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J. Embryol. exp. Morph., Vol. 17, l,pp. 171-175, February 1967
With 1 plate
Printed in Great Britain
Inductive influence of testosterone upon central
sexual maturation in the rat
By W. N. ADAMS SMITH 1 & M. T. PENG 2
From the Department of Human Anatomy, University of Oxford
INTRODUCTION
The influence of the testis and of testosterone upon the development of the
male genitalia has been extensively investigated and a number of reviews of this
work have been published (Jost, 1960; Burns, 1961). However, Witschi (1957)
has stressed the need to distinguish between adult sex hormones, such as testosterone, and the secretions of the immature gonad. The formation of corpora
lutea in the ovaries transplanted to adult male rats which had been castrated at
birth, and the absence of corpus luteum formation in ovaries transplanted to
male hosts bearing transplanted testes in the neck from birth, was reported by
Pfeiffer in 1936. Similar observations have been reported by Yazaki (1960) and
Harris (1964). A single injection of testosterone propionate has been found to
lead to permanent sterility and a loss of corpus luteum formation in the
ovaries of mice (Barraclough & Leathern, 1954) and rats (Barraclough, 1961).
The present investigation was undertaken to see whether testosterone propionate
would suppress corpus luteum formation in ovarian tissue transplanted to male
rats castrated on the day of birth.
METHODS
Male rats of an inbred Lister colony were castrated on the day of birth and
were injected subcutaneously with 500 fig of testosterone propionate in ethyl
oleate and arachis oil on the fifth, tenth or twentieth day from birth. A control
group of castrates was not injected. When the rats were 3 months old, immature
(18-22 day) ovarian tissue was placed in the anterior chamber of one eye. The
development and activity of the ovarian grafts was observed for 2 months and
the rats were then killed and examined for testicular remnants. The ovarian
grafts were fixed in Bouin's fluid, embedded in paraffin wax and serially sectioned at 6 fi. The sections were stained with haematoxylin and eosin and studied
microscopically.
1
Author's address: Department of Anatomy, Medical College of South Carolina, 80
Barre St, Charleston, South Carolina, U.S.A.
2
Author's address: Department of Physiology, College of Medicine, National Taiwan
University, Taipei, Taiwan.
172
W. N. ADAMS SMITH & M. T. PENG
RESULTS
No testicular remnants were found in testosterone-treated castrates. One
control rat was found to have a small part of one testis (confirmed by histology)
remaining and the activity of the ovarian graft in this rat is described below.
Castrates injected with testosterone at 5 days of age. There were thirty-four
animals in this group. The ovarian grafts became vascularized quickly and
growth of the tissue was apparent within 6-10 days of transplantation. There
was a rapid increase in the size of the follicles, which became considerably
larger than those seen in the grafts borne by control animals. The follicles did
not appear to rupture and no formation of corpora lutea was observed. After
3-5 weeks a few follicles in several of the rats became surrounded by a ring of
solid tissue which had a pale orange colour, rather than the pearly white colour
of the interstitial tissue. The general appearance of the grafts was of large,
vesicular follicles interspersed with thin strands of interstitial tissue. None of the
grafts occupied more than half of the anterior chamber of the eye.
In histological section all of the grafts displayed follicles at all stages of
development, but follicles with abnormally large antra predominated (Plate 1,
fig. 1). In sections from seventeen different grafts one or two clearly demarcated
solid bodies of luteinized cells were seen. Sections from most of these seventeen
grafts and from some of the other grafts contained follicles in which there
appeared to be luteinization of the theca interna (Plate 1,fig.2). The solid bodies
of luteinized cells were generally smaller than typical corpora lutea and their
vascularity was less obvious, but sections from two of the grafts contained
aggregations of luteinized cells which were similar in appearance to typical
corpora lutea. Both of these grafts also contained follicles undergoing luteinization of the theca interna.
