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Xerox University Microfilms
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Elvers ole, Wilburn J
Effect of steroid hormones on sexual
development in fish (Lebistes reticul atus)...
New York, 1940.
60 typewritten leaves,
Thesis (Ph.D.) - New York university,
Graduate school, 1940.
Bibliography: p . 54-62.
Shelf List
University Microfilms,
Ann Arbor, Michigan 48106
N, Y. Uwrv.
Effect of Steroid Hormones on Sexual Development
in Fish (Lebistes reticulatus)
Wilburn J. Bversole
April, 1940
"Submitted in partial fulfillment of the requirements for the degree
of Doctor of Philosophy at New lork University".
Table of Contents
Introduction and Statement of Problem - - - - - - - - - - - Historical Survey - - - Material and Methods
---- - - - - - - - - - - - - - - - -
Experimental Observations I. Normal External. Sexual Differentiation in
Untreated A n i m a l s ---------- - ---- ----- - - - - II. Effects of Pregneninolone on the Secondary
Sex Characters---------------------------------III. Effects of Pregneninolone on the Gonads
- - - - - - - - -
IV. Animals Treated v/ith Testosterone propionate
V. Steroids Lacking any Effect
39 - 4.0
- -- -- -- -- -- -- --
------------ ---------- --- -
4. O - 4I
42 - 50
Effect of Steroid Hormones on Sexual Development
in Fish (Lebistes reticulatus)
Introduction and Statement of Problem
The availability of steroid substances in crystalline form, similar
or identical to those produced in the gonads and the adrenal cortex, has
given great impetus to investigations of their possible roles in verte­
brate sexual determination and differentiation.
A uniformity of response,
however, has not been obtained when identical compounds were administered
to various vertebrate groups, and it is clear that phylogenetically dif­
ferent mechanisms have been used in the control of sexual characteristics.
That the fish gonad secretes a hormone or hormones having a control­
ling effect an the secondary sex characters has been indicated by a
limited number of castration experiments (Kopec, 1918} Boch, 1928}
Tozawa, 1929} Glaser and Haempel, 1931} Kinoshita, 1935; and others).
More decisive and satisfactory results, in the analysis of the control of
sexual development in fishes, have been obtained by studying the effects
of hormone overdosage.
A few studies of this type have been made with
crystalline estrogens (Berkowitz, 1937, 1938} Padoa, 1937} Regnier, 1937,
1938) and androgens (Castelnuova, 1937} Regnier, 1937, 1938} Witschl and
Crown, 1937} Eversole, 1939} Kinoshita, 1939} Baldwin and Golden, 1939).
The steroid hormones, including the sex and adrenal cortex hormones,
are known to serve different primary functions in mammals but the possi­
bility that different steroids have a common effect upon sexual manifests-
tions in lower animals cannot be precluded since it has been shown by
numerous investigators that these substances can substitute one for
the other under various circumstances (Shapiro, 1936; Nelson, 1937;
Gaunt and Hays, 1938; Salmon, 1939; Greene, Burrill and Ivy, 1939;
van Heuverswyn et al., 1939: Hooker and Collins, 194-0; and others).
The first purpose of this work was to study the effects of syn­
thetic and naturally occuring steroids on the development of secondary
sex characters of both sexes of the guppy (Lebistes reticulatus).
was intended to determine which,if any, of the sexually dimorphic traits
depend upon hormones known to control such phenomena in higher verte­
brates and which, if any, were responsive to substances chemically
related to the sex hormones.
The second question of greater interest with which this work was
concerned involved the effects of steroid hormones on the differentiation
and development of germ cells.
It is known that there are influences,
apparent under various atypical circumstances, which will reverse the
genetically predisposed direction of primordial germ cell differentia­
This has been attributed to sex hormones (Lille, 1917; Burns, 1925-
1939; Humphrey, 1927-1938) and to embryonic inductors (Witschi, 1914.-1937).
Berkowitz (1938), in this laboratory, has clearly shown that estrogenic
substances can effect a partial, reversal in the guppy but Eversole (1939)
was unable to obtain reversal with androgens.
In this work we have
observed the effects of six different steroids on gonad development in
Lebistes treated throughout all stages of reproductive life.
The author wishes to express his gratitude for the helpful criticism
of his adviser, Dr. Robert Gaunt, under whose direction this work was
The author is also grateful for the advice and suggestions
of Dr. Barry A. Charipper.
He is further indebted to Dr. Max Gilbert
of the Schering Corporation for supplying testosterone propionate,
desoxycorticosterone acetate, progesterone and pregneninolone.
Historical Survey
Normal development and spontaneous sex inversions in Lebistes.
The ovoviviparous fish, Lebistes reticulatus (Guppy), was
selected for this study and a more detailed consideration will be given
to this species than to other forms.
Vaupel (1929) gave the first account of spermatogenesis in
Lebistes, finding germ cells enclosed in cysts, the earlier stages
being toward the periphery of the testis.
He described typical matura­
tion stages and the transformation of the spermatid into the mature
Dildine(l933, 1936) and Goodrich et al. (1934) describe the
post-natal external sexual differentiation and the origin of germ cells
and gonad differentiation.
There is a period of embryonic bisexuality
which begins soon after the formation of the genital ridge and continues
until birth.
The development is first ovarian, as in Anura, the evidence
being that many germ cells are in typical ovarian synapsis.
The gonads
of one-half of the embryos soon become testicular while the other one-
half develop into ovaries.
In testicular gonads all synaptic germ cells
undergo pycnotic dissolution as the gonad hilus organizes into a pro­
spective medulla.
In ovaries the germ cells in synapsis continue to
enlarge as definite ovocytes but occasional pycnotic cells are found;
indicating the operation of male factors even in the female gonad.
embryology of gonads in Lebistes suggests that in teleosts, as in amphi­
bians, birds and mammals, there is a testis-producing medulla and an
ovary-producing cortex.
The gonad is bilateral at birth and protrudes into the coelom on
each side of the gut lateral to the dorsal mesentery.
Sex is distinguish­
able before birth although the gonad, especially the testis, may retain a
bisexual character.
Early bisexuality, present in all vertebrates, to a
greater or lesser degree, is here seen in a somewhat exaggerated form.
It probably indicates a near balance between sex determining factors.
This fact, we think, makes the guppy a favorable form in which to study
extragenetic sex-differentiating influences.
From one to three weeks after birth, the paired ovaries fuse to form
a single gonad enclosing the ovarian cavity which connects directly with
the oviduct.
Maturity results about three months after birth.
the two juvenile testes partially fuse and later the fusion is complete
so that at two and one-half months after birth the testis is mature and
ripe sperm are abundant.
Purser (1938) made a study of embryos developing within the ovary,
finding that each embryo is covered by two membranes, one of which is de­
rived from the egg and the other from the ovary.
Disruption of the
ovarian membrane leaves the egg membrane intact so that each individual
is in a separate ovarian compartment.
The follicles containing the
embryos have an orifice opening into the ovarian cavity so that at time
of "ovulation" the hilum of the follicle opens and allows entrance of
sperm but not escape of egg.
The fertilization membrane forms and the
embryo leaves the follicle at hatching in readiness for birth.
Blacher(1926) met with some spontaneous divergences in Lebistes
from the usual sex dimorphism.
In some individuals the coloration, shape
and size of body resembled that of the female but the gonads were atro­
phied, missing or hermaphroditic.
Faller (1926) also made similar obser­
From this it was concluded that atrophy of the testis is
parailed by disappearance of male sex colors, that female
coloration is
the characteristic coloration of asexual forms, and that development of
black and especially the red and yellow pigment spots depend upon the
male sex hormone produced in the testis.
Hinge (1923), however, concluded
that color characters of the male depend on genes located in the sex chromo­
somes, especially the I-chromosome.
He is substantiated by Aida(l921) who
derived the same conclusion from work
on another form, Aplocheilus latipes.
Winge further states that the Lebistes female, even if its X-chromosome
contains genes of color characters, does not phenotypically exhibit the
Effect of hormones in Lebistes
Berkowitz (1937, 1938) working in this laboratory found that estrogenic
hormone (Progynon) fed to male guppies from the time of birth completely
prevented the development of any male secondary sex characters and caused
the assumption of female secondary sex characters; withdrawal of hormone
treatment then permitted male characters to appear coexistent with induced
female characters.
The testes of animals treated for two months showed
partially suppressed spermatogenesis and if treatment was continued for
three months there was, in a high percentage of cases, a development of
Estrogenic hormone treatment started after sexual maturity
had been reached had no appreciable effect on the secondary sex characters
of the male but caused the testis to become hermaphroditic.
He found fur­
ther that estrogens had little if any effect upon the ovary.
obtained somewhat similar results with intramuscular injection of estra­
diol but did not report the production of ovotestes.
Estrogenic hormone
(estrone) has been shown to cause the formation of an hermaphroditic gonad
in amblystoma (Bums, 1938; Ackart and Leavy, 1939) and also (theelin and
theelol) in chicks (Kozelka and Gallagher, 1934; Willier et al. 1935;
Wolff and Ginglinger, 1936; Willier et al., 1937; Domm, 1939).
The author (Eversole, 1939) reported that female fish fed or injected
pleuroperitoneally with testosterone propionate assumed some but not all
of the male secondary sex characters.
