The effects of gonadotropic and estrogenic hormones on the teleost, Xiphophorous helleri Heckel

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THE EFFECTS OF GONADOTROPIC AHD ESTROGENIC HORMONES ON THE TEIEOST, XIPHOPHORUS HELLERI HECKEL

A Thesis Presented to the Facility of the Department of Zoology University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science

by Min Hsin Li May 1942

UMI Number: EP67149

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T h i s thesis, w r i t t e n by

.......... MIN-MM..LI................... u n d e r the d ir e c t io n o f h .. % 3 F a c u it y C o m m itte e , a n d a p p r o v e d by a l l its m e m b e r s , has been p r e s e n t e d to a n d a c c e p t e d b y t h e C o u n c i l on G ra d u a te S tud y a nd Research in p a r ti a l f u l f i l l ­ m e n t o f the r e q u ir e m e n ts f o r the degree o f

MASTER OF SCIENCE

S ecretary D a t e ...

F a c u lty C o m m itte e

C h a irm a n

ACKNOWIEDGMBNTS The writer wishes to express his thanks for the helpful criticism of Dr, Francis M. Baldwin, under whose direction this work was undertaken, as well as the advice and suggestions of Dr, Bruce M. Harrison and Dr, Arthur J, Tieje. The writer is indebted to the Schering Corporation in supplying hormones, Pranturon and Progynon-B, which were used in this investigation.

For the photographs and photomicro­

graphs illustrated in this thesis, the writer also wishes to acknowledge the photographic skill of Mr. Gr. Aschcraft and Mr. Ozro B. Wiswell.

TABES OP CONTENTS CHAPTER

PAGE

ACKNOWIEDGMENTS...............................

ii

I.

INTRODUCTION...................................

1

II*

REVIEW OP L I T E R A T U R E ............ .............

4

A* Gonadotropic H o r m o n e s ...................

5

B. Estrogenic H o r m o n e s .....................

21

III.

MATERIALS AND M E T H O D S .........................

40

IV,

OBSERV A T I O N S ...................................

50

A. Male P i s h .................... *..........

50

1, Gonads of the control males •»•••-••«

50

2. Gonads after the gonadotropic hormone administrations......

57

5. Gonads following the estrogenic hormone injections ............ B. Female P i s h ........... 4. Gonads of the control f e m a l e s ......

65 88 88

5. Hormonal induction of the sex-reversal in f e m a l e s ......... , V.

VI.

93

D I S C U S S I O N S ....................................

119

A. Actions of the G o n a d o t r o p i n ............

119

B. Actions of the E s t r o g e n ..............

127

S U M M A R Y ........................................

B I B L IOGRAPHY..........................

185 137

TABLE OF PLATES PLATE

PAGE

1.

Konnal Control Female (FC-61).

Scale in mm.

2.

Normal Control Male (MC-6).

3.

Cross-Section of A Control Male Gonad (MC-8).

Scale in mm.

...

41

.....

43

x 400 .......................................... 4.

Cross-Section of A Control Sesame Oil Injected Male Gonad (MSO-2). x 400 ....................

5.

58

Cross-Section of A Gonadotropin Treated Male Gonad (MPU-17). x 500 .................

7.

54

Cross-Section of A Gonadotropin Treated Male Gonad (MPU-ll) . x 400 ...................

6.

52

60

Cross-Section of A Gonadotropin Treated Male Gonad, Showing the Recovery of Hormonal Effects After Discontinued Treatment for Seven Weeks. (MPU-4) . x 400 ....................

8.

Cross-Section of An Estrogen Treated Male Gonad, Phase 1 (MPB-4). x 400 .......................

9.

73

Cross-Section of An Estrogen Treated Male Gonad, Phase 3 (MPB-l). x 400 .......................

12.

70

Cross-Section of An Estrogen Treated Male Gonad, Phase 3 (MPB-3). x 400 ......................

11.

67

Cross-Section of An Estrogen Treated Male Gonad, Phase 2 (MPB-9). x 400 .......................

10.

63

76

Cross-Section of An Estrogen Treated Male Gonad, Phase 4 (MPB-17). x 400 ......................

79

V

TABLE OB PLATES (Continued) PLATE 15.

PAGE Cross-Section of An Estrogen Treated Male Gonad, Phase 5 (K PE-5), x 400 ...........

14.

Cross-Section of An Estrogen Treated Male Gonad, Phase 5 (MPB-12). x 500 ........

15.

90

Developing Gonopodium of A Gonadotropin Treated Female (FPU-37), x 80 .............

17.

84

Cross-Section of A Control Normal Female Gonad (EC-14). x 400 .......

16.

82

95

Developing Gonopodium of An Estrogen Treated Female (FPB-ll). x 80 ..........

18.

Control Male Gonopodium (MC-5). x 80 ........

19.

A Gonadotropin Treated Female, Showing the

98 100

Transformation of Anal Fin into A Gonopodium (FPU-37). Scale in m m ...................... 20.

A Gonadotropin Treated Sex-reversed Female Gonad (FPU-37), x 400 .....................

21.

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

107

A Gonadotropin Treated Sex-reversed Female Gonad (FPU-42), x 400 .....................

23.

105

A Complete Sex-reversed Female (FPU-32). Scale in mm.

22.

102

110

An Estrogen Treated Female, Showing the Transformation of Anal Fin into a Gonopodium (FPB-13). Scale in m m ......

114

vi TABLE OF PLATE (Continued) PLATE 24.

PAGE Ovotestis of An Estrogen Treated Female (FPB-13). x 500 ..........................

116

CHAPTER I INTRODUCTION With the discovery of an increasing number of sex hormones in mammals, and an increasing diverse series of physiological activities in which they are Involved, it has become of great interest to examine the specific action and relative potency of such substances on the various parts of the reproductive system of lower vertebrates.

Many of the exploratory works, however,

have been discouraging because the results of mammalian gonadotropins injection have particularly not been clearcut, or because of the relatively massive dosages required to yield a minimum response.

The relative inefficiency

of mammalian gonadotropic preparations in many lower vertebrates has led some investigators to suggest that a species specificity of gonadotropic hormones might exist. Other investigators having found some species which will respond to certain

doses of mammalian gonadotropins,

make opposite claims.

In fishes, the stimulation of

gonads with extracts of fish pltuitaries or with homoplastic implantation of pituitaries from other fishes has been reported by several investigators (Houssay, 1931; Cardosa, 1934; Pereira and Cardosa, 1934; Ihering, 1935;

2

Hasler et al., 1939; Matthews, 1940; Burger, 1941).

The

administration of mammalian gonadotropic hormones to fish has, however, produced inconsistent and often negative results (Saphir, 1934; Kleiner et al., 1935; Koch and Scheuring, 1936; Hasler et al., 1939; Johnson and Riddle, 1939; Matthews, 1939; Landgrebe, 1941)• Positive effects have been reported by others (Calvet, 1932; Damas, 1933; Boucher et al., 1934; Owen, 1936; Berkowitz, 1941). The rble of estrogenic hormones in sexual differ­ entiation, including the development of the gonad, has been studied in many forms, including fish (Berkowitz, 1937, 1941a; Regnier, 1938; Bullough, 1940; Noble and Borne, 1940).

Similar work concerning androgen effects

in fish has also been reported (Witschi and Crown, 1937; Regnier, 1938; Baldwin and Goldin, 1939, 1941; Eversole, 1939, 1941; Bullough, 1940).

Baldwin and Goldin (1939,

1941) found that the androgenic substance, testosterone propionate, not only transformed the secondary sex characters but caused sex-reversal in approximately b0% of the female of an ovoviviparous teleost, Xiphophorus helleri Heckel. Even from the above findings, there still can be drawn no general conclusion on the effects of the mammalian gonadotropic preparations in fish.

We under­

3

took, therefore, to study further the responses of the mature male and female of the Xlphophorus hellerl (swordtail), to a gonadotropic hormone of human chorionic origin*

The purpose is to reveal first whether this

species of fish is responsive to the pregnancy urine preparation, and secondly, if so, to determine what elements in the gonads could be stimulated and what, if any, distinct follicle-stimulating and luteinizing effects could be separately elicited.

Attention is also paid to

the influences of mammalian gonadotropin on the primary and secondary sexual differentiations of the fish.

The

present investigation of the estrogenic substance, alpha-estradiol benzoate, is complementary to that of Baldwin and Goldin (1939, 1941) in this same species. The particular effort is to determine what manner of action is the estrogen on the mature gonads and secondary sex characters of either sexes, especially under prolonged injections.

It is hoped that experimental

results of these sex hormones in the Xiphophorus hellerl can lend some more light on the physiological relations of the sex and internal secretions.

CHAPTER II REVIEW OF LITERATURE A little more than a decade ago, the direct influence of the anterior hypophysis on the gonads and the indirect influence of this gland on the accessory reproductive organs was established.

This work had been

preceded by several interesting and instructive papers which had revealed that the gonads were not independently functioning organs, but were dependent on some unknown but essential substance for their maintenance.

Investi­

gation revealed that this unknown substance was supplied by the anterior hypophysis as well as some other sources. The isolation and experiment of estrogenic substances were also new to endocrinology.

The existence of an

ovarian hormone was not known in 1910.

Although history

of the study of these sex hormones is very short, an immense amount of work has been done and the literature is extensive and, to a considerable degree, confusing. In this chapter we review separately the experimental results of previous investigators on the physiological activities of these two closely related hormones, namely, human chorionic gonadotropin and estrogen.

Only litera­

ture dealing with our experiments is being considered.

5 A.

Chorionic gonadotropic hormone.

The demon­

stration of the gonad-3timulating action of the anterior hypophysis was followed by the discovery, in rapid succession, of three other rich sources of gonadotropic substances;

(1) A gonadotropic principle in human

pregnancy urine.

This is called "the pregnancy urine

factor” or in a nomogram, PU.

(2) The substance found

in the blood serum but not in the urine of pregnant mares. This is referred to as PMS.

These two substances, though

qualitatively different, have the common factor of occurring only during the presence of living chorionic tissue.

This relation together with a certain similarity

of biological reactions led Hamburger (1935) to call them “chorionic hormones.”

(3) A large amount in the blood

serum and in the urine of castrate or postmenstrual women.

This gonadotropic hormone of castrate urine is

designated as CU.

It appears that the same substance is

present in the urine of both men and women in lesser amounts throughout life. Ascheim and Zondek (1923) first announced the gonadotropic action of human pregnancy urine (PU) and established the practical Ascheim-Zondek test for pregnancy.

In Zondek1s Prolan A reaction only follicle

stimulation resulted, while with Prolan E luteinization occurred.

The reaction of the 6-8 gram mouse or 22 day

6

old rat gives these typical reactions which easily lead to the concept that the effects of anterior pituitary implants or PU injections were identical, and that the source of all gonadotropic substances was the anterior hypophysis. On hypophysectomized mature or immature female rats, however, Selye, Collip and Thompson (1933) report changes in the thecal cells with thecal luteinization. Estrus did not result in the immature, but was continuous in the mature animal.

