.T. Popa, avea de jucat un rol important in politica nationala ~i internationala loco-regionala: ,,E singura universitate 1

2

Ion T. Ro~culescu, op. cit., pp. 52, 59. I. Iancu, foe. cit., p. 4 7.

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20

romaneasca in care s-a format un fel de traditie culturala, in care puterile critice ale spiritelor fine s-au dezvoltat mai mult, ~i in care invatatura a capatat un inteles mai inalt. Epoca pregatitoare unei mi~cari de creatie intensa a trecut deja peste Ia~i, unde s-ar putea - mai bine am zice s-ar fi putut - incepe adevarata creatie" (Starea trecuta :ji actuala a la:jului). Din pacea bibliotecilor, universitarul are, in consecinta, de coborat in agora cetatii pentru a-~i supune fantezia probei realitatii, pentru a oferi solutii viabile societatii, intrucat, avertiza Gr.T. Popa: ,,Degradarea se face incepand cu creierul". Opera sa insa~i se poate oricand metamorfoza intr-un virtual Institut de Neuro~tiinte, pe care il credea atat de necesar: ,,Exagerand intrucatva, am putea spune ca in locul atator conferinte ~i asociatii mondiale ar fi mai nimerit sa se intemeieze un uria~ institut de cercetare a sistemului nervos ~i prin el sa se descopere ~i sa se difuzeze cuno~tintele privitoare la eel mai teribil explozibil cunoscut ~i care este substanta nervoasa. in tot cazul, pana azi eel putin, se pare ca savantii sunt mai ingrijorati de 1 energia atomica ~i mai putin de energia cerebrala, ceea ce ni se pare a fi o gre~eala" • Civilizafie §i cultura

Dupa cum mentiona in Evolufia unui om :ji evolufia unei :jliinfe, problemele de invatamant ~i procesul asimilarii de cuno~tinte 1-au preocupat in tot timpul activitatii universitare. Deja, ca student, fusese desemnat de colegi secretar general (1912-1913), vicepre~edinte (1913-1914) ~i apoi pre~edinte (1914-1919) al Societatii Generate a Studentilor din Ia~i2. Nu intamplator, Profesorul Popa a indeplinit apoi functii de conducere in invatamantul superior atat la Ia~i, cat ~i la Bucure~ti. Ca ~i marele Victor Babe~, era convins ca ~tiinta ~i universitatile sunt cea mai percutanta expresie a patriotismului ~i dorea trezirea con~tiintei conationalilor, inscrierea natiunii sale in noua ordine mondiala nu doar prin ,,starea de spirit momitareasca", prin imitatia automata a altora, ci prin asumarea unor realitati moderne ~i prin implicare civica sustinuta (Starea trecuta :ji actuala a la:jului). Gr.T. Popa a criticat, a~adar, fara crutare ~i fara partinire, dupa o atenta observatie ~i studiu concret al problemei, ineficienta reformelor dese, limitate ~i nesubstantiale - ,,pruritul legislativ", cum il numea -, voluptatea schimbarilor aleatorii in sistemul instructiei publice din tara, precum ~i incompatibilitatea dintre atributiile didactice ~i alte functii sociale, politice ori chiar profesionale clientelare, care impiedica profesorul universitar de la dedicarea sa intru totul misiunii educative, universitatii. Ca un ecou peste vreme, verdictul Profesorului Popa rasuna implacabil ~i azi: ,,invatamantul e ca ~i distrus. Cererea asta a mea de-a se stabili o incompatibilitate intre politica ~i ~coala, desigur nu va fi inteleasa de nimeni, nici de acei care

1 Grigore T. Popa, ,,Boala secolului", in Reforma spiritului, Editura Viaµ Medicala Romaneasca, Bucure~ti, 2002, p. 241. 2 Ion T. Ro~culescu, op. cit., p. 52.

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momentan nu fac politica, fiindca in sinea lor aproape toti nutresc speranta ca vor -face-o candva" (Problema fnva/amantului superior). inalta stima pe care o avea fata de educatia prin ~tiinta ~i pentru ~tiinta, conditie de civilizatie, de evolutie creatoare, o va marturisi constant, pana in cele din urma conferinte, evidentiind legatura dintre cunoa~tere, actiune ~i perfectionarea ,,substratului biologic al progresului" - creierul ~i, cu deosebire, cortexul cerebral. A~a a fost lectia rostita in 1945, la deschiderea anului universitar, la Facultatea de Medicina din Bucure~ti, al carei decan tocmai devenise (Substratul biologic al progresului). $i, cum cultura prin excelenta este un fapt personal ~i doar civilizatia are anvegura sociala, in viziunea lui Gr.T. Popa, daca nu toti putem fi oameni de cultura, prin vocatie, toti putem fi ~i suntem oameni civilizati, prin formatie. Decalajul dintre cultura ~i civilizatie, insa, i se parea periculos, deoarece, in drumul evolutiv de la arhipallium catre paleopallium ~i neopallium, impulsivitatea colectiva salbatica a creierului reptilian a reu~it sa fie controlata doar cultural 1• Inca din eel dintai articol publicat in ,,insemnari ie~ene", argumenta: ,,Cand civilizatia pierde tutela culturii, se produc dezordini spirituale care due la anarhia actelor (sociale)". Disecand amenintarile pseudo-culturii, accentua ca ,,singurul remediu intr-o astfel de vreme critica este sa se intensifice cultura, pentru a o pune la nivelul civilizatiei" ( Civiliza/ie §i cultura). Estima conflictul dintre civilizatia sociala ~i cultura personala ca generator de crize ale valorilor menite a repune temeiurile, ,,tezele'' societatii ~i individului in antiteza, spre a redimensiona - hegelian - sintezele innoitoare. Pentru Popa, intr-o actualitate continua: ,,Criza actuala este o criza a culturii" (Substratul biologic al progresului). in rostirea de mai tarziu a filosofului Constantin Noica, acest tip de cultura ar fi echivalat intelepciunii, unei intelepciuni cultivate, desigur: ,,Despre intelepciune prin cultura. Nu ca intelepciunea sfinteniei; nu ca intelepciunea indiana. Aceasta intelepciune, prin cultura, ca forma ultima a culturii, cultivarii de sine, ne e data noua"2 . ,,Formator de personalitati", cum definea statutul profesorului, Gr.T. Popa credea, neclintit, in inalµirea omului prin cultura. ,,Nevoia de cultura - accentua Magistrul -, daca este simtita, merge inaintea tuturor celorlalte, chiar ~i inaintea nevoii de aparare, fiindca cultura e la baza tuturor celorlalte manifestari" (Facultatea de Medicina din la§i ca tip de problema universitara provinciala). Iata, prin urmare, rolul hotarator al ~colii ~i plasmuitorilor de con~tiinte, al personalitatilor, in aceasta ,,antropologie redagogica" la care Gr.T. Popa a trudit toata viata, daruindu-~i-o cauzei cdor multi ~i anonimi ai neamului sau, din care se ridicase ~i pe care i-a pretuit mereu: personalitatile, crescute ,,pe o masa de anonimi ~i de neprecizati, ( ... ) comunica celor distin~i puterea lor de evolutie, putere pe care imprejurarile o fac exceptionala. Din aceasta pricina este o stransa legatura intre 1

Mihai Draganescu, ,,Cultura ~i societatea cunoa~terii", in Dan Tufi~, Florin Filip, Limba romanii in societatea iriformafionalii - societatea cuno~terii, Editura Expert, Bucure~ti, 2002, pp. 441-442, 459-472. 2 Constantin Noica, Carte de infelepciune, Editura Humanitas, Bucure~ti, 2009, p. 19.

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masa $i intre personalitati. Nu se pot gasi popoare cu totul ignorante care sa abunde in genii $i nici popoare pe un inalt etaj de civilizatie a caror figuri reprezentative sa fie simple mediocritati sau personalitati $terse". (Starea ttecuta §i actuala a la§ului). Fata de metamorfozele imposturii, mai ales academice, acest om de cuvant $i al cuvantului a fost neiertator, fapt atestat de paginile polemice publicate sub titlurile Cu prilejul unui tratat de anatomie umana (cercetare critica) ori Despre ,,Paternitate ", ,,Prioritate" §i... ,, Mahalagism ". Stapan pe meserie $i asumandu-$i riscul de a fi incomod, de o probitate desavar$ita, considera de dreptul $i datoria sa sa apere adevarul, sa fie justitiar, sa se implice, fie ca lupta impotriva plagiatului, fie ca argumenta meritul sau fundamental in descoperirea sistemului port-hipotalamo-hipofizar, fie ca declara razboi dictaturilor, ,,manelismelor" teoretice $i practice de orice fel. Ultima luare de pozitie a lui Gr.T. Popa a fost la 15 octombrie 1946, cand a depus la Senatul UniversitaJ:ii din Bucure$ti un Memoriu ce ataca Legea 658 din 24 august 1946. Preconizand ,,revolutionarea" cadrului organizatoric al procesului educational in Romania postbelica, prevederile anihilau practic autonomia universitara (articolele 24 $i 64 bis). Fidel principiilor unei eteme Magna Carta Universitaria, Profesorul a rectionat prompt: ,,Universitatile noastre nu merita blamul care li se da prin Legea 658. (... ) Sa apelam la forurile in 1 drept pentru a o indeparta" • Lec(ia de experien(a

,,Lectia de experienta'', exemplul personal, a fost fundamental in orientarea pedagogica, atat de clara, documentata $i avansata, a lui Gr.T. Popa (Problema fnvatamantului superior). ,,Tinerii trebuie sa vada pe maistrul lor lucrand pentru a putea lucra $i ei" ... ,,Peste tot gasim definit rostul profesorului din invatamantul superior ca un rost de cercetator intr-o specialitate, propagator $tiintific $i formator, nu numai de profesioni$ti dar $i de noi cercetatori", sublinia cu tarie. ,,Starea actuala a cuno$tintelor omene$ti impune, oricui are de participat la educatia altora, convietuire sustinuta cu ideile specialitatii sale. Formularea $i verificarea adevarurilor $tiintifice nu pot fi straine celora care le transmit mai departe." Concluzia era unica $i ferma: ,,Contest cu toata energia posibilitatea de existenta a profesorului bun, fara ca el sa fie totodata $i cercetator. Pentru universitate acest adevar este incontestabil". Profesorul bun ,,poate fi erudit $i savant, pe cand cercetatorul poate fi descoperitor $i inventator". Gr.T. Popa insu$i era con$tient de nevoia personalizarii principiilor. ,,La amandoua categoriile - adauga -, valoarea oamenilor de $tiinta poate merge de la ucenicie pana la geniu." in fond, dezbatand Problema fnva/amantului superior, el insu$i alesese drept motto o reflectie a lui Elliot Smith, care intarea rostul creator al invataturii: "All true assimilation of knowledge is of the nature of original research". Cu un student obi$nuit, $i profesorul ramanea unul 1

p. 38.