The gonopod could be produced pre­
cociously in immature males and at any stage of development in females.
Testosterone did not induce in females the typical male red, yellow and
orange body pigment but did cause, in fifty percent of the cases, the
appearance of typical male black spots behind the pectoral fins.
gravid spot, a distinctive female trait, could not be suppressed in
Regnier (1938) on the contrary, found a disappearance of the
gravid spot and later development of masculine colors by intramuscular
injections of testosterone.
The author found further a suppression of
growth in females to such an extent that male size was approximated in
genetic females.
Berkowitz had previously been able to stimulate the
growth of genetic males with estrogens.
In agreement with Regnier it
was found that testosterone treatment in females uniformly inhibited
gametogenic activity, causing a resorption of yolk and embryos, if
In only three cases did the author observe ovotestes (obvious­
ly transformed ovaries).
This was an insignificant number in view of
the occasional spontaneous occurrence of such gonads (Blacher, 1926;
Faller, 1926).
The adult male gonad m s apparently unaffected by
treatment of short duration but males receiving injections exceeding a
period of three months showed indications of inhibited spermatogenesis.
Testosterone evidently lacks the sex reversing potency of estrogens
in fishes and in this respect the guppy responds to crystalline sex hor­
mones in a fashion somewhat similar to amblystoma (Burns, 1938, 1939).
However, in Anura, testosterone promotes testis differentiation in the
female and estrogens have little effect on the structure of the testis
in Rana clamitans but stimulate ovocyte development in testes of Rana
pipiens (Foote, 1938; Foote and Witschi, 1939).
In the chick Willier
(1937) has shown that synthetic androsterone and dehydrosterone pro­
duce ovotestes in both female and male embryonic chicks, while testo­
sterone has a similar effect on the female only.
These effects, there­
fore, are such that generalizations as to the relative sex reversing
potencies of crystalline hormones are difficult to apply to different
vertebrate classes and interpretations must necessarily vary with the
species employed.
The site of sex hormone production
That the interstitial cells are responsible for sex hormone
production in fish is not definitely known.
Stephen and Clavert (1933)
found cells in the intertubular spaces of the adult Lebistes testis
which were characteristically different from other cells and made their
appearance at the time of installation of spermatogenesis and develop­
ment of definite secondary sex characters.
These cells came from cells
which appeared non-functional before maturity and a similarity of these
to the cells of Leydig of mammals was suggested.
Champy (1923) could
find only a trace of interstitual tissue in Tinea vulgaris and there
was no increase in interstitium with the assumption of male secondary
sex characters.
Courrier (1921) from his studies on the stickleback,
Gasterosteus aculeatus, came to the conclusion that interstitial gland
is responsible for secondary sex characters because at the spawning
season, when certain of the secondary sex characters appear, the inter­
stitial cells are present, but during quiscence v/hen nuptial coloration
is absent the interstitium is lacking.
Unlike mammals this animal dis­
plays the interstitial gland only during certain periods.
In direct
opposition to this interpretation is the work of van Oordt (1925) who
reported that in the stickleback and Xiphophorus helleri, the occur­
rence of a well developed interstitium testis does not coincide with
the development of secondary sex characters.
Rasmussen (1928) stated that interstitial cells have been found
in the testes of over a dozen species of fish, while, on the other
hand, they have not been demonstrated in many other species.
Castration experiments, etc.
That the fish gonad secretes a hormone has been demonstrated
by a number of investigators.
Kopec (1918) castrated Phoxinus laevis,
keeping the animals alive for three weeks, and found they failed to
exhibit their usual nuptial colors.
Bock (1928) obtained more con­
vincing results with castrated male sticklebacks, Gasterosteus aculeatus.
These animals lived several months and failed to exhibit second­
ary sex characters which are normally present during the breeding
Tozowa (1929) found in the Japanese bitterling, Acheilognathus
intermedium, that the secondary sex characters are modified by gonadectomy but their appearance could not be completely suppressed.
The caudal ocellus is present in all Immature bowfin, Amia calva,
but with maturity it persists only in the male.
Ovariectomy results
in the reappearance of the ocellus while castration in the male results
in persistence; therefore, the black spot is potentially present in
both sexes and evidently its appearance in the normal female is inhibited
by an ovarian endocrine principle (Zahl and Davis, 1932).
Kinoshita (1935) found in Holichoere, as did Courrier in the stickle­
back, that the development of interstitial cells parallels directly the
appearance of secondary sex characters.
He further demonstrated that
the color pattern of the totally castrate male becomes just like that
of the female while in unilateral castrates the color pattern partially
fades and becomes lessened in intensity.
It seemed conclusive then that
the color pattern of the male depends on the secretion of the testis and
that the male sex hormone has a quantitative effect upon secondary sex
characters in this species.
Hansen (1931) removed the gonads of immature goldfish, Carassius
auratus, and found that both sexes developed characteristic coloring of
the adult.
It was concluded that secretions of the gonad, in this form,
have no influence upon color change.
The male stickleback, Gasterosteus aculeatus, develops nuptial
colors and the kidneys hypertrophy and secrete a mucilagenous material
which is used in making a nest at the time of breeding.
Ikeda (1933)
found, in this form, that castration during the breeding season resulted
in return of the kidneys to a non-breeding condition and fish lost ability
to build nests.
He found further that complete castration after partial
development of nuptial colors inhibits the development of erythropores
and hastens their disappearance.
Spayed Betta splendens can regenerate a gonad from the cut end of
the oviduct, the regenerated gland invariably being a testis (Noble and
Kumpf, 1937)*
Animals containing regenerated gonads are typical males
in every respect and are fertile as shown by mating them with normal
That the fish ovary contains a principle similar to that of the
mammalian female sex hormone was demonstrated by Weissman et al. (1937).
An extract from swordfish ovaries when injected into properly prepared
rats was able to produce an estrous reaction after 96 hours.
A minute
quantity of hormone was obtained from a large number of fish ovaries
indicating that the hormone content of fish ovary is lower than the
hormone content of mammalian ovary.
Bitterling, Rhodeus amarus, ovar­
ies transplanted into spayed mice will also evoke estrous (Fleishman
and Kann, 1932).
As in higher vertebrates, the fish pituitary evidently has a
controlling effect on the gonad.
Matthews (1938) was able to hypophy-
sectomize successfully male Fundulus and found that gonads failed to
enlarge as they normally should.
These testes contained practically
no spermatocytes indicating that the pituitary plays a role in control­
ling the seasonal cycle of the male and is apparently of particular
importance in the maturation of male germ cells.
Stimulation of the
fish gonad with mammalian and fish anterior pituitary preparations
has been reported by a number of investigators including Cardoso(1934),
Calvet (1932), Khan (1938), Matthews (1939), Hasler et al. (1939),
Gerbilsky (1938) and Pereira (1934).
On the contrary, Johnson and
Riddle (1939), Young and Bellerby (1935) and Hansen (1939) were unable
to stimulate the gonads of fish with mammalian pituitary extracts.
Hormone treatment
1. The bitterling, Rhodeus amarus.
A great deal of work has been done on ovipositor lengthening in the
female bitterling by overdosing with many different hormone preparations.
The injection of human ovarian follicular hormone into adult females,
during their resting period, led to growth of the external oviduct to
the length characteristic of the spawning season.
tive (Flei^hmamand Kahn, 1932).
Prolan was ineffec­
Barnes et al. (1936) were convinced
that male hormones were responsible for ovipositor lengthening but
after using extracts of a number of tissues, excluding gonads, they
found that the only extract giving a positive response came from the
It was later shown by Fleidhraann and Kann (1938) that cry­
stalline corticosterone, an adrenal cortical hormone, gave the same
Owen (1937) found it difficult to break ovipositor dormancy in
female bitterlings by treatment with sex hormones but substances caus­
ing an occasional lengthening of the ovipositor were dessicated anterior
pituitary, growth factor of the pituitary, urine from aged males,
aqueous testicular extracts, and high dosages of testosterone acetate.
Kleiner et al. (1937) believed ovipositor lengthening is occasioned
by the presence of male sex hormone.
They obtained a positive reaction
with androsterone whereas Barnes et al. could find no evidence for a
stimulative action with this substance.
Kanter et al. (1934) working
with the Japanese bitterling, Achelognathus intermedium, were able to
obtain ovipositor lengthening by adding pregnant urine to the surround­
ing water.
The authors believed lengthening to be due to estrogenic
substances because boiled urine which is lacking in gonadotropic factors
gave positive results.
Tests carried out with commercial products gave
positive reactions with estrogenic preparations but negative results
with gonadotropic factors.
Macht and Bryan (1936) obtained an antagonistic action of fol­
licular and corpus luteum extracts on ovipositor lengthening.
ular hormone in aquaria water containing female bitterlings resulted
in a marked elongation of the ovipositor in 24 to 48 hours.
luteum extract had no stimulating action and follicular hormone plus
corpus luteum extract resulted in no stimulation of the ovipositor.
It has been shown recently by de Wit (1938, 1939, 1939a, 1939b) that
ovipositor-lengthening of the female bitterling can be induced by
administering separately about 27 different steroids belonging to the
estrogen, androgen, and progesterone-like hormone groups.
the way in which the ovipositor-lengthening became manifest was
specific for each of these three groups of substances.