Leonard and Smith (1933, 1934a)

observed the responses in a considerable series of hypophysectomized rats, both mature and immature, in which treatment with PU was in certain groups instituted at once, and in others only after a period of gonadal atrophy extending from 16 to 78 days.

They suggested

that capacity to form corpora lutea with PU treatment immediately after hypophyseetomy depends on the age of the rat.

That is, unless the ovary of the test animal

already has maturing follicles, formation of corpora lutea will not follow PU treatment. Cytological studies of the pituitary of rodents led Sevringhaus (1934) to assume that injections of PU led to a rapid discharge of the gonadotropic substance. The hypophyses of PU treated animals showed a decreased potency in normal treated animals but not in castrated

7 females (Leonard, 1933).

The evidence from all experi­

ments on female rodents, therefore, Indicates that the human chorionic hormone, PU, is never a follicle stimu­ lator.

Tho apparent effect of follicular stimulation in

intact test animals may be produced by other factors; the most apparent of which is the activation of the animal* s own pituitary gland, the action being in some ways mediated through the ovary. Masculinization of infantile female rats by treatment with gonadotropic extracts from PU and PMS has been reported by several workers.

The treatment of PU

causes gross enlargement of the clitoris and the develop­ ment of an os priapi (Greene and Burrill, 1939)•

Bradbury

and Gaensbrauer (1939) made similar observations in female rats and, in addition, noted that continuation of injection beyond 30 days of age caused no further growth of the clitoris.

On the other hand, a gonadotropic

extract from anterior pituitary did not cause any of this response.

Greene and Burrill (1939a) implanted

ventral prostates from male littermates into female rats 10 days of age.

Daily treatment with PU produced no

evidence of androgenic stimulation in the prostates when the females were castrated.

When the ovaries were not

removed the prostates of the treated animals showed evidence of stimulation at 18 to 25 days, but not after

8

28 days of age.

The ovaries of masculinized suckling

rats were lacking in corpora lutea, but they contained an excessive mass of thecal or interstitial tissue, usually in the luteinized form.

Marx and Bradbury (1940)

concluded that when the ovary is incapable of undergoing corpus luteum formation under gonadotropic stimulation, it may produce enough androgenic substance to cause a marked masculinization. Engle (1929), Brouha and Simonnet (1929) reported that the action of the PU principle in immature male rats or mice is primarily on the interstitial cell mass, and thus on the accessory organs.

This type of response to

PU extracts is generally accepted.

There is some

discrepancy of opinion as to whether precocious spermato­ genesis is induced with PU. genesis was not accelerated.

Engle stated that spermato­ Brouha and Simonnet believed

that precocious spermatogenesis occurred, as did Boeters (1931), Neumann (1931), and others.

Moore (1936) was

unable to find any precocity effect in immature rats. Others have reported failure to obtain this acceleration in normal rats or mice (Engle, 1932; Molieu, D ’Amour and Gustavson, 1933).

Moore (1936) adopted as a standard,

the appearance of ”the elongated nuclei of metamorphosing spermatids, here designated *sperm heads’.”

With this

physical criterion, Moore found that in his colony, at

9 all periods of the year, this stage normally appears in rats from day 33 to 35.

He concludes that the discrepan­

cies between those who found precocity and those who did not may be due to a number of factors, of which "the utilization of different criteria for judging precociousness" may be the most important. The response of the hypophysectomized male rat has been studied by several groups.

Collip, Selye and

Thompson (1933) reported that treatment of immature or mature hypophysectomized rats with the PU factor did not prevent degeneration of germinal epithelium, although there was over development of the interstitial cell mass. Smith and Leonard (1933, 1934a) in a larger series of mature rats report maintenance of tubular structure, spermatogenesis and fertility when injections were begun immediately after hypophysectomy.

Injections begun after

a period of atrophy restored spermatogenesis In only one animal, although the general condition and size of the testis was improved.

Regression occurred in both tubules

and interstitial cells during long treatment.

The mating

reaction of hypophysectomized male rats when treated with PU extracts was also reported by the same workers (1933a). The earlier records showed considerable destruction of germinal epithelium (Engle, 1929, 1932; Neumann, 1931). More recent experience with more highly purified products

10 Indicates that this type of response was due to toxicity or impurity, and not to a direct hormonal action.

The

change in tubule size is not significant in the youngest rats treated (6 day3 old), nor in the mature onoo (Engle, 1958). Friedman (1929) demonstrated that by a single intravenous injection of PU in the adult female rabbits in heat, ovulation could be induced in from 10 to 15 hours. This finding has been widely employed as a rapid and accurate test for pregnancy.

The corpora lutea resulting

from this type of induced ovulation seem to be perfectly normal in producing characteristic early pregnancy changes in the genital tract (Friedman, 1952a).

However, if ripe

Graafian follicles are ruptured by puncture, corpora lutea do not form; but if a small amount of PU is introduced into a single follicle, it will induce transformation into lutein tissue without influencing neighboring follicles (Friedman, 1952). In the ground squirrel, normal young of the season could be induced to form spermatozoa much earlier than usual (Wells and Moore, 1956) and adults likewise are stimulated to produce spermatozoa during the period of relative testicular inactivity (Baker and Johnson, 1956).

Booster and Hi saw (1935) although concerned mainly

with anterior pituitary fractions, also report that in

11 the anestrous cat follicular development can be stimu­ lated with PU, and that ovulation has been obtained. This effect of induction of mating and ovulation in the oat with PU treatment has been confirmed by Windle (1939). Papanicolaou and Falk (1934, 1936) reported a masculinization of female guinea pigs due to treatment with PU substance.

This effect consisted of growth and

enlargement of the clitoris.

Masculinization on the

temporal muscles of the skull was also noted by them (1938).

They assumed that the hypertrophied interstitial

tissue of the ovary produces an androgenic substance. Jares (1931) reported the inability to induce ovulation in guinea pigs by intravenous injections of a PU prepa­ ration; in this Loeb (1932) agreed.

On the contrary,

King (1933) obtained positive effect of ovulation in immature guinea pigs with PU and other gonadotropins. Adult monkeys (Engle, 1933) when taken during the first half or estrone phase of the cycle when the estrous sex skin reaction is increasing, are treated with PU, either by subcutaneous or intravenous injections, a marked blanching occurs within 2 or 3 days.

The sex skin

is a sensitive indicator of estrogenic action.

In none

of the animals treated with PU substance has any signifi­ cant development of the sex skin occurred.

The ovaries

indicate the reason for the arrest of estrogenic action,

12 since the follicles invariably show signs of degeneration. Many of the animals complete hyalinization of the theca interna, with cytolysis of the granulosa occurs.

These

hyalinized follicles are considered to be the terminal phases of atretic involution.

No luteinization appeared,

although infrequent luteoid changes can be seen.

The

male monkey responses with increase of the interstitial cell mass, uniform and pronounced increase of the testis weight and size, and by descent of the testes (Engle, 1932, 1932a; Aberle and Jenkins, 1934).

However, they

failed to stimulate the spermatogenic development in young monkeys. The earliest gonadotropic material used in the modern era of endocrine therapy in gynecology was chorionic gonadotropin, PU, formerly called “anterior pituitary-like” (APL) because it was thought to resemble the hormone produced by the anterior pituitary gland.

It

is now known that this was erroneous, and that the chorionic gonadotropins does not stimulate human ovaries to normal types of activity consistent with regular cycles and fertility (Council Pharm. Chem., Am. Med. Assoc., 1940).

Although the capability of the human

testicle to exhibit germ cell stimulation has been demonstrated by Brosius and Schaffer (1933); Schering Corporation recommends the PU extract (Pranturon), which

13 the writer used in experiments, only for the treatments of cryptorchidism and menorrhagia. Birds respond with remarkable rapidity and intensity to substances derived from pituitary sources but appear rather refractory to gonadotropic agents from pregnancy urine (Schockaert, 1933),

Lahr, Riddle and

Bates (1936, 1941) induced growth of testis, increased intertubular tissue and accompanied with complete spermatogenesis in a two months dove or pigeon testis by daily injection of a PU preparation.

In the females, it

decreases the size of the oviduct (uterus) and stops or retards growth in the ovary (Riddle and Polhemus, 1931), Breneman (1936) reported it increases the gonad weights of new hatched chickens.

Early PU extracts containing extreme

ly little of the follicle-stimulating factor, therefore, give no effects on the testes of dove, pigeon (Riddle and Bates, 1933; Evans and Simpson, 1934), and fowl (Martins, 1934).

Adult male sparrows treated with pituitary deriva­

tives during winter produce spermatozoa months before the controls (Witschi and Keck, 1935). The possible role of gonadotropic hormones in ovariogenic masculization in the immature female chick has been demonstrated by Domra (1932, 1937) and Uotila (1939). Injections of hypophyseal as well as PMS hormones produced some medullary cord and interstitial cell hypertrophy,

14 which accompanied by the masculinization of head furnish­ ings in pullets*

Results show that PU extract is

comparatively weak in this effect. Among the reptiles, Ilerlant (1953) injected PU preparation into female blindworm (Anguis) during the non-breeding period which provoked the sexual segments of the renal collecting tubules and the epididymis to active secretory condition and an increase in interstitial tissue.

Administration of the same substance to a male

lizard (Lacerta) was followed by hypertrophy of the inter­ stitial tissue.

Evans (1935, 1935a) increased the weight

of gonads and accessory sex organs in both male and female lizards (Anolis), and brought to breeding season condition in females, by injections PU in the period of sexual quiescence.

Same results in male Eumeces were observed by

Turner (1935).

Mellish and Meyer (1937) increased the

weight of horned lizard (Phrynosoma) ovaries by injecting the anterior pituitary extract of a hog in addition to PMS. However, in all cases the spermatogenesis in male lizards was not definitely stimulated by PU treatments.

Risley

(1941) found that the gonadotropic agents of the hypophysis induced similar effects in male juvenile terrapins; while in the females, gonadotropins gave variable responses. Adams (1931) failed to induce the ovulation in toads(Bufo vulgar!s) by means of PU preparation, although

15 she succeeded In frogs (Banja vulgaris) and toads by pituitary inoculations and injections of mammalian pitui­ tary extracts.

Hugh (1934) claimed that in amphibia,

there was no species or sex specifity when a certain amount of amphibian pituitary suspensions was injected.

Mammalian

pituitary extracts, whole sheep pituitary, and the PU material have proven to be entirely ineffectual in respect to the ovulation in Kana pipiens.

Kuyper et al. (1933)

reported neither the PU agent nor the PU combines with estrin is capable of inducing ovulation in toads, Bufo amerlcanus. Burns and Buyse (1931, 1934) found that the gonads of larval or recently metamorphosed salamanders are greatly stimulated in size and in proliferation of the germinal tissue, with precocious spermatogenesis, by extracts of mammalian hypophyses.

Both the whole sheep pituitary

extract and the PU preparation have been useful in inducing ovulation with Amblystoma. although the latter seems to be more potent (Buyse and Burns, 1931).