Grigore T. Popa, Reforma spiritului, Editura Viata Medicala Romiineasca, Bucure~ti, 2002,

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obi~nuit, bun ~i eel mult sever, insa studentul cu aplecare spre intelegere aprofun-data ~i experiment trebuia sa afle ~coala in care sa se formeze, omul de la care sa deprinda ,,lectia de experienta", trecand, totodata, de la individualism ~i ,,spiritul de multime la spiritul de echipa" (Problema tineretului). De aceea, modelul este etalonul universitar, ,,pentru oricine vrea sa traiasca pe linia progresului, folosindu-~i puterile toate". in figurile paradigmatice ale oamenilor mari, ,,cautam indreptari pentru viata ( ... ), urmarim felul cum au trait, cums-au dezvoltat ~ice au lasat dupa ei", explica universitarul ie~ean (Focul sacru al $fi1n/e1. I. Cantacuz1no ). in paginile sale, Gr.T. Popa a schitat portrete ale unor astfel de mentori, pe care invatamantul romanesc ~i el insu~i i-au avut: loan Cantacuzino, animat de ,,focul sacru al ~tiintei", Francisc Rainer, savant de o ,,cultura uria~a" (Profesorul Fr.I. Rainer), sir Grafton Elliot Smith, membru al Academiei Britanice, ,,vestit antropolog ~i cercetator al creerului" (,Jnsemnari ie~ene", Or1g1nile civil1za/1ei, Despre ,,Paternitate ", ,, Prioritate" $1... ,, Mahalag1sm"), dar ~i Rene Leriche, eel care i-a validat unele rezultate experimentale (Scr1itori stra1n1), ori genialul Claude Bernard, creatorul filosof al metodei experimentale (Claude Bernard $1 metoda experimentala). 0 frumoasa pledoarie pentru ~tiinta ~i descoperirea unor noi universuri, care aduc pe investigator in preajma dumnezeirii, este deopotriva realizata odata cu evocarea lui Pasteur. in discursul de receptie la Academie, Pasteur destainuise ca notiunea de infinit, care atrage ~i se cere mereu explicata, reprezenta de fapt, pentru el, expresia supranaturalului, care sala~luie~te in inima fiecaruia, ideea de Dumnezeu nefiind decat o forma a ideii de infinit: ,,La notion de l'lnfini dans le monde, j' en vois partout !'inevitable expression. Par elle, le sumaturel est au fond de tous les creurs. L'idee de Dieu est une forme de l'idee de l'Infini" (L 'esprit d'aventure en science). Cuvinte de lauda a avut Gr.T. Popa ~i pentru Ivan Petrovici Pavlov, laureat Nobel pentru medicina, contemporan lui, care inaugurase era psihofiziologiei, aplecandu-se cu har asupra activitatilor nervoase superioare, departajand primul ~i eel de al doilea sistem de semnalizare, cautand intelegerea instinctelor ~i intuitiei, fanteziei ~i jocului tert al ideilor, prin evocarea semnalelor. Gandirea ~i imaginatia se profilau prin prisma reflexelor conditionate ~i neconditionate, a limbajului ~i memoriei. Pavlovismul i-a oferit ~i el elemente de prim ordin in conturarea rostului de clectie al scoartei cerebrale: ,,Transformari lente, complicatie progresiva ~i joc antagonist de forte, astea sunt proprietatile fundamentale ale scoartei cerebrale, a~a cum le-a evidentiat Pavlov. Aceste proprietati, creerul uman, care este instrumentul gandirii ~i al actiunii, le-a impus ~i societatilor pe care le-a format ~i din care a facut mediu obligator pentru om" (Substratul biologic al progresului). Marturisirea de credinµ a lui Gr.T. Popa, formulata simplu, concentra valentele ontogenetice ale umanismului academic: ,,Universitatea trebuie sa fie o adevarata mare familie, in care stimulul trebuie sa vie in chip natural de la cei mai in varsta catre cei mai tineri ~i in care nivelul intelectual trebuie sa fie mereu in urcare.

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Mediul universitar trebuie sa fie atragator, datator de satisfactii inalte tuturor celor cari se afla in el ~i starnitor de admiratie pentru cei din afara. Asemenea mediu ~i asemenea atmosfera nu se pot insa stabili numai pe rutina uscata sau simpla disciplina impusa. Mai este nevoie acolo ~i de suflet, de simtire adanca comuna, care sa piece din iubire omeneasca ~i care sa aspire mereu spre inaltari nobile" (Profesorul I. Cantacuzino).

Or, acest mediu capata vitalitatea empatica a personalitatilor - adica a modelelor sale -, tot atatea ,,motive de transformare" interactiva a intelectului, apte sa ,,scanteie franturi din sufletul" lor, daruindu-se operei (Evolu/ia unui om ~i evolu/ia unei $fiin/e). Curentul nazuin(elor bune

De un accentuat militantism cntic, realizat plenar in plan profesional ~i spiritual, Profesorul Popa nu a putut fi agreat de toti, trezind invidii, resentimente, ostilitati. A fost atacat atat de legionari, cat ~i de comuni~ti, renegat de fo~ti 1 colaboratori, ca ~i de simpli ne~tiutori . La Ia~i, in deceniile '20-'30 ale secolului trecut, Gr.T. Popa, alaturi de curnnatul sau, profesorul Radu Cernatescu, adunase 2 ,,bani pentru ajutorarea luptatorilor fasci~ti plecati in Spania" , iar, prin conferinte ~i randurile publicate in ,,insernnari ie~ene", prin atitudine, se opusese mi~carilor de dreapta aparute in propoqie ingrijoratoare ~i printre studenti. ,,Suntem azi la o mare cotitura a spiritului omenesc", avertiza savantul, demonstrand ,,falsitatea ~i incompatibilitatea rasismului cu principiile ~tiintei" ( ... ) ,,Se pune intrebarea rezuma Profesorul - daca exista putinta egala de perpetuare pamanteasca pentru toti oamenii sau trebue sa se faca o selectie sangeranda, cu disparitie de popoare intregi. Aceasta este problema cea mai insemnata a zilelor noastre." ( ... ) ,,in alte domenii de preocupari umane" - sublinia Gr.T. Popa, care califica teoriile rasiste ca puerile ~i nefondate -, o asemenea baza de discutie ar fi cu totul ridicola ~i orice teorie cladita pe asemenea baze s-ar narui sub hohote de ras." Aceasta incercare de . ,,antropotehnie", pe care o compara cu zootehnia, ,,aceasta selectiune rasiala, care confera superioritati innascute, (... ) face ca spiritul contaminat sa devie surd pentru toate legile ratiunii" 3. Apoi, antrenat din convingere in combaterea dictaturii antonesciene, in aprilie-iunie 1944, Gr.T. Popa a fost printre initiatorii ~i sernnatarii ,,Memoriului intelectualilor" pentru ie~irea tarii din razboiul antisovietic, document 4 adresat mare~alului Antonescu . A luat in mod similar atitudine contra dictaturii comuniste. Astfel, la 15 ianuarie 1945, in lectia de deschidere a anului academic la Universitatea din Bucure~ti, proaspat instalat decan al Facultatii de Medicina, G~.T. Popa ~i-a expus punctul de vedere asupra Substratului biologic al progresului. In aula Facultatii de 1

Ion T. Ro~culescu, op. cit., pp. 38-39. Traian Gheorghiu, op. cit. 3 Gr.T. Popa, ,,Introducere la un studiu al raselor", in Viafii ~i societate, p. 108, 126. 4 Ion T. Ro~culescu, op. cit., pp. 64-68. 2

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Drept, cu acest prilej, erau prezenti Regele Mihai ~i Principesa Elena, alaturi de ·rector, profesorul Simion Stoilov. Facand apel la legile ~i devenirea biologiei ca ~tiinta, la logica viului, in care evolutiile decurg lent ~i nu provin dintr-un fixism catastrofic precum la Cuvier, raportandu-se la orizontul de fagaduinte al neocortexului bine instruit, a lamurit ca transformarea revolutionara, adica brutala, a societatii nu prea avea soqi de izbanda. Savant democrat, apara nepartinic libertatea, con~tient fiind ca aceasta ,,nu poate fi efectiva daca nu-i protejata de lege pentru a nu se abuza de ea". Dar: ,,Protectia ~i controlul libertatii trebuesc facute printr-un sistem de guvemare liber", el insu~i ,,pus sub controlul cetatenilor" ( ... ) ,,0 societate libera insa, este, mai ales, produsul unei minti libere ~i inainte de a stabili libertatile in alcatuiri sociale trebue sa le stabilim in scoaqa cerebrala a cetatenilor" (Substratul biologic al progresului). in acest macrocosmos biocenotic dainuind dupa chipul ~i asemanarea microcosmosului antropic, totul era dictat de creier, substratul biologic al progresului, deci al mintii ~i discernamantului omenesc: ,,in tot cazul insa procesul de transformare al societatii, expresie a transformarilor din scoarta cerebrala, trebue sa poarte pecetea principiilor de organizatie ale acesteia. Si societatea ~i scoaqa creierului au ajuns sa fie alcatuiri extrem de complicate, care nu se mai pot modifica brose, fara primejdia distrugerii. Progresul care este evolutiv ~i nu catastrofal cere sa nu intrebuintam violenta pentru a determina primeniri, ci prin expresie libera sa introducem, treptat-treptat, elementele datorita carora transformarea se face dela sine". Dar, creierul insemna ~tiinta ~i con~tiinta. A~a s-a facut ca, in 3 februarie 1947, in Consiliul Profesoral al Facultatii de Medicina din Capitala, impreuna cu Prof. Ion T. Niculescu, Prof. Gr.T. Popa a luat apararea profesorului de biochimie, coleg de stagii Rockefeller ~i prieten literat Vintila Ciocalteu, care il ~i precedase in functia de decan. Vestitul biochimist, al carui nume apare inca pomenit in istoria ~tiintei mondiale, in special in denumirea reactiei Folin-Ciocalteu - de evaluare a capacitatii totale reducatoare a unei probe ~i parte a reactiei Lowry de dozare a proteinelor -, i~i disputa catedra cu farmacistul Simion Oeriu. in mod tragic, sub presiunea psihica exercitata asupra sa in ultima perioada, Vintila Ciocalteu a cazut in plenul ~edintei, rapus de un atac cerebral. Gr.T. Popa a fost nu doar afectat de acest episod, ci ~i acuzat de a-1 fi favorizat. ,,Chestiunea e serioasa", nota in propriile insemnari Profesorul Popa. ,,Ne aflam in fata unui caz de constrangere 1 morala ~i de presiune politica unica in Analele Universitatii" • Membru in Comisia de Armistitiu, Oeriu era un privilegiat colaborator al puterii recent instaurate, reu~ind rapid, peste cadavrul profesorului Ciocalteu, sa dobandeasca ravnita pozitia universitara. La 14 martie 1947, o noua disertatie in Aula Academiei Romane venea sa incununeze rezistenta intelectuala activa a lui Gr.T. Popa, prin conferinta-manifest despre ,,Tensiunea nervoasa ~i boala secolului". Vorbind despre ,,boala secolului", 1

Ibidem, pp. 11-13; 149.

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stresul care, inevitabil, i1 oprima personal fara scapare, Magistrul a dezbatut, in ultima parte a discursului, urmatoarele subpuncte, atat de actuale atunci: Tensiunea cerebrala; Renunpirea la personalitate; Contrazicerea evolutiei; Frica ~i neincredere; Minciuna - principiu politic ~i social; Orientarea spre ura ~i violenta; 1 Atentatul contra libertatii; Statul organism • Se consemneaza ca, in acel moment al sustinerii conferintei, i s-a zis ,,nebun": ,,Il est foul 11 a perdu son esprit de conservation!", ar fi exclamat acad. Stefan S. Nicolau, pe cand asistenta se imbulzea spre 2 ie~iri, ca semn al desolidarizarii ~i de orator, ~i de mesajul sau . Acest ,,du~man al poporului" de formatie anglo-saxona trebuia respins, iar cine nu era cu el trebuia vadit sa-i fie impotriva. Accentul transcendental al con~tiintei ~tiintifice a raze~ului din Surane~tii Vasluiului ce razbise, nesperat de repede, prin har ~i tenacitate, pana in aula Academiei Romane, se dovedea a fi de sorginte cre~tina. Rostit la 15 aprilie 1947 la Ateneul Roman, cuvantul Profesorului Popa intitulat ,,Morala cre~tina ~i timpurile actuale", venea sa incline balanta spre faptul ca ,,mai este posibila azi credinta in lsus Hristos". in fata libertatii de a face orice, a ~tiintei slujind gre~it societatea, a creierelor diabolice in actiune, criteriul de adevar absolut se dovedea, o data in plus, a fi eel cre~tin: ,,Mai ales cele doua simplificari pseudo-~tiintifice «rasismul» ~i «marxismul» au produs ~i continua sa produca ravagii in sufletele oamenilor ~i catastrofe in viata sociala. Alt drum de ie~ire, mi se pare mie, nu · avem acum decat indemnul lui Poincare: «mai multa ~tiinta», dar mai ales mai multa morala cre~tina"3 . Era o morala a dragostei ~i respectului faµi de semeni, care respingea insa neconditionat ~i misticismul, ~i superstitia, ~i ignoranta: ,,Misticismul este semnul neputintei ~i al inferioritatii; e manifestarea celor care au renuntat la ce poate fi mai dumnezeesc in firea omului: creatia" (Orizonturi neelastice §i misticism). De aceea, spre a inlesni progresul: ,,ne trebue o schimbare de spirit"4, unde ceea ce Gr.T. Popa numea ,,spiritualismul teologic" ~i ,,spiritualismul biologic"5 trebuia sa se intalneasca. Explorand Spiritualismul in biologie, Profesorul conchidea, intr-o scriere complementara: ,,Aceasta personalitate divina exista in noi ~i noi existam in ea, perfectionandu-ne impreuna". Parea o confirmare a moralei anticului Xenofan: ,,Doar Zeii nu au dat/ Muritorilor totul de la inceput/ Ci ei in~i~i cauta ~i gasesc treptat/ Lucruri tot mai bune" ... Din timpul recluziunii fortate a lui Gr.T. Popa, in cele din urma luni de viaµi, s-au pastrat, pana de curand exclusiv in manuscris, gandurile referitoare la Limitele

1

Grigore T. Popa, Reforma... , pp. 240-272. Ibidem, pp. 38, 280. 3 Gr.T. Popa, Mora/a cre~tinii ~i timpurile actuale. Mai este posibilii azi credinfa fn lsus Hristos? (conferinta prezentatii in 15 aprilie 1947, la Bucure~ti, la Ateneul Roman), ,,Revista Romana", 1996,an2,nr. l,2,3. 4 Gr.T. Popa, ,,Substratul biologic al progresului", in Viafii ~i societate, p. 304. 5 Gr.T. Popa, ,,Spiritualismul in biologie", in Viafii ~i societate, pp. 40-46. 2

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cunoa:jlerii biologice (intre certitudine §i ipoteza) 1, limite pe care intr-un fel le traia · atunci el insu~i, frant - trup, minte ~i suflet - intre certitudine ~i deplina nesiguranµ. Redactat in casa doctorului Razvan Djuvara, textul era un adaus la cele expuse in Substratul biologic al progresului, unde creierul ~i neopallium-ul sau mai ales, conform celor avansate ~i de Elliot Smith, detineau ponderea, trasau coordonatele directoare. Daca ,,nimic nu a fost in intelect mai inainte de a fi fost in simturi", dupa aseqiunea lui John Locke, ,,azi am putea complecta constatarea asta cu alta: n'.guliCn:rnent cette prcssion.

t

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Raport publicat in Analele Academiei Romane. ,,Memoriile Sectiunii Stiintifice" 7, 1940, Seria III, Tom. XVI.

