Inducing Extraseasonal Ovulation in Rana Pipiens With Steroid Harmones

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FORDHAM UNIVERSITY GRADUATE SCHOOL

June.....................19..4S

This dissertation prepared under my direction by

William B» Langan...................... entitled

Inducing Bxtraseaaonal Ovulation in

....... ,Rana,..p.ipi,ens.,,witfe,,,,St

...

has been accepted in partial fulfilment o f the requirements for the

Degree o f

............................................

(Faculty A d viser)

INDUCING EXTRASEAS OKAL OVULATION IN RANA PIPIENS WITH STEROID HORMONES

BY WILLIAM B. LANGAN M.A., COLUMBIA UNIVERSITY 1957

DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF BIOLOGY AT FORDHAM UNIVERSITY

NEW YORK 1942

ProQuest N um ber: 10992507

All rights reserved INFORMATION TO ALL USERS The q u a lity of this re p ro d u c tio n is d e p e n d e n t u p o n the q u a lity of the co p y su b m itte d . In the unlikely e v e n t that the a u th o r did not send a c o m p le te m a n u scrip t and there are missing p a g e s, these will be n o te d . Also, if m a te ria l had to be re m o v e d , a n o te will in d ic a te the d e le tio n .

uest P roQ uest 10992507 Published by ProQuest LLC(2018). C o p y rig h t of the Dissertation is held by the A uthor. All rights reserved. This work is p ro te cte d a g a in s t u n a u th o rize d co p yin g under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

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TABLE OF CONTENTS

LIST OF TABLES ACKNOWLEDGMENTS INTRODUCTION

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MATERIALS AND METHODS

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OBSERVATIONS AND DISCUSSION OF TABLE I OBSERVATIONS AND

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DISCUSSION OF TABLE II ..............

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OBSERVATIONS AND DISCUSSION OF TABLE III OBSERVATIONS AND

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DISCUSSION OF TABLE IV

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NATURE OF STIMULUS FOR O V U L A T I O N ...................... 15 SUMMARY

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CONCLUSIONS ...........................

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LITERATURE CITED

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LIST OF TABLES Page TABLE I

Experiments, dosage, and percent ovulation for the hormone estradiol. ....

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TABLE II Experiments, dosage, and percent ovulation for the hormone progesterone............................

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TABLE3III Experiments, dosage, and percent ovulation for the hormone testosterone• II TABLE IV Experiments, dosage, and percent ovulation for the hormone desoxycorticosterone• .... ...••....... .

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ACKNOWLEDGMENTS The author is indebted to Mr. R. C, Mautner, Ciba Pharmaceutical Products Incorporated, for supplies of progesterone (Lutocylin) and estradiol (Bi-ovocylin)• The author is equally indebted to Doctor William H. Stoner, Sobering Corporation, for supplies of testosterone (Oreton) and desoxycorticosterone (Cortate). It was only through the kindness of the above named that research "was made possible.

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INDUCING EXTRASEASDIAL QYULATION IK SANA PI PI M S WITH STEROID HORMONES

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*1 INTRODUCTION

Wolf (1S29) and Houssay, Guisti, Lascano-Gonzalez (1929) independently and simultaneously demonstrated that ovulation could be induced in amphibia by implantation of the anterior pituitary gland.

Houssay (1929) found that pituitary implants from various

vertebrate classes would not duplicate the phenomenon of inducing ovulation as produced in the toad by homoplastic implants.

He was

the first to advance the hypothesis of gonadotropic hormone specificity as an interpretation of these results.

This concept

was somewhat devaluated by Willis, Riley, and Stubbs (1933), by showing that large doses of pituitary from the garpike Lepidosteus, induced the toad to ovulate.

Since this time it has become

definately established (Review by Creaser and Gorbman, 1939) that there exists a quantitative variation of anterior pituitary hormones from species to species.

Yet in some cases, especially those where

the donor and recipient are of widely divergent taxonomic groups, a qualitative variation like that postulated by Houssay, must be considered.

Creaser and Gorbman, (1935) tested the ovulatory response of Rana pipiens to large doses of mammalian gonadotropic materials. They concluded that gondotropic hormone specificity had been demon­ strated, for the high doses used and the variety of preparations employed, obviate any explanation of these facts on a threshold basis.

The ovary of Rana pipiens was responsive to hyphophyses of

other Anura (Rana eland tans, Rana catesbeiana, Rana sphenocephala,

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Bufo marinus) but when large doses of fish pituitary were used, the same responsivness could no longer be elicited.

Creaser and Shcolnek

(1939) showed that Rana pipiens will not respond to doses of eighteen pituitaries of the urodele Heoturus maculosus.

However, Adams and

Granger (1938a) induced ovulation in Rana pipiens with a high dose of forty to ninety pituitaries from Triturus viridescens. This general refractoriness of Rana pipiens to pituitary donors outside of the taxonomic group Anura, and the complete re­ fractoriness to mammalian gonadotropic factors attracted considerable attention. ments.