In adult Triturus,

a striking enlargement of ovaries and ovulation can be induced by injections of the sheep pituitary substances (Mayo, 1937). The duality of the hypophyseal gonadotropic hormone was claimed by Zondek (1930), Fevold, Hisaw and Leonard (1931), and some others.

One of these fractions was stated

16 to produce follicle growth, and the other luteinization. A concept has been formulated by Fevold and Hisaw (1934) in regard to the action of these two substances in the sex cycle.

From all the previous literature, it may be seen

that the pregnant urine gonadotropin (PU) from chorionic origin when applied to the vertebrates higher than fishes gave more or lees definite effect of luteinization.

This

luteinizating factor of the gonadotropic hormone, in all cases, stimulates the interstitial tissue in the affected male animals.

It is also evidenced by the response of the

accessory reproductive organs as a result of the increased output of the male sex hormone.

The effect of gametogenic

stimulation with PU treatments, however, is rather variable. Masculinization of immature female mammals and birds was considered to be an effect of chorionic hormone treatments which mediated through the affected ovary. In fishes, Houssay (1931) found that injection of saline suspensions of the hypophyses from large fish, Micropop;on. into small ones, Gnesterodon, was followed by the expulsion of eggs in from 1 to 3 days, although saline suspensions of muscle, or saline solution alone produced normal spawning by only 15 days.

Cardosa (1934) Injected

saline suspensions of the pituitary body of Pimelodus clarias Into other individuals of the same species.

In

both sexes he noted an Increase in weight of the gonads

17 over those of control animals, the effect was very much greater in the female.

Pereira and Cardosa (1934) working

with Prochilodus. injected saline suspensions of pituitary glands of the adult into females.

In all cases ovulation

occurred, varying from 24 to 96 hours after injection, but the injections of suspensions of nervous tissue into control animals did not result in ovulation.

Von Ihering (1935)

repeated these experiments by injecting suspensions of the pituitary glands of the Hoplias malabaricus into species Astyanax.

Mating activity was stimulated with an

increase in size of both ovary and testis over the untreated controls.

When injections were made into immature

Prochilodus of both sexes eggs and spermatozoa were obtained. More recently, Hasler and his collaborators (1939) obtained mature egg and sperm from rainbow trout, Salmo. 6 to 7 weeks in advance of the onset of the normal spawn­ ing period by injections of fresh or acetone dried pitui­ tary glands of the carp, Cyprinus carpio .

While they

failed to induce the spawning prematurely in trout by injecting the PMS and the follicle-stimulating fraction prepared from sheep pituitary.

Matthews (1939) observed

practically no stimulation of the immature testes of Fundulus with the injection of an unfractionated extracts of sheep pituitary.

In the female, records were obtained

from 35 cases of which only 4 delivered eggs earlier than

18 did control animals, one case delivering ripe eggs 6 weeks, the other 3 cases about 4 weeks before the normal breeding season.

He demonstrated that removal of the pituitary in

adult Fundulus is followed by inactivity or atrophy of the gonads.

In a further note, Matthews (1940) found that

the implantation of Fundulus pituitary into non-hypophysectomized immature Fundulus induced gametogenic activity, especially in the male.

Burger (1941) secured the positive

effects from pituitary implantations in hypophysectomized adult male Fundulus. The chorionic gonadotropins and extracts of other mammalian gonadotropic hormones have likewise been used in fishes with some success.

Calvet (1932) placed young

petromyzon in aquaria containing PU and found that the ovary Increased markedly in size over that of the controls. Damas (1933) injected 30 lampreys with PU and obtained expulsion of eggs in all cases.

He also employed extracts

of the anterior pituitary with similar results.

Boucher

et al. (1934) injected PU into eels and reported enlarge­ ment of the gonads in both sexes and evidence of the maturation of spermatozoa in testes.

Saphir (1934) found

that injection of PU (Zondek’s Prolans)

into male

Chromosomus erthrogaster caused Inconstant response of “wedding dress”.

Owen (1936) produced this chrotnatophore

reaction in 75^ of male Rhodeus amarus by employing the

19 same hormone in aquarium water.

Using the ovipositor

lengthening as an indicator, Kanter, et al. (1934) suggested the use of Japanese bitterling (Acherlognathus intermed1n m ) substituting a rabbit or a mouse for the pregnancy test.

But this idea of pregnancy urine test

was soon disapproved by Kleiner and others (1935, 1936). Recently Berkowitz (1941) injected four different preparations of mammalian gonadotropins, i.e., extracts of whole sheep pituitaries, PMS, PU, and normal male urine into the guppy, Lebistes reticulatus.

All these substances

stimulated the precocious development of male secondary sex characters, as well as the precocious but normallypatterned development of the immature male gonads.

The

response of adult male Lebistes« if at all, is by an inter­ stitial stroma hypertrophy and a decreased gonad size.

The

former phenomenon persists only in some and not all of his fishes for the longer treatments (30 days).

There were no

effects on the secondary sex characters or gonads of immature female Lebistes.

He failed to mention the effect

of gonadotropins on the adult female of this species. However, it was suggested that one mammalian gonadotropic hormone is enough to complete the activition of the testis of the immature fish. On the other hand, wholly negative results were obtained by Young and Bellerby (1935) in both young and

20 adult lamprey with the injection of anterior pituitary extracts.

Koch and Scheuring (1936) failed in injecting

Phoxinus with PU and other preparations.

Johnson and

Riddle (1939) tested the action of follicle-stimulating principle from beef pituitaries, powdered PMS, and prepara­ tion of PU on 90 rainbow trout, Salmo.

Both in ovary and

testis were uneffected, as did Easier et si. (1939) and Landgrebe (1941).

They concluded that the gonadotropic

hormone elaborated by pituitary of fish differs qualita­ tively from that of mammals. From the above findings, the administrations of mammalian gonadotropins to immature fishes do have some effects in precocious maturation in both sexes.

However,

no general conclusions can be drawn in the mature fishes. We undertook, therefore, to study further the effects on the adult female and male of a teleost fish, Xiphophorus helleri (Swordtail), to a gonadotropic hormone of human chorionic origin.

It was hoped to reveal first whether

this species of fish is responsive to the PU preparation, and secondly, if so, to determine what elements in the gonads could be stimulated and what if any distinct follicle-3timulating and luteinizing effects could be elicited.

Attention was also paid

to the influences of

PU on the sexual differentiation of the fish.

21 B.

Estrogenic hormone.

Estrogen is a collective

term for all substances producing estrous growth in vagina, uterus and mammary glands and female secondary sexual characters of the mammal.

At least nine natural estrogens

of closely related structure have been isolated.

In a

recent paper (1941) Doisy summarized the bio- chemical works in a table as follows:

Date

Investigators

Urine, preg.women

1929

Urine, preg.mares Urine, men Urine, stallion

1931 1938 1934

Placenta, human Testes, stallion Adrenal, beef Palm kernels Urine, preg.mare

1938 1940 1939 1933 1938

Urine, preg.women

1930

Placenta, human Pussy willows? Urine, preg.women

1931 1933 1935

Doisy et al., Butenandt Laqueur et al. Laqueur et al. Haussler; Deulofeu Westerfeld et al. Beall Beall Butenandt Schachter & Marrian Marrian; Doisy et al • Browne and Collip Skarzynski Cohen 5c Marrian

Ovaries, sow

1935

Urine, preg.mare

1935

b-Estradiol

Urine, preg.women Placenta, human Urine, preg.mare

1939 1940 1939

Equilin Hippulin Equilenin 17-Dihydroequilenin

Urine, Urine, Urine, Urine,

1932 1932 1932 1936

Estrogen Theelin or Estrone

Estrone sulfate Theelol or Estriol Estriol glycuronide Dihydrotheelin or a-Estradiol

Source

preg.mare preg.mare preg.mare preg.mare

MacCorquodale, Thayer, & Doisy Wintersteiner et al. Smith & Huffman Huffman et al. Hirschman Sc Wintersteiner Girard et al. Girard et al. Girard et al. Wintersteiner et al.

22 Since the estrogens in purified form became avail­ able, their effect on sexual differentiation and develop­ ment has been studied simultaneously in different labora­ tories.

It is generally agreed that injections of

estrogenic hormones cause an atrophy of the gonads (Moore and Price, 1930, 1932; Biskind, 1941; others) and an increase in the weight of hypophysis with a decrease In its gonadotropic power (Leonard et al., 1931; Wolfe, 1935; Pevold et al., 1936; others).

In immature male and female

rats daily injection of estrogen definitely inhibits the spermatogenesis and ovogenesis (Spencer, 1932).

Gonads

did not develop normally but remained small and Infantile. Degenerating ova are found in the ovaries and no corpora lutea are present.

The prostate glands and seminal

vesicles of male animals are in a castrated condition. Spencer et al. (1932) also found that estrogen treated young rats did not grow as rapidly as did their littermate controls.

Similar results were reported by Clausen,

Clay and Freudenberger (1939).

However, they added that

the vagina Is prematurely opened and the size of uterus is increased.

Although the body weight of injected immature

female rats is considerably decreased, neither the weight of ovary nor the body length is affected.

Talbot (1939)

observed a significant advance in the skeletal age of Immature female rats but not of the males by the injections

23 of an estrogen. The effects of estrogen upon reproductive organs of the adult rats have been studied by Halpern and D*Amour (1934).

After 3 to 4 weeks of injections, estrogen caused

the loss of weight in testes of male animals.

There are

only a few primary spermatocytes left, and numerous mitotic figures are present in the spermatogonia.

The interstitial

tissue of the testes is considerably reduced.

As in male,

the ovaries of adult female rats also showed a progressive loss of weight, whereas the diameter and weight of uteri are increased.

Castrated uterus is restored to the

approximately size of the normal female.

Results showed

that the secreting activity of the mammary glands in both sexes is stimulated.

The size of male mammary glands is

induced to a condition simulating that of the normal female.

Gradually decrease of body weight is observed in

normal males and castrated females, while it is maintained in the normal treated female rats. Ball (1938) reported that the injections of estrogen into adult castrated female rats produced the anatomical and behavior changes characteristic of estrus.

If adequate

amounts of the hormone is injected into the intact females it prevents the ovarian changes upon which normal estrus is dependent.

Behavior during the injection period showed

a fairly constant low degree of sexual receptivity.

However,

24 this is believed due to the persistence of functional corpora lutea in the ovaries of injected normal female rats (Donahue, 1938; Merekel and Nelson, 1940).

The fact

of estrogen injections inducing the liberation of luteinizer, and corpus luteum formation has also been reported in rabbits (Heckel and Allen, 1938; Robson, 1939)* It is well known that estrogenic activity may be shown by extracts of testes and by extracts of urine from the males of certain species.

Conversely, angrogenic

activity can readily be demonstrated in the urine and ovarian extracts of normal women (Parkes, 1937),

This

paradoxical effect is first showed by the restoration of atrophic seminal vesicles and prostates of castrated mice (Hill, 1937) or rats (Deanesly, 1938) with ovarian grafts. They believed that the androgen output Is directly related to the amount of thecal luteinization present in the grafted ovaries.

The androgenic potency of the progesterone has

been demonstrated in the rat (Greene, Burrill and Ivy, 1939).