THE INFLUENCE OF THE SYMPATHETIC ON THE PIGEON'S WING BY GREGOR T. POPA AND

FLORICA POPA INSTITUTE OF ANATOMY, JASSY, ROMANIA FROM THE INSTITUTE OF ANATOMY, UNIVERSITY COLLEGE, LONDON

The results described in our preliminary note to the Physiological Society (Proc. Dec. 1928, Journ. ofPhysiol., vol. LXVII), in which we confirmed the effects of sympathectomy upon skeletal muscle described by John I. Hunter, have been questioned by A. E. Coates and 0. W. Tiegs (Australian Journ. of Exp. Biol. and Med. Sci., vol. VII, 1930, p. 37) who suggest another explanation in these words: G. and F. Popa find that if, in the pigeon, from one to five sympathetic ganglia are severed from their attachments to the brachia! nerves (C. 11-15) and then removed, there occurs a droop in the wing, thus confirming the conclusion of Hunter that the sympathetic nerves subserve plastic tone in the wing musculature. Hunter based his experiment on Langley's statement that the first thoracic nerve is the first by which preganglionic fibres emerge from the cord; so that severing the sympathetic chain immediately in front of this root should suffice to abolish all tone due to sympathetic action. We ourselves failed to obtain Hunter's effect. Meanwhile Popa states that "Langley's account of the sympathetic in birds ignores the presence of white rami th communicantes (preganglionic fibres) above the 14 spinal nerves", thus depriving the bird's wing of the special anatomical peculiarity upon which Hunter relied for his demonstration. Hunter's own observation (if Popa's statement be correct) must therefore clearly be wrong. But it is only necessary to make a dissection of the pigeon's brachia! plexus to realise that a droop in the wing following removal of the attached sympathetic ganglia cannot afford valid evidence to prove that sympathetic nerves play any direct part in maintaining muscular tone, for the ganglia are so firmly attached to the spinal nerves that it is quite impossible to remove them without doing injury to the adjacent spinal nerve fibres ... The sympathetic ganglion is so intimately associated with the main nerve trunk that its removal must

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inevitably produce injury to the latter. When it is remembered that there are five such ganglia, that they are about the size of a pin's head, and that they are comparatively inaccessible, it seems clear that injury to the brachial nerves, with consequent droop in the wing, could not possibly be avoided. (a) In this passage, Coates and Tiegs claim that our results are due to injury of the brachial nerves. We submit photographs of pigeons (Figs. 1, 2 and 3) operated upon by the method described in our note and quoted by Coates and Tiegs. Fig. 1 shows a pigeon, distinguished in our note-book by the numbers 33-927. On 12 November, 1927, its left stellate ganglion was removed. Figs. 2-8 show another pigeon (270-929) from which, on 10 October, 1929, the five left sympathetic ganglia of the brachial region were removed. Both pigeons are still alive at University College, London. The photographs were taken in October, 1930, that is, in the case of pigeon 33-927, 35 months after the operation, and of pigeon 270-929, 12 months after.

Fig. 1.

Fig. 2.

Fig. 3.

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The immediate effects described by Hunter still persist after all these months. Even when the animals walk about in their cage the wing on the operated side droops slightly. After an effort, however, the drooping becomes emphasised and is quite unmistakable. How can this be explained by the assumption of injury to the brachial nerves? Even if half the fibres had been cut they would be regenerated during the one and two years that elapsed in our experiments. As a matter of fact no brachial (somatic) fibres were cut. It might perhaps be argued that damage may have been inflicted on the nerve roots by pressure while lifting the roots. This eventuality we eliminated by purposely handling the brachial roots on the opposite side in the same way without, of course, actually removing the ganglia. No appreciable effect could be detected, however. The relationship of the sympathetic ganglia to the brachial nerves is not so close as the criticisms suggest. These ganglia are surrounded by a connective tissue capsule and their enucleation is very easy. Pending the publication of our work in extenso, we may quote what we have written with reference to the method of removing ganglia: By using a fine hook the roots of the brachial plexus are gently lifted, the corresponding sympathetic ganglia are displayed and with a fine ophthalmic knife the ganglia can be separated without any direct damage to the spinal nerves. After separation the sympathetic chain is pulled out gently and cut with scissors at both ends. (b) The existence of preganglionic fibres above the limit described by Langley is fully demonstrated in our work. Langley states that "the last large nerve to the brachial plexus, i.e. the last cervical nerve, is the first to cause the movements of feathers and other effects of cervical sympathetic stimulation". The last cervical nerve is the 14 th spinal nerve in the pigeon and according to Langley, above this level no nerve fibres from the spinal cord pass to the sympathetic ganglia. Here we present two pictures of brachial nerves and of corresponding sympathetic ganglia together with their reciprocal relations. Fig. 4 shows the 13 th spinal nerve, one segment above the upper limit of the sympathetic origin as described by Langley. A large contribution of preganglionic fibres (Pr.) is made to the corresponding sympathetic ganglion by the somatic nerve (its motor root) and from this sympathetic ganglion a large bundle of fibres (postganglionic fibres (Po.)) joins the somatic nerve. Fig. 5 shows the 12t1i spinal nerve, two segments above the upper limit described by Langley for the origin of the sympathetic. One can see preganglionic fibres (Pr.) coming from the motor root of this nerve to the corresponding sympathetic ganglion. These fibres scatter in the ganglion and some of the postganglionic fibres (Po.), originating in the same ganglion, are seen joining the somatic fibres of the same spinal nerve.

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Fig. 4.

(c) How the positive results of Hunter, which we confirm, agree with this finding of unknown preganglionic fibres is explained largely in our paper (which is not yet published). We quote again a paragraph of this lengthy manuscript: The numerous results reported so frequently in the literature by different observers are due to their adherence to Langley's descriptions and diagram. The white rami have been cut below the level of the 14th spinal nerve, only the larger part of the sympathetic innervation to the skeletal muscles of the left wing has been left intact. It is not surprising therefore that positive results were not obtained constantly. Hunter himself was guided in his experiments by Langley's statement, but he soon observed that the results were more certain if the sympathetic trunk is cut between 13 th and 14th spinal nerves. "In sea gulls I invariably cut the dorsal and ventral trunks immediately behind, or below, the penultimate (13 th) cervical nerve as well1J,,_ In the very place where Hunter cut the sympathetic chain we have found that the sympathetic trunk is really a ganglion. Instead of a simple intemodal ramus, consisting only of nerve fibres, there is in the pigeon a long ganglion containing nerve cells which extend in between the nerve roots. Fig. 6 shows exactly the interganglionic communication of the sympathetic chain between the 13 th and 14th brachia! nerves, the place where Hunter performed the majority of his experiments.

I) John I. Hunter: ,,Lectures on the sympathetic innervation of striated muscle". Delivered by Professor Elliot Smith at University College, London, 1925, p. 33.

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Eventually Hunter, operating on this most important contribution of the sympathetic to the innervation of the skeletal muscles of the wing, obtained almost constant positive results.

Fig. 5.

Fig. 6.

Lucrare publicata in Journal ofAnatomy, 1931, vol. 65, part 4, pp. 407-410.

,....

CERTAIN FUNCTIONS OF THE MIDBRAIN IN PIGEONS BY

GR.T. POPA AND

FLORICA GR. POPA, UNIVERSITY OF JASSY, ROMANIA (FROM UNIVERSITY COLLEGE, LONDON) (COMMUNICATED BY G. ELLIOT SMITH, P.R.S.- RECEIVED SEPTEMBER 15, 1932)

I.- In 1928, in the course of our investigations on the sympathetic system, we found that electrical and mechanical stimulation of the pigeon's brain revealed some striking peculiarities. During that time, a communication to the Physiological Society of the results obtained by Professor W. H. Wilson* of Cairo (presented by Dr. H. A. Harris), which have not yet been published, made us acquainted with phenomena exhibited by the lizards Varanus and Uromastix, which are identical with those seen by us in pigeons. Dr. Wilson found that in the cortex of the colliculus in lizards there is a kind of sensory-motor area, with definite localization for various movements and postures of the body, roughly comparable with those determined by the precentral convolution in the cerebral hemispheres of mammals. In fact, he obtained in Varanus and in Uromastix by electrical stimulation definite areas for flexion of the head on the neck; abduction of the head; extension of the head and neck; rotation of the head; movements of the tail, of the fore-limb and the hind-limb, and contraction of the sterno-mastoid. All these movements were produced by stimulating certain definite points of the cortex of the colliculi and the effects were displayed on the side stimulated. Excepting the latter characteristic, it seems that the functions analogous to those of motor cortex of mammals are performed in the midbrain of lizards by the cortex of the colliculi. We found exactly the same thing in the pigeon. Briefly, the localizations which we noted, and repeatedly confirmed, on the pigeon's colliculi are as follows:

• Quoted with the consent of Professor W. H. Wilson.

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(a) Only a limited area on the surface of each colliculus is excitable. This area is indicated in figures 1 to 4 by cross-hatching, and it extends from the anterior (inferior) to the posterior (superior) pole of the colliculus, which occupies the medial position. (b) In this area electrical, as well as mechanical, stimulation produces: From area (1) movements of the neck (especially extension). ,, (2) ,, neck and wing. ,, (3) wing and leg. ,, (4) tail. All these movements may be produced by a very weak induction current, or merely by touching the surface of the colliculus. The same current, and even one ten times as strong, did not produce any movement or notable reaction when applied to the rest of the colliculus, to the cerebral hemisphere or to the cerebellum. (c) From the areas noted on figs. 1 and 4 by the numbers 1 and 2, we obtained also movements of the beak (opening and closing) and of the larynx (raising and lowering). These movements are determined, however, by deeper structures in the colliculus and not by the surface as we shall explain later. All these movements are on the same side as the excitation but, when the current is increased in strength, they become bilateral. (d) By plunging the electrode into the depths of the colliculus we found, in addition to the movements already described, a region marked 6 in fig. 1 and xx in fig. 4 where excitation produced the whole complex of the movements for the act of" crowing" and actual, persistent "crowing" of the bird. (e) In the same bird (subsequently to obtaining responses by mechanical or electrical stimulation) after complete decerebration, when the bleeding has stopped, the same areas of the colliculus when excited, again yield the same responses, viz., movement of the neck (1), of the wing (2), of the leg (3), of the tail (4), of the beak and larynx (5), and "crowing" (6). (f) For the purpose of localizing these areas with greater precision we exposed the colliculi on both sides and, with a red-hot spatula, quickly destroyed the surface of the colliculi by superficial burning all round. In the same sequence the characteristic responses to electrical stimulation were suppressed one by one. After this operation, electrical stimulation still caused movements of the beak and larynx (5 on fig. I and x on fig. 4). (g) In a second operation in the same bird (with decorticated colliculi) the whole mass of the colliculi was destroyed by burning and then no movement of the beak and no crowing were obtainable. The bird became completely blind and "speechless". (h) Mechanical and electrical stimulation, carefully applied in succession to every millimeter of hemispherical cortex and cerebellum gave no motor response, even when the current was increased to ten times the strength used for obtaining reactions from the midbrain. Needles or electrodes plunged deeply into these

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structures, or actual transpiercing of the whole mass of the hemispheres or cerebellum, produced no motor effect. We were quite unable to obtain from the cerebral cortex such responses as Kalischer (1900) claimed to have evoked in the parrot, or those more recently recorded by Rogers (1922-23). II.- More recently (1932) one of us, assisted by Dr. A. Abbie (of University College, London) has repeated the above experiments with the same results. The cortical localizations of the colliculi were clearly shown and the more deeply placed centres for crowing, and for movements of the beak and larynx were also constant. Some new observations were made during this recent series of experiments: 1. The birds with punctured or partially destroyed colliculi are very excitable, very frightened and have brisk, exaggerated movements. 2. The birds in which both colliculi have been destroyed have dilated, fixed pupils which do not react to light or anaesthetics, but they react to pain and especially to asphyxia. 3. After destruction of both colliculi, the general muscle-tone (skeletal) is decreased, the flapping of the wings is weak, the power of the legs is diminished and the birds tire very rapidly. The co-ordination of movements, however, is perfectly maintained.