He found pro­
gesterone to elicit a maximum response in 5 hours; estrogens were with­
out effect in this period of time and although androgens produce a
similar reaction to progesterone about fifty times as much material
was required.
He concluded then that ovipositor lengthening of the
female bitterling is specific for the group of steroids
sex and adrenal-cortex hormones also belong.
to which the
Kleiner (1940) found
that although progesterone causes lengthening of the ovipositor in
greater dilution than does testosterone propionate, this effect is
relatively slight as compared with that of male hormone and that the
maximum progesterone action takes place during the first 6 to 8 hours
while that of testosterone is observed only in 24.-72 hours.
The effect of the steroids on ovipositor-lengthening is somewhat
similar to the action of these substances in causing ovulation in the
South African clawed frog, Xenopus laevis, in that specificity is lack­
Shapiro (1936, 1939) and Zwarenstein (1937) have shown that
practically all of the steroid hormones, with the exception of estrogens,
will induce ovulation in Xenopus.
Similarly, in mammals, Gaunt and Hays
have shown that progesterone can substitute for the adrenal cort­
ical hormones in adrenalectomized ferrets and rats; Greene, Burrill and
Ivy (1939) found progesterone to have androgenic activity in castrate
male rats and Hooker and Collins (194-0) observed androgenic activity of
desoxycorticosterone on the capon comb and the prostate and seminal
vesicles of the mouse; Salmon (1939) found desoxycorticosterone to be
estrogenic in the human female and van Heuverswyn et al.^ found it to
resemble progesterone in that it produces progestational proliferation
in the endometrium of immature rabbits.
Glaser and Haempel (1931) experimentally produced the nuptial
dress, which consists of certain color patterns exhibited only during
the mating season in male bitterlings, in castrates by injection of
crude male sex hormone within 4- or 5 hours.
a similar effect.
Yohimbine hydrochloride had
Wunder (1931) also succeeded in inducing the "mating
coat" in sticklebacks, carp and bitterling with epinephrine and Yohimbine.
Wunder thought that the probable action of these substances was by way
of the sympathetic nervous system.
Owen (1937) likewise easily induced
nuptial coloration in males try administering several endocrine prepara­
tions including testosterone acetate, testicular extracts and dessicated
anterior pituitary.
Muller (1937) believed the activity of testicular,
ovarian and posterior pituitary extracts in bringing out the wedding
dress may be ascribed to their control of melanophore hormone.
2. Other fish
The female, Rivulus uropthalmus, displays a perennial velvetyblack ocellus in the dorsal region of the caudal fin.
The ocellus is
present in all juvenile fish but in the male it fades to extinction with
the advent of sexual maturity while in the female it becomes somewhat
Zahl (1934) reported that crude male sex hormone had an in­
hibitory action on the ocellus in the immature and adult female fish.
Saphir (1934-) was able to induce the appearance of the "mating coat" in
Chrosomus erythrogaster by injection of Yohimbine hydrochloride and pro­
lan prepared from urine of pregnant women.
The conclusion was reached
that prolan has either a stimulating effect upon the sex glands of the
fish or a direct effect similar to that of the drug Yohimbine.
In Chloea sarchynnis the female develops nuptial coloration which
lasts for about 60 days in the spring of each year.
During the breeding
season the interfollicular spaces are filled with interstitial cells but
after the breeding season these cells decrease markedly in number and
connective tissue fills up the spaces.
Ovahormone (estrin ?) in females,
before breeding season, results in appearance of nuptial colors.
Spermatin (nature of compound not stated) and prolan the effect is a
positive one of medium grade.
Yohimbine brings out a deep coloration
while adrenalin results in a paling of the body.
The effects of in­
jection after the breeding season is weaker than before it and the
effect of prolan and Spermatin is almost negative.
Furthermore, the
reaction of the same preparation varies according to its quantity;
the smaller the quantity the slower the appearance of the nuptial
coloration and also the shorter its duration (Kinoshita, 1939).
Regnier (1937, 1938) was able to induce the characteristic
changes (gonopod, sword, and body size and shape) associated with the
development of males by injecting testosterone in immature Xiphophorus hellerij estrogens inhibited the development of these characters
in males.
An interesting observation was made by Grobstein and Bellamy
(1939) who found that thyroid feeding of immature Platypoecilus maculatus and P. variatus resulted in decreased growth rate and altered
body proportions in addition to precocious sex maturity as indicated
by the early but atypical differentiation of the male gonopod.
The effects of hormone preparations on the fish gonad have
received little attention as compared with effects on the secondary
sex characters.
However, an increasing number of investigators have
found recently that sex hormones exert a striking effect on germ cell
Padoa (1937) working on the trout, Salmo irideus,
found that by adding ''Crlstollovar" to aquaria water, in which fish
were kept, the males subsequently contained gonads in which the
testicular portions were inhibited and ovocytes made to develop.
gonads of females were little effected with low dosages but with higher
dosages genetic ovaries were inhibited.
As previously discussed,
Berkowitz (1938) made similar observation by administering Progynon to
Witschi and Crown (1937) and the author (Fversole, 1939), work­
ing on Xiphophorus helleri and Lebistes respectively, found that testo­
sterone inhibited the ovaries, but produced no sperm cells.
(1937, 1938) working on Lebistes and Xiphophorus reported that intra­
muscular injections of testosterone and estradiol into females and
males respectively resulted in an inhibition of the gonads; no gonad
reversals were observed.
Baldwin and Golden (1939, however, found
that Xiphophorus females when treated with testosterone contained gonads
in 50$ of the cases, which were characterized either by a resorption of
gonad elements or some phase of spermatogenesis.
Castelnuovo (1937) injected, separately, preparations of fol­
licular, testicular, and anterior lobe pituitary into immature male
carp, Cyprinus carpio, and produced an acceleration of spermatogenesis
in each instance.
Knowles (1939) found in the river lamprey, Lampetra
fluviatilis, that gonads of larvae were not affected by mammalian anter­
ior pituitary extracts and gonad hormones (testosterone propionate and
oestrone) but in the adult male spermatogenesis was slightly accelerated
if changed at all.
Similarly, the stimulation of spermatogenesis in
reptiles vdth mele sex hormone has been reported by Forbes (1940).
author found that Implantation of pellets of testosterone in adult
male lizards (Sceloporus) further stimulated spermatogenesis and
that implanted estrone pellets prevented this process.
These find­
ings are of particular interest since it is well established in
mammals that the hormones secreted by the sex glands have a depress­
ing effect on the gonad of either sex when administered to normal
animals in large amounts.
Materials and Methods
The fresh water, ovoviviparous top minnow, Lebistes reticulatus,
(commonly known as the "guppy”/which belongs to the family Poecillidae,
was the animal employed in the present experiments.
The fish were
kept in 2 1/2 gallon aquaria, in groups of from 8 to 12 (if adults)
and in groups never exceeding 18 (young broods).
All animals were fed
a basic diet of tropical fish food (Blue Ribbon) supplemented with
Daphnia or with worms (Tubifex) and brine shrimp (Artemia) if Daphnia
were not available.
The temperature of the water was thermostatically
controlled at approximately 78 degrees F., and each aquarium, in addi­
tion to containing living plants for aeration, was continuously sub­
jected to a thin stream of air try means of an aeration pump.
The fish were treated with 6 different steroid preparations,
namely; pregneninolone (Pranone, ethinyl testosterone, anhydro-hydroxyprogesterone), testosterone propionate (Oreton), progesterone (Proluton),
desoxycorticosterone acetate (Cortate), pregnanediol and adrenal cortical
Pregneninolone, a synthetic compound having progestational
activity per os in mammals, was in the form of tablets, each of which
contained either 5 or 10 mg. of crystalline material.
into the aquaria the fish ate the tablets freely.
When crumbled
The five other substances
were dissolved in oil of sesame and a special injection technique was em­
ployed in their administration.
The preparations used are steroid in nature and the chemistry of
all, with the exception of adrenal cortical extract, is known.
They have
the following chemical structures:
C t\j
Fregneninolon e
Testosterone Propionate
— crt
A total of 70 fish were fed pregneninolone.
Four groups consist­
ing of 51 animals were treated from hirth for periods varying from
14. days to 56 days.
Two of these groups (23 fish) were each subjected
to approximately 10 mg. of pregneninolone per week; another group (16
fish) to 5 mg. per week, and the fourth group (12 fish) to a total of
5 mg. which was added to the aquarium at birth.
Two more groups con­
sisting of 9 mature males and 10 mature females were each subjected to
10 mg. of pregneninolone per week over a period of 44 days and 50 days
Twenty animals were raised, handled and their tissues
sectioned in a manner identical with the immature treated fish except
pregneninolone treatment was not employed.
Weighed and sectioned
gonads of twelve normal mature males and 9 normal mature females served
as controls for the mature experimental fish.
Animals receiving injections were immobilized by transitory immer­
sion in ice water; this treatment inactivated them for about one minute.
They were then placed on absorbent cotton and injected in the abdominal
cavity by means of a micro-injection circuit attached
to a binocular
Twenty-one fish were injected with testosterone propionate.
of these were allowed to develop normally, after birth, for a period of
fourteen days and at the end of this period 0.001 cp.(0.025mg.) of
hormone, in oil of sesame, v/as injected and weekly injections continued
thereafter for a period of 60 days.