These studies were repeated and extended by Turner

and Haffen (1940).

Angrogenic ovaries in the rats can be

produced by postnatal injections of estrogen.

The follicles

become atretic before reaching full maturity.

Thecal cells

become epithelioid and persisted after other elements of the follicle have deteriorated.

The ovaries from adult

animals of this type are capable of Inducing secretion in

25 the male sex accessories. Furthermore, Greene et al. (1939a, 1940) conducted a series of experiments on intersexuality of rats.

They

found that when large doses of estrogens were administered to pregnant rats, in 21 newborn females there was a stimu­ lation of certain female structures, e.g., uteri and nipples and inhibition of other female structures, e.g., lower vagina and ovarian capsules.

Wolffian ducts were

partially or completely preserved in these masculinized females.

On the other hand, feminizing effects of estrogen

on the sexual differentiation of genetic male rats by ante­ natal treatments have also been reported by them (1938). The male offspring were feminized in that development of the epididymis, vas deferens and seminal vesicle was inhibited and prostates were absent; a vagina, parts of the uteri and nipples were present.

The testes were

retained in the position typical of the female gonads. Franzier and Mu (1935) observed the development of female characteristics in adult male rabbits following the prolonged injection of an estrogenic substance.

Not only

the nipples of treated males showed greatly hypertrophied, but the milk could be expressed.

Testes decreased in size

and ascended from the shortened scrotum.

After 100 days

of injections it eventually became atrophy.

The coats of

the feminized male rabbits were usually heavy and clean

26 except over the areas where the injection were given. Their behavior was more passive and docile than the normal males• Moore (1939, 1940) and Burns (1939, 1939a) have attempted to modify sexual development with sex hormones in a lower type of mammal, the opossum.

At birth this

animal is sexually undifferentiated (Moore) or almost undifferentiated (Burns).

Estrogens inhibit normal develop­

ment of the urogenital sinus in the male opossum as shown by the absence of prostates.

The Wolffian duct of both

sexes, however, is directly stimulated, according to Moore, and at least not inhibited, according to Burns.

Both

investigators found that Mullerian duct is stimulated.

The

phallus of the male shoivs evidence of feminization (Burns). Either ovaries or testes are unappreciably modified in their development. Feminization and demasculinization in a case of a 17-year-old girl by injections of an estrogenic hormone has been reported by Lisser (1940).

Heckel and Steinmetz

(1941) produced the result of azoospermia in a 72-year-old man by injections of massive doses of an estrogen. Schering Corporation suggests the use of Alpha-estradiol benzoate (Progynon-B) for the therapy of hypogonadism, which in the female may assume the forms of uterine hypoplasia, amenorrhea (frequently primary), underdeveloped

27 breasts, vagina and vulva, and sexual indifference, as well as infertility, gonorrheal vaginites and dysmenorrhea. The birds exhibit a variety of responses when treated with, sex hormones at a stage prior to the beginning of sexual differentiation (Willier, Gallagher and Koch, 1937)•

Upon the injection of the estrogenic hormones, the

ovaries of genetic female chicks as a rule remain unaffect­ ed.

The left testis of the genetic males is markedly

altered in form and structure, appearing most often as a flattened ovary-like body consisting of both ovarian and testicular tissues (ovotestis).

The right testis remains

unchanged except in those cases exhibiting complete or nearly complete reversal of the left testis, whereupon it appears reduced in size, flattened, and contains ovarian rneduallary tissue; its testicular character is never entirely lost.

Mullerian ducts in the males persist partially or

completely. increased.

In the females the length of right oviduct is However, the Wolffian ducts appear unmodified.

The degree of sex-reversal attained is roughly proportional to the quantity of estrogen injected. The influences of embryonic injection of estrogens on sexual modification of brown leghorn have been studied further by Doram (1940, 1940a).

Some cockerels are normal

while others display vary degrees of feminization in plumage.

Masculine behavior is subnormal in many.

Although

28 crowing is common, treading is less so.

Pullets derived

from the treated eggs revealed no evident modification in secondary sexual characters.

Post mortem usually showed

norma]] ovaries. Hoskins and Koch (1939) stated that Injection of 0.5 mg. of estrogen daily for 2 or 3 weeks into cockerels and Incompletely castrated cockerels produced a temporary shrinkage in the comb with resumed growth as soon as the injections were discontinued. those of the controls.

The testes were smaller than

In immature pullets (Uotila, 1939),

a relatively small dosages of estrogens results in a slight inhibition of comb growth, a marked atrophy of the ovaries and an increase in the weight of the oviducts.

The

development of the ovarian cortex Is slightly retarded, whereas pronounced atrophic changes are induced in the medulla.

These effects are generally confirmed by Munro

and Kosin (1940).

However, they observed the loss of body

weight in the treated baby chicks and the combs of pullets are unaffected. In adult male brown leghorns (Emmens, 1939) with prolonged dosage of estrogens the treatment caused extreme atrophy of the testes and comb, and also thyroid disturb­ ances.

Unlike the rodent, no obvious abnormality of the

adrenals is seen, nor are the pituitaries of treated birds enlarged.

Keck (1934) and Hingoen (1940) observed the

29 injections of estrogenic hormone into female English sparrows, as in starling (Viitschi and Fugo, 1940) , during the fall and winter months induced oviduct hypertrophy* The gonads of the fall treated females showed no indication of having been affected, whereas those of the winter treated birds showed suppression of the regular seasonal development of the sex glands.

Such inhibitions of

ovarian growth has previously been reported in pigeons (Riddle and Tange, 1928). Among the reptiles, Dantschakoff (1938) observed effects of estrogenic hormones and found partial sex reversals from male to female in Lacerta embryos*

Estrogen

produced a marked reduction in testicular size in lizards, Eumeces laticeps. but caused enlargement of the epididymis and \¥olffian duct (Turner, 1935).

In Seloporus (Gorbman

1939; Forbes, 1941), no significant effect of treatment is found in the thyroid, adrenal, and ovary*

Testes,

epididymidal and seminiferous tubules are atrophied in males*

V;olffian ducts of the males are increased in size,

but not in the females.

An enlargement and increase in

thickness of oviducts are produced with the estrogen. Evans and Clapp (1940) observed that estrogen injections caused hypertrophy of the oviducts of female lizards, Anolis carolinensis. the ductus deferentes of males and the cloacal epithelium of both sexes*

It caused marked

30 atrophy of the testis, slight atrophy of the ovary of mature females but a slight enlargement of ovaries of immature females* In immature alligators C3trogon stimulated Mullorian ducts, both male and female, also the ovary and testicular cortex but has no effect on the medullary portion of the gonad (Forbes, 1938).

Very similar actions were reported

in juvenile terrapins (Hisley, 1941).

A marked decrease in

testis size is produced, but the cortical remnant is increased in relative extent; ovaries relatively uninflu­ enced.

Slight decreased in size of glans and corpora

fibrosa of both sexes may be interpreted as feminization tendencies.

The urogenital folds are specifically femin­

ized by increased width in both sexes. In 1936 Padoa announced a paradoxical masculinizing effect of a female sex hormone from pregnancy urine extract, Cristallovar, in amphibian gonad development.

Newly

hatched Rana esculenta tadpoles are kept in water which contained the hormone.

All animals subjected to this

treatment differentiated into males.

Witschi and Crown

(1937) using the same method of hormone administration treated R. pipiens tadpoles but were unable to obtain the same result.

However, they found that ovocytes are formed

in the testes of males, and androgen treatment caused testes formation.

Foote (1938)

treating R. pipiens

31 tadpoles with sex hormones, reported results in accordance with those of Witschi and Crow, while when the Cristallovar is used all males were obtained.

In a more recent

paper, Podoa (1939) reported that estrogen administrated to R. esculenta tadpoles appears to have a feminizing effect in genetic males. In sexually differentiated larvae of R. clamitans (Foote and Witschi, 1939) transformation of ovaries into testes is induced by an androgenic hormone.

On the other

hand, estrogenic hormones have little effect on the structure of the testes.

The tubules of the rete testis

showed a tendency to become somewhat inflated, or develop­ ment which homologued of the ovarial sacs of the ovary. While the slowly metamorphosing forms, R. catesbiana. gonadotropin is a necessary aid to the reactions before the sex hormones could exert their effects (Puckett, 1940), since estrogen or androgen alone failed to effect gonadol modi fi ca ti on s . The first report on the effects of estrogenic hormones on gonads of urodeles was made by Burns (1938). He stated that estrogen injected Into larvae of Ambystoma punetaturn caused testes of genetic males to be transformed into ovaries.

Ackart and Leavy (1S39) injecting A.

tigrinum with an estrogen, reported that ovotestes resemb­ ling retarded ovaries are formed in genetic males.

32 Treating A* maculaturn and A. tigrinum larvae with estrogens, Foote (1940) found that a large proportion of the animals possessed typical ovaries.

In a small group

of larvao which died relatively early, hermaphroditic gonads with testicular and ovarial features of varying proportions were found.

In the same study of A. opacum

(Foote, 1941), he observed that the testis is affected by the hormone and assumed typical ovarial structure.

It

appears that the estrogenic substances, previous to or during the time of sex differentiation, suppress medullary development and permit cortical development to progress without directly accelerating it. In a discussion of developmental physiology, Witschi (1941) summarized:

"It appears that salamanders,

reptiles, and birds react mainly to female sex hormones and by changes appearing as feminization; frogs and mammals (the latter in a more limited extent) respond more to male sex hormones by showing effects of masculinization.,r In fishes, Saphir (1934) obtained negative effect on the wedding dress reaction of Chromosomus erythrogaster with the injections of estrogenic substances.

Kanter

et al. (1935, 1936) and Owen (1936) have attempted to produce ovipositor lengthening with sex hormones in the female bittering, Rhodeus amarus.

They found that

estrogens do not give this reaction or do so in a very

35 slight degree.

Positive response is secured when treated

with the androgenic substances.

They have never noticed

spontaneous ovipositor lengthening during the breeding season when the females were kept segregated from the males. Upon extracting the ova of whitefish and the ovaries of 30 mature herrings in the resting phase of the ovulatory cycle, Weisman and his co-workers (1936) were unable to find the evidence of existence of an estrogenic hormone by using immature female mice for the test animals.

They

assumed, therefore, that fish ovaries either do not contain the estrogenic hormones or that the hormones are not present in large enough amounts to produce the estrous reaction.

However, a year later they (1937) extracted less

than 6 R.U.':c* of estrogen from 10 lb. of swordfish ovaries as shown by the positive result on freshly spayed rats. Blacher (1926) observed the natural atrophy of testes in 6 male Lebistes reticulatus which is paralleled by the disappearance of the male sex colors and a case of hermaphrodite possessing the ovotestis*

Based upon these

facts, he concluded that the intensiveness, shape and development of the pigment spots depend upon the hormones

The threshold dose of a given estrogen for producing estrus, as determined by the Allen-Doisy technique, is called one rat unit (R.U.) or one mouse unit (M.U.)--depending on which animal is used.

