René Habert

Transforming Growth Factor β1 and β2 Reduce the Number of Gonocytes by Increasing Apoptosis1

Transforming growth factors beta1 and beta2 (TGFbetas) have recently been detected by immunohistochemistry in the fetal and neonatal rat testis, and the aim of the present study was to determine whether these factors can act as local regulators to control the number of gonocytes. Testes were kept in organ culture, and TGFbeta1 was found to have dose-dependent inhibitory effect on the number of gonocytes in testes explanted on fetal day 13.5. Either TGFbeta1 or beta2 at 10 ng/ml reduced the number of gonocytes by half after 2 days culture. TGFbetas did not decrease the BrdU labeling index of gonocytes or Sertoli cells, whereas these factors significantly increased the DNA fragmentation in gonocytes (TUNEL method). The other testicular cell types showed no positive TUNEL reaction. TGFbeta1 did not reduce the number of gonocytes in testes explanted on fetal day 17.5 (i.e. during the quiescent phase), but it did so in testes explanted on postnatal day 3 (i.e. stage of resumption of mitosis). To determine the potential cell type targets for TGFbetas, type I and type II TGFbeta receptors were immunolocalized in developing testis from fetal day 13.5 to postnatal day 3. Both receptors were present in the gonocytes throughout the whole period studied, and in the Leydig cells from fetal day 16.5 onward, but they were not detected in the Sertoli cells. Taken together, these results suggest that TGFbetas directly increase apoptosis in gonocytes without changing their mitotic activity during the developmental phases of proliferation.

Multiple Effects of Retinoids on the Development of Sertoli, Germ, and Leydig Cells of Fetal and Neonatal Rat Testis in Culture

Abstract We investigated the effect of retinoids on the development of Sertoli, germ, and Leydig cells using 3-day culture of testes from fetuses 14.5 and 18.5 days post-conception (dpc) and from neonates 3 days postpartum (dpp). Addition of 10−6 M and 3.10−8 M retinoic acid (RA) caused a dose-dependent disruption of the seminiferous cords in 14.5-day-old fetal testes, without any change in the 5-bromo-2′-deoxyuridine (BrdU) labeling index of the Sertoli cells. RA caused no disorganization of older testes, but it did cause hyperplasia of the Sertoli cells in 3-dpp testes. Fragmentation of the Sertoli cell DNA was not detected in control or RA-treated testes at any age studied. The cAMP produced in response to FSH was significantly decreased in RA-treated testes for all studied ages. Both 10−6 M and 3.10−8 M RA dramatically reduced the number of gonocytes per 14.5-dpc testis. This resulted from a high increase in apoptosis, which greatly exceeded the slight increase of mitosis. RA caused no change in the number of gonocytes in testes explanted on 18.5 dpc (the quiescent period), whereas it increased this number in testes explanted on 3 dpp (i.e., when gonocyte mitosis and apoptosis resume). Lastly, RA and retinol (RE) reduced both basal and acute LH-stimulated testosterone secretion by 14.5-dpc testis explants, without change in the number of 3β-hydroxysteroid dehydrogenase-positive cells per testis. Retinoids had no effect on basal or LH-stimulated testosterone production by older testes. In conclusion, RE and RA are potential regulators of the development of the testis and act mainly negatively during fetal life and positively during the neonatal period on the parameters we have studied.

Ontogenesis of the in vitro response of rat testis to gonadotropin-releasing hormone.

The age-related evolution of the in vitro effects of a gonadotropin releasing hormone agonist ([D-Trp6]-GnRH) on the secretion of testosterone by the testis, cultured during 3 days on a Millipore filter floating on M199 medium, was studied during the perinatal period in the rat. The basal and luteinizing hormone (LH)-stimulated secretions by testes explanted on fetal day 14.5 were unaffected by the agonist. With fetal testes explanted on days 16.5 and 18.5 post-conception, the agonist inhibited, in a concentration-dependent manner, both basal and LH-stimulated secretions from the second or the third day of treatment onwards. With fetal and neonatal testes explanted on days 20.5, 21.5 and 31.5 post-conception, the GnRH agonist also had a long-term inhibitory effect on LH-stimulated secretion, but increased basal secretion. This stimulatory effect was already observed after 4 h of culture, and was maintained for 3 days. These results suggest that, during fetal development, the cellular mechanisms involved in the negative testicular response to GnRH are differentiated 3-5 days before those involved in the positive response. Lastly, after 3 days of preculture in hormone-free medium, fetal testes explanted on day 14.5 displayed long-term GnRH agonist inhibition of in vitro basal secretion of testosterone. This observation points out a spontaneous differentiation of the negative responsiveness to GnRH in the cultured fetal testis.