The hormone dosage was gradually
increased to 0.01 cc. (0.25 mg.) with increase in size of the fish.
mature females were injected biweekly with 0.01 cc. (0.05 mg.) of
testosterone propionate over a period of six weeks; hormone treatment
vias then discontinued and the fish observed over a period of 4 months.
At the end of this period three of the females were sacrificed and
their gonads inspected.
A total of 44 guppies were injected vfith progesterone.
six of these animals were two weeks of age when the weekly injections
began and the dosage was gradually increased from 0.001 cc. (0.01 gm.)
to 0.01 cc. (0.1 gm.) as development progressed.
The fish were
treated over periods varying from 56 days to 79 days.
Two mature males
and six mature females were injected weekly with 0.01 cc. (0.1 gm) of
progesterone in oil of sesame over a period of 56 days,
Desoxycorticosterone acetate was injected weekly into 32 fish;
the first injection of 0.001 cc (0.005 gm.) was given to 14 day old fish
and the dosage increased to 0.01 cc. (0.05 gm) as development progressed.
All animals were treated for a period of 56 days.
Cortical extract was Injected weekly into 13 fish in a manner
similar to the progesterone treated animals.
however, varied from 45 days to 57 dayB.
The length of treatment,
Six normal mature females and
2 normal mature males were injected weekly with 0.01 cc. of cortical
extract over a period of 56 days.
Six normal mature females and two normal mature males were in­
jected weekly with 0.01 cc. (0.25 mg.) of pregnanediol over a period
of 56 days.
As controls for the fish which were Injected from the immature
to the mature state 42 litter mates were segregated at birth and
raised in a manner identical with the experimental animals except
that instead of injecting hormone substances the solvent of these
preparations, oil of seBame, was employed.
In most cases the
control animals were raised in the same water as the experimentals
by partitioning the aquaria with glass plates arranged so that
water freely circulated between the two compartments.
The lengths of most animals, both experimental and control,
were measured at 42 days of age; a period in normal development
just previous to external sex differentiation and divergence in
size of the two sexes.
The animals were again measured and also
weighed when sacrificed.
The dimorphic secondary sex characters studied were size,
shape, coloration, sex behavior and condition of the anal fin.
The animals which were Immature when the injections began were
sacrificed at a stage in development when the externally dimorphic
traits were clearly distinguishable (60-79 days).
When the animals
were sacrificed the abdominal cavity was slit open with fine scis­
sors and the gonads removed under a dissecting binocular microscope
by stripping away the gut and lifting out the gonad with watch maker
The gonads were weighed when possible.
If yolk eggs were
present they were dissected free of the ovary so as to avoid diffi­
culty in sectioning.
After clearing in xylol, infiltrating and embedding in tissue
mat, all sections were cut at 8 mu. and stained with Delafield's
hemotoxylin and counterstained in eosln.
Experimental Observations
Normal External Sexual Differentiation in Untreated Animals.
At birth the sexes of Lebistes reticulatus are externally indis­
tinguishable but on microscopic examination the gonad is differentia­
ted, the ovary being organized to a greater extent than the testis.
At about 40-50 days after birth there is a divergence in size of
the two sexes, from which time the females normally become larger
than the males.
The anal fin is modified, if the animal is to be
male, to become the gonopod (intromittant organ) and thereafter assumes
a horizontal position parallel to the long axis of the body; however,
the gonopod formation is not fully completed until the time of sexual
maturity (60-70 days after birth). The anal fin of the female under­
goes no modification and maintains its more or less vertical position.
A further distinction found in the female is the development of a
puberty spot at the base of the anal fin due to a concentration of
black pigment in this area.
This characteristic is usually evident
before anal fin modification in the male so that females are almost,
but not always, invariably distinguishable at an earlier stage of
The male assumes coloration 55 to 65 days after birth,
having a full, complement of sex characters at 70 days.
At this stage
the male becomes sexually active and is in almost constant pursuit
of fleeing females.
The adult male Lebistes possesses, as its second­
ary sex characters, a gonopod, various color patterns and small size.
The female possesses a well formed gravid spot, uniform gray body
color and large size.
The average length of the male (from tip of
snout to base of caudal fin) is 17 mm.; the adult female is about 30
The female being ovoviviparous receives the sperm at mating
and has the capacity of harboring the seminal elements for months at
a time so that one single mating may be sufficient to produce several
Fertilization and early development occurs within the ovary
of the female.
As well as being a constant breeder, thereby exhibiting
no nuptial changes, the guppy is very prolific producing 18-30 young
every thirty days under favorable environmental conditions.
observations in general confirm those of Goodrich et al. (1934) and
Dildine (1933, 1936).
XI. Effects of Preeneninolone on the Secondary Sex Characters
Effects on the Anal Fin
In normal development the anal fin of immature Lebistes is com­
posed of 10 rays which when spread out have a fan shaped appearance
(Plate I, fig. 4j plate II, fig. 1C).
There is no modification of
this basic arrangement in females (Plate I, fig. 1; Plate II, fig. 1A)
but at approximately 4-0 days after birth the fin undergoes some trans­
formation in males; the third, fourth, and fifth rays elongate and
become clearly serrated.
The third ray is most prominent and with
sexual maturity (approximately 70 days) develops on its anterior
side a membranous flap of tissue (Plate I, fig. 2; Plate II, fig. IB).
All treated fish, regardless of age, sex and dosage, exhibited
a transformation of the anal fin in the male direction.
In fish
treated from birth the modification occurred within 5 days and as the
precocious development proceeded the anal fin became very elongate
and extended almost to the caudal fin (Plate II, fig. 2B). Further
development into a complete gonopod was very rapid, requiring 20 days
in fish receiving high dosages (5 or 10 mg. in tank per week) and 30
days in fish receiving low dosages (total of 5 mg. in tank).
elongating rays had a tendency to curl in a dorsal direction (Plate I,
fig. 5) but as the animal increased slightly in size the completed
gonopod straightened and assumed the position and proportions as found
in the mature male.
Concurrent with these changes in the anal fin the
pelvic fins became extremely elongate.
feature females required a relatively longer period of treatment
(10 days) before the anal fin became modified; however, females treated
for AO days developed a thickened and serrated third ray and a membran­
ous flap.
The transformation was not a complete one, but the induced
structure looked more like a male gonopod than like a female anal fin
(Plate I, fig. 3; Plate II, fig. 2A).
Pregneninolone had no effect
upon the gonopodb of sexually mature males.
The normal condition was
apparently the maximal one which could be induced.
Effects on Coloration
Most fish receiving treatment from birth, regardless of sex and
dosage, exhibited masculine yellovr, orange, and red color patterns
within 15-20 days after treatment began.
In normal development these
colors develop 55-65 days after birth in males only.
The precocious
development of color in treated fish was not a uniform response as
regards amount and intensity of pigment.
Some fish were more respon­
sive than others and hence were more brilliantly colored.
Males on the
whole were more responsive than females but in most cases the sexes
were externally indistinguishable and the gonads had to be studied
histologically before sex could be definitely established.
Of the
sixteen males receiving treatment exceeding 20 days all exhibited some
red coloration whereas of the U3 females receiving treatment exceeding
20 days T7% displayed red coloration.
The inducement of red coloration in adult female Lebistes was
difficult to obtain, thus indicating that these fish were more resist­
ant to male color development than immature females.
Of the ten fish
treated only 4- exhibited color throughout the 50 days of treatment and
only two of these were brilliantly colored.
However, male black pig­
ment spots and streaks were present in all fish after 30 days of treat­
Pregneninolone treatment had no effect on coloration of mature
males, indicating that, as in the case of the gonopod, the normal
condition was the maximal one which could be induced.
In normal females, at 30-35 days of age there is evident an
area of black pigment at the base of the anal fin known as the
"gravid spot".
This gravid spot was completely suppressed in all
females receiving treatment from birth but in the mature female it
could not be made to disappear completely; however, its prominence
was less than in the normal mature female.
Effects on Body Size and Shape
Normally, a divergence In size of the two sexes becomes apparent
at the time of external sexual differentiation so that at 60-70 days
of age the female is longer than the male and otherwise larger in all
Pregneninolone had an inhibitory effect on somatic growth
of all immature fish and also on mature females.
The growth of im­
mature fish was inhibited to such an extent that'4-2 day old fish were,
on the average, 3.3 mm. shorter than control fish of the same age (Ta­
ble l). Furthermore, subsequent treatment resulted in a continued
Blow growth rate so that animals rarely exceeded a length of 12 mm.
Concurrent with this, as one would expect, the weights of treated fish
were much lower than control weights.
Pregneninolone completely
stopped growth in growing sexually mature females.
The animals were
exactly the same length after treatment as before, whereas some in­
crease in length would normally have been expected during this period.
Pregneninolone has no effect on body size of mature males.
is to be expected since the growth of males stops abruptly with the
advent of sexual maturity.
All treated fish assumed a male body shape in that the fins were
carried more closely to the body, the rotund female shape was lacking,
and the animals had a "streamlined" appearance (Plate II, fig. 2A).
Effects on Sex Behavior
The normal mature males are sexually very active.
They are almost
continuously in pursuit of females and upon mating swing their gonopods
in an anterior direction so as to connect with the urogenital sinuses
of females.