Langan (1941) conducted a series of preliminary experi­

The results corroborated the previous data on refractoriness

of Rana pipiens to mammalian gonadotropic factors, but introduced a new concept, namely, that certain steroid hormones are capable of inducing ovulation in Rana pipiens. The experiments herein reported are an attempt to check and advance this concept.

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MA.TEBIALS AND METHODS Fresh hibernating adult female Rana pipiens were obtained directly from Vermont every three weeks, from November to April, and stored at 10°€. with just enough water to maintain a moist body. The frogs were removed to room temperature for experimentation and kept individually in one gallon glass bowls containing a one inch depth of water.

The animals were not fed, since the length of ex­

periments were not sufficient to deplete the fat bodies. Hypophysectomy was accomplished under ether anesthesia. It was effected through the following stepst

(1) an incision ap­

proximately one centimeter long was made in midline of roof of mouth, thereby exposing the parasphenoid bone, (2) using a bone drill driven by a small hand electric drill, a cavity was made through the parasphenoid bone thus exposing the pituitary, (3) a pipette, partially filled with sterile ringers solution was in­ serted into the bone cavity and by alternately applying a pressure and suction action the gland was freed and lifted into the pipette. By careful manipulation pituitaries may be extracted in their en­ tirety thus insuring complete removal.

Another advantage of this

method is the reduction of bleeding and surgical shock. Hypophysectomised frogs were returned to the 10°C. storage chamber for three to five days to allow recuperation from any operative effects.

Extracted pituitaries were preserved for control experi­

ments by placing in a five percent alcoholic Holtfreter solution and maintaining a temperature of 10°€.

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^ The steroid hormones tested for ovulation inducing proper­

ties were, (1) testosterone (Oreton), (2) progesterone (lutooylin), (3) estradiol (di-ovocylin), and (4) desoxycorticosterone (cortate). They were carried in sesame oil.

Hormones were administered by in­

jecting, with a small hypodermic, directly into the lower left quadrant of the abdominal cavity. greatest and most rapid absorption.

This technique is conducive to the (Hugh 1934).

After injecting a

given dosage the degree of ovulation was determined at twenty four hour intervals. methods?

This was ascertained by either or both of two

(1) Attempted stripping, and (2) sacrificing frogs for

dissection.

In some cases of persistent negative response to

stripping, homoplastic pituitaries were injected to determine whether or not the specimens were capable of responding.

'When an animal was

sacrificed for exploration by dissection, portions of ovaries were tested ttin vitro”.

Such a test is performed by removing portions of

ovaries, each containing approximately one hundred eggs, rinsing in Ringers solution and then immersing in a pituitary suspension made by mashing one anterior lobe in twenty five cubic centimeters of Ringer solution.

Portions of ovaries placed in plain Ringer solu­

tion serve as controls.

After twelve, eighteen, and twenty four

hours immersed ovarian fragments are observed for extruded eggs. Several eggs extruded from such fragments constitute a positive re­ action.

Usually twenty five percent ovulation was completed before

being impeded by cytolysis.

Ryan (1940) demonstrated that ovulation

produced in this manner is identical with the same process induced "in vitro"* L

Portions of oviducts and ovaries from all experiments j

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(1) normal and hypophysectoraised

frogs injected with homoplastic pituitaries; (2) normal and hypophysectoraised frogs injected with sesame oil (vehicle for hormenes); and (3) normal and hypophysectoraised frogs maintained at experimental conditions.

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OBSERVATIONS AND DISCUSSION Experiments, dosage and reactions for estradiol are sum­ marized in table 1; for progesterone in table 11; for testosterone in table 111; and for desoxycorticosterone in table IV.

Experiments

recorded in each table are arranged chronologically, the first repre­ senting early December, the concluding being the middle of April. This order parallels the seasonal decrease in ovarian threshold to ovulatory stimulus.

Ovulatory response is recorded on a percentage

basis representing the relative number of eggs liberated from ovaries* TABLE 1A. A significant fact emerging is the consistent refractoriness. 1941).

This corroborates previous observations (Langan

Experiments A-2a and A-2b deserve special consideration in

terms of checking ovulatory capacity of treated frogs.

Each re­

ceived two injections of 1 cc. on the first and fourth days.

Re­

maining negative until the twelfth day, A-2a received two homo­ plastic pituitaries.

Maintaining negativity, on the fifteenth day

each received 2 pituitaries.

During the period of the eighteenth

to the twenty-second day, A-2a ovulated approximately 60%, while A-2b responded about 10^.

Contrasted to controls, which ovulate

completely from two pituitaries, A-2a and A-2b require at least double the stimulus for ovulation.

This increase in threshold may

be attributed to one or both of two factors*

(l) estradiol or

sesame oil, or both, affects ovary; (2) ovaries undergo resorptive changes due to lack of food.

Evidence for action of sesame oil

is gained from a sesame oil control, which required a pituitary

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