Inhibition by TGF-beta 1 of the in vitro thymulin-stimulated proliferation of gonocytes from fetal rat testes.

The cytokine transforming growth factor beta 1 (TGF-beta 1) inhibits the growth of certain cells and the differentiation of others. A thymus hormone, thymulin, stimulated the proliferation of fetal male germ cells in explants of testes from 13.5 gestation day rat fetuses. The way in which thymulin acts is unknown. Adding TGF-beta 1 to the culture medium blocked the response of the fetal male rat germ cells to thymulin. We suggest that TGF-beta 1 and thymulin may thus influence the same metabolic chain of events.

Development of the gubernaculum and processus vaginalis in freemartinism: further evidence in support of a specific fetal testis hormone governing male-specific gubernacular development.

BACKGROUND: Freemartinism occurs in some species of ruminants and affects most female bovine fetuses in heterosexual, multiple pregnancies owing to fusion of the chorionic blood circulations soon after implantation. Maldevelopment of the ovaries and Müllerian ducts have been described and recognized as resulting from exposure of their respective primordia to an excess of anti-Müllerian hormone. The present study aimed to analyse the prenatal growth and development of the gubernaculum in freemartins to find out its possible affliction through foetal testis hormones derived from their male co-twin. METHODS: Histological sections of young and drawings and photographs of further developed freemartins and control male and female bovine foetuses were analysed. The specimens had been collected earlier for analysis of the time course of male and female gonadal and genital development and its impairment associated with freemartinism. RESULTS: The gubernaculum of 35-40-day-old male and female fetuses was in the initial stage of development and of similar appearance in all specimens. Gubernacula of 60-70-day-old male fetuses differed from those of females of similar age in various respects: the male gubernaculum size was larger and extension of the processus vaginalis was deeper. Freemartins showed an intermediate development with some individuals resembling male and others resembling female agemates. During further development, gubernacula in males developed into muscular cremaster sacs, whereas those in females generally did not develop beyond the size and structural complexity of 70-day-old foetuses. Beyond day 70 of fetal life, gubernaculum development in freemartins definitely showed male characteristics with respect to size and growth of a processus vaginalis with a cremaster muscular wall. The male-like pattern of the outgrowth of the processus vaginalis changed during the second half of prenatal life. Rather than its further deepening as in males, this structure became inverted to become emerging as a papilla-like structure from the inguinal abdomen bottom. An explanation is proposed for this unprecedented inversion, taking into account: (1) the faster and higher reaching rightsided ascent of the kidneys and gonads, (2) the femalelike outgrowth of the cranial gonadal suspensory ligaments, and (3) the absence of scrotum development. The ovaries and mesonephric remnants in developing freemartins, during their ascent together with the kidneys while remaining attached to the bottom of the developing processus vaginalis sacs via the gubernaculum ligament, are proposed to act together to pull up the bottom of the processus vaginalis sacs. From this action, "inverted hernia sacs" result as the irreversible consequence. CONCLUSION: The data support the concept that foetal testes act, via as an yet unidentified third hormone, to establish malelike development of gubernacula into muscular cremaster sacs. Further work is required to reveal the identity of this hormone. Furthermore, the apparent similarity of the freemartins' inverted processus vaginalis sacs and the fetal rodents' gubernacular cones suggests that the ruminants' and rodents' processus vaginalis are essentially similar structures. Thus there is no longer an urgent need to distinguish between two different types of gubernaculum development and testis descent in rodents and ruminants, respectively, and involving or not fetal gubernacular cones. The present observations may thus contribute to the development of a unified hypothesis for sexually dimorphic development of the gubernaculum throughout the mammalian class.

Effects of thymulin on in vitro incorporation of 3H-thymidine into gonocytes of newborn rat testes.

3H-Thymidine incorporation into gonocytes was investigated by means of quantitative autoradiography in organ cultures of testes from newborn rats between day 0 (day of birth) and day 7, in the presence or absence of thymulin. The data indicated that thymulin had no effect on gonocyte incorporation on day 0 or on days 1 and 7; in contrast, thymulin had a strong effect on day 2. At day 2 the percentage of labeled gonocytes was 10-fold higher in experimental animals than in controls. Between days 2 and 6, thymulin had moderate activity. Thymulin thus appeared to have an age-dependent effect upon incorporation of 3H-thymidine into germ cells. On the other hand, in spite of a high incorporation rate of 3H-thymidine into gonocytes after incubation with thymulin, the number of mitoses remained low, suggesting that thymulin may affect DNA duplication in gonocytes of newborn rat testes.