At the same time the body becomes tense and exhibits vibra­
tory movements.
The females, on the other hand, seem to lack any
positive mating behavior pattern and are continuously avoiding the
Pregneninolone treatment induced in mature females the male
mating behavior after 20 days of treatment and also brought out this
characteristic precociously in all immature fish vrithin the same length
of time.
Normal Control Pish
Twenty immature and 21 mature animals were sectioned and used as
controls for this series besides observations made on normal fish over
a period of three years.
The immature males (sex determined histological­
ly) exhibited no changes in secondary sex characters throughout the 4-2
day observation period; they still retained their immature type of
anal fin, color was not apparent and they exhibited no sex behavior.
The immature control females showed no modification of the anal fin
and no sex behavior but all displayed a clearly defined gravid spot.
The 9 mature female controls and 12 mature male controls exhibited
normal secondary sex characters throughout the 50 day observation
Effects on Gonads
Effects of Pregneninolone on the Testes of Young Males
When treatment was started at birth, there was a marked
stimulation of the testis as indicated by a precocious maturation
of the male germ cells but the gonad did not increase in size suffic­
iently to be weighed.
Fourteen day old Fish
TREATED - (Total of 20 mg. pregneninolone in tank)
These fish had a small but well organized immature
type of testis which was composed of an abundance of precocious prim­
ary spermatocytes arranged in cysts.
There was also present a few
spermatogonia, a well developed interstitium, occasional cysts of
cells with pycnotic nuclei and rather large testicular ducts, the
epithelium of which was composed of low columnar cells (Plate II,fig.4-).
The control gonad was not as advanced in development;
no primary spermatocytes were present but primary spermatogonia were
congregated around the periphery of the testis and some of these by
division had given rise to nests of secondary spermatogonia which
were surrounded by connective tissue sheaths derived from the abun­
dant stroma cells (Plate II, fig. 3).
2. Twenty day old fish (Total of 30 mg. pregneninolone in tank)
These testes,were larger than those of the 14 day old treated
They contained primary spermatocytes, and secondary spermato­
cytes in cyst formation had made their appearance.
Occasionally a
group of primary spermatocytes could be seen in a cyst composed almost
entirely of secondary spermatocytes.
in normal testes.
The duct system was further stimulated as evidenced
by increase in height
This condition was not present
of cells but a well developed interstitium was
Spermatogonia were relatively scarce as compared with the
control testis which had essentially the same structure as the 14 day
old control testis (Plate II, compare fig. 5 with fig. 3).
3. Twenty-nine day old Fish
TREATED - (Total of 30 mg. pregneninolone in tank)
There was a further increase in size of the gonad and
all stages of spermatogenesis, up through spermatid formation, could
be found.
However, spermatogonia and primary spermatocytes were rela­
tively scarce, the gonad being composed primarily of nests of secondary
spermatocytes and spermatids.
Occasionally, abortive, abnormal sperma­
tids were present within the greatly hypertrophied testicular ducts.
There was an abundance of gonad stroma and occasional cells with pycnotic
nuclei were found (Plate III, fig. 1).
The control gonad was beginning to show better organization
in that formation of peripheral cysts of spermatogonia had forced the
earlier ones inward and some of these had become primary spermatocytes.
Secondary spermatocytes and later stages were lacking.
The testicular
ducts were well developed but the cells of the epithelial lining were
quite flat and cuboidal in nature.
Interstitial tissue was present
but to a less extent than in the treated animals (Plate III, fig. 2).
4. Thirty-six day old Fish
TREATED - (Total of 35 mg. pregneninolone in tank)
The testes pf males treated for 36 days were somewhat
similar to the testes of males treated for 29 days except they were
more advanced in germ cell development.
Besides containing spermato­
cytes and spermatids there was also present an occasional group of
sperm cells in the process of aligning themselves in spermatophore
The cells of the ducts were hypertrophied to such an
extent that in some cases the lumen was obliterated and in others the
small luraina contained eosinophilic colloid (Plate III, fig. 3).
These testes were much smaller than treated ones
and contained an abundance of primary spermatocytes and well developed
Interstitial tissue was present but in reduced amounts as
compared with 29 day old normal fish (Plate III, fig. 4).
5. Forty-two day old Fish
TREATED - (Total of 35 mg. pregneninolone in tank)
All stages of spermatogenesis could be found but
later stages were more abundant than early ones.
The center of the
testis was composed of inter-tubular, hyperplastic areas of connec­
tive tissue and great numbers of testicular ducts in which were
found abortive spermatids and somewhat atypical spermatophores.
The epithelium of the ducts was definitely hypertrophied and the
cells were tall and distended with a vacuolated cytoplasm which was
heterogenous in character.
Occasional groups of cells containing
pycnotic nuclei were seen but on the other hand a few spermatogonia
exhibit mitotic figures.
The gonad was abnormal in all respects but
degenerative changes were not clearly evident (Plate IV, fig. 4).
This testis was quite similar in all respects to
the 36 day old control; there was an abundance of spermatogonia,
primary spermatocytes, some interstitial tissue, and well developed
ducts (Plate IV, fig. 3).
6. Forty-two day old Fish
TREATED - (Total of 5 mg. pregneninolone in tank)
The gonads of males receiving small dosages of pregnen-
inolone were not as abnormal as those receiving high dosages.
The gonad
was a mature testis in miniature, in that it was extremely small but
contained all stages of spermatogenesis including mature sperm in typical
Bpermatophore arrangement.
However, late stages of spermatogenesis were
more abundant than early ones.
The testicular ducts were slightly, if
at all, hypertrophied and there were relatively more gonad stroma than
found in the normal mature testis (Plate IV, fig. 5).
The control was the same as for the 42 day old fish in
group 5.
7. Fifty day old Fish
TREATED - (Total of 55 mg. pregneninolone in tank)
These testes were extremely abnormal.
In some, a few
terminal stages of spermatogenesis were present in certain regions but
near the center only an occasional atypical sperraatophore could be seen.
Hyperplastic areas of gonad stroma were present but most of the gonad
was composed of highly ramifying duct remnants whose epithelium was in
the process of dismemberment.
In the lumina of other ducts large
of cellular debris were found.
In other gonads germ cells were
entirely absent and the gonad remnant was composed of duct vestiges,
stroma, and cellular debris (Plate IV, fig. 1).
These gonads contained primary spermatocytes, a few
secondary spermatocytes, a number of well defined ducts, a fairly
well developed interstitium; all of which were surrounded by a peri­
pheral layer of nests of spermatogonia (Plate IV, fig. 2).
Effects of Pregneninolone on the Mature Testis
TREATED - (50 mg. pregneninolone in tank over 44 day period)
The testes of all males showed a marked divergence from
the normal but some were affected to a greater degree than others.
Macroscopically, the gonads were much enlarged and occupied a greater
portion of the abdominal cavity than mature controls.
The extremely
"ripe" intact testis was difficult to remove from the pleuroperitoneal
cavity due to the escape of gonad elements (probably sperm) from the
cut end of the main testicular duct.
The average gonad weight of these
fish exceeded that of controls by 2 mg., a difference which when sub­
jected to statistical analysis was seen to be of probable significance
(Table l).
The abnormal testes, when studied histologically, were lacking
in spermatogonia and only a very few cysts of spermatocytes were
There was, in all cases, an overabundance of terminal sperma-
togenic stages and the organization of mature sperm in normal spermatophore arrangement, wherein the sperm heads are aggregated around the
periphery of a cyst-like structure with tails directed toward the center
in a whorl arrangement, was almost entirely lacking.
Abortive immature
sperm were found in great numbers within the large lumen of the primary
sperm duct.
The epithelium of the smaller ducts was made up of hyper­
trophied high columnar cells while the epithelial cells of the main
ducts were mostly cuboidal in nature.
Hyperplastic areas of gonad
stroma were present throughout all regions of the gonad and filled
in the wide spaces between peripheral spermatophores and ducts.
increase in gonad stroma probably accounted for the increased size
and weight of the testes, more so than did an increase in spermatogenic elements (Plate V, fig. 3).
The normal mature testis was composed of peripheral
spermatogonia and primary spermatocytes, and central secondary sperma­
tocytes, spermatids and mature sperm.
The main testicular duct had
a wide lumen which contains eosinophilic colloid and scattered typical
to columnar.
The epithelial cells of the ducts ranged from cuboidal
Interstitial cells were relatively scarce (Plate V, fig./*).
Effects of Pregneninolone on the Ovaries of Young Fish
The immature female gonad was not as responsive to experimental man­
ipulation as was the male gonad.
Significant effects were not apparent
until the time of yolk deposition (approximately 30-35 days) at which
period this process was inhibited.
There was no indication of sex re­
versal in the gonads but prolonged treatment (50 days) resulted in an
abnormal accumulation of fluid within the ovarian cavity.
Of the 27
gonads sectioned and studied, only two representative cases will be
1. Thirty-four day old Fish (Total of 50 mg. pregneninolone in tank)
Both treated and control gonads had essentially the same histo­
logical appearance except that small ovocytes could more readily be dis­
tinguished in the controls.
All ovaries were rather compact and had an
abundance of large ovocytes in the process of yolk formation.
ovarian cavity of each gonad was clearly distinguishable and the epi­
thelial lining of the cavity in the treated gonad was quite normal but
showed a slight indication to hypertrophyj however, this was not a uni­
form condition and varied with individuals (Plate V, compare fig. 1
with fig. 2).