Castrates injected with testosterone at 10 days of age. There were fourteen
rats in this group and their ovarian grafts developed in the same way as those
in hosts injected with testosterone at 5 days of age. The histological appearance
of the grafts was of a preponderance of cystic follicles with some follicles showing luteinization of the theca interna (Plate 1, fig. 3). Solid bodies of luteinized
cells were seen in four grafts, but none of them looked like typical corpora
lutea.
Castrates injected with testosterone at 20 days of age. There were fourteen
rats in this group. The grafts became estabhshed in the first post-operative week
and follicles developed normally, with ovulation and corpus luteum formation
first occurring at 10-14 days after operation. Further follicular development and
corpus luteum formation occurred in a cyclical fashion. The grafts came to
occupy almost all of the anterior chamber of the eye and only a few follicles
could be seen between the many large, salmon-coloured corpora lutea.
Histological sections of the grafts were mainly occupied by typical corpora
lutea, with developing follicles lying between them. Two of the grafts contained
/. Embryo], exp. Morph., Vol. 17, Part J
PLATE 1
The scale mark represents 1 mm in all figures.
Fig. 1. Ovarian graft from a male rat castrated on the day of birth and injected with testosterone at 5 days of age. Abnormally large follicles and two theca lutein bodies are present.
Fig. 2. A similar section to that infig.1, showing a pale ring of thecal luteinization surrounding
the darker granulosa in the follicle indicated.
Fig. 3. Graft from a castrate injected with testosterone at 10 days of age. There are numerous
large follicles, and the follicles marked display thecal luteinization. The torn follicle is not
an ovulation site.
Fig. 4. Graft from a non-injected castrate. Some follicles and many corpora lutea are present.
Fig. 5. Graft from the non-injected castrate in which a testicular remnant was found. The
host was a litter-mate of the rat whose graft is shown infig.4.
Fig. 6. Section of the tissue removed post mortem from the host whose graft is shown in
fig. 5. The appearance is similar to that of a cryptorchid testis.
W. N. ADAMSSMITH&M.T. PENG
facing p. 172
Testosterone and central sexual maturation
173
fewer corpora lutea, and some larger follicles, than were found in grafts from
control animals. No thecal luteinization was found in a careful study of all the
sections from all grafts.
Non-injected castrates. There were twenty-five rats in this group and the grafts
of twenty-four of them developed in the same way as those in hosts injected
with testosterone at 20 days of age, with cyclical maturation of follicles and
development of corpora lutea. The other graft developed large follicles, but no
corpora lutea were seen to form, and the rat bearing this graft was subsequently found to have a testicular remnant.
The sections of all but one graft were largely composed of typical corpora
lutea and also contained follicles in various stages of development (Plate 1,
fig. 4). No luteinization of follicular theca interna was seen. Sections of the other
graft, from the host bearing a testicular remnant, displayed large follicles but
no corpora lutea (Plate 1, fig. 5). The tissue suspected of being a testicular
remnant was examined histologically and presented an appearance similar to
that of a cryptorchid testis (Plate 1, fig. 6).
DISCUSSION
It is of interest that the ovarian grafts in rats treated with testosterone propionate at 5 or 10 days of age did not attain a size comparable with that of
grafts of control hosts or hosts injected with testosterone propionate at 20 days
of age. The rate and degree of vascularization of the grafts could account for a
variation in size, but factors such as this do not explain the consistent size
difference found, which is in agreement with the findings of Barraclough (1961)
and Swanson & van der Werff ten Bosch (1963) that the ovaries of androgensterilized female rats are smaller than those of comparable normal females.
This can be accounted for by the absence of corpora lutea.
The majority of the epithelioid cells in the mammalian corpus luteum are
derived from the stratum granulosum (Corner, 1945), but follicular atresia is
often accompanied by the proliferation of the theca interna (Corner, 1938).