Ul'ER!Ol\ GhNGl.lOIL

l ··-·

)

-~/

LUCRARI ~TIINTIFICE

,

461

Fig. 1 - Left lateral view of the whole brain of a pigeon. The excitable area of the cortex of the colliculus is indicated by cross-hatching: (1) neck; (2) neck and wing; (3) wing and leg; (4) tail; (5) beak and larynx; (6) crowing ("speech").

Fig. 2 - Dorsal aspect of colliculus, cerebellum and medulla oblongata. The excitable cortex of the colliculus is indicated by cross-hatching.

Fig. 3 -Ventral aspect of colliculus, cerebellum and medulla oblongata. The excitable cortex is again indicated.

Fig. 4 - Left colliculus cut across at the limit of the excitable cortex. The notations of localizations on the cortex are as in fig. 1. "x" indicates the area where mechanical or electrical stimulation produces movements of the beak and larynx, and "xx" the centre for speech.

4.

5.

6.

7. 8.

After every muscular effort the feathers remain ruffled, but at the slightest touch they suddenly become depressed on the body more quickly than in the normal bird. It seems that the motor control of the feathers is disturbed, and that the colliculi play some part in this effect. The birds become absolutely blind when both colliculi are destroyed and when flying they do not avoid obstacles. Auditory memory is retained. Thus when the birds hear the sound of falling wheat in the cage, they listen, become attentive and orient themselves according to the direction of the sound. The memory for performing such complicated acts as picking up corn or drinking water is lost. If placed in a large tin full of wheat, the birds walk around upon the wheat for hours and, although their crops are empty, they do not pick a single grain. When the sound of falling grain is heard, however, the birds become attentive to it and turn towards the direction of the sound which probably reminds them of a pleasant experience. They pay no attention, however, to the wheat under their feet and, if not fed, would die of starvation in the midst of plenty. Some of the complicated motor actions, however, are still retained. The birds clean themselves and preen their feathers as normal birds do. Excitation of the cerebral hemispheres and of the cerebellum, if the correct current is used, never elicits movements of any kind. If the current is greatly increased it may cause various effects by spreading to other parts of the brain.

III. We do not attempt here a review of the literature relating to this subject. Many authors have noted movements caused by excitations of the colliculi. In relation to our own observations, we would, however, recall Allen's (1927) observation that there is no area in the cerebral cortex of the rabbit stimulation of

462

CON$TIINTA $I CUNOA$TERE

which excites movements of the hind limb, such as can still be evoked by electrical stimulation of the superior colliculus. If this is confirmed the rabbit represents a phase in evolution in which the transference of motor functions from the midbrain to the cerebral cortex has only been partially effected. IV. Conclusion. The scattered facts recorded in the literature, correlated with our experiments, show definitely that in birds there is a real motor cortex with definite localization in the colliculi, but no such thing in the cerebral hemispheres. In the central mass of the same colliculi there is a centre for voice production and for movements of the beak and larynx. REFERENCES Allen (1927). J. Comp. Neural., vol. 43, p. 509. Kalischer (1900). Fortsch. Med., vol. 18, p. 1001. Rogers (1922-23). J. Comp. Neural., vol. 35, p. 61.

Lucrare publicata in Proceedings of the Royal Society, B. 1933, vol. 113, pp. 191-195.

A LIPO-GEL REACTION EXERTED BY FOLLICULAR FLUID UPON SPERMATOZOA AND ITS SIGNIFICANCE (LILLIE'S REACTION) GREGOR T. POPA

1

UNIVERSITY OF BUCHAREST, ROMANIA

I. INTRODUCTION Some time ago Professor F. R. Lillie observed that when sperm suspensions are mixed with follicular fluid taken from the Graafian follicles a coagulum is obtained (unpublished). As this phenomenon may be significant for the process of fertilization, Professor Lillie suggested that I study the reaction and determine the exact conditions under which it appears. I desire to express my thanks to Professor Lillie for suggesting this subject of research and for placing at my disposal the facilities of the zoological laboratories of the University of Chicago. II. MATERIALS AND METHODS

Several species were used in the study, chiefly cattle and sheep: Fresh organs obtained at the slaughter-houses of Swift and Company, Chicago,2 were always employed. For comparison material was also taken from the goat, opossum, pig, and guinea-pig. The testes and ovaries were usually taken from the bodies of animals just killed and carried to the laboratory in a thermos bottle. Under these conditions the organs remained at a temperature of 25° -30° C. until they were used. The experiments were almost always completed within two to four hours after the removal of the organs from the animals. To establish certain points which required 1 From the Hull Zoological Laboratory and Whitman Laboratory for Experimental Zoology, University of Chicago. 2 The author wishes to show his thanks to the officers of Swift and Co., who provided the material necessary for this work.

464

CONSTIINTA SI CUNOASTERE

absolutely fresh material, the same series of experiments was performed twice, once at the slaughter-house, within a quarter of an hour after killing, and again in the laboratory. · The spermatozoa were generally removed from different parts of the epididymis by transverse cuts through that structure. Sometimes they were also taken from the vas deferens and from the testes directly and also from the seminal vesicles. The follicular fluid was obtained by pricking the follicles at their most transparent points by means of a fine pipette into which the follicular fluid was drawn by suction. All organs and especially the female genital tract were taken from the freshly killed animal by myself and examined immediately. Other details of the technique will be described and explained in connection with the experiments. III. EXPERIMENTS 1. When follicular fluid is mixed with spermatozoa taken directly from the epididymis or other parts of the male genital tract a coagulum is obtained in ten to twenty minutes. This coagulum is whitish, semi-opaque, friable, and of a specific gravity greater than that of water; when dissociated with needles, the coagulum breaks up into blocks with sharp edges, indicating a firm consistency. Small fragments of the coagulum when viewed under the microscope appear as homogeneous and transparent masses in which the spermatozoa are imbedded, scattered or in groups. After more complete dissociation of the coagulum into minute fragments one may isolate here and there bundles of fibers coated by a hyaline substance. The surface of the coagulum shines like a fatty surface and does not adhere to water. If therefore the coagulum is set carefully on the surface of water, it will float. When submerged, it sinks, showing that its specific gravity is greater than that of water. This coagulum is produced at all temperatures between 10° C. and 56° C.; higher temperatures are unfavorable for the reaction. After its formation the coagulum persists at temperatures to 100° C. The fluid around the coagulum produces a second coagulum when heated to 72° C. 2. When follicular fluid is mixed with spermatozoa only, the results are not uniform. For example in one series of 90 tests, the result was negative in 43 cases, positive in 36 cases, and uncertain in 10 cases. But if the spermatozoa are mixed with 0.9 per cent, sodium chloride solution the reaction is more certainly obtained. For instance in a series of 242 tests, 41 were negative, 198 positive, and 3 uncertain. Furthermore in many cases in which the reaction failed to appear, using follicular fluid and spermatozoa alone, the coagulum was produced upon the addition of 0.9 per cent, sodium chloride solution to the mixture.

LUCRA.RI STIINTIFICE

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This result suggested that the salts which are normally present in the follicular fluid play an important role in the reaction. The effect upon the reaction of several salts which commonly occur in organic liquids was thereupon tested. The following salts singly or in mixtures were tried: NaCl, KCl, CaCli, MgC12, LiCl, NaHCO3, KHCO3, and CaCO3. All of these gave a negative result. Only sodium chloride is able to favor and accelerate the reaction. Since, however, there were a number of negative cases even after the addition of sodium chloride, an explanation for the failure of the reaction in these cases was sought. It was found that the reaction always occurs when the constituents are employed in certain definite quantities. After numerous tests, the following proportions were found to be successful in nearly all cases: 0.9 per cent, sodium chloride, 0.1 cc; follicular fluid, 0.2 cc; dry sperm, 0.1 cc. In a series of 128 tests using these proportions only four tests were negative. The role of these proportions in the reaction may be illustrated graphically as in Fig. 1. If too much sodium chloride is added to the same amount of sperm, then the dilution of the sperm is too great and the reaction fails to appear. On the other hand if the quantity of sperm is excessive, then the action of the salt solution upon the sperm - necessary for the reaction - is inadequate and the coagulum is not formed. With an excess of follicular fluid, the coagulum appears surrounded by a large quantity of fluid; if the quantity of follicular fluid is insufficient, the reaction fails also. From this we must conclude that some substance in the follicular fluid is essential for the formation of the coagulum. A minimum quantity of follicular fluid is thus necessary; this quantity (0.2 cc. to 0.1 cc. dry sperm) is indicated at the point a in the diagram. The line from a to a' indicates an indefinitely increasing quantity of follicular fluid in which the reaction is still possible, providing the proportions of dry sperm and sodium chloride solution remain constant in the ratio of 1 : 1. We must assume also an active maximum quantity for the sodium chloride solution, namely, a quantity equal to that of the amount of dry sperm. From this amount the proportion of sodium chloride may be decreased indefinitely to zero, at which the reaction is still possible, providing the follicular fluid contains sufficient sodium chloride. The four negative cases obtained with correct proportions stated require explanation. The following possibilities are suggested: (a) Possibly the essential substance in the follicular fluid was insufficient in quantity or lacking altogether. (b) Possibly the quantity of sodium chloride normally present in the follicular fluid was lacking or reduced so that even after the addition of 0.1 cc. sodium chloride solution the amount of this salt present was still insufficient. (c) Possibly the proportions of the three substances used was not perfectly correct as they were measured by drops usually.

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466

Follicular fluid 0.2cc

For all practical purposes the proportions of follicular fluid, sperm and sodium chloride solution stated above are sufficiently accurate and seldom fail to yield the reaction. 3. After the conditions necessary for the production of the coagulum had been determined, a new series of experiments was undertaken to establish more definitely the nature of the reaction. The results may be summarized briefly. (a) The reaction is not produced by other liquids of the organism. I added follicular fluid or dry sperm to the following body fluids: amniotic fluid, blood taken from the jugular vein, defibrinated blood, smear of the mucous membrane of the uterus and tube, urine, cerebrospinal fluid, aqueous humor of the eye, vitreous body of the eye, peritoneal fluid, and pericardia! fluid. In all cases the result was negative. (b) The reaction is interspecific. The following combinations were tried : Follicular Fluid.

Dry Sperm. ram

X

cow

bull

X

sheep

goat

X

cow

goat

X

sheep

opossum

X

cow

guinea-pig

X

cow

ram

X

pig

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In all of these tests the result was positive. (c) Materials from different sources. Follicular fluid taken from one follicle of the cow was tested with sperm taken from the epididymes of ten different bulls. The result was positive in all cases. Follicular fluid from ten different ovaries of the cow was tried with dry sperm taken from the same epididymis of a bull with positive results in seven cases only. Probably some of the follicles had not reached the proper stage of concentration of the necessary constituents. However, follicular fluid taken from follicles of ten different sizes (cow and sheep), from the smallest to the largest gave a positive result in all cases. The age of the follicle does not appear to be of importance for the reaction. (d) Time of reaction. In a series of ten tests (cow and sheep) observed after ten minutes, six were positive and four were negative. In ten tests observed after twenty minutes, all showed the coagulum. In a third series of ten tests, five of which were observed after one hour, five after 24 hours, all were positive. Thus the minimum average time necessary for the reaction lies between ten and twenty minutes. But the reaction is often observable after one minute. When dry sperm is mixed with follicular fluid on a slide, the reaction appears instantly. A delicate layer of coagulum is formed on the surface of the fluid. (e) Variations in the time of addition of one of the components. In these experiments two of the three components were mixed and the third constituent added after a time interval, varying from one to twenty minutes. The following are the possible combinations: (1) Follicular fluid mixed with salt solution; sperm added later. (2) Sperm mixed with salt solution; follicular fluid added later. (3) Sperm and follicular fluid mixed; salt solution added later. No noticeable difference in the time of appearance of coagulum was noted in all three cases counting from the time of addition of the third constituent. In the third case the beneficial effect of salt solution on the reaction was again verified. (f) Variations of temperature. Dry sperm and follicular fluid were brought to various temperature and mixed (with the addition of salt solution as usual). The results are summarized on opposite page. These results show that there is in the follicular fluid a thermo-labile substance which is responsible for the reaction. If the follicular fluid is heated to 56° C, the reaction fails. If the sperm is heated to a high temperature, even to 100° C, the reaction is obtained as long as follicular fluid is not heated above 55° C. (g) Freshness of material. Dry sperm and ovaries were kept in the refrigerator and used for the test at various intervals. It was found that sperm fifteen days old and follicular fluid eight days old still yield a positive reaction either when tested with each other or with fresh sperm or follicular fluid

468

CONSTIINTA SI CUNOASTERE

respectively. The age of the materials therefore within the limits stated does not affect the reaction. 4. In order to obtain a better understanding of the ·nature of the reaction follicular fluid and dry sperm have been examined microscopically and the literature has been consulted as to the possible constituents present in these two fluids. I found that dry sperm may contain spennatozoa, spermatocytes, blood, and lipoid substances. Follicular fluid contains or may contain blood (red and white corpuscles and many substances in solution or suspension), granulosa cells, proteins, sodium chloride and other salts, and pigment. I attempted to detennine which of these constituents are involved in the reaction. Result.