34 produced in the testis of Lebistes* In older minnows, Phoxinus laevis, Bullough (1940) described 10 hermaphroditic gonads which showed various stages of a transformation from the female to the male type*

The estrogenic hormone, when injected into normal

male Phoxinus * caused a breakdown in the structure of the testes so that only the primary germ cells and the inter­ stitial cells remained*

Light female body pigmentation is

developed in the treated fish.

However, estrogen induced

no sex-reversal in his experiments.

Three months after

the end of injection, the testes had completely recovered and were normal for the time of the year in structure and in volume. Berkowitz (1937, 1941a) demonstrated that the feeding of estrogenic substances to newly born Lebistes reticulatus. inhibited the development of male secondary sex characters (gonopodia andmale coloration) and stimulated the development of female sex characters (gravid spot, general body shape, and large size) so that all such treat­ ed fish took on a female appearance.

There is an inhibi­

tion of the testis together with its transformation into an ovo-testis in nearly all of the genetic male fish if treatment was begun at 14 days of age.

The effects on

the ovary were less marked and irregular.

Injection of

estrogens into sexually immature fish produced more gonad

35 inhibition but less sex reversal than estrogen feeding. Large doses of estrogens administered to adult males generally stimulated the transformation of existing germ cells into mature sperm and with atypical extrusion of these sperm into ducts. On the contrary, Eversole (1939) was -unable to effect any sex-reversal with androgenic substance in the ovary of female Lebistes. and he observed suppression only.

In a further experiment (1941), he administered

the pregneninolone (androgen) and other related steroids on the same species.

Pregneninolone fed to immature

guppy from birth completely prevented the development of any female secondary sex characters and caused the precocious assumption of male secondary sex characters. Prolonged treatment resulted in an exhaustion of testes to such an extent that only gonad remnants remained.

However,

pregneninolone and testosterone propionate have no modify­ ing effect on the ovary but caused an inhibition of yolk deposition.

The latter substance produced an estrogen-like

subpression of male red color In the male fish. Scott (1941) noted that most parts of the skeleton Lebistes were not responsive to either estrogenic or androgenic hormones, except in size.

Pregneninolone

caused a generalized reduction in skeletal size in both sex, while estrogen increased the size of males but not

36

the females.

Skeletal constituents associated with the

anal fin differ markedly in the two sexes.

Male-like

condition was fully developed by pregneninolone in either sex, and tho female condition was likewise induced by estrogenic substances.

The development of anal find and

the influence of androgen on it have been studied by Grobstein (1940, 1940a, 1942) and Cohen et al. (1941) in Platypoecilus maculatus , and Turner (1941, 1941a) in Gambusia affinis affinis. Vshile working on another problem, Essenberg (1926) observed and recorded 2 cases of natural complete sexreversal in Xiphoph orus hellerl, from female to male.

He

theorized that about 50% of all females might undergo sexinversion, but did not necessarily have to do so.

Anal fin

of the normal female consists of 10 pairs of rays, all of which are approximately of the same length and diameter. During the period of natural sex-reversal, he observed a medial thickening of the third pair of rays, as well as elongation of the third, fourth, and fifth pairs of rays, to approximately twice the length of the original fin. The first, second, and sixth to tenth pairs of rays remained constant in length.

The apex of the third pair of rays

together with the fourth and fifth pairs formed hooks and counterhooks for anchorage to the female genital pore during copulation.

After making a study of sex-differentia-

37 tion in this species of teleost, Essenberg (1932) conclud­ ed that any agent or condition which tends to decrease the capacity for female sex hormone secretion beyond a certain limit, became an immediate factor in the possibility of the sex-reversal in the female of the X* helleri. Witschi and Crown (1937) added an androgen to the water in aquaria in which they kept pregnant X. helleri and obtained abortion and resorption of the young.

In

non-pregnant adult females, under the above conditions, all the large eggs underwent resorption.

All treated

females developed ovaries that resembled testes, but spermatogenesis was not observed, although the treated fish assumed gradually but completely the male secondary sex characteristics.

Regnier (1938) reported extensively

on experiments with Xiphophorus of an undifferentiated race and also on some experiments with Lebistes.

Androgenic

hormone induced development of male secondary sex characters in the females while estrogen suppressed them in the males. Estrogen also inhibited spermatogenesis but formation of ovocytes in the testes has not been observed.

Androgen

reduced the size of ovaries, but sex-reversal had not occurred. The effect of sex hormones on the social hierarchy of X- helleri has been noted by Noble and Borne (1940). Androgenic substance induced female fishes to rise in the

38 "pecking order."

A sword began to grow after the dominance

relationship had changed.

The anal fin was modified more

slowly into a gonopodium.

Elements of male behavior

appeared in a definite sequence after the rise in order. Attempts at copulation occurred while the gonopodium was only rudimentary.

Injections and implantations of estro­

genic substance in both gonadectomized and intact females brought no change in social hierarchy.

They concluded

that sex hormones cause female fishes to rise in their social hierarchies only by bringing a reversal of sex. An extensive Investigation on the problem of hormonal influences of sex-differentiation In the X* helleri has been carried on in our physiological labora­ tories.

Baldwin and Goldin (1939, 1941) found that not

only does the experimental administration of androgenic substance cause the regular morphological changes in all cases to simulate the male in body color, formation of a caudal sword, and the development of the anal fin into the copulatory organ, the male gonopod, but these changes are accompanied by histological changes In the primary sex organs in approximately 5 0% of the experimentally treated virgin female fish.

Females which respond to such treat­

ment in these experiments are characterized as showing either (a) resorption of the gonad, or (b) some phase of spermatogenesis.

Individuals which are completely altered

39 histologically apparently pass from normal female gonadal structure through progressive resorption stages and finally show histological features resembling those of the normal male gonad*

These results substantiate

Essenberg1s postulation, previously mentioned.

However,

they do not conclude that the appearance of changes in secondary sex characteristics are to be taken as a certain index of complete sex-reversal in this form. Burns (1938) stated that synthetic sex hormones should be used widely among various vertebrate species in order to elucidate and correlate further the effects on various urogenital organs at differing stages of development.

At the present time, as witschi (1941) points

out it is still impossible to interpret the conflicting notes and incomplete reports on effects produced by sex hormones in fish.

For these reasons, aside from the many

interesting problems concerned with the manner of action of the hormones, and other as yet unclarified points, the following experiments with an estrogenic hormone on adult X. helleri were undertaken.

CHAPTER III MATERIALS AND METHODS The swordtail fish, Xiphophorus helleri Heckel, that served as material for the experiments belong to the common green type.

They are characterized by an olive

green body color with one prominent red lateral stripe from the eye to the end of the body.

The olive green

appearance is due to the presence of many xanthophores and melanophores in the integument.

Genetics of this particu­

lar species of poeciliid fish has been studied by Gordon (1935, 1937, 1938) and Goodrich et al. (1941). All the fish were past the undifferentiated stage and were definitely sexually mature as evidenced by the structure of primary reproductive organs and external secondary sex characters.

The adult female (Plate 1)

has a perfectly normal caudal fin and an unspecialized anal fin.

Besides the central lateral body stripe there

were two additional faint lines parallel above and below to it.

The adult male (Plate 2) showed slimmer bodily

contours and exhibited a long sword-like “tail” extending from the ventral portion of the caudal fin.

The anal fin

was modified into an intromittent organ, gonopodium.

The

color of the lateral stripe in adult males was generally

Plate 1 Scale in ima. Normal Control Female (FC-61) A

Anal Fin

C

Caudal Fin

Plate 2 Scale in mm, Hormal Control Male (MC-6) C

Caudal Pin

G

Gonopodium

S

Sword

45 deeper than those in the females. The male and female animals were kept in separated aquaria.

All specimens were maintained at a temperature

of 18° to 23° C. during the experiments. at 21° C.

Tt was usually

Retroperitoneal injections were made approxi­

mately 2mm. anterior to the urogenital pore, ventrally and laterally.

A B-D Tuberculin Syringe, double scale: minims

and l/lOOth cc., No. i LT, i cc. was used with a B-D No. 27 needle. The chorionic gonadotropic substance, "Pranturon,” is extracted from human pregnancy urine.

It is supplied

by Schering Corporation in dry, stable form.

The prepara­

tion is dissolved in the sterile diluent immediately before using.

The estrogenic substance, Progynon-B, is the only

estrogen thus far definitely chemically identified.

The

estrone (theelin) and estriol (theeiol) with known lesser activity per milligram are degradation products.

The active

principle in the Progynon-B is alpha-estradiol (a-dihydroxyestrin).

Alpha-estradiol benzoate in sesame

oil for injection has a more protracted action, which slow­ ly hydrolyzed In the tissue with the liberation of the free hormone. Of 123 adult fish, the total number used, 61 were male and 62 were female.

They were treated as follows:

46 1.

Gonadotropic series, a.

A control group consisting of 23 animals,

15 virgin females (designated as FC) and 8 males (MG). The body length of these animals was measured at the beginning of this experiment with a divider. 27.5 to 33.5 mm., mean

Females:-

29.5 mm.; Males:- body length 31

to 45 m m . , mean 36 m m . , total body length including the '’tail'* of the caudal fin 33 to 56 mm., mean 41 mm.

Ten

of these females were injected with 0.05 cc. of pure distilled water weekly for 10 to 20 weeks.

Another 5

females and 8 males were not treated. b.

A hormonal group consisting of 40 animals,

20 virgin females (FPU) and 20 males (MPU).

The body

length of females was 28 to 33.5 mm., mean 33.5 mm.; male body length was 33 to 46 mm., mean 36 mm., total body length was 33 to 58 mm., mean 42 mm. injected for 10 to 20 weeks.

Twenty females were

Each fish received 25 I.U."

(2.5 mg.) gonadotropic hormone (Pranturon) in 0.05 cc. of distilled water weekly.

The males were kept separately in

5 aquaria, each containing 1500 cc. of water.

Seven hundred

and fifty I.U. (75 mg.) of the hormone was added weekly to

" The international unit (I.U.) is defined as the specific gonadotropic activity of 0.1 mg. (100 gammas) of the standard preparation established by the Permanent Commission on Biological Standardization of the Health Organization of the League of Nations.

47 each aquarium.

From the surrounding water the hormone

administered was absorbed by the gill tissue of the fish. The treatment was conducted over a period of 4 months. 2.

Estrogenic series. a.

A control group, consisting of 15 animals,

7 virgin females (FSO) and 8 males (MSO), injected with pure sesame oil. dosage.

Each animal received 0.02 cc. in weekly

At the beginning of the experiment their body

lengths were measured.

Female;-30 to 47 mm., mean 31 mm.;

Males;-body length 31 to 46 mm., mean 35.5 mm., total body length 37 to 55 m m . , mean 40 mm. b.

A hormonal group, consisting of 45 animals,

20 virgin females (FPB) and 25 males (MPB), each received weekly injections of 0.02 cc. alpha-estradiol benzoate in sesame oil (Progynon-B).

Thus the weekly dosage consisted

of 20 R.U. or 0.3 mg. of the active estrogenic principle. Autopsy was made at interval times during the 7 months of experiments.