2. Fifty day old Fish
TREATED - (Total of 50 mg. pregneninolone in tank)
Most animals treated for this length of time contained
a gonad which was greatly enlarged due to an accumulation of fluid
within the ovarian cavity.
Macro scopically, the ovary had the appear­
ance of a clear vesicular sac with occasional white spots (ovocytes)
scattered over its periphery.
Puncture of the ovary with a fine glass
needle resulted in complete collapse due to an outflowing of the con­
tained clear, serous fluid.
Upon histological examination of an un­
punctured ovary, the rather sparse, small ovocytes were seen in a thin
layer of connective tissue which formed the wall of a large hollow
This cavity represented the ovarian cavity whose epithelial
lining was indistinct due to stress caused by the accumulated fluid.
No oogonia were present (Plate V, fig, 5).
The control ovary was well organized and contained
rather large ovocytes in the process of yolk deposition.
The ovarian
cavity was lined with a 7fell developed columnar epitheliumsand near
the proximal borders of the epithelial cells, within the gonad stroma,
were found oogonia exhibiting mitotic figures (Plate V, fig. 6).
D, Effects of Pregneninolone on the Ovaries of Mature Females
Yolk deposition was almost completely inhibited and the condition
of the ovary varied with the presence or absence of developing embryos.
If large yolk eggs were r,resent, when treatment began, there was a re­
turn of the gonad to a juvenile condition and if embryos were present
the gonad shows degenerative changes due to resorption of embryos.
The gonad weight was greatly reduced so that the average weight of
controls (ovaries weighed immediately after giving birth when they were
smaller than at any other time) exceeded the average weight of the
treated by 4-6 mg. (Table l).
Histologically, the juvenile type of ovary had a normal appear­
ance except for the presence of a hypertrophied ovarian cavity epithliura, the cells of which were extremely vacuolated and had a thin
heterogenous cytoplasm.
Cellular debris was present within the ovarian
cavity but rather large normal appearing ovocytes, encased in follicle
cells, made up most of the gonad (Plate VI, fig. l).
Ovaries containing resorbing embryos were composed primarily of
a heterogenous mass of focal areas of degeneration around remaining
strands of embryos.
Leucocytic infiltration, necrotic tissue, pycnotic
nuclei and all signs of degeneration were present.
On the periphery
was found a small number of ovocytes and ovarian ducts (Plate VI, fig.2).
The control ovary was composed primarily of large yolk
eggs, ovocytes, and well developed ovarian ducts (Plate VI, fig. 3).
IV. Animals Treated with Testosterone Propionate
In previous experiments (Eversole, 1939) it was shown that
testosterone propionate caused the transformation of the anal fin
into a gonopod in immature fish and in all females at any stage
of development.
Furthermore, testosterone caused the appearance
in some females of the male-like black pigment spots but did not
stimulate the male orange, red and yellow pigmentation.
Grovrth in
females was suppressed but partially masculinized females did not
show male mating behavior and the gravid spot persisted.
sterone given to females at any stage of development caused an in-r
hlbition of ovogenesis and the retention of an immature type of
With improvement in injection technique coupled with admin­
istration of optimal dosages over a uniform period of time, it was
thought that masculinizing effects could be more accentuated.
proved to be true but to a lesser extent than anticipated.
A. Effects on the Secondary Sex Characters
As well as causing the precocious appearance of the gono-
pod, testosterone inhibited the development of a well formed gravid
spot and induced male mating behavior in all immature fish.
sexes were indistinguishable throughout treatment and there was an
indication that somatic growth was inhibited (Table l).
black spots were apparent in all fish when sacrificed but in onlyone case was there any red coloration.
Apparently testosterone in­
hibited the development of red, orange and yellow color patterns a surprising fact since this color is one of the most definite male
secondary sex characters.
B, Effects on Gonads
1. Effects on the Male Gonad
Macroscopically, these testes were very small and could not
be accurately weighed.
Upon histological examination they had some­
what the same appearance, except for size, as the adult testes treated
with pregneninolone.
The testes were composed primarily of greatly
hypertrophied ducts containing spermatophores and a few abortive
There was also an abundance of hyperplastic areas of
gonad stroma.
Early stages of spermatogenesis were almost entirely
absent (Plate VI, fig. 5).
The control gonads were large and contained all stages of sperma­
There was little gonad stroma but testicular ducts, made
up of cells ranging from cuboidal to columnar, were numerous.
In the
lumina of these ducts were found many typical spermatophores scattered
throughout eosinophilic colloid (Plate VI, fig. 4).
2. Effects on the Female Gonad
Examination of the minute ovaries under the binocular micro­
scope, immediately after splitting the abdomen, revealed small whitish
ovocytes instead of normal large yolk eggs as found in controls.
Histological examination showed not only an inhibition of formation of
yolk eggs and ovogenesis in general but also a definite tendency toward
involution or maintenance of the juvenile condition.
The stroma cells
located between the ova, as well as the cells lining the gonaduct were
slightly, if at all affected by treatment and as a consequence the
oviduct retained a normal appearance (Plate VI, compare fig. 6 with
fig. 7).
The ovary can recover from testosterone effects as observed
in mature females which received treatment for six weeks.
Four months
after stoppage of treatment the ovaries contained large normal yolk
eggs and embryos in the process of development.
V. Steroids Lacking any Effect
Progesterone, desoxycorticosterone acetate, cortical extract and
pregnanediol had no effects upon sexual development in Lebistes.
treated animals, as compared with the 4-2 fish injected with oil of
sesame, showed no difference in growth rate, times of appearance of
different secondary sex characters, and in the development of
Fish treated with these preparations, from the immature
state, showed a uniform sexual divergence at approximately the
same age as hundreds of normal fish observed over a period of
three years.
The gravid spot was evident in most females at 30-35 days
of age, the gonopod at 42-50 days of age and color at 60-70 days of
There was very little difference, among the various groups,when
lengths «re measured at 42 days of age and also when sacrificed.
The testes and ovaries that were large enough to weigh, and the per­
cent body weights of the gonads were approximately the same for all
groups (Table 1).
An occasional fish was found in both experimental
and control groups which varied from the normal.
These fish were
invariably of a larger size than males of the same age, but had neither
gravid spots nor gonopods.
Histological examination, in each case,
revealed a small juvenile testis.
The cause of this delay in develop­
ment is not known.
Mature males and females injected with progesterone, desoxycorticosterone and pregnanediol showed no changes in secondary sex
characters throughout the entire experimental period and most of the
females gave birth during treatment.
Histologically, the testes were
perfectly normal in all respects and the ovaries exhibited yolk eggs
or live embryos as evidenced by heart beat.
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The work reported here together with that of Berkowitz (1938
and unpublished results) concerning estrogen and gonadotropic overdosage permits a partial analysis of the control of primary and
secondary sex characters in Lebistes.
The responsiveness of the secondary sex characters of fish to
steroid substances would indicate that in these lower forms com­
pounds of this general type are the natural sex hormones, as in
higher vertebrates.
Our results, ho?/ever, are not easily interpre­
ted in terms of the theory that testosterone, the most effective
androgen of mammals and generally regarded as the male sex hormone,
is the endogenous male hormone of the fish used here.
It is a re­
latively weak androgen in the fishes, and as pointed out later, has
certain properties that should be termed estrogenic.
a synthetic compound having progestational and some estrogenic activ­
ity in mammals, and having very weak androgenic properties in birds
at least, fulfills much more satisfactorily the expectations of a
fish androgen.
This is not to imply that this synthetic substance,
not known to be produced in the tissues of any vertebrate, is the
male sex hormone of fishes.
An analogous compound, other than testo­
sterone, however, may be the true hormone.
Small or large doses of pregneninolone administered to immature
females inhibited the development of female secondary sex characters
and in most cases caused the development of a full complement of
male secondary sex characters.
Furthermore, all male secondary sex
characters could be produced precociously in males end in 77# of
the females.
The other 23# of the females developed gonopods and
male mating behavior but the male red color patterns were not induced.
The complete control of pigmentation is evidently complex and one in
which both genetic and endocrine factors are probably closely linked.
It is much more easy to induce male color in immature males than in
isne striking orange, yellow and red colors always present in
the sexually mature male are characters which one would expect to be
under male hormone control.
Berkowitz found that estrogens given to
immature males completely suppressed them and high doses of estrogens
in mature males caused a fading but not a complete disappearance of
these colors.
We found that testosterone prepionate 7/as unable to
bring out such color in females and caused its suppression in males.
In this respect testosterone had an estrogenic effect.
on the contrary, caused all males and most females to assume preco­
ciously the adult type of male coloration.
The inference v/ould be,
therefore, that most females and all males have the genetic potency
for such pigmentation and that activation of this potentiality ne­
cessitates the presence of fish male sex hormone.
This inference is
substantiated by the fact that Berkowitz (unpublished results) was
able to cause the precocious development of color in males by admin­
istration of gonadotropic substances.
Since the male gonad is
stimulated by such treatment it is inferred that this response is a
result of fish male hormone production.
The greater difficulty of
eliciting this color in females probably implies the existence of a
complicating genetic factor.
The gravid spot, a distinctive female trait, has apparently a
somewhat different control.