Expression and Effect of Insulin-Like Growth Factor I on Rat Fetal Leydig Cell Function and Differentiation

Insulin like growth factor I (IGF-I) is believed to be a potent para/autocrine stimulator of Leydig cell function in adult testis. We investigated whether IGF-I is also an intratesticular regulator of fetal Leydig cell function by measuring its production in the fetal testis and its ability to affect testicular steroidogenesis during fetal development. Northern blot analysis revealed one major IGF-I transcript of 7–7.5 kb and two minor transcripts of 3.8 and 1.8 kb in 20.5 day fetal testis. IGF-I was detected by RIA in 16.5 fetal day testes, and the amounts of IGF-I secreted by 16.5 and 20.5 fetal day testes in vitro were much greater than the amounts contained in the testes, indicating active synthesis in culture. The secretion of IGF-I by the fetal testis in vitro was increased with testicular age and time in culture. It was not modified by gonadotropins or (Bu)2cAMP. Testosterone secretion by fetal testes explanted 13.5, 16.5, 18.5, and 20.5 days after conception and cultured in the presence or absence...

Immunohistochemical localization of transforming growth factor-β1 in the fetal and neonatal rat testis

The localization of transforming growth factor-beta 1 in the fetal and neonatal rat testis (from day 13.5 of fetal life to postnatal day 20) was investigated by an immunohistochemical staining method employing a polyclonal anti-TGF-beta 1 antibody that does not cross react with either TGF-beta 2 or TGF-beta 3. In testis and mesonephros tissue, immunostaining for TGF-beta 1 was undetectable on fetal day 13.5 and appeared exclusively in the primordial Sertoli cells on fetal day 14.5. Staining in Sertoli cells was still clearly observed on days 15.5 and 16.5 of fetal life and became faint from fetal day 18.5 onwards. In fetal Leydig cells, a positive reaction for TGF-beta 1 appeared on day 16.5 and became very intense during late fetal life. After birth, fetal-type Leydig cells, which were still observed on postnatal days 4 and 20, also exhibited a very strong immunostaining for TGF-beta 1, whereas adult-type Leydig cells, observed on day 20, showed a slight staining. No immunoreactivity for TGF-beta 1 was found in germ cells and peritubular cells on any day studied. In conclusion, TGF-beta 1 is present very early in the fetal rat testis and its prevailing localization shows age-related changes, which suggests that this factor plays an autocrine/paracrine role in the regulation of testicular function and differentiation, during early development.

Transforming growth factor beta1 inhibits steroidogenesis in dispersed fetal testicular cells in culture.

TGF beta1 has been detected by immunohistochemistry in the rat fetal testis. Therefore, we attempted to determine whether this factor can act as a local regulator of Leydig cell function during fetal development. An inhibitory effect of TGF beta1 on basal and luteinizing hormone (LH)-stimulated testosterone secretion by fetal testes in vitro was observed only with testes from 13.5 day-old fetuses and not with testes from older stages. The lack of effect of exogenous TGF beta1 in organ culture after day 13.5 might be related to an elevated intratesticular concentration that would already exert maximal biological effect. On the contrary, in a model of dispersed testicular cells in culture, TGF beta1 was able to inhibit LH-stimulated testosterone production by fetal Leydig cells from 16.5 and 20.5 day-old fetuses. This inhibition of LH-stimulated testosterone production was dose- and time-dependent and was maximal after 48 h of treatment with 1 ng/ml TGF beta1, with testosterone secretion being reduced to 25% of control values. Inhibition of testosterone secretion was also observed in basal and dbcAMP-stimulated conditions, suggesting that one site of action of TGF beta1 is located after the production of cAMP. However, TGF beta1 was also able to inhibit LH-induced cAMP production. As demonstrated by the transformation of steroidogenic precursors into testosterone, TGF beta1 did not significantly alter 3beta-hydroxysteroid dehydrogenase (3beta HSD) activity but induced a strong inhibition of cytochrome P450 17alpha-hydroxylase/C17-20 lyase (P450C17) activity which was associated with a marked diminution of cytochrome P450C17 mRNA levels (26% of control values) but not of cytochrome P450scc mRNA. In addition to its effect on steroidogenesis, TGF beta1 exhibited morphogenic actions on the fetal testicular cells, inducing spreading when the cells were adherent and aggregation when the cells were cultured in conditions of lesser adherence and without any significant effect on either total cell number or 3beta HSD positive cells. Taken together these results suggest that TGF beta1 likely plays a morphogenic and physiological role very early in the fetal testis via paracrine/autocrine mechanisms.