The range of female body length was same as

in the controls.

Male body length was 40 to 62 mm., mean

45.5 mm., total body length was 45 to 86 mm., mean 67.5 mm. The length of the sword-like "tail” in all cases of the adult males was measured as the distance the "tail” extended from the posterior end of the caudal fin.

The

anal fin is another important criterion in the sexual differantiation.

It was removed from the experimental

animals and passed through the various grades of alcohol

43 up to 95>b for the purposes of dehydration.

Then the

euparol, a mounting medium for this type of work, was used directly. The fish woro killod by pithing at tho desired times and dissected laterally so as to remove the gonad intact.A detailed study of the histological structures of gonads were made in order to determine the essential effects of the sex hormones administration and to correlate the changes, if any, of the external secondary sex characters. Microscopic slides used for study were prepared according to the following procedure: 1) Fixing agent: All of the gonads were fixed in Bouin*s solution for a period of at least one week. 2) Dehydrating:

The Bouinfs solution was washed away

with 35/6 alcohol, and the tissues were given 30 minute changes of 50 and 70 % alcohols.

A change of 80 % alcohol

followed for a minimum of 24 hours.

Thirty minute changes

of 95 % and absolute ethyl alcohol completed this stage. 3) Clearing:

The tissues were left in pure xylene for

a period of 5 minutes. 4) Infiltration:

Nine parts paraffin and one part

bayberry were used for a paraffin bath for a period of 4 hours with one change. 5) Imbedding:

Bayberry-paraffin, of the above propor-

49 tions, was used for imbedding. 6) Sectioning;

Serial sections were cut at 6 or 8

microns by using a Spencer Rotary Microtome. 7) Mounting;

The usual procedure of mounting the

sections on the slide was used, with Meyer*s Albumen as fixative.

The mounted slide was dried in a low tempera­

ture incubator for at least 2 days. 8) Staining:

The slides were stained with Delafieldfs

haematoxylin for 20 minutes, followed by eosin as counter­ stain for 1 minute. 9) Covering:

After staining, the sections were covered

with gum damar or Canadian balsam and regular cover glasses. Average measurements of various structures were made by ocular micrometer in order to verify histological find­ ings and for purposes of comparing the effects appearing in the various experimental groups.

The gametogenic

stages as discussed by Kssenberg (1923) and Bailey (1933) were used as the basis for certain interpretations.

CHAPTER IV

OBSERVATIONS The experimental results to be presented in this section are divided into two main parts according to sexes male and female, of the animals.

In each part a descrip­

tion of the control normal genital organs is given first and followed by the detailed observations on the actions of the gonadotropin and estrogen respectively.

The

internal histological responses of the primary sex organ, gonad, is considered and then correlated to the external changes, If any, In the secondary sex characteristics. Am

1.

Male Fish

Gonads of the control males.

The sixteen males

used as controls for the investigations were equally divided into two groups, eight treated controls, and eight untreated controls.

The former group was injected with a

weekly dosage of 0.02 cc. of the estrogen-solvent, sesame oil.

Five animals received four injections each, and

other three animals were each subjected to six Injections. After the injection periods, no significant changes in the gonads nor In the secondary sex characteristics were noted They showed similar to those of the untreated normal males

51 (Compare Plates 3 and 4). The control male gonads are typically testicular. They are white elongate bodies and are to be found in a position postei'ior to the intestine stretching in a diagonal line, just beneath the dorso-pleuroperitoneal epithelium.

The anterior end of the testis is attached

dorsally to the swim-bladder, and the posterior end terminates at the urogenital orifice.

Generally the

posterior part of the testis shows more or less complete bilaterality; while the anterior part is smaller and in a fused state, thus producing a bilobed condition.

The two

halves of the testis are somewhat separated by a mesorchium, which is composed of the adipose, blood vascular, and some other connective tissues. In a transverse section, the bilobed testis is commonly showed the butterfly-3hape.

Each half may be

roughly compared to a bunch of grapes with the sperm duct as the main stem.

The sperm duct consists of an outer

muscular, a middle connective tissue, and an inner epithelial layers.

The cells of the flagellated epithelium

are cuboidal or columnar In shape.

They are averaging

0.018 mm. to 0.02 mm. in diameter.

The sperm duct

associates with the sperm tubules forming the medullary portion of the testis.

In the periphery of the testis

various stages of germ cells constitute the cortical

Plate 3 X 400 Cross-Section of A Control Male Gonad (MC-8) IT

Interstitial Tissue

LSD

Lumen of Sperm Duct

M

Mesorchium

SD

Sperm Duct

SpC

Spermatocyst

SPh

Sp ermat ophor e

SpT

Spermatids

SSp

Secondary Spermatocytes

ST

Sperm Tubule

k$T>

c»

Plate 4 X 400 Cross-Section of A Control Sesame Oil Injected Male Gonad

(MS0-2)

IT

Interstitial Tissue

LSD

Lumen of Sperm Duct

PGC

Primitive Germ Cells

PSp

Primary Spermatocytes

SD

Sperm Duct

SpC

Spermatocyst

SPh

Spermatophore

SpT

Spermatids

SSp

Secondary Spermatocytes

si

'wa/r.

D.

:Oo2 "6 p

o 0 ;A ft

**N

Plate 5 X 400 Cross-Section of A Gonadotropin Treated Male Gonad

(MPU-ll)

BV

Blood Vessel

IT

Interstitial. Tissue

LSD

Lumen of Sperm Duct

SD

Sperm Duct

SDE

Sperm Duct Epithelium

SPh

Spermatophore

SpZ

Spermatozoa

ST

Sperm Tubule

♦•°°oV

t9 'o*«wm OoO^

§■

*•V

(ft*

2

*

»

^

56 portion.

Germ cells in same spermatogenic stage are

clustered together and forming numerous cysts. structures are called the spermatocysts.

These

The appearance

of such a testis is generally known as the acinus type. The external epithelium of the testis is flattened and has some connective tissue and blood capillaries beneath it.

There are some small clumps of primitive

germ cells and primary spermatocytes observed near the periphery.

The primitive germ cells showing partially

clear cytoplasm and light staining.

The cell dimensions

are hard to measure because the entire acinus presents the aspect of a syncytium.

The nuclei average 0*007 mm.

in diameter, and most of them are spherical or ovate.

The

primary spermatocytes averaging 0.006 mm. in diameter. Prominent mitotic chromosomes are observed In these cells. The nuclear membrane is not well defined.

Many larger

cysts of the secondary spermatocytes are showed next to them.

These secondary spermatocytes measure on the average

0.004 ram. in diameter.

Their nuclei are reduced in size,

averaging 0.0035 mm., and contain less chromatin material as compared to the primary spermatocytes. average 0.003 mm. in diameter.

The spermatids

The size of the nucleus

has diminished to 0.0025 mm. In the medulla,

the lumina of the sperm duct and

tubules are filled with spermatophores and are distended

57 with spermatozoa.

These spermatophores are scattered

rather than clumped together within the lumina.

The heads

oT spermatozoa average 0.003 ram. by 0.001 mm. in size. They are closely pressed together and appear like s single layer of epithelial cells.

All sperm tails are extended

toward the core of the cyst. Occasional blood capillaries are seen among the spermatocysts.

An interlacing thin network of connective

tissue is found between sperm tubules and beneath the sperm duct. cells.

It is, however, quite hard to locate the interstitial Thus, the intertubular spaces appear rather trans­

parent in histological sections. 2. trations.

Gonads after the gonadotropic hormone adminis­ Examination of testes (Plates 5 and 6) from fish

which were treated for ten to twelve weeks as against controls showed that the gonadotropin has increased the diameter of the medullary portion. been greatly accelerated.

The spermatogenesis has

Lumina of the sperm tubules are

crowded with spermatophores.

The number of spermatophores

in a single tubule sometime reached as many as one hundred and so clustery that their shape changed from ovoid to polyhedral (Plate 5). phores has not reduced.

However, the size of the spermato­ It is indeed difficult to find

spermatids and spermatocytes in these treated testes.

Peri­

pheral primitive germ cells of the cortex are infrequently

M

W.X

Plate 6 X 500 Cross-Section of A Gonadotropin Treated Male Gonad

(MPU-17)

IT

Interstitial Tissue

SDE

Sperm Duct Epithelium

SPh

Spermatophore

:: t p

62

found.

Certain degree of damage on the epithelial lining

of sperm ducts and tubules are seen. In some instances, the gonads (Plate 6) appear to have undergone a state of precocity indicative of exhaustion, where some tubules even contain the desquamated epithelial cells and debris only.

In this condition there

is evidence of hypotrophy and reduction in size. Besides the hastened effect of mature sperm forma­ tion, there is a marked increase in the interstitial tissue, which is accompanied by the vascular tissue growth.

Yvell differentiated interstitial cells, averaging

0.005 mm. in diameter, are deposited in the interstitial stroma.

Their nuclei average 0.0035 mm. in diameter, and

are well stained with eosin.

In all cases, the gonadotro­

pin treated testes reveal dense and compact in the tran­ sect! ons as contrast to those in the controls.

This

interstitial hypertrophy of the stroma is considered to be a direct result of an attempt on the part of the testes to counteract the excess of the gonadotropic hormones. After ten weekly hormonal treatments, a group of four fish were kept alive in fresh water and discontinued to give the hormone.

They were killed in seven weeks later.

The histological pictures of their testes (Plate 7) show somewhat recovered from the hormonal effects as evidenced by the reappearance of the mitotic germ cells and a

Plate 7 X 400 Cross-Section of A Gonadotropin Treated Ifiile Gonad, Showing the Recovery of Hormonal Effects After Discontinued Treatment for Seven Weeks. (MPU-4)

BV

Blood Vessel

IT

Interstitial

PGC

Primitive Germ Cells

PSp

Primary Spermatocytes

SD

Sperm Duct

SPh

Sp ermat ophor e

SpT

Spermatids

SSp

Secondary Spermatocytes

Tissue

0

Z' (

o " T— JfcrT-

o^Bra

x3^;! *£>

'•U'.

J^r^n,.

P

O

P cj. Ok

— Spti

65

relatively decreased amount of the interstitial tissue. The spermatocytes in these gonads seem to be derived from the remaining primitive germ cells which attached to the peritoneal epithelium.

The number of these primitivo germ

cell cysts and the number of these cells within the cysts are both increased. In no instance is there noted any effect on the already well developed male secondary sex characters.

The

established male gonopodium is not affected. 3. tions.

Gonads following the estrogenic hormone injec­

The effects of the estrogenic substance upon the

testes of adult male fish vary with the individual and the amounts of the hormone with which they are administered. However, the length of time is apparently an important factor for the reactions.

In general the treated gonads

show certain sequence of atrophy.

At first the hormone

hastens the spermatogenesis and increases the interstitial stroma.

After prolonged injections it finally causes

exhaustion, suppression, and pathological involution. Judging by the histological responses and the duration of treatments, the twenty-five animals in this series of experiment may be roughly divided into the following five arbitrarily phases. Phase 1.

Nine fish are used as the basis of

observation in this phase of reaction.