It can be brought out in males with
estrogens and we v;ere able to suppress it in females vfith testosterone
and pregneninolone.
It must, therefore, be genetically present in
both sexes and depends upon some ovarian activity for its expression.
We would interpret the suppression of such traits by androgens as
being due to a suppression of estrogen production, in view of the
obvious gonad inhibition observed, rather than to an androgen-estrogen
antagonism in the reactor organs.
This is further indicated by the
finding (Berkowitz, 1936) that male and female traits can co-exist.
In males the anal fin develops into a gonopod.
The control of
this trait is apparently dependent entirely upon the male sex hormone,
since it can be produced in females at any stage of development and
can be brought out precociously in both males and females by testo­
sterone or pregneninolone administration.
Likewise, Berkowitz was
able to induce a precocious gonopod in males by administration of
gonotropic preparations which by stimulating the testis apparently
caused the secretion of male sex hormone and this in turn acted upon
the anal fin.
This effect on the anal fin is certainly the most
sensitive and quickest androgenic response observed in this species.
It could probably be standardised for assay purposes.
Curiously, how­
ever, the anal fin of Xipophorus is less responsive than the caudal
fin to androgens
(Regnier, 1938).
An unusual effect of testosterone and especially pregneneinoione,
since the opposite occurs with androgens in mammals (McEuen, Selye,
and Collip, 1937), is their suppression of grov/th.
Males are normally
much smaller than females, and v/e v/ere able to suppress the growth of
the latter with testosterone.
Pregneninolone had a more marked effect
on growth than did testosterone in that the grov/th of young fish (male
and female) was slowed down to a greater extent.
This effect was not
due to the toxicity of pregneninolone because the treated fish all
lived and were just as viable as untreated controls.
They displayed
more activity after treatment due to the induction of male mating be­
Similarly, in normal development, growth of males stops
abruptly at the time of testis maturity.
Probably the neutral asexual
size, then, is that of the female, although it is theoretically equally
possible that estrogens stimulate grov/th.
Growth of males is enhanced
by estrogen treatment (Berkowitz) and this could be a direct effect01due
to testis inhibition.
The mating behavior of males is evidently entirely dependent upon
the hormone or hormones produced by the testis because in normal devel­
opment testis maturity parallels mating behavior and this activity can
be produced precociously in all fish treated from birth with testo­
sterone and pregneninolone and also in mature females treated with
the same substances.
The question of the role of sex hormones in the gonad dif­
ferentiation of the guppy is still left in some confusion.
found that estrone would cause the development of an ovotestis in a
high percentage of males.
The expected result with androgenic treat­
ment would then be the production of ovotestes in treated females.
However, this was not the case.
The effects of pregneninolone and
testosterone on the fish ovary were purely ones of inhibition, the
former compound having a more marked effect than the latter.
observations in regard to testosterone are somewhat similar to those
of Regnier (1938) in Lebistes and Xipophorus, and to the findings of
Witschi and Crown (1937) in Xipophorus.
Baldwin and Golden (1939) }
however, apparently were able to cause testicular development in some
ovaries by treating Xiphophorus females with testosterone.
results, on the whole, were unlike the sex reversing effect of testo­
sterone as reported for amphibians by Burns (1939) and for the chick
embryo by 7fillier (1937).
Berkowltz found that estrone had little effect, if any, on the
ovary and he was unable to stimulate the ovary with gonadotropic pre­
These findings suggest that the female gonad is more
resistant to experimental manipulation than the male gonad which
probably indicates a near balance of sex determining factors in the
male but not in the female.
Furthermore, the ovary in LebisteB is
better developed and differentiated at birth in the female than is
the testis in the male.
This factor alone would be sufficient as
a partial explanation for the inability to produce ovotestes in
the females.
The effects of pregneninolone on the male gonad are not clear
and are difficult to interpret on the basis of effects in higher
The few studies with this compound in mammals indicate
that it is almost entirely lacking in androgenic properties but due
to its high androgenic potency in fish it is best to consider it here
as acting as a male sex hormone.
The ability of this compound to
stimulate spermatogenesis in the male gonad rather than inhibit it,
as one would expect in a comparable situation in mammals, i.e.,
where the substance would, exert its effect through an hypophyseal
inhibition (EJoore and Price, 1932), implies that in fish this syn­
thetic steroid acts directly on the male gonad and not by way of the
Even in mammals, however, there are cases where male sex
hormone seemingly has a direct effect on the gonad.
Y/ells and Moore
(1936) found that androsterone or bull testis extract would cause a
precocious spermatogenesis in young ground squirrels and would stim­
ulate spermatogenesis in adults during the hibernation period,
thermore, Walsh et al. (1933) found that androgens,extracted from
urine, would maintain
the testes of hypophysectomized rats.
Nelson and Gallagher (1936) confirmed this finding.
Instances of
gonad stimulation with androgens in other classes of vertebrates
have been reported by Castelnuovo (1937) who produced an acceler­
ation of spermatogenesis in the immature male carp with testicular
preparations and by Forbes (1940) who found that implantation of
testosterone pellets in adult male lizards further stimulated
On the basis of these observations it is more
likely that the action of pregneninolone on the male gonad of
Lebistes is a direct one.
Exactly why the testis of young fish is at first greatly
stimulated and later regresses and shows degenerative changes is
not clear.
Evidently, the action of pregneninolone on the testis
is to stimulate maturation of existing spermatogenic elements but
to prevent proliferation from early stages.
If this is the case,
as it appears to be, the gonad should eventually become devoid of
germ cells except for the very earliest stages.
This is exactly
what happens because the testis, with prolonged treatment, is only
a gonad remnant.
Similarily, treatment with testosterone over a
relatively long period of time and treatment of mature males with
pregneninolone results in a testis which contains only the later
stages of spermatogenesis and an abundance of gonad stroma.
owitz found that estrogens had a similar effect on the testes of
mature males and in this respect androgens and estrogens have a
common effect upon the male gonad.
Results similar to these have
never been described in the literature.
Berkowitz was able to obtain a precocious developing of the
male gonad of Lebistes with mammalian pituitary preparations.
However, the stimulation was not as striking and drastic as that
obtained with pregneninolone.
This might further indicate that
the action of pregneninolone is not by way of the hypophysis.
The progesterone-like action of pregneninolone, as described
by Inhoffen and Hohlweg (1938), Ruzicka, Hoffman, and Meldahl(1938),
Zondek and Rozin (1939), Emmens and Parkes (1939, 1939a) and Salmon
(1939) suggests that progesterone might likewise have the same
activity as pregneninolone in lower vertebrates and that the two
compounds might substitute one for the other under a given set of
In substantiation of this possibility, it is known that
pregneninolone (sometimes referred to as anhydro-hydroxy progesterone
and ethinyl testosterone) and progesterone are chemically very similar.
Furthermore, Lamar (1937) and Green, Burrill and Ivy (1939) obtained
androgenic activity with progesterone in castrate rats; Hooker and
Collins (1940) found desoxycorticosterone to have androgenic activity
on the capon comb and the prostate and seminal vesicles of the moxise;
Wells and Green (1939) and van Heuverswyn et al. (1939) obtained pro­
gestational activity with desoxycorticosterone in the rabbit and
Salmon (1939) obtained an estrogenic activity with this compound in
the human female.
The substitution then of one steroid compound for
another oan occur under certain conditions in mammals and the expec­
tation of a similar type of substitution might seem feasible in lower
This is true for the bitterling in which ovopositor
lengthening can be elicited by many different steroids, including
the adrenal cortical and gonad hormones.
It is also true in the
South African clawed frog in which ovulation can be induced with
all steroid hormones with the exception of estrogens.
in the present experiment, progesterone, desoxycorticosterone,
cortical extract, and pregnanediol are lacking in androgenic or
estrogenic properties and hence it is impossible to draw generali­
zations as to the relative effects of similar compounds in differ­
ent vertebrate classes.
The inability of these steroids, especially progesterone,
to modify sexual development in Lebistes is quite surprising because
of the chemical similarity of these compounds to pregneninolone and
because they do have a common effect on sexual manifestation in other
fish and also in amphibians.
Progesterone is the most effective
steroid in causing ovipositor lengthening in the bitterling (deWit,
1939) and can substitute for testosterone in maintaining the testes
of hypophysectomized rats (Nelson, 1937), but it has no effect on
the primary or secondary sex characters of the guppy.
The multiple biological properties of pregneninolone warrants a
unique classification of this compound as having the properties of
most of the steroid hormones.
In fish it is, or can substitute
the male sex hormone but other similar compounds cannot completely
substitute for it, although testosterone does parallel its action in
producing some, but not all, of its effects.
1. Pregneninolone fed to immature guppies from the time of birth com­
pletely prevented the development of any female secondary sex charac­
ters and caused the precocious assumption of male secondary sex
Genetic females were as responsive as genetic males in
developing a gonopod and male mating behavior but less responsive in
the development of male red color patterns.
The female gravid spot
was completely suppressed in fish treated from birth.
2. Mature females treated with pregneninolone required a relatively
longer period of treatment before male characters could be induced.
In only a few cases could red color pattern be induced but male black
spots, mating behavior, and gonopod formation could readily be pro­
duced in all females.
The prominence of the gravid spot was less in
treated fish than in normal mature females.