Deanesly (1938) has observed, in non-cycling ovarian transplants in the ears
of male rats castrated when adult, that the fully developed granulosa, failing to
luteinize, commonly begins to degenerate, and that under these circumstances
luteinization of the theca interna cells often takes place. Proliferation of the
theca interna and degeneration of the granulosa has also been described in the
ovary of the prepubertal rat (Dawson & McCabe, 1951). Since the ovarian
grafts in the castrate controls and in castrates injected with testosterone at 20
days of age did not show degeneration of the granulosa or thecal luteinization,
it is probable that the bodies of luteinized cells in these grafts were normal
corpora lutea. In view of the finding of thecal luteinization in most of the
ovarian grafts in castrates injected with testosterone at 5 or 10 days of age, it is
suggested that the few aggregations of luteinized cells seen in these grafts
originated from cells of the theca interna and were not true corpora lutea.
174
W. N. ADAMS SMITH & M. T. PENG
The administration of testosterone at 5 or 10 days of age to male rats castrated on the day of birth led to an abnormal functioning of ovarian grafts
borne by these rats. There appeared to be a loss of cyclical activity and an
inability to form corpora lutea. Testosterone administered at 20 days of age
failed to cause changes in the host rat which would lead to abnormal function
of the ovarian graft. It would appear that the male rat is sensitive to the masculinizing influence of testosterone for only a short time after birth, which is in
agreement with the reports of testosterone sensitivity in the female rat (Barraclough, 1961; Adams Smith, 1967). Testosterone administered to the castrated
male rat within the sensitive period leads to a sexual maturation of the male
type, as measured by ovarian function, but its administration after the sensitive
period is unable to reverse sexual maturation of a female type. The results of this
study indicate that the inductive influence of the testis upon sexual maturation
as reported by Pfeiffer (1936) is paralleled by testosterone propionate.
The masculinizing influence of testosterone upon the female rat during
the sensitive period appears to be due to the action of this hormone upon the
central nervous system (Adams Smith & Peng, 1966) and the results of the
pituitary transplantation experiments of Harris & Jacobsohn (1952) in the rat
indicate that the anterior pituitary does not become sexually differentiated. It
would seem possible that the masculinizing effect of testosterone in the male rat
is the result of an inductive influence upon the central nervous system, resulting
in an acyclical release of gonadotrophins.
SUMMARY
1. The influence of testosterone propionate upon the central sexual maturation of the male rat has been investigated.
2. Male rats castrated on the day of birth and injected with testosterone propionate at 5, 10 or 20 days of age had immature ovarian tissue transplanted to
the anterior chamber of one eye when 3 months old.
3. Ovarian grafts in castrate males injected with testosterone at 5 or 10 days
of age did not show cyclical activity. Grafts in non-injected castrates or hosts
injected with testosterone at 20 days of age showed cyclical activity.
4. The site of action of testosterone is discussed, and it is suggested that it
may have an inductive influence upon the sexual maturation of the rat brain.
RESUME
Influence inductrice de la testosterone sur la maturation
sexuelle centrale chez le rat
1. L'influence du propionate de testosterone sur la maturation sexuelle
centrale du rat male a ete etudiee.
2. Des rats males sont castres le jour de la naissance puis recoivent des
injections de propionate de testosterone a l'age de 5, 10 ou 20 jours. Du tissu
Testosterone and central sexual maturation
175
ovarien immature est greffe dans la chambre anterieure d'un ceil de ces animaux
lorsqu'ils ont atteint trois mois.
3. Les greffons ovariens places chez les males castres recevant de la testosterone a l'age de 5 ou 10 jours ne montrent pas d'activite cyclique. Chez les
castrats non traites ou recevant la testosterone a l'age de 20 jours les greffons
montrent une activite cy clique.
4. Le lieu d'action de la testosterone est discute et il est suggere que l'hormone
pourrait exercer une action inductrice sur la maturation sexuelle du cerveau du
rat.
We wish to express our gratitude to the Nuffield Dominions Trust (W.N.A.S.) and the
Population Council (M.T.P.) for support during this work. We would also record our
thanks to Professor G. W. Harris, F.R.S., and our indebtedness for technical assistance to
Mr R. F. White.
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{Manuscript received 11 May 1966)