Follicular Fluid.

Dry Sperm.

10°

C .................... Positive

IOO

28° 370

c .................... ♦ c... .. ♦

400

c.... ..... ' . . . . . . . . . . .

''•

450

c ....... ~.............

,,

28° 370 560 57°

500 c.....................

''

•

•

•

..

•

•

•

•

•

•

•

•

•

•

,,

.,

54-0 c .. ♦ ♦• 550 C,~.................. '' 56° C. ................... Negative 28° C... . ............... Positive ••••••••••

•

•

•

•

•

28° c....................

,,

,,

56° C. ................... Negative 560 c .................... ~ .,

C. C. C. C. C.

560 C. 560 C. 560 56° 56° 72°

C. C.

100°

C.

C. C.

28° C.

(a) Blood. It is very difficult to avoid the admixture of blood into either sperm or follicular fluid owing to the cutting or pricking of bloodvessels in obtaining these substances. In my earlier experiments blood was constantly present in my tests and I therefore was inclined to ascribe the reaction to the presence of blood. Later, however, I took very 1 minute precautions to avoid admixture of blood and found that the reaction was always obtained in the complete absence of blood. I also while at the slaughter-house repeatedly mixed blood from the jugular

1

Ovaries taken directly from the body; washing them very carefully in laboratory before experiments; after that drying by absorbent paper; dissecting the follicles with a sharp knife until they appear like transparent vesicles. Only after that treatment puncture of follicles.

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vein with follicular fluid and never obtained the reaction 1• It thus appears that the blood, and by implication the various constituents of the blood are not involved in the reaction. (b) Proteins. A series of experiments performed with egg albumin gave completely negative results. This result would be expected from the negative finding with blood. Another proof that proteins are not involved may be derived from the fact that the reaction fails at a temperature of 56° C, although the fluid so heated and incapable of producing the reaction still contains proteins which do not coagulate until a temperature of 72° C is reached. (c) Sodium chloride. This salt and salts in general never produce the coagulum when mixed with either spermatozoa alone or follicular fluid alone. (d) Granulosa cells. Follicular fluid was centrifuged and the clear fluid filtered to remove all cells. The absence of cells was verified by microscopic examination. The clear fluid was then violently agitated with glass granules for fifteen minutes with the idea of possibly altering the degree of dispersion of colloidal material that might be present. The fluid was then filtered through four sheets of soft filter paper. The fluid so treated still produces the reaction, indicating that cells or other formed constituents are not responsible for the coagulum. It may be mentioned here that filtered follicular fluid was always employed in all experiments. (e) Spermatocytes. I was unable to separate the spermatocytes from the spermatozoa and therefore could not test their effect on the reaction separately. But I used material taken directly from the testes of animals of various ages and always obtained a positive reaction. Material from the testis may be supposed to contain a larger percentage of spermatocytes or cells in other stages of spermatogenesis than material taken directly from the epididymis or vas. No difference can be seen in the reaction however. (f) Spermatozoa. Spermatozoa taken from any part of the epididymis, testis, vas deferens, or seminal vesicle always yield the reaction under the conditions previously described. Spermatozoa have been ground in a mortar for ten minutes, then mixed with salt solution, centrifuged, and filtered through four sheets of filter paper. The fluid obtained in this fashion was observed under the microscope; no spermatozoa or fragments of spennatozoa could be distinguished. Such a fluid however 1 In this case the anticoagulant effect of the sperm or of follicular fluid can be explained by the properties of NaCl and fatty substances, both able to stop the normal coagulation of the blood. Gilbert et Weinberg, Traite du sang, 1913-1021, p. J 5-16.

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gives the reaction both with untreated follicular fluid and with follicular fluid that has been agitated, centrifuged, and filtered. Thus all of the experiments to this point show that a ·coagulum is produced normally by a particular substance present in the follicular fluid, a product of the ovary, when mixed with products of the testes, especially the spermatozoa. The follicular substance is thermolabile and resistant to the action of mechanical factors. The experiments also show that the spermatozoa as formed cells are not essential to the reaction. Up to this point in the experiments the reaction appeared to be perfectly specific: only products of the ovary act upon products of the testes. 5. Role of lipoids. Through certain microscopical observations, however, a new line of experimentation was suggested to me. Whenever spermatozoa treated with sodium chloride or with follicular fluid were studied under the microscope it was noted that after a short time the field of the microscope became filled with numerous small droplets, refractile, perfectly round, of the dimensions of cocci, and engaged in active brownian movements. The droplets increase in number with 1 the degree of concentration of the salt solution • When the suspension begins to dry, the droplets run together into larger and larger drops which are also refractile and homogeneous. Their appearance is very similar to that of a fatty substance. These droplets persist in normal hydrochloric acid but dissolve in sodium hydroxide (0.9 per cent). When treated with a saturated solution of Sudan III in 96 per cent, alcohol, all of the droplets tum red and appear as bright red points. These facts indicate the lipoid nature of the droplets. The final analysis of this substance is a task for the chemist. The important fact in connection with these experiments is that the spermatozoa are always accompanied by a variable quantity of a lipoid substance2. This lipoid substance is responsible for the reaction with which this paper is concerned. The lipoid is in the state of a colloidal sol. When mixed with salt solution its state of dispersion is gradually changed but it still remains in the sol state. Upon the addition of follicular fluid, the thermolabile substance in this fluid exerts an influence upon the lipoid changed by salt solution and causes it to pass into the gel state. A coagulum

1 This phenomenon of drop production by action of salt solution is very clearly explained in Aschoff, L.. "Zur frage der tropfigen Entrnischung", Verh. deutsch. path. Gesell., 17. Tagung Mlinchen, 1914, p. 103-109. The influence of NaCl solution upon lipoids was observed also by Runnstrom, J., "Weitere Studien uber die Verenderungen der Lipoide bei der befruchtung Seeigeleies", Arch. Zoo!., 16, 1924, p. 1. The same effect was obtained instead of NaCl solution with thymol by Bidermann, W., "Uber Wesen und Bedeutung der Protoplasma lipoide", Pfluger 's Archiv, Bd. 202, 1924, p. 223. 2 The same idea of a substance surrounding the spermatozoa is considered by Braus-Redenz and Redenz, H. E., Nebenhoden u. Samenfaden. Anat. Anz. Erg. 58, 1924, p. 121-131. But these authors merely postulate that such a substance exists and give no evidence concerning its nature. As regards the role of this hypothetical substance, the study of Redenz does not touch the essential meaning of it because the nature of the substance is not explained.

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(s thus formed. Because of these characteristics of the reaction, I have designated the latter as a lipo-gel reaction. The question to be next considered is whether the lipoid substance which surrounds the spermatozoa is specific for the reaction as is the thermolabile substance present in the follicular fluid. An article of Kolliker's suggested to me that the lipoid accompanying the spermatozoa might be similar in nature to 1 myelin , found in the central nervous system. I therefore mixed follicular fluid with a cell-free extract obtained by grinding up spinal cord (cow) in salt solution in a mortar and filtering. The result was exactly the same as when follicular fluid is mixed with spermatozoa, except that the coagulum was more translucent. Other organs which contain lipoid substances were then tested in the same way. The following were tried: thyroid, parathyroid, pancreas, adrenal gland, hypophysis, corpus luteum, skeletal muscle, heart, kidney, cerebrum, cerebellum, subcutaneous fat, subperitoneal fat, uterine mucous membrane, stomach, and lung. A positive reaction was obtained with all of these except the last three. The coagulum in all of these cases was quite similar and appeared under the same conditions as the coagulum produced by sperm and follicular fluid. Size, color, and time of formation vary a little with the organ used but the general result is the same. Numerous experiments of this kind were performed; in each case tests were made in the slaughter-house with organs from freshly killed animals but without salt solution, and again in the laboratory in the manner described above. Sometimes when the reaction failed to occur, when the tests were made at the slaughter-house, it succeeded in the laboratory after the addition of sodium chloride. Thus the reaction with different organs and follicular fluid has the same characteristics as the reaction between spermatozoa and follicular fluid. Furthermore the typical reaction is again obtained upon mixing a commercial lipoid product kephalin (made by Armour and Company) with follicular fluid. Extracts which I have prepared from spermatozoa and medulla oblongata2 likewise give the coagulum when mixed with follicular fluid, although the coagulum is rather thin. None of the extracts mentioned nor kephalin give any reaction with amniotic fluid, aqueous humor of the eye, or hydatic fluid. The reaction in all of these cases invariably fails to occur if the follicular fluid is heated to 56° C. Thus the supposition that the coagulum results from the action of a particular thermolabile substance in the follicular fluid on the lipoid material surrounding the spermatozoa is demonstrated to be correct. 1

A. Kolliker, "Physiologische Studien iiber die Samenfliissigkeit", Zeitschr. far wiss. Zoo!., Bd. VII., 1856, p. 201. 2 Spermatozoa or ground nervous tissue were kept 24 hours in 96 alcohol, than 24 hours more in ether; evaporation on water bath. The residue dissolved in salt solution 0.9; centrifugation for elimination of cells themselves. The filtrates used for tests. Cf Loewe, S., "Zur physikalische Chemie der Lipoide", I.-IV., Biochemische Zeitsch .. Bd. 42, 1912.

. 472

CONSTIINTA SI CUNOASTERE

6. The question next to be considered is the role of sodium chloride in the reaction. It has been shown that sodium chloride accelerates the reaction. Microscopical observation of the action of sodium chioride on spermatozoa indicates that the sodium chloride causes the minute droplets of lipoid to aggregate into larger droplets. We can conclude that the sodium chloride prepares the way for the action of the follicular fluid by producing some change in the colloidal state of the lipoid. Further research would be required to elucidate this point. 7. An additional point has been determined - namely, that the lipoid substance occurs on the surface of and between the spermatozoa. This is shown by the following experiments: (a) When spermatozoa are washed once with 0.9 per cent, salt solution and filtered, both the washed spermatozoa and the filtrate produce the reaction in five to ten minutes. (b) When such spermatozoa are washed a second time and filtered the washed spermatozoa and the filtrate from the second washing yield the reaction only after 24 hours. (c) The same spermatozoa after being washed a third time do not give the reaction in 24 hours nor does the filtrate from the third washing yield any coagulum in this time. (d) These spermatozoa after being washed three times were ground in a mortar with salt solution and filtered again. The reaction again failed. From these experiments it appears clear that the lipoid substance is not contained inside of the spermatozoa but occurs on their surfaces and between them. The question of the role of this lipoid for the life of the spermatozoa and in the process of fertilization will be the subject of new researches now under way. IV. THE PHYSIOLOGICAL SIGNIFICANCE OF LILLIE'S REACTION

The first point to be considered is whether the reaction is organ specific. The experiments show that only follicular fluid produce the reaction and so it is organ specific in this sense. Stricto sensu, the reaction is however not specific for I have shown that it can be obtained with products from a large number of organs and tissues. But as the reaction occurs in nature it may be said to be specific, for the follicular fluid under natural conditions does not meet any other fluid with which it produces a coagulum except spermatozoa. It has been shown that the reaction does not occur with peritoneal fluid, smear of the mucosa of the genital tract, or urine. The reaction is produced with lipoids but the only lipoid with which the follicular fluid would ordinarily come in contact is that accompanying the spermatozoa. We may next discuss the possible physiological purpose of this coagulation phenomenon. At first sight the phenomenon appears to be of a paradoxical nature. In order to perform their function of fertilizing the egg the spermatozoa must retain their motility when introduced into the genital tract; yet it appears from my