Sight of them

66

(MPB 4, 14, 15, 21-25) each Injected with a single dosage of 0.5 mg. of the estrogen.

MPB 11 subjected for two

injections with a total amount of 0.6 mg.

When killed

after an elapsed period of 7 to 10 days following the injections, the histological preparations (Plate 8) show that these gonads resembled testes which recovered from the ten weekly treatments of the gonadotropic hormone (Compare Plate 7).

The only exception is the presence of

normal number of the primitive germ cell cysts.

As noted,

the lumina of ducts of these testes are crowded with spermatophores.

The size of the spermatophores vary in

diameter from 0.04 mm. to 0.048 mm., so that they are not much beyond the normal range in contrast to the controls. In the cortical portion, cells of various spermatogenic stages are found, i.e., primitive germ cells, primary spermatocytes, secondary spermatocytes, spermatids, and some spermatophores.

In the ducts, however, these sperma­

tophores are scattered in the sperm tubules and are fewer in number.

Some of the tubules show necrosis of their

epithelial cells.

A striking feature is the increased

amount of the interstitial tissue, the stromal tissue surround the ducts and tubules and fill the space among them.

Numerous well-definded interstitial cells are

deposited on the tissue so that the gonads are compact in the sections.

So far, there is no external modification

Plate 8 X 400 Cross-Section of An Estrogen Treated tt&e Gonad, Phase !•

(MPB-4)

IT

Interstitial Tissue

PGC

Primitive Germ Cells

PSp

Primary Spermatocytes

SD

Sperm Duct

SPh

Sp ermat ophor e

SpT

Spermatids

SSp

Secondary Spermatocytes

■ — [MrJ

r

c\ •1

—

;—

■» ».* /*>*r'2r®v

.

69 of the sex characters observed in this phase of the treated fish. Phase 2.

Two treated males offer the material to

be presented for this phase.

MPB 2 received five weekly

injections, and MPB 9, received three weekly injections. Gonads of these treated animals are slightly reduced in size • Following the tendency of the hastened sperm forma­ tion, these testes (Plate 9) contain masses of spermatophores.

They distend both the sperm ducts and tubules.

The number of the spermatophores in a single clump is frequently over sixty.

Other stages of germ cells are

under suppression, and are at the point of dissappearance. The wall of sperm ducts and tubules show the necrotic phenomenon.

Due to the expansion of sperm tubules the

cortical portion of the treated testes is greatly decreased. The cell proliferation is seen in the external epithelium. Most of the peripheral primitive germ cell cysts have migrated to a position subjacent to the tubules.

The

general appearance of gonads of this phase is quite similar to that of the ten-week treatment (Plate 5).

Perhaps the

only difference is the latter possessed relative greater amount of the interstitial stroma. Phase 3. phase of study.

Seven treated males are used for this The gonads exhibit a very marked inhibition

Plate 9 X 400 Cross-Section of An Estrogen Treated Male Gonad, Phase 2.

(MPB-9)

IT

Interstitial Tissue

PGC

Primitive Germ Cells

SD

Sperm Duct

SPh

Spermatophore

ST

Sperm Tubule

72 of spermatogenesis, atypical release of germ cells, and interstitial tissue stimulation.

According to the degree

of these histological reactions, these treated gonads may be separated into two groups. The first group consists of two fish, MPB 3 and 10. The former received six weekly injections with a total amount of 1.8 mg. of the estrogen.

The latter received

seven weekly injections with a total amount of 2.1 mg. of the same substance. An excessive growth of sperm ducts is observed in treated testes of this group (Plate 10).

They occupy

about one-fourth of the total area of the gonad.

The wall

is in normal condition, and measuring from 0.008 mm. to 0.02 mm. in diameter. spermatozoa.

The lumina of ducts are filled with

Instead of arranging in the form of ordinary

spermatophores, they show atypical intrusion. tophores are found only at the periphery.

The sperma-

Their number is

not more than fifty in any cross-section of these testes. Cysts of spermatids and secondary spermatocytes are rare. However, the primitive germ cells are grouped beneath the external epithelium.

The stimulation of interstitial

stroma is less pronounced in this group. Another group consists of four fish.

MPB 6 and

18 received five weekly injections, and MPB 1, 16, and 19 received six weekly injections.

The treated testes of

Plate 10 X 400 OroBs-Section of An Estrogen Treated Male Gonad, Phase 3.

(MPB-3)

BV

Blood Vessel

IT

Interstitial Tissue

PGC

Primitive Germ Cells

SB

Sperm Duct

SpT

Spermatids

SpZ

Spermatozoa

SSp

Secondary Spermatocytes

yf

SSiSE 2S3st t.1^1

- —

-

'■^T^i~i_**r WJW'I. mmFm

75 this group (Plate 11) are of small size.

Microscopic

examinations show that the hormone has caused a pathogenic hypertrophy of the interstitial stroma.

The interstitial

cells appear as cords of large mononuclear cells with dark staining nuclei.

These cells measure approximately

0,008 mm. in diameter, with relatively large nuclei which average 0.005 mra. in diameter.

These are appreciably

larger than those in the gonadotropin treated testes.

The

hyperplasia of these interstitial cells progress in growth so as to practically completely compress the sperm tubules. The lumina of the ducts are mainly filled with atypical released spermatids and containing a very small and few spermatozoa.

In some parts, they contain also the

desqumated cytoplasm from the necrotic epithelium of the wall.

Hence, the gonads are very opaque in sections.

Spermatogenesis has been inhibited.

Usually fewer than

thirty of spermatophores are seen in a testicular tran­ section and they are scattered in the cortical region. Cysts of primitive germ cells, however, are still visible here and there. Pishes after five or more weekly hormonal injections usually appeared to be very inactive in the aquarium.

The

red lateral body stripe fades and turns to chocolate color. Moreover, the sword-like tail of the caudal fin frequently shows some degree of atrophy.

Weekly body measurement

Plate 11 X 400 Cross-Section of An Estrogen Treated Male Gonad, Phase 5.

(MPB-l)

IT

Interstitial

Tissue

PGC

Primitive Germ Cells

SD

Sperm Duct

SPh

Spermatophore

SpT

Spermatids

78 reveals that the total body length of these prolonged treated fish are from 1 mm. to 3 ram. shorter than those of the controls. Phase 4.

The observations of this phase of reaction

are made from three treated males.

MPB 8 were injected

weekly for fifteen weeks, and received a total amount of 0.45 rag. (300 R.U.) of the estrogen.

MPB 17 and 20 were

injected weekly for twelve weeks with a total amount of 3.6 rag. (240 R.U.) of the estrogen.

Such treatments

reduced the size of the gonads in both. Histological sections (Plate 12) of these treated testes show that they simulate the first group of the phase 3 by the excessive growth of sperm ducts, atypical release of spermatozoa, and comparative less pronounced stimulation of the interstitial stroma.

However, they

possess some newly formed testicular tubules which arrange themselves in many cords. A pathogenic formation of testicular tumor is seen in the testis of the MPB 17. to a sperm duct.

The tumor is located subjacent

It consists of a mass of large mononuclear

irregular arranged cells with a characteristically vacuo­ lated or pigmented cytoplasm. 0.012 mm. in section. in diameter.

These tumor cells average

Their nuclei measure about 0.004 mm.

The vacuole occupies almost the whole cell,

and pushes the nucleus to the side of the cell.

It Is

Plate 12 X 400 Cross-Section of An Estrogen Treated Male Gonad, Phase 4.

(MPB-17)

c

Tumor Nodule

IT

Interstitial Tissue

PGC

Primitive Germ Cells

SD

Sperm Duct

SPli

Spermatophore

SpZ

Spermatozoa

ST

Sperm Tubule

—W

81 about 0,008 mm. in diameter.

In routine heamatoxylin-

eosin preparations, a small amount of pigment forms diffuse, yellowish-brown deposits in the cytoplasm.

It

seems that this is a malignant sarcoma arising from the interstitial cells of the stroma.

Further observation on

the cases of the testicular tumor induced by the prolonged treatments of estrogen will be described in the next phase. Phase 5. of this phase. tions.

A group of four fish served for the study They underwent prolonged estrogen Injec­

MPB 5 were injected for sixteen weeks, MPB 13 for

twenty-four weeks, and MPB 7, and 12, for twenty-five weeks each.

The estrogenic hormone has not only inhibited the

testicular activity but caused the atrophy of these treated testes.

The size of testes is greatly reduced.

In tran-

sections, for instance, MPB 13 testis measured 0.9 mm. in diameter. None of the typical spermatophores are observed in these testes (Plates 13 and 14).

Lunina of sperm ducts

contain the atypical released spermatozoa.

In the lumina

of the newly formed tubules as seen in the phase 4 are networks of desquamated cytoplasm. germ cells, however, are present. is increased.

Many cysts of primitive The size of these cells

They average 0.012 mm. in diameter, which

is about double the si?e

of those In the controls.

Their

nuclei are also enlarged to average 0.006 mm. in diameter.

Plate 13 X 400 Cross-Section of An Estrogen Treated Male Gonad, Phase 5.

(MPB-5)

BV

Blood Vessel

IT

Interstitial Tissue

LSD

Lumen of Sperm Duct

SPh

Spermatophore

/

■- V

Plate 14 X 500 Cross-Section of An Estrogen Treated Male Gonad, Phase 5,

(MPB-12)

BV

Blood Vessel

C

Tumor

IT

Interstitial Tissue

PGC

Primitive Germ Cells

SD

Sperm Duct

SpZ

Spermatozoa

ST

Sperm Tubule

Jm&r .• C

• ’

\V *»’;••?■!** Vf?*/ •

* thdt'

■,$*' j a a r

/*■

\

-%-------------------

■ 11'

If

. ^ n --

V v y V " W

S*^r *$Lmr

o

86

These germ cells apparently are in a state of growth and reach the leptonema stage of the prophase of primary spermatocytes.

Within the nucleus are leptotene spiremes

and deoply stained nucleoli. The most significant result is the pathologic histology of these prolonged treated testes.

Except the

MPB 5, all other three gonads and their adjacent pleuroperitoneum showed the growth of tumors.

The testicular

tumors seem to be derived from the abnormal proliferation of the interstitial tissue cells of these testes.

They

compose of a mass of large and round or polyhedral cells with relatively big, vesicular nuclei and well-defined nucleoli.

These cells are capsulated by fibrous stroma

tissue in the form of nodules.

The largest testicular

tumor nodule measures 0.36 mm. in diameter.

In the case

of MPB 17 in the phase 4, these tumor cell3 contain big cytoplasmic vacuoles.

However, the difference is the

increased amounts of the yellowish-brown pigment.

The

pigment is laid down in the cytoplasm in the form of fine granules which accumulate to such an extent as to ultimate­ ly obscure the nucleus and cause considerable enlargement of the cell.

Here and there throughout the tissue are

generally seen small quantities of extracellular melanin liberated by the rupture of overloaded cells.

Associated

with such liberation of pigment Is the frequent occurrence

87 of round or oval cells, average 0*01 mm. in diameter, swollen sometimes to a very marked extent with melanin and lying isolated or in groups among the tumor cells. These are histiocytes or melanoplioreg which have ingested freed pigment. vessels.

