G. Livera

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.

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.

Retinoid Receptors Involved in the Effects of Retinoic Acid on Rat Testis Development

Abstract We have previously shown that retinoic acid (RA) is able to act on the development of Leydig, Sertoli, and germ cells in the testis in culture (Livera et al., Biol Reprod 2000; 62:1303–1314). To identify which receptors mediate these effects, we have now added selective agonists and antagonists of retinoic acid receptors (RARs) or retinoid X receptors (RXRs) in the same organotypic culture system. The RAR α agonist mimicked most of the effects of RA on the cultured fetal or neonatal testis, whereas the RAR β, γ, and pan RXR agonists did not. The RAR α agonist decreased the testosterone production, the number of gonocytes, and the cAMP response to FSH of fetal testis explanted at 14.5 days postconception (dpc). The RAR α agonist disorganized the cords of the 14.5-dpc cultured testis and increased the cord diameter in cultured 3-days-postpartum (dpp) testis in the same way as RA. All these RA effects could be reversed by an RAR α antagonist and were unchanged by an RAR β/γ antagonist. The RAR β agonist, however, increased Sertoli cell proliferation in the 3-dpp testis in the same way as RA, and this effect was blocked by an RAR β antagonist. The RAR γ and the pan RXR agonists had no selective effect. These results suggest that all the effects of RA on development of the fetal and neonatal testis are mediated via RAR α, except for its effect on Sertoli cell proliferation, which involves RAR β.

Retinoid-Sensitive Steps in Steroidogenesis in Fetal and Neonatal Rat Testes: In Vitro and In Vivo Studies

Abstract Retinoic acid (RA) was recently shown to modify testosterone secretion of the fetal testis in vitro. We characterized this effect by culturing rat testes explanted at various ages, from Fetal Day 14.5 to Postnatal Day 3. In basal medium, RA inhibited, in a dose-dependent manner, both basal and acute LH-stimulated testosterone secretion by testes explanted on Fetal Days 14.5, 15.5, and 16.5. It had no effect on testes from older animals. The negative effect of RA did not result from a diminution in the number of Leydig cells but from a decrease in P450c17 mRNA levels and in LH-stimulated cAMP production. However, the RA-induced decrease in P450C17 mRNA levels was also observed with neonatal testes, suggesting that this enzymatic step is no longer rate limiting at this developmental stage. To study the physiological relevance of RA effects, we used fetuses and neonates issued from mothers fed a vitamin A-deficient (VAD) diet, resulting in a threefold decrease of plasma retinol concentration. On Fetal Day 18.5 and on Posnatal Day 3, testosterone secretion by the testis ex vivo was significantly increased in VAD animals. This shows that the endogenous retinol inhibits differentiation and/or function of fetal Leydig cells before Fetal Day 18.5 and is required for the normal regression of fetal Leydig cell function that occurs after Fetal Day 18.5. In conclusion, our results show that retinoids play a negative role on the steroidogenic activity during the differentiation of rat fetal Leydig cells.

Development of the foetal and neonatal testis

The foetal testis originates from a proliferation of the mesonephric and the coelomic epithelia which are colonized by the primordial germ cells. In the foetal testis, the development and functions of the three main cell type precursors (Leydig, Sertoli and germ cells) do not depend upon gonadotropins. Numerous intra‐ and extra‐testicular factors are candidates for the control of its development and functions. To study the potential involvement of these factors, we developed an organotypic culture system. In absence of any growth factors or hormone, this system allows a development of the three main cell types which mimics that observed in vivo. The effects of different regulators (gonadotropin‐releasing hormone, follicle‐stimulating hormone, transforming growth factor‐β, insulin‐like growth factor‐I, anti‐Mullerian hormone, retinoic acid, oestrogens) were tested in this system. Whether or not some of the effects observed in vitro have a physiological relevance was evaluated using appropriate transgenic mice. It is concluded that the foetal testis cannot be considered as an adult mini‐testis since it has a specific physiology which largely differs from that of the immature or adult testis.