LUCRARI $TIINTIFICE

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experiments that in the genital tract they meet a fluid, namely, the follicular fluid, 1 which destroys their motility • Without discussing this matter at too great length the following explanations of the utility of the reaction may be suggested. 1. Lillie has shown that the eggs of certain invertebrates contain and produce a substance which he designates as fertilizin which has the property of activating and agglutinating the spermatozoa and is of importance in the fertilization of the egg. If fertilizin is produced by the mammalian egg it would certainly occur in the follicular fluid. I could not determine the place of origin of the substance in the follicular fluid which gives the coagulating reaction with spermatozoa. It might originate from the ovum, or the granulosa cells. My experiments (p. 230) indicate that the granulosa cells (including presumably the ovum) do not contain sufficient of the substance to give the reaction. It was also shown that blood does not yield the reaction. It is therefore necessary to suppose that the substance responsible for the reaction is a modified product of either the blood or the granulosa cells plus the ovum, or of both, and does not exist as such in any of these objects; or that it is a substance which gradually accumulates in the follicular fluid. In favor of the hypothesis that the active substance originates from the cells of the follicle including the egg is the observation that follicular fluid from atretic follicles does not yield the reaction. The ovary of the cow frequently contains follicles filled with a clear colorless liquid; such follicles do not contain any granulosa cells or any ovum. They are readily recognized by the colorless watery appearance of the contained fluid. This fluid however invariably fails to yield the coagulating reaction, eleven cases having been tested. The active substance in the follicular fluid does not appear to be identical with the fertilizin of Lillie in its properties. For instance, fertilizin is stated to be very resistant to heat, while the follicular substance as shown above loses its coagulating property when heated to 56°C. 2. Whatever may be the role played by the coagulating substance in the process of fertilization I believe that it may have another secondary function which does not necessarily exclude the first suggestion. I am inclined to think that the follicular fluid may serve to protect the peritoneum against possible infection by infected spermatozoa. 1

It is very surprising that in the whole literature one cannot find the slightest indication of this phenomenon. A tremendous number of workers have tried the reaction upon spermatozoa of different kind of substances excepting the follicular fluid and this seems the most logical substance to be tried. Professor Lillie was the first to try this reaction and thus opened an entirely new field of experimentation. The origin of the whole question concerning the lipo-gel reaction is included in the long series of works published by Professor Frank R. Lillie. Compare, e.g., ,,Studies in Fertilization", V. ,,Mechanism of Fertilization in Arbacia", The Journ. of Exp. Zoo/., vol. 16, 1914, p. 523; ,,Studies on Fertilization". V. ,,The Behavior of the Spermatozoa of Nereis and Arbacia with Special References to Egg-extractives", The Journ. of Exp. Zoo/., 1913, vol. 14. As a general review of the entire problem in which my work must be integrated see Frank R. Lillie, ,,Problems of Fertilization", Univ. of Chicago Press. One can find there also a very rich literature concerning the subject.

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I have made a number of observations on the life and behavior of spermatozoa in various media and particularly in follicular fluid. From these experiments it appears that the spermatozoa furnish a very 'good medium for the growth of bacteria. After standing for three or four hours at room temperature sperm suspensions were seen to contain many cocci and bacilli: and after twentyfour hours they were swarming with microorganisms. For this reason spermatozoa live much longer at low than at higher temperatures. Spermatozoa will live for several days in small glass tubes if they are kept in the refrigerator, but only for 24 to 30 hours at room temperature. On two occasions spermatozoa that had been kept in the refriferator for nine days showed motility when brought into room temperature . Spermatozoa removed and kept under aseptic conditions may remain alive for eight days in the refrigerator and for four or five days at room temperature. Even infected spermatozoa retain their motility for some time and move through the field of the microscope carrying bacteria with them. I have injected spermatozoa from the ram into the excised uterus of a sheep. In two hours the spermatozoa were found to have penetrated into the uterine tube but they were greatly damaged and fragmented and many of them had been phagocytized. Those spermatozoa which were still alive were in a state of agitation and covered with bacteria. It appears that the surface of the spermatozoa is very sticky and that all 2 kinds of particles therefore adhere readily to them • The spermatozoa are placed normally in a very infected organ, namely, the vagina. The penis itself bears some species of bacteria and at all events at the moment of copulation many bacteria are introduced from the outside into the vagina. These bacteria are carried up into the uterine tubes by the spermatozoa and constitute a menace for the peritoneum which is well known to be exceedingly sensitive to infection. The agglutination of the spermatozoa by the follicular fluid would hold these bacteria and permit them to be phagocytized more readily, thus protecting the peritoneum from possible infection. This hypothesis of the protective role of the coagulating substance in the follicular fluid does not exclude the possible importance of this fluid in fertilization; also it is not possible, at the present time, to estimate more accurately the practical Lucrare publicat in Biological Bulletin, 1927, vol. 52, nr. 4, pp. 223-237.

1

The same observation on favorable effect of low temperature upon longevity of spermatozoa has been repeatedly made e.g. by Redenz in the paper already mentioned; Cf also Mettenheimer, M., "Sperma und kunstliche Befruchtung bei Mensch und Tier", Munch. Med. Wach. schr. 72, Jahr. g. 1925, p. 977; Mettenheimer, M., Archf Gynak., 1923, Bd. 162, p. 215. 2 I found after I finished my work, that the proof that spermatozoa may transport bacteria was already furnished by Rotter, "Wie ascendiert die Gonorrhoe", Archiv.f Gynakologie, Bd. 117, S. 153. This author has proved that spermatozoa can transport Bacillus subtilis and gonococcus.

THE DISTRIBUTION OF SUBSTANCES IN THE SPERMATOZOON (ARBA CIA AND NEREIS) DR. GREGOR T. POPA' UNIVERSITY OF BUKAREST, ROMANIA STUDIED BY INTRA VJTAMSTAINS AND BY STAINS OF LIPOIDS ACCORDING TO THE METHOD OF SCHUMACHER

I. INTRODUCTION In a previous paper2 I come to the conclusion that the spermatozoa of mammals are surrounded by a layer of a lipoid substance which is responsible for a reaction observed by Prof. Frank R. Lillie, and called by me the lipo-gel reaction. With other methods and in different animal species, I have been able to analyze much more completely the distribution of this lipoid substance in and between spermatozoa. The following description is a record of these new facts. In a recent paper Josef Schumacher has devised methods for the identification of lipoids, lipoproteids and fats in the tissues and cells by means of combining different strong reagents with the action of stains. One conclusion of his work is that there is a series of stains (above all Victoria blue and fuchsin) which have a special affinity for lipoids, giving a salt by combination with the lipoidic acid. After destroying the proteins by different macerating reagents, one can isolate the lipoids and in this case the staining with the mentioned substances is a specific stain. I shall not review this method in detail. Everyone who needs to use it must read the original paper: Josef Schumacher: ,,Zur Chemie der Zellfaerbung VIII. Mitteilung. Ueber die Nachweis der Lipoide in Zelle und Gewebe", Chemie der

1

From the Marine Biological Laboratory, Woods Hole, Mass. Gregor T. Popa, 1927, ,,A Lipo-gel Reaction Exerted by Follicular Fluid upon Spermatozoa (Lillie's Reaction) and Its Significance", Biol. Bull., vol. 52, p. 223. 2

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Zelle und Gewebe, Zeitschr. f die Probleme der Gaerhung, Atmung u. Vitaminforschung, Bd. XII., Heft 5, 1926, S. 433. , I have used his methods (see page 24 7) but most of the observations were made by simpler methods, using stains recommended by him as specific stains, and in addition various intra vitam staining methods. In the latter case the staining was done under the cover slip as in the method of Koltzoff "Studien ueber die Gestalt der Zelle", Arch.f Zellforschung, Bd. II., S. 1, 1909.

II.RESULTS 1. THE USE OF STAINS WITH FRESH SPERMATOZOA (a) Victoria Blue and Fuchsin Victoria blue in 1 per cent, solution in distilled water: Stain under the cover slip: one drop of sperm suspension (one drop dry sperm to 10 cc. sea-water) is placed on the slide, and after covering with a cover slip, with a fine pipette one adds at the margin of the cover slip a drop of staining solucion. The fluid penetrates by capillarity, advancing more and more slowly. In this way, one gets in the same preparation all degrees of staining, from the strongest color to the complete lack of stain. At the one end of the slide the spermatozoa are overstained, and on the opposite side the spermatozoa are still active. The first impressive fact is a remarkable change of the shape and size of the spermatozoa. The stain is in distilled water and the spermatozoa are in sea-water: there is a great difference in the osmotic pressure of the two fluids. Thus, a rapid penetration of the staining solution takes place into the spermatozoa, which stain immediately. Shortly after, the heads of the spermatozoa swell, become round and then burst; the following diagrammatic figures show some of the variations in distribution of the stain (Figs. 1-3).

•• •• • ••• • ... •• f!.

Figs. I, 2, 3. Fresh spermatozoa of Arbacia stained with 1 per cent, solution of Victoria blue in distilled water.

••P.

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When the reaction is too strong, i.e., when the contact of the spermatozoa (in sea-water) with Victoria blue (in distilled water) is immediate, the tails swell also from place to place and one may find shapes as in Figure 4. Frequently, when the reaction is still stronger, the tails twist round the heads and shapes very similar to those described by Koltzoff result (Joe. cit., pp. 9-12). Victoria blue, fuchsin, and the dyes from the same series (Gentian violet, Malachite green, etc.) always stain substances in the acrosome region, in the middle piece, and in the tail. The reaction with Victoria blue, and with the stains in the same class, gives strong reasons for postulating the presence of lipoids in the composition of the acrosome, middle piece, and tail. Victoria Blue in Alcohol (I cc. of the distilled water solution in 10 cc. alcohol, 70 per cent): The solution fixes the spermatozoa at the same time that it stains, and the stain being more dilute, one can see the gradual transformations of the spermatozoa. The heads swell slowly; the tails never swell. The distribution of the stain in the spermatozoa is exactly the same; tip, middle piece, tail. Also, the stain in the region of the acrosome does not always occur, and the middle piece varies in size. By variations of concentration one can have a series of pictures more and more similar'to those obtained by Victoria blue in distilled water. Fuchsin (1 per cent, distilled water solution): The affinity of the fuchsin for the lipoids is less than that of Victoria blue. The staining is accordingly slower and one can follow this process more gradually in the parts of the slide where the effect of distilled water is moderated by a suitable mixing with sea-water.

a

Fig. 4. Arbacia. Fresh spermatozoon in 1 per cent. Victoria blue in distilled water Swelling of tail and twisting around head. o. middle piece; 6, hydrophilic substance; c, lipophilic substance.

The new fact obtained by this method is the possibility of observing changes of shape and variations of size and form of the middle piece. One can see in its region only granules varying in number (2-6), or a ring varying in size from one

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1

spermatozoon to another, and in the same spermatozoon during the observation until at the end of the reaction almost all spermatozoa show pie middle piece as a compact mass, red-stained.

Fig. 5. Spermatozoon of Nereis to show sensillae amreboidea.

When the slowly moving spermatozoon shows its tip, one can see that there is really a minute opening. The tip of the spermatozoon is extremely sticky and adheres to everything which touches it: granules, slide, cover slip, eggs, or to another spermatozoon. Frequently it sticks to the tail of the same spermatozoon. Victoria blue or Fuchsin in sea-water solution: These substances are very feebly soluble in sea-water; but if we keep an excess of stain for several hours in sea-water, a weak solution is obtained in which the spermatozoa can live for ten to thirty minutes and one can see the changes which occur more easily than in distilled water solution. The regions of the spermatozoon already mentioned take the color. But for a short time there is a slight stain on the surface of the head also, before the middle piece is stained. After a variable time the head loses the color gradually and one can see a slow enlargement of the middle piece and the formation of delicate protoplasmic filaments therefrom, which move sporadically. These deserve a more precise description. 1

The instability of this region was very clearly observed by Retzius and Ballowitz, but they believe that it occurs because of the technique. However, without staining, under conditions approximately normal, the same variations of shape can be noticed. Gustav Retzius, "Die Spermien von Aurelia aurita L.", Biol. Unters., N. F., XIV., 1909, S. 67. Gustav Retzius, "Die Spermien der Nereiden", Biol. Unters., N.F., XIV., 1909, S. 69. E. Ballowitz, "Uber die Komige Zusammensetzung des Verbindungsstiickes der Samenkorper der Knochen-fische", Arch. Zellforschung, 14 Bd., 1917, p. 355.

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Fig. 6. Spermatozoon of Arbacia showing sensilla amceboidea.