[Fetal thymus and thymuline stimulate in vitro proliferation of gonocytes in the fetal testis in rats]. / Le thymus foetal et la thymuline stimulent la prolifération des gonocytes dans le testicule foetal de rat in vitro.

Testes from 13.5-day-old rat foetuses were cultured in vitro for 1 or 2 days: 1) in synthetic medium; 2) in thymulin- or corticosterone supplemented medium; 3) in co-culture with fragments of foetal thymus; 4) in co-culture with fragments of foetal thymus in corticosterone-supplemented medium. The numbers of germ cells were about 2-fold greater in testes cultured with fragments of thymus, or in thymulin-supplemented medium than in all the other experimental conditions. These results indicated that thymulin can stimulate foetal gonocytes proliferation and that corticosterone inhibits thymulin-stimulated gonocytes proliferation. The data suggest a functional relationship between corticosterone, thymulin and the foetal gonocytes in vivo.

[Increase of the number of oogonia in the ovary of fetal rabbit hypophysectomised by decapitation. Probable role of thymus]. / Augmentation du nombre des ovogonies dans l'ovaire de foetus de lapin hypophysectomisé par décapitation. Rôle probable du thymus.

A significant increase in ovarian volume and in the numbers of germ cells was observed in rabbit foetuses hypophysectomised by decapitation on day 19 and killed on day 28 when compared with unoperated females of the same litter. It seems that the hypophysis acts indirectly while the foetal thymus has a likely direct role.

[Fetal thymus and thymuline stimulate the in vitro proliferation of oogonia in the fetal rat ovary]. / Le thymus foetal et la thymuline stimulent la prolifération des ovogonies dans l'ovaire foetal de rat in vitro.

In rat ovaries explanted on day 13.5 p.c. and cultured in vitro for up to 6 days, the number of germ cells is enhanced in thymulin-supplemented medium and/or after co-culture of the ovarian explants with foetal thymic tissue compared to ovaries cultured in synthetic medium. Corticosterone added to the medium prevents the secretion of thymulin by the foetal thymus and in that condition the thymus does not influence the proliferation of oogonia. These results provide additional evidence that the pituitary-adrenal-thymic axis might be involved in the control of oogonia proliferation in vivo, taking into account our previous experimental finding that the number of germ cells is increased in ovaries of hypophysectomized foetuses.

Influence of age and medium on formation of epithelial cords in the rat fetal ovary in vitro.

Fetal ovaries of 14.5-day-old rats were cultured for periods of up to 19 days in control medium or in medium conditioned by the preliminary culture of testes from fetal or young rats. In all ovaries, after 12 days of culture in either medium, epithelial cords were noted having an aspect identical to that of seminiferous cords present in fetal testes explanted at 14.5 days and also cultured for 12 days, i.e. the epithelial cords appeared in ovaries when there was no 'male' or testicular influence. The appearance of histological preparations suggested that the disappearance of the germ cells might bring about a reorganization of the follicular cells in epithelial cords during the differentiation period of the first follicles. With ovaries cultured in conditioned medium, degeneration of the germ cells was more marked, follicles were rare and intra-ovarian cords were greater in number than in ovaries cultured in control medium. The ovaries thus transformed produced the anti-Müllerian hormone (AMH) although they lacked the "germinostatic activity" normally developed by testes of fetal or young rats. This germinostatic activity prevents the multiplication of oogonia when the testes and ovaries are co-cultured in vitro. The transformed ovaries therefore do not have all the functional capacities of fetal testes.

[Limitation on the number of germ cells in the fetal ovary of the rat by older ovaries, in vitro]. / Limitation du nombre des cellules germinales dans l'ovaire foetal de rat par des ovaires plus âgés, in vitro.

Ovaries from 9-day-old rats co-cultured for 4 days in vitro with ovaries from 13.5-day-old fetal rats prevent the proliferation of the oogonia in the fetal ovaries (germinostatic effect) and produce the anti-Müllerian substance. The hypothesis that the germinostatic and anti-Müllerian activities are not due to the same factor is discussed.

[Isolation by binding to ultrafiltration membranes of a testicular factor limiting the number of germ cells in fetal ovary of rats in vitro]. / Isolement par liaison à des membranes d'ultrafiltration, d'un facteur testiculaire limitant le nombre de cellules germinales dans l'ovaire foetal de rat in vitro.

The medium used for culturing in vitro fetal or neonatal testes, when used subsequently to culture for 4 days ovaries from 13.5 day old rat fetuses, has the property of severely limiting the number of ovarian germ cells. The non-dialysable factor(s) responsible for the observed effect binds to ultrafiltration membranes (Diaflo, Amicon) and can be eluted from these membranes with fresh medium added 1 M NaCl.