Expression of type I and II receptors for transforming growth factor β in the adult rat testis

Abstract After having established the specificity of the antibodies for the rat testis by western blot analysis, the potential target cells for transforming growth factors (TGFβs) were identified by immunohistochemical detection of both type I (TβRI) and type II (TβRII) transducing receptors for TGFβs in the adult rat testis in situ. Leydig cells showed a strong TβRII immunoreactivity whereas the TβRI staining was weak. Only TβRII was detectable in Sertoli cells. In germ cells, staining for TβRI was stronger than for TβRII and the expression of both receptors depended on the seminiferous cycle stage. TβRI first appeared in pachytene spermatocytes and was absent in elongated spermatids from stage XIV onwards. Labelling for TβRII was observed as early as the spermatogonia stage; it increased in pachytene spermatocytes at the onset of TβRI and disappeared in elongating spermatids from stage XI onwards. These results show that TGFβs can affect somatic cells functions and suggest that these factors are involved in the control of meiosis and early spermiogenesis, exerting a direct effect on germ cells.

Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors.

Fetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk.

Luteinizing hormone-dependent activity and luteinizing hormone-independent differentiation of rat fetal Leydig cells

Addition of 5x10(-2) U/ml recombinant luteinizing hormone (LH) to testes from fetuses at 16.5 day post conception (dpc) cultured for 5 days increased the number of Leydig cells by 34% and the acute LH-stimulated testosterone production by 600%. To determine whether these positive effects of LH in vitro are physiologically relevant in vivo, fetuses were decapitated on days 16.5 pc (before the onset of LH expression in the hypophysis) or 18.5 pc (before the surge of LH in the fetal plasma) and removed at 21.5 dpc. The number of fetal Leydig cells per testis and the acute LH-stimulated testosterone production by the testes ex vivo were unaltered by decapitation. Since, in all groups, the number of Leydig cells doubled between 16.5 and 18.5 dpc and between 18.5 and 21.5 dpc, these results suggest that neither the appearance of new fully differentiated fetal Leydig cells nor the maintenance of differentiated functions in existing fetal Leydig cells depend on LH during late fetal life, although this hormone is present in the plasma. Decapitation reduced the testosterone concentrations in the plasma (-56%) and in the testis in vivo (-67%) and the basal testosterone secretion of the testis ex vivo (-70%). This suggests that LH is required to maintain the physiological activity of the Leydig cell during late fetal life. However, the decrease of the in vivo testosterone production after decapitation was not sufficient to impair the growth of the Wolffian ducts and the lengthening of the anogenital distance. In conclusion, during late fetal life in the rat, Leydig cells are LH-independent for their functional differentiation and LH-dependent for their activity.

Effects of retinoids on the meiosis in the fetal rat ovary in culture.

We investigated the effect of retinoids on the entrance of female germ cells into meiotic prophase and their progression through it, using explants of rat ovaries from 14.5 days post coïtum (dpc) fetuses cultured with or without 10(-6) M retinoic acid (RA) or 10(-9) M retinoic acid receptor alpha (RARalpha) specific agonist. The percentages of oogonia and of oocytes at each meiotic stage in the ovary were evaluated at explantation (D0) and after 3 (D3), 5 (D5) and 9 (D9) days of culture and on equivalent stages in vivo (i. e. 17.5 and 23.5 dpc). The number of germ cells per ovary were counted at D0, D3 and D9. Newly explanted (D0) ovaries contained no germ cell in meiosis. In control medium some germ cells had spontaneously reached the stage leptotene and very few the zygotene on D3. The first pachytene were observed on D5 and the first diplotene on D9. This pattern mimicked that which occurs in vivo although with a slight delay. RA reduced the percentage of oogonia by more than half and increased the percentage of zygotene by more than 22-fold on D3, showing that it accelerated entrance into meiosis. This effect was also observed in response to RARalpha agonist. RA increased the percentage of zygotene and reduced the percentage of pachytene on D9, showing that it can also delay the zygotene/pachytene transition. Lastly, RA reduced the total number of germ cells present on D3 but not on D9. This may be the result of a double effect of RA on the number of germ cells: negative when the cells are in proliferation (D0 to D3) and positive when they entered in meiotic prophase (after D3). Thus, RA is a potential regulator of germ cells meiosis and number in the fetal ovary.