The basal part of the head has the appearance of a ring, somewhat thicker than the rest of the head. When the spermatozoon turns in the fluid in such a manner as to show its larger end, one can see very clearly even on fresh preparation, not stained, the presence of this ring. It has different sizes, according to the species (Arbacia and Nereis) and different outlines on the same spermatozoon. Here and there it is thicker or thinner; sometimes it becomes fragmented in granules and again, it forms a compact mass. In Nereis (Fig. 5) there are normally two swellings placed symmetrically, outgrowths of the ring, on which one can distinguish two long filaments as in Fig. 5. In Arbacia (Fig. 6), the ring shows in the same way various thickenings (3-5) and these, not so constantly as in Nereis, support (usually only one of them) a prolongation ended by a kind of knob. As the spermatozoon floats in the fluid, these filaments show protean movements, elongation, shortening and thickening, very similar to amoeboid movements. They are constructed of the same material as the basal ring and they are to be considered as prolongations of this. Provisionally they may be called 1 sensillce amceboidece • In all changes of osmotic pressure, in all media which change or kill the spermatozoa, these sensillae retract upon the ring where they produce granules projecting a little above the general level. 1

It is very interesting that the presence of these filaments was noticed by Mischer "Die Spermatozoen einiger Wirbeltiere. Ein Beitrag zur Histochemie", Verh. der naturf Gesl/sch. in Basel, 1878, Bd. VI.; the interpretation of them was wrong. E. Ballowitz, in the same species, denies Mischer's observations "Uber die Samenkorper des Lachses. Ein weiteres Beitrag zur Kenntnis der Spermien der Salmoniden", Archj Ze!!forschung, 14 Bd., 1917, S. 451; but in an older paper and in other species", Untersuchungen fiber die Struktur der Spermatozoen, zugleich ein Beitrag zur Lehre von feineren Bauder Kontraktilen Elemente. Die Spermatozoen der Insekten", Zeitschr.f wiss. Zoo!., 1890, Bd. 50. S. 317, he himself has observed such filaments and even their movements. He even calls them Wimpelfasem. The meaning of them is not explained, and he says that they are "riitselhaft". The conditions of observation were bad because he employed always "maceration", after killing the spermatozoa. In fact, the sensillae are to be observed much more clearly on fresh, slowly motile spermatozoa, without stain.

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(b) Janus Green; Trypan Blue; Neutral Red (1 Per Cent, in Sea-water) These substances are frequently used for staining intra vitarn. The penetrating power is different for each. Janus green gave the best results in our experiments. It penetrates very slowly, and shows excellent contrasts. By these stains one obtains more clearly the same results as by Victoria blue in sea-water. With Janus green especially, one obtains an entire series of pictures, which prove clearly that there is a migration of the same substance which stains in Victoria blue, to the connecting region between head and tail and a large accumulation there of the migrating substance (Fig. 7 a-e). But here and there one can find 1 spermatozoa in which the lipochromatic substance migrates towards the apex. Compare the action of Victoria blue and fuchsin in distilled water (Figs. 2, 3, and 4) in which one sees the lipochromatic substance also accumulated on various regions of the head. These facts show the presence in the spermatozoon of an active cytoplasmic layer, external to the nucleus, loaded with lipoids and taking various forms. This layer flows in and out of middle piece and forms the sensillal

~- a. oc o. 6.0Q

Fig. 7. a-e, sperm heads of Arbacia showing stages of staining in Janus green in sea-water; movements oflipochromatic substance, fk, Victoria blue in sea-water, various conditions. The lipochromatic substance is shown black.

1 I use this term lipochromatic substance for a succinct description, implying only that the substance mentioned takes very strongly the stains reputed by the work of Schumacher to be lipophiles: Victoria blue and fuchsin especially. 2 I have strong reasons to believe that Ballowitz has observed also the migration of the peripheral substance of the spermatozoa, "Untersuch. iiber die Struktur der Spermatozoon der Fische. Amphibien, Reptilien", Arch.f mikr. Anal., 36 Bd., 1890, S. 225. His explanation is not satisfactory. He believes that the change of color in various parts of the head of the spermatozoon is a consequence of stain diffusion in the medium (p. 239). But the changes can be better observed by the method of Victoria blue, fuchsin and Janus green than by his method; and one can also observe the accumulation of the substance in the connecting region even on spermatozoa not stained at all.

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2. STAINING METHODS AFTER FIXATION Spermatozoa of Arbacia and Nereis were also studied by the more usual methods of fixing and staining. The fixing methods used were: l. Corrosive sublimate, saturated solution in distilled water, 100 parts, plus glacial acetic acid, 8 parts. 2. Vapor of osmic acid, 2 per cent, aqueous solution. 3. Flemming, strong solution. 4. Formalin, IO per cent. 5. Heat, after rapid evaporation. The following stains were employed: 1. Iron-haematoxylin (Heidenhain) and eosin. 2. Iron-haematoxylin (Heidenhain) and safranin. 3. Fuchsin. These procedures applied to the study of the spermatozoa, when united with the observations on living material, give us the conviction that there is no one fixing fluid which preserves perfectly the form and the structure of the 1 spermatozoa •The fastest acting fixing agents (as vapor of osmic acid) and the most penetrating fluids (as acetic acid plus K-bichromate) change the size, the configuration of the spermatozoa and the mutual relations of the substances in it. The outlines of the head slightly change, the heads swell, the sensillae disappear, the ring changes its shape, and the lipochromatic substance migrates. 3. SCHUMACHER'S METHODS

A. One makes a smear with sperm as thick as possible, and before the sperm is dried the slide is plunged into HCl solution 1: 4. The slides remain in the acid 24 hours. After that time, wash in distilled water and stain for 5 minutes in 1 per cent. Victoria blue in distilled water. Again 5 minutes in distilled water. Mount after usual treatment in cedar oil, or, in order to have clearer pictures (but more unstable) one can mount directly in water and seal with paraffin. The idea of this treatment is as follows: The various chemical components of cells show a different resistance to the various chemical agents. By combining the destructive agents and the time of acting, it is possible to isolate by turn the chemical components of the cell and detect them afterwards. Schumacher uses strong acids in order to destroy the proteins of the cell. The remainder, after maceration, must be some substance not influenced by acids, 1

Students of spermatozoa note the same thing. This fact is demonstrated particularly clearly by C. Pictet, "Recherches sur la sperrnatogenese chez quelques invertebres de la Mediterranee", Mitteil a. d. Zoo!. SI. Neapel., 10 Bd., 1891-93, S. 75.

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essentially lipoproteins (proteins united with lipoids) and lipoids. If this is true, the stains characteristic for proteins, such as methylen blue, will stain no more, and on the contrary, the stains of the fuchsin series still will give a coforation. This method was used by Schumacher in studying leucocytes, yeast cells, liver, lung, and skin. I have applied this method in studying the spermatozoa.

m.p.

ci

a

Fig. 8. Spermatozoa of Arbacia prepared according to method A of Schumacher and stained in Victoria blue.

With the above method one obtains with Victoria blue the following pictures (Fig. 8); staining with methylene blue fails completely. This demonstrates that the various portions of spermatozoa stained with Victoria blue by the procedures already described, resist the action of strong acids, while the other portions are destroyed. Therefore, according to the theory of Schumacher, we can say that in the construction of the tail, middle piece, and acrosome of the spermatozoon there is a great quantity of lipoid substance.

B. For deciding if in an organic construction, which resists the action of acids and still is stained by Victoria blue, there are free lipoids or lipoids associated with proteins (lipoproteins ), Schumacher uses another procedure: Smears made as in A (above) are placed for 24-36 hours into a mixture of alcohol and ether. Afterwards the slides are transferred for 24 hours to HC 1 1:4. The staining following is identical with that described in A (above).

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. The idea is this: The alcohol-ether dissolves and washes out free lipoids, but does not touch the lipoproteids; afterwards HCl destioys the proteins, and again the lipoproteids remain intact, and thus they are isolated. If, after such a treatment, we still obtain a coloration, we can say that there is a lipoprotein component. Applied to the study of spermatozoa, this method gives the pictures shown in Fig. 9. A pale reticulum built by interlacing tails, and here and there some knobs at the ends of fibers, inferred to be the remains of middle pieces. It is to be noticed also that the fresh preparations immediately after staining with Victoria blue, are much clearer than the preparations mounted in balsam, and particularly after treating with xylol.

Fig. 9. Spermatozoa of Arbacia prepared according to method B of Schumacher and stained in Victoria blue.

Summarizing the facts obtained by the method of Schumacher, one can say that in the structure of the spennatozoon there are free lipoids and lipoproteids which are located normally in the tail, middle piece, and acrosome. The facts described in 1 and 3, connected with the facts observed in living spermatozoa, demonstrate that the lipoids and lipoproteids of the tail are located on the su,face. 4. OSMIC ACID (I PER CENT. AQUEOUS SOLUTION 24 HOURS OR 2 PER CENT AQUEOUS SOLUTION FIXING BY VAPOR 1-5 MINUTES) Osmic acid shows in Arbacia very similar pictures to those obtained by the lipophilic stains. One can see very clearly the basal ring, the tail, the middle piece; the conical part of the head appears darkened and unbumished, which gives us the impression that the substance darkened by osmic acid is located on the surface of the spermatozoon. The substance between spermatozoa is darkened too. On fresh preparations this substance can be distinguished still more clearly. If spermatozoa are shaken in sea-water, and from the clc:ir fluid, which separates in the upper part of the test tube, a drop is taken and submitted to the action of osmic acid vapor, one sees under the microscope very fine round droplets, black and refractile, evidently originating in the intercellular substance. The preparations by osmic acid give us the impression that there is on the surface of and between the spermatozoa a substance which contains fat and which surrounds the cells as a mantle.

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5. SUDAN III Sudan III shows, in the same way as osmic acid, the presence of fatty globules accumulated between the spermatozoa after treating them with the reagents. 6. THE MACROCHEMICAL TEST WITH VICTORIA BLUE If 1 per cent Victoria blue in distilled water is mixed with ether, one obtains, after shaking, an emulsion which, after 10-30 minutes, separates in two distinct layers: the non-stained ether above, and the strong colored Victoria blue below. If to the same mixture one adds dry sperm, after shaking, the ether separates again, but now it carries with it the fatty substances from the spermatozoa. These substances are already stained by Victoria blue, and, therefore, the ether in the upper part of the test tube is blue. If, instead of spermatozoa, we use only the fluid separated after centrifuging a sperm suspension, we may have a complete transfer of color into the ether, the lower layer, representing the water of the Victoria blue solution, being now uncolored. This reaction corroborated by the tests of Schumacher and the reactions with Sudan III and osmic acid demonstrates that around and between spermatozoa there is a substance rich in unsaturated and neutral fats.

7. OBSERVATIONS ON LIVING SPERMATOZOA IN SEA-WATER Observations are made with the oil immersion lens. By varying the focus, the strength of the light and the eye pieces, one can see quite clearly many of the facts obtained by staining methods. Without doubt, the value of this kind of observation is very great, especially when combined with the above mentioned methods; more and more one reaches the conviction that there are in the spermatozoa two different zones: a darker one at the periphery, which becomes thicker towards the connecting region between head and tail, where it constructs a large ring. The central zone is more refractile. It is larger towards the connecting 1 region and diminishes towards the apex . Almost all transformations of the ring can be seen in fresh spermatozoa; the sensillae amoeboideae show most clearly. The migration of substances can be deduced by seeing the changeable sizes of various parts of the same spermatozoon during the observation. In the preceding observations, there is one fact which deserves more emphasis: this is the stickiness of the pointed part of the head, and of the connecting region too. Very frequently, and chiefly in some particular reactions, taken into consideration below, the spermatozoa bunch together, sticking by these 1

Mischer (foe. cit.) noticed the same thing. Ballowitz in Untersuch. u. die Struktur d. Sp. (Fische, Amph. Rept.), p. 249, distinguishes in the head of the spermatozoon two different zones, and he says that in the process of swelling "the internal zone leads". In fact, it alone swells the external substance merely changing its place.

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two regions. By careful and patient observations of fresh spermatozoa, during longer time and in various media, one gets the impression that the spermatozoa eliminate through the point of the head (where really there is an exceedingly minute opening) very small amounts of an extremely sticky substance. 8. THE EFFECTS OF EGG-WATER ON THE SPERMATOZOA The mutual relations of the various constituents of the spermatozoon, being established and knowing the !ability of these relations, a large field for studying the topographical changes of the principal substances in various media is opened. I shall limit myself here to one point: the changes which occur in the spermatozoa in eggwater, which might be expected to resemble changes preliminary to fertilization. 1 This is the classical phenomenon studied by Lillie • It is very well known at the present time that there is exerted an agglutinative action. Upon spermatozoa by the sea-water in which seaurchin eggs have been for a short time. The reaction is instantaneous. The spermatozoa exhibit great activity and bunch together, for a while, in compact clusters of various sizes. What is the immediate cause of the agglutination residing in the spermatozoa? Is it due to a change in the arrangement of the substances, or to an active elimination of the sticky substance observed in fresh suspensions of spermatozoa?