Number of germ cells and meiotic prophase stages in fetal rat ovaries cultured in vitro.

Ovaries from 13.5-day-old rat fetuses were cultured in vitro in a hormone-free medium for up to 8 days. The number of germ cells increased during the first 4 days and then sharply decreased. The initial decrease was concomitant with the first leptotene stages. All stages of meiotic prophase progressively appeared in the remaining germ cells.

[Effect of the fetal testis on the number of germ cells in the fetal ovary of rats in vitro]. / Action du testicule faetal sur le nombre des cellules germinales de l'ovaire du faetus de rat, in vitro.

When ovaries from 13.5-day-old fetuses are explained and cultured in vitro for 4 days in a synthetic medium, the number of germ cells increases 6 fold, on average. This increase is only approximately 2 fold if a pair of 16.5-day fetal testes is cultured together with the ovaries or if the ovaries are cultured in a medium in which testes have previously been grown for 4 days. The effect of the latter medium persists if it is dialysed against fresh medium, which suggests that the conditioned medium contains one or several substance(s) of molecular weight superior to the cut-off of the membrane. The testicular effect seems to be effective mainly during the final phase of intense multiplication of the germ cells.

[The first manifestations of freemartinism in the calf fetus do not depend on XX/XY chromosomal chimerism]. / Les premières manifestations du "freemartinisme" chez le foetus de veau ne dépendent pas du chimérisme chromosomique XX/XY

In order to investigate the eventual role of XX/XY chimerism in freemartinism the vascular anastomoses between twin fetuses were surgically suppressed in litters with several fetuses, before the appearance of the first sexual anomalies. In three female fetuses isolated from their co-twins on days 37 and 45, the initial freemartin effect of gonadal and Müllerian inhibition was absent, in spite of an important XX/XY chimerism in the liver.

[Meiotic prophase in germinal cells of rat ovarian primordium cultivated in vitro in anhormonal medium]. / Prophase méiotique dans les cellules germinales de l'ébauche ovarienne de Rat cultivée in vitro en milieu anhormonal

Gonads of female Rat fetuses were cultured in vitro in an anhormonal medium with the whole or parts of the mesonephros, at an age of 12, 13 or 14 days, i.e. 2 to 4 days before the onset of meiosis. Under these conditions the meiotic prophase takes place and proceeds to the dictyate phase, obeying a somewhat delayed chronology in comparison with controls in vivo.

[Chronology of development of the genital tract of the calf fetus]. / Chronologie du développement de l'appareil génital du foetus de veau

The chronology and the modalities of the differentiation of the genital apparatus were studied in 187 calf fetuses whose insemination age was exactly known (between 32 and 110 days). Male. In the male, the first seminiferous cords form around days 41-42, the interstitial cells appear 2 or 3 days later. From days 60-70 on, the rete testis is made of tubules with an open lumen, which connect the seminiferous cords. The masculinisation of the external genitalia begins as early as day 47 by a rapid increase of the anogenital distance: on day 60, the penis opens under the umbilicus and the scrotum is well differentiated. The regression of the Müllerian ducts starts at the level of their anterior (tubal) part on day 50, when their diameter decreases. From day 58 on, the oviducts become discontinuous and they are almost completely absent by day 63; the uterine horns and the vagina have dissappeared by day 80. The masculinization of the internal genitalia occurs during two phases: 1) between days 56 and 58: the early buds of the seminal vesicles and of the prostate appear, as well as the first differences at the level of the urogenital connections and of the Cowper's glands; a supra-urethral diverticulum develops at the level of the posterior prostatic urethral flexure; the non sexual urethra remains short. 2) After day 70 take place: the differentiation of the epididymides the branching of the seminal vesicles and the stabilization of the Wolffian ducts (after a transitory diminution of their diameter between days 60 and 80). Female. In the female, sexual organogenesis proceeds later than in males. The Müllerian ducts (which show a transitory reduction of their diameter at their tubal level between days 50 and 60) develop steadily after day 60 at the level of their uterine horns and of the vagina; simultaneously the non sexual urethra lengthens rapidly. A suburethral diverticulum, which is absent in the male, develops at the level of the urogenital connections, between days 60-70. The regression of the Wolffian ducts takes place after day 70 when the mesonephros disappear; they first become discontinuous in their median part (at the uterine level) between days 77-80, but important remains of these ducts still persist on day 110, in the posterior part of the vagina. The first primordial ovarian follicles form only after day 100 approximately.