They seem to be migrated from the blood

In all cases the blood vessels in the pleuro-

peritoneum are filled and distended with these black dark histiocytes. Many of the same type of tumors are observed along the connective tissue of the pleuro-peritoneum.

These

tumors are possibly not metastases of the testicular tumors, but rather may be considered to be a contemporary growth by the toxic action of the estrogen. The distribution of the animals in the five phases in this series of hormonal administration may be summar­ ized as follows;-

88

Subject

MPB 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Total number of weekly injection 6 5 6 1 16 5 25 15 3 7 2 25 24 1 1 6 12 5 6 12 1 1 1 1 1

B. 4.

Amount of estrogen injected 1.8 1.5 1.8 0.3 4.8 1.5 7.5 4.5 0.9 2.1 0.6 7.5 7.2 0.3 0.3 1.8 3.6 1.5 1.8 3.6 0.3 0.3 0.3 0.3 0.3

mg. " n u M " 11 M ” " n

" 11 11 11 11 u " 11 tl ” " 11 w 11

(120 R.U. ) (100 " ) (120 n ) ( 20 " ) (320 " ) (100 " ) (500 " ) (300 M ) ( 60 " ) (160 " ) ( 40 !i ) (320 " ) (300 " ) ( 20 ” ) ( 20 " ) (120 lt ) (240 u ) (100 H ) (120 !t ) (240 " ) ( 20 11 ) ( 20 w ) ( 20 H ) ( 20 “ ) ( 20 w )

Phase of reaction 3 2 3 1 5 3 5 4 2 3 1 5 5 1 1 3 4 3 3 4 1 1 1 1 1

Female Fish

Gonads of the control females.

adult virgin females were used as controls.

Twenty-two

Ten of them

were each injected weekly with 0.05 cc. of pure distilled water; seven were each injected weekly with 0.02 cc. of sesame oil; and another five were left untreated.

Those

controls treated with distilled water were given ten to twenty injections, while those treated with sesame oil

89 were subjected to nine or ten injections each.

After the

elapsed periods of the administration, no changes in the secondary sex characters were noted.

Histological prepara­

tions showed that these injections have no significant effect on the ovaries. Xiphophorus helleri is a viviparous teleost.

The

ovary (Plate 15) of control fish is a large unpaired body which in mature fish occupies most of the posterior part of the pleuro-peritoneal cavity.

Enveloping it is a thin,

slightly pigmented peritoneal membrane.

When this is

removed the follicles or ovisacs appear lying quite separately making up the bulk of the cortex.

Ova of

varying sizes can be found in the normal ovary.

Each

ovum is contained in a follicle, consisting of a single layer of cuboidal cells.

This follicular epithelium of

a young ovum reaches its greatest height, about 0.015 mm., during the period of oil secretion before yolk granules appear.

The follicles are surrounded by a very slightly

developed theca folliculi which emerges indistinctly into the surrounding stroma.

Immature ova, approximately

0.08 mm. in diameter, are most commonly observed in the region of the epithelium of the ovarian cavity.

Yolk is

deposited at the time when the ova have reached median size, about 10.3 mm. in diameter.

At this time the young

ovum has a beautiful alveolar structure in sections due

Plate 15 X 400 Cross-Section of A Control Normal Female Gonad.

(FC-lft)

FE

Follicular Epithelium

FOCE

Folds of Ovarian Cavity Epithelium

N

Hucleus

HO

Nucleolus

0

Oocyte

001

Ovarian Cavity Epithelium

FGC

Primitive Germ Cells

FE

OC E

HC

pac FOCE N

0

92 to the presence of oil globules which have been dissolved by reagents.

The round or oval nuclei usually measure

0.08 ram. to 0.09 ram. in diameter.

Many spiremes of

chromatin material are observed within the nucleus, while the rest of the nuclear material is diffuse.

One or

several darkly stained nucleoli are present, they average 0.01 mm. in diameter.

About two to six of big mature ova

are not infrequently seen in the control ovaries. measure on the average 1.6 ram. in diameter.

They

These ova

are filling with the yolk which stains pink color in the haematoxylin-eosin method.

The edge of these ova usually

show many round holes where the oil globules were formerly located. The ovarian cavity connects with a short oviduct forming the axis of the ovary.

The cavity is lined with

columnar epithelial cells which vary in height from 0.008 mm. to 0.020 mm.

Nuclei of these cells are oval in

shape and average 0.006 mm. in diameter.

The prominent

folds of the epithelium serve the purpose of admission of the sperm into the ovum and creation of a space of rupture for the escaping young.

Groups of primitive germ cells

are found beneath the epithelium of the ovarian cavity. These cells average 0.01 mm. in diameter with vesicular nuclei about 0.005 mm. in size.

Connective tissue, show­

ing spindle-shaped cells and an occasional nucleus, is

93 diffuse throughout the ovary. The secondary sex characters of the control normal females will be considered in the following group of experiments. 5. females.

Hormonal induction of the sex-reversal in A

total of forty adult virgin females are used

for two series, gonadotropin and estrogen, of the hormonal investigations.

The most striking result in these adminis­

trations is the induction of sexual inversion. paradoxical reaction is observed in six animals.

This Pour of

them (FPU 32, 37, 42, and 49) are caused by the injec­ tions of the gonadotropic hormone, and the other two (PPB 11 and 13) are induced by the estrogenic hormone. They show partial or complete changes in the secondary sex characters.

Histological studies of gonads of these

animals reveal that they have transformed from ovaries to ovotestes or, in most cases, to testes.

Indications of

the sex-reversal are first noted by the changing of sexual behavior and the modification of the anal fin.

Thirty-

four others similarly treated females are practically unaffected, both in the primary and secondary sex organs. However,

the ovaries of these unaffected females sometime

do show certain degree of decreasing in size by either prolonged injections of the gonadotropin or estrogen. While various stages of the ovarian cycle and other ovarian

94 structures have not modified.

So far as could be observed,

the hormones have no marked effects on the secondary sex characters in these unaffected adult females as contrast to the controls. The female bodily contours (Plate 1) are well rounded, and a dull chocolate color centro-lateral body stripe is observed horizontally along the length of the body proper.

This centro-lateral body stripe is usually

parallel with two more faint lines, one above and the other below.

The caudal fin of the normal females has a

round posterior margin.

The anal fin consists of ten

pairs of rays of approximately the same length and diameter.

These rays are bifurcated (dichotomy) at about

the half way of the total length, and are segmented like the stem of a bamboo. The first noticeable thing in sex-reversal is the transforming of the female anal fin into an intromittent or gonopodium.

The third pair of rays thickened to

several times in diameter.

As the thickening of this

pair of rays continues, the third, fourth, and fifth pairs of rays elongate to approximately double the original length of the fin.

In addition,

the tips

of

the last-named rays form knob-like projections which are to be transformed into copulatory hooks in later develop­ ment (Plate 16).

The first,

second, sixth to tenth rays

Plate 16 X 80 Developing Gonopodium of A Gonadotropin Treated Female

(FPU-37)

Enoh-like Projections of Third Pair of Rays R3

Elongated and thickened Third Pair of Rays

R4

Elongated Fourth Pair of Rays

R5

Elongated Fifth Pair of Rays

KP3

\

\

97 are subjected to no special changes and remain rudimen­ tary.

During the time of these modifications, the

animals usually show some elements of male behavior. Attempts at copulation are also observed.

Subsequently,

the knob of the ends of the third and fifth pairs of rays develop into primitive copulatory hooks which soon become fully developed (Plate 17).

The knob-like pro­

jections of the fourth pair of rays appear to have under­ gone an early development to form accessory copulatory hooks, or counterhooks, and fifth pairs of rays.

to supplement those of the third These accessory hooks which

began their development just previously, soon appear as fully formed accessory copulatory hooks.

Furthermore,

the third and the dorsal part of the fourth rays form two rows of symmetical " teeth” which project backwards and outwards.

There are about eight to nine 11teeth" on each

side of the third rays and ten in the fourth.

The two

members of the fifth rays in the secondary growth region, proximal to the hooks, have fused and broadened laterodorsally to form a concave spoon-like groove on the dorsal margin of the anal fin.

It is by the above processes, the

hormones have transformed the normal female anal fin into that of a male-like gonopodium (Plate 18; compare Plates 2 and 21). After five weekly gonadotropin injections, the

Plate 17 X 80 Developing G-onopodium of An Estrogen Treated Female ACH4

(FPB-ll)

Accessory

Copulatory Hook

of Fourth Pair of Hays CH3

Primitive

Copulatory Hook

of Third Pair of Hays CH5

Primitive

Copulatory Hook

of Fifth Pair of Hays R3

Elongated

and Thickened Third

Pair of Hays H4

Elongated

Pourth Pair of Hays

H5

Elongated

Fifth Pair of Hays

TP

Teeth-like Projections

Plate 18 X 80 Control Male Gonopodium (MC-5)

ACH4

Accessory Copulatory Hook of Fourth Pair of

CH3

Copulatory Hook of

Rays Third Pair

of Rays GH5

Copulatory Hook of Fifth of Rays

R3

Third Pair of Rays

R4

Fourth Pair of Rays

R5

Fifth Pair of Rays

TP

Teeth-like Projections

Pair

Plate 19 Scale in mm* A Gonadotropin Treated Pemale, Showing the Transformation of Anal Pin into A Gonopodium,

(PPU-37)

C

Caudal Pin

G

Developing Gonopodium

104 modification of anal fin toward a male-like gonodium is observed in four (FPU 32, 37, 42, and 49) of the treated females (Plates 19 and 16). after the initial injection.

FPU 37 is killed

sixty

days

Histological examination

reveals a small unilateral maturing testis (Plate 20). Almost all germ cells are in the processes of maturation divisions.

The gonad is mainly filled with many poly­

hedral cysts, average 0.08 mm. in diameter, of the primary spermatocytes.

They show the clear darkly stained

chromosomes of the metaphase.

These chromosomes arrange

like a round mass in each cell, which averaging 0.004 mm. in diameter.

The cell membrane of these primary spermato­

cytes is indistinct. spermatocytes.

Same appearance is the secondary

However, the chromosome mass in a single

cell is approximately 0.0035 mm. in diameter.

The number

of chromosomes is apparently undergone the mitotic reduction.

A

few cysts of spermatids are seen among them.

The spermatids averaging 0.0035 mm. in diameter.

Each

spermatid has a mass of chromosomes about 0.002 mm. in diameter.

Cysts of newly transformed spermatozoa are

infrequently found in sections of this inverted gonad. The typical spermatophores, however, are not present. There are many small cysts of primitive germ cells beneath the external epitheliun of the gonad.

These cells are

large and about 0.01 mm. in diameter.

They show partially

Plate 20 X 400 A Gonadotropin Treated Sex-reversed Female Gonad

(FPU-37)

BV

Blood Yessel

PGC

Primitive Germ Cells

PSp

Primary Spermatocytes

SSp

Secondary Spermatocytes

ST

Sperm Tubule

f3V -?