Fig. 10. Arrangement of agglutination experiment; a, egg-water, b, clusters. c, sperm-suspension, d, drop of Janus green.

a

t:

One drop of egg-water and one drop of sperm suspension are placed on a slide (Fig. 10). A very narrow bridge is established between the two drops. Immediately the agglutination takes place where the drops meet, and slowly spreads, so that we have again various stages of a phenomenon on the same slide. At the left end of the egg-water drop (Fig. l 0) the fluid is still clear; at the middle, agglutinated clusters of spermatozoa in great activity; at the right side of the sperm suspension drop, the spermatozoa in various degrees of motion, are in a homogeneous suspension.

1

Frank R. Lillie, "Studies of Fertilization. VI. The Mechanism of Fertilization in Arbacia", 1014, Journ. of Exp. Zoo!., vol. 16, p. 523. Frank R. Lillie, "Studies of Fertilization. V. The Behavior of the Spermatozoa of Nereis and Arbacia with Special Reference to Egg-extractives", Journ. ofExp. Zoo!., vol. 14, 1913. p. 515.

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In these conditions, one keeps the slides various lengths of time ( 1-10 minutes). Afterwards one puts very carefully a cover slip with another drop of eggwater underneath (to mitigate the power of capillarityf Many bunches of spermatozoa are spread out, but many other resist, and one can observe, under the oil immersion lens, what happens in the clusters.

Fig. 11. Behavior of the spermatozoa of Arbacia in eggwater; intra-vitam staining with Janus green. See text.

A long series of transformations of the individual spennatozoa occur, represented by the next sketch (Fig. 11 ). These transformations take place under the influence of egg-water only. But in order to see them more clearly, one may add at the margin of the cover slip a drop of l per cent Janus green in sea-water. The changes shown diagrammatically in Fig. 11 are: (a) A normal spermatozoon; (b) Initial changes in the arrangement of lipochromatic substance; (c) The nucleus swells a little and moves towards the base of the head. At the same time the lipochromatic substance divides into two, a part follows the nucleus, covering it, another part remains in the region of the basal ring. Thus one can see in fresh preparations on the connecting region of the spermatozoa two transversal dark zones, between which there is a refractile one. On stained preparations there are two colored strips and one light band; (d) The same process, much more advanced; (c) The lipochromatic substance accumulated in greater amount, shifts from the connecting region; (f) A round or slightly oval body is thus extruded from the head, and it takes a lateral position, where it persists without any modification, and the whole spermatozoon becomes perfectly quiet. It is to be noticed that the spermatozoa which have extruded this lateral body do not swell. On the other hand, the spennatozoa which have not extruded it, swell and become round.

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III. DISCUSSION

Summarizing all the facts revealed by the various methods above described, concerning the topographical distribution of the material in the spermatozoon (in Nereis and Arbacia) there is to be distinguished a substance stained by lipophilic stains, the lipochromatic substance; and another substance very avid for water, the hydrophilic substance. The lipochromatic substance is located at the periphery of the spermatozoon, and its thickness is variable in different regions; towards the connecting region it constructs a large ring and the sensillae; in the tail it takes part in the construction of the tail mantle. This substance is not avid for water, and therefore does not change its volume in various hypotonic solutions. On the other hand, it shifts position readily, and it accumulates normally in the connecting region of the spermatozoon (Fig. 11 ). The hydrophilic substance is located in the axis of the spermatozoon, and does not take the lipophilic stains. In the head it includes the nucleus or may be represented exclusively by the nucleus. It is extremely hydrophile, swells in water (hypotonie solutions) and thus enlarges the head of spermatozoon until more than three times diameter. This substance is centrally located in the head and also in the 1 axis of the tail • Apparently the hydrophilic substance of the head is nothing else than the nucleus and the lipochromatic substance must be the cytoplasmic component of the spermatozoon loaded with lipoids and lipoproteids. The cell is surrounded by a very thin membrane, the presence of which is obvious when the heads swell and explode in hypotonie solutions. The membrane must be very elastic and permeable to water. The whole complex is protected externally by a thick layer offatty substance, darkened by osmic acid and detected also by Sudan III and by macrochemical analysis with Victoria blue2 . Its chemical composition is still not clearly demonstrated.

1

Concerning the sensitiveness of these cells to the changes in the osmotic pressure see: Richard Goldschmidt, "Kleine Beobachtungen und Ideen zur Zellenlehre III Die Bedeutung der atypischen Spermatozoen", Arch. J Ze/!forsch., 14 Bd., 1921, S. 290. Richard Goldschmidt, "Versuche zur Spermatogenese in vitro", Arch. f Zel!forsch., 14 Bd., 1921, S. 421. Josef Speck, "Neue Beitrage zum Problem der Plasmastrukturen", Zeilschr. f Zellen- u. Gewebelehre, I Bd., 1924, S. 278. 2 This layer outside the spem1atozoon has been frequently noticed recently by various authors: Redenz, E., "Versuch einer biologischen Morphologie des Nebenhodens", Arch. f Mikr. Anal. u. Entw. Mech., 1924, 103 Bd., S. 391. Gellhom, E., "Physiologische Untersuchungen an Spermatozoen und Eiem. Ein Beitrag zum Befruchtungsproblem. Sammelreferat", Arch. f Mikr. Anal. u. Entw. Mech., 1924, 101 Bd., S. 437. Redenz, E., "Versuch einer biologischen Morphologie des Nebenhodes. IL Die Bedeuting elektrolytarmer Losungen fiir die Bewegung der Spermien", Wilh. Roux' Arch. f Entw. mech. d. Org., I 06 Bd., 1925, S. 290. T. von Lanz, "Uber Bau und Funktion des Nebenhodens und seine Abhangigkeit von der Keimdruse", Zeilschr.f die ges. Anat. Z.f Anat. u. Entw. g., 80 Bd., Festschr. f S. Mollier, 1926, S. 177.

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Mixing dry sperm with sea-water one can clearly see this substance spreading out on the surface film like oil. In the dry sperm under the microscope, one can see how abundant this intercellular substance is. In this medium'the spermatozoa live longer than without it. Presumably the contact of the spermatozoa with the water is very much mitigated by the surrounding fatty medium, so that changes of osmotic pressure influence the spermatozoa gradually, and the modifications of form occur very slowly. This layer of fatty substance may be considered on the whole as a buffer between a very sensitive structure (spermatozoon) and a salt solution at a very high concentration (sea-water). Certainly the fatty substance may have some other important significance, too, not yet taken into consideration. As soon as the concentration of the medium is changed and the fatty medium is modified, the spermatozoa react first by motility, and secondly the mutual relations of the two substances (hydrophilic substance - lipochromatic substance) undergo changes. As a result we have the various pictures of the spermatozoon already demonstrated. For completion of the general sketch of the speimatozoon there is to be remarked the presence at the distal end (that is to say, the tip of the head) of a minute opening, which it is convenient to call by analogy micropyle; and also at the connecting region there is another small ring very easily colored by stains which have affinity for chromatin, especially by carbol-pyronine. This is the classical proximal centriole 1• We have seen above the various changes of the mutual relations of the two substances in the head of spermatozoon. The end result of these changes is the elimination of the lateral body. This elimination phenomenon occurs, also, under various other circumstances, for instance, when spermatozoa are in weak formaldehyde solution, or in some staining solutions, but the process takes place very slowly and quite irregularly. On the other hand, in the egg-water after five minutes almost all spermatozoa show this lateral body. Fresh preparations of spermatozoa of Arbacia in egg-water also show quite clearly the elimination of a substance through the pointed apex of the spermatozoon. This appears as a small granule on the points of almost all spermatozoa. The adhesion of the spermatozoa to one another or to other objects is made by means of 2 this granule . In the middle of some clusters one can see groups of such granules very refractile, round and close together. For this observation only fresh preparations are to be trusted, because stains may form very fine precipitates which cannot be distinguished from drops of the eliminated substance. Very probably, under the influence of some chemical substances included in the egg-water (Lillie's fertilizin?) spermatozoa eliminate through the micropyle a 1 Here also is the opening observed by Mischer and Ballowitz (toe. cit.) and called by the former worker microporus. 2 Ballowitz, E. (Zeilschr. f wiss. Zoo/., 1890, Bd. 50, S. 317) called this granule "Spitzenknopf' and noticed its instability (p. 375)

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~ticky substance, which so long as it still adheres to the apex of spermatozoa keeps them agglutinated. After it is lost from the tip, the spermatozoa spread out again in the fluid. I cannot decide if this substance exists as such in the spermatozoon or if it is produced by a secretory process at the moment of stimulation by egg-water, as in 1 the sense of Bowen . The spermatozoa also undergo in the egg-water more extensive alterations, which are expressed by changes in the arrangement of the substances and as the end result we have the formation of the lateral body as described above. ADDITIONAL REFERENCES I. Bowen, R. H. '20 Studies on Insect Spermatogenesis. I. The History of the Cytoplasmic Components of the Sperm in Hemiptera. Biol. Bull., Vol. XXXIX., p. 316. 2. Bowen, R. H. '23 On the Idiosome, Golgi Apparatus and Acrosome in the Male Genn Cells. Anat. Rec., Vol. 24, p. 159. 3. Alexeieff, A. '24 Comparaison entre la structure des spermatozoYdes et celle des flagelles. Arch. f. Protistenkunde, XLIX., p. I 04. 4. Chambers, R. '23 The Mechanism of the Entrance of Sperm into the Starfish Egg. Journ. of Gener. Physiol., V., p. 821. 5. Tennent, D. H. '24 Specificity in Fertilization. Science, p. 162. 6. Vies, F. '24 Recherches sur les proprietes physico-chimiques des produits sexuels d'Oursin. Arch. de physique biol., III., p. 42. 7. Godlewski, E. '24 Sur !'inactivation du sperme d'oursin par le sperme d'especes etrangeres. C.R. Soc. Biol., XCI., T. IL, p. 84. 8. Iwanow, E. '24 Recherches experimentales a propos du processus de la fecondation chez Jes poules. C. R. Soc. Biol., XCI., p. 54. 9. Najera, F. C. '23 Contribution a l'etude de la sterilite. Rev. Mexicana. Biol., III., 159. l 0. Gellhorn, E. '24 Befruchtungsstudien. IV. Ueber den Einfluss von Nichtelektrolyten auf die Permeabilitat der Spennatozoen. Pflliger's Arch., 206 Bd., S. 250.

1 According to R.H. Bowen, who adopts the theory ofNassonov concerning the role ofGolgi apparatus, "Das Golgische Binnennetz und seine Beziehungen zu der Sekretion. Untersuchungen iiber einige Amphibiendrusen" Arch./ mikr. Anal., 97 Bd., 136, the acrosome should be a secretory organ and the granule on the tip of the spermatozoon might be the product of its secretion, "On the Acrosome of the Animal Sperm", Anatomical Record, vol. 28, 1924, p. I.

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11. Yensen, 0. S. '86 Ober die Struktur der Samenkorper bei Saugetieren, Vogeln und Amphibien. Anat. Anz., I. Jahrg., 1886, S. 251. 12. Voinov, D. '25 Les elements sexuelles de Gryllotalpa vulgaris Lati. Arch, de Zool. exper. et gen. Tome 63, p. 437. 13. Toedtmann, W. '24 Die Spermatozoen von Vesperguo mourus Bias. Zool. Anz., Bd. 58. S. 59. 14. Toedtmann, W. '34 Die Spermatozoen von Formica rufa L. Zool. Anz., Bd. 68, S. 52.

Lucrare publicata in Biological Bulletin, 1927, vol. 52, pp. 238-257 (cu 11 figuri).

THE MECHANO-STRUCTURE OF THE PERICARDIUM 1 BY GREGOR T. AND

POPA

EUG. LUCINESCU UNIVERSITY OF JASSY, ROMANIA

I. INTRODUCTION

In the literature, as far as we know, the structure of the pericardium is hardly considered, and enough attention has not been paid to the orientation of collagen fibres within the pericardium. A. MacAlister (I) says: "In structure the parietal layer of the pericardium consists of two almost inseparable strata, an outer fascial layer composed of several laminae of mostly longitudinal white and yellow fibres, continuous into the sheaths of the vessels which pierce it, and an inner representing a serous membrane ... etc." Rauber-Kopsch (2), Henle (3), Poirier (4). Testut (5), Cruveilhier (6), Sappey (7), and in general all the authors of text-books, consider the pericardium as being a fibrous structure built up of fibres orientated in all directions. The connections of the pericardium with the diaphragm and blood vessels (aorta, 1

This is a part of a series of studies which one of us started six years ago. These studies are concerned with the orientation of connective tissue. The first study published in this series is entitled: Gr.T. Popa, "Structure fonctionnelle de la