3. Pregneninolone inhibited the growth of young fish to such an extent
that at forty-two days of age theywere on the average 3.3 mm. shorter
than control fish of the same age.
Pregneninolone completely stopped
growth in growing sexually mature females and caused the assumption of
male body shape in both immature fish and in mature females.
4. Pregneninolone induced no change in or accentuation of the secondary
sex characters of adult male fish.
5. Pregneninolone induced a marked precocious spermatogenesis in the
young male fish and probably accelerated spermatogenesis in the mature
Prolonged treatment in young males resulted in a regression
of the testes to such an extent that only gonad remnants remained.
Pregneninolone stimulated the development of testicular ducts and
gonad stroma.
6 . Pregneninolone had little effect on the ovary of young fish but
prolonged treatment resulted in an inhibition of yolk deposition and
ovogenesis and an accumulation of fluid v/ithin the ovarian cavity.
Pregneninolone caused the resorption of yolk eggs and embryos If pres­
ent in mature females.
7. Testosterone propionate caused the precocious appearance of the
gonopod, inhibited the development of a well formed gravid spot in
females and induced male mating behavior in all mature fish.
black spots were induced in all fish but testosterone inhibited the
development of red, orange, and yellow color patterns.
8. Testosterone propionate probably accelerated maturation of germ
cells in the male but caused a decrease in size of the gonad.
sterone stimulated the testicular ducts and caused an increase in
gonad stroma.
9. Testosterone propionate given to females not only inhibited the
formation of yolk eggs and ovogenesis in general but also caused a
definite tendency toward involution or maintenance of the juvenile
10. Testosterone induced no changes in or accentuation of the second­
ary sex characters of adult male fish.
11. Progesterone, desoxycorticosterone, adrenal cortical extract,
and pregnanediol had no effects upon sexual development in Lebistes.
Treated fish shov/ed a normal growth rate, a normal divergence of sexes
and contained normal gonads*
12. It is suggested that testosterone, the most effective androgen of
mammals and generally regarded as the male sex hormone, is not the
endogenous male hormone of the guppy.
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Plates I - VI
All histological material illustrated has been fixed in
Boilin's, stained with hematoxylin and eosin, and
sectioned at 8 micra.
Plate I
Explanation of Figures
Anal fin of a normal mature female.
X 6.
t- Gonopod of a normal mature male showing a membranous
flap on the thickened serrate third ray.
X 6.
Induced gonopod of a mature female treated for 4.0 days
with pregneninolone showing a membranous flap on the
thickened serrate third ray.
X 6.
Compare with
figs. 1 and 2.
Anal fin of a normal immature fish.
X 6.
Induced gonopod of an immature fish treated with
pregneninolone for 30 days. X 6.
Compare with fig. 4.
FI G. 5
Plate II
Explanation of Figures
Fig. -1-
A) Normal mature female exhibiting typical grayish body
color, unmo d ifie d anal fin and characteristic body
X 1 1/3,
B) Normal mature male showing characteristic body shape,
color patterns and a gonopod. X 1 l/3.
C) Normal immature fish lacking external sexual differ­
entiation. X 1 1/3 .
Fig. -2-
A) Female treated 30 days with pregneninolone, showing
a well formed gonopod, color patterns and male body
shape. X 1 l/3. Compare with figs. 1A and IB.
B) Immature fish treated from birth for 15 days with
pregneninolone shoving an induced gonopod. X 1 l/3.
Compare with fig. 1C.
Fig. -3-
Cross section of a portion of a normal immature testis
taken from a 14 day old fish showing little organiza­
tion of spermatogonia and gonad stroma. X 375.
Fig. -4-
Cross sectionof a portion
of a testis of a 14 day old
fish treated with pregneninolone from birth, showing a
precocious cyst of primary spermatocytes and v/ell formed
ducts. X 375. Compare with
Fig. -5-
Cross sectionof a portion
fig. 3.
of a testis of a 20-day old
fish treated with pregneninolone from birth, showing
cysts of primary and secondary spermatocytes and well
developed ducts. X 375- Compare with figs. 3 and 4.
Plate III
Explanation of Figures
Fig. -1-
Longitudinal section of a portion of a testis of a
29 day old fish treated with pregneninolone from
birth, showing large size, abundant gonad stroma,
cysts of spermatids and abortive spermatids within
hypertrophied testicular ducts.
X 125.
•with fig. 1.
Fig. -2-
Longitudinal* section of a portion of a well organ­
ized testis of a normal 29 day old fish showing
peripheral spermatogonia and central primary sperma­
tocytes and ducts. X 125.
Fig. -3-
Compare with fig. 1.
Cross section of a portion of a testis of a 36 day
old fish treated with pregneninolone from birth,
showing large size, abundant gonad stroma, spermatocyteB, spermatids, mature sperm and greatly hypertro­
phied testicular ducts. X 125.
Fig. -A-
Compare with fig. 4.
Cross section of a testis of a normal 36 day old fish
showing little gonad stroma, primary spermatocytes and
small well developed testicular ducts. X 125.
with fig. 3.
Plate IV
Explanation of Figures
1- Cross section of a testis of a 50 day old fish treated
with pregneninolone from birth showing an abnormal gonad
remnant composed of duct vestiges and a few sperm cells.
X 125. Compare with fig. 2.
2- Cross section of a portion of a testis of a normal 50
day old fish showing primary spermatocytes, a few second­
ary spermatocytes, a number of well defined ducts and a
fairly well developed interstltium. X 125. Compare with
fig. 1.
Fig. -3- Cross section of a testis of a normal 42 day old fish
showing peripheral spermatogonia, primary spermatocytes,
some interstlal tissue and small well developed ducts.
X 125. Compare with fig. 4.
Cross section of a portion of a testis of a 42 day old
fish treated with high dosage of pregneninolone from
birth, showing a few spermatocytes, spermatophores, and
a few abortive spermatids within greatly hypertrophied
testicular ducts. X 125. Compare with fig. 3.
Cross section of a testis of a 42 day old fish treated with
low dosages of pregneninolone from birth showing an essen­
tially normal, miniature, mature testis.
All stages of
spermatogenesis are present but later stages predominate.
X 125. Compare with figs. 3 and 4.
Plate V.
Explanation of Figures
Fig. -1- Cross section of an ovary of a 34 day old fish treated
from birth with pregneninolone, showing a normal ap­
pearing compact gonad containing large ovocytes and a
slightly hypertrophied duct. X 125.
Compare with fig.2.
Fig. -2- Cross section of a normal ovary of a 34- day old fish
showing a distinct ovarian cavity lined with cuboidal
epithelium and large ovocytes in the process of yolk
deposition. X 125. Compare with fig. 1.
Fig. -3- Cross section of a testis of a mature male treated with
pregneninolone for 44- days showing an abnormal abundance
of terminal spermatogenic stage*, hyperplastic areas of
gonod stroma, and abortive immature sperm within the large
primary sperm duct lumen. X 50.
Compare with fig. 4.
Fig. -4- Cross section of an untreated mature testis, shov/ing all
stages of spermatogenesis. X 50. Compare with fig. 3.
Fig. -5- Cross section of an ovary of a 50 day old fish treated
from birth with pregneninolone,showing a few small ovo­
cytes in a thin layer of connective tissue which forms
the wall of a greatly distended hollow cavity. X 45.
Compare with fig. 6.
Fig. -6- Cross section of an untreated ovary of a 50 day old
female, shov/ing rather large ovocytes in the process of
yolk deposition and an ovarian cavity lined with columnar
epithelium. X 325.
Compare with fig. 5.
*8 M
Plate VI.
Explanation of Figures
Fig. --1- Cross section of an ovary of adult female treated with pregnen- .
inolone for 50 days, showing small size, ovocytes lacking yolk
and hypertrophied ovarian cavity epithelium.
X 25.
with figs. 2 and 3.
Fig. -•2- Cross section of an ovary of adult female treated with pregnen­
inolone for 50 days, showing focal areas of degeneration around
remaining strands of embryos and a' few peripheral ovocytes and
ducts. X 25. Compare with figs. 1 and 3Fig. --3- Cross section of an untreated mature ovary,showing large yolk
eggs, ovocytes and ovarian ducts. Xl6. Compare with figs.l and 2.
Fig. -•4-- Cross section of an untreated mature testis, showing testicular
ducts, little interstitium and all stages of spermatogenesis.
X 50. Compare with fig. 5.
Fig. -■5- Cross section *of a testis of a fish treated with testosterone
propionate for 65 days from 2 weeks of age. Note the small size,
abundance of gonad stroma and absence of early spermatogenic
stages. X 50. Compare with fig. 4.
Fig. -ib- Cross section of an ovary of fish treated with testosterone pro­
pionate for 65 days from two weeks of age.
The histological
appearance of ducts, etc., was normal but the size of the gonad
was abnormally small (less than 1 mg.) due to inhibition of yolk
deposit in the ovocytes. X 125. Compare with figs. 3 and 7.
Fig. -7- Cross section of a select?*region of an ovary of a 79 day old
untreated fish illustrating histological normality of the ovary
shown in fig.6. This ovaiy(wgt.6 mg.) however,was about 10 times
the size of that shown in fig.6, due to the presence of large
yolk eggs only fragments of which are seen in region photographed.
X 125.
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