Paracrine control of Leydig cell activity by FSH dependent proteins from Sertoli cells: an in vitro study

The regulating effect of follicle-stimulating hormone (FSH) on Leydig cell function was studied using a model of immature porcine Leydig and Sertoli cells cultured in a hormone supplemented defined medium. FSH pretreatment for 2 days of Leydig cells cultured alone was with no effect. FSH pretreatment of Leydig cells cocultured with Sertoli cells increases Leydig cell activity in an FSH dose-dependent manner with a maximal effect observed at 50 ng/ml porcine FSH (pFSH). Leydig cells cultured for 2 days in conditioned medium (CM) by FSH stimulated (FSH-CM) Sertoli cells, as compared to CM by unstimulated (control) (C-CM) Sertoli cells show an increase of their activity with a maximal effect observed at 50 ng/ml pFSH. Leydig cells cultured in CM as compared to non CM, show a marked development of organelles (smooth endoplasmic reticulum and mitochondria) involved in the steroidogenic activity. The activity of FSH-CM as compared to C-CM on Leydig cell function was non dialyzable and trypsin sensitive. These data suggest that Sertoli cells exert a regulatory action on Leydig cell steroidogenic activity via FSH dependent secreted proteins.     

In vitro interactions between Sertoli cells and steroidogenic cells

Both the cell and the species specificities of the steroidogenic potentiating activity (SPA) of Sertoli cells on Leydig cells were studied using a coculture system. Coculture of purified pig Leydig cells with rat or pig Sertoli cells in the presence of FSH led in both cases, to a significant increase in hCG receptor number and in hCG-stimulated testosterone production. Similarly, coculture of bovine adrenal cells with rat or pig Sertoli cells enhanced the steroidogenic response of adrenal cells to ACTH and angiotensin II. Such effects were not observed when pig Leydig cells or bovine adrenal cells were cocultured with bovine aortic endothelial cells. Coculture of Sertoli and Leydig cells in the presence of hCG, resulted in a significant increase in FSH receptor number and in FSH-induced plasminogen activator activity. Such effects did not occur when Sertoli cells were cocultured with either adrenal or aortic endothelial cells.     

Interactions between immature porcine Leydig and Sertoli cells in vitro

Summary Interactions between Leydig and Sertoli cells, as well as a stimulatory effect of FSH on Leydig cell activity, have been reported in many studies. In order to investigate these interactions, the ultrastructure of immature pig Leydig cells under different culture conditions has been studied. When cultured alone in a chemically defined medium, there is a marked regression of the Leydig cell smooth endoplasmic reticulum and a swelling of the mitochondria. Addition of FSH or hCG does not prevent these phenomena. Co-culturing of Leydig cells with Sertoli cells from the same animal maintains the smooth endoplasmic reticulum at the level seen in vivo and in freshly isolated Leydig cells. The addition of FSH to the co-culture stimulates its development and increases Leydig cell activity, as assessed by an increase in hCG binding sites and an increased steroidogenic response to hCG. These results suggest that Sertoli cells exert a trophic effect on Leydig cells, and that the stimulatory effect of FSH on Leydig cell function is mediated via the Sertoli cells. These results reinforce the concept of a local regulatory control of Leydig cell steroidogenesis.Post-Doctoral fellow supported by CIRIT, Generalitat de Catalunya, Spain     

Paracrine role of Sertoli cells

Data from several experimental approaches strongly suggest that Sertoli cells exert a paracrine control of the two main testicular functions, androgen secretion and spermatogenesis. Further evidence supporting this role of Sertoli cells was obtained by coculture of Sertoli cells with other testicular cells. Coculture of pig or rat Sertoli cells with pig Leydig cells produces an increase in the hCG receptor number and an increase in the steroidogenic activity of Leydig cells. Pretreatment with FSH further increases the values of these two parameters. These biochemical changes were associated with ultrastructural changes in Leydig cells. The effects of Sertoli cells on Leydig cells depend upon the ratio of the two cells and on the substrate in which the cells are cultured. Moreover, Leydig cells produce an increase in the FSH receptor number and in the FSH stimulation of plasminogen activator production by Sertoli cells. Coculture of rat or pig Sertoli cells with rat germ cells, induces an increase in the RNA and DNA biosynthetic activities of germ cells. Most of the stimulatory effects seemed to be mediated by diffusible factors, secreted by Sertoli cells, but full expression of the stimulatory action was observed when germ cells were in contact with other cells. In this coculture system, a fraction of rat germ cells containing mainly mature forms of spermatocytes inhibited rat Sertoli cell RNA and DNA synthesis, but had no effect on pig Sertoli cells. On the contrary, a fraction of rat germ cells richer in spermatogonias and preleptotene spermatocytes, stimulated rat Sertoli cell DNA synthesis but was without effect on pig Sertoli cells. These results clearly show that the stimulatory effects of Sertoli cells on Leydig and on germ cells which are not species specific are mediated mainly by diffusible factors, the secretion of which is regulates by FSH.     

Evidence for a Sertoli cell, FSH-suppressible inhibiting factor(s) of testicular steroidogenic activity

By using a model of immature porcine Leydig and Sertoli cells cultured in serum free defined medium, we evidenced a paracrine control of Leydig cell steroidogenic activity by Sertoli cells via a secreted inhibiting protein(s). This protein(s), partially purified using gel filtration (M.W. 20,000-30,000) suppresses the steroidogenic responsiveness to LH/hCG by decreasing the specific LH/hCG binding (52% decrease) and hormone steroid biosynthesis (73% decrease) at a level(s) located between cAMP production and pregnenolone formation. The suppression of this inhibitor(s) by FSH, in a dose dependent manner, is one mechanism by which FSH "sensitizes" Leydig cell response to LH/hCG stimulation.     

Inhibitory effect of Sertoli cell-cultured media on LH binding to mouse Leydig cells in culture

The aim of this study is to examine the influence of Sertoli cells on LH binding to Leydig cells in culture in immature mice. Leydig cells and Sertoli cells were obtained from the testes of immature C57BL/6Ncrj mice and were cultured in serum-free medium for 7 days. The LH binding to Leydig cells and the FSH binding to Sertoli cells were dependent on incubation time, the number of cells, and the amount of labelled hormone added. The dissociation constant for LH binding to Leydig cells was 7.3 x 10(-10) M. Co-culture of Leydig cells with Sertoli cells for 7 days decreased LH binding to Leydig cells. The binding was 34.9% of that to Leydig cells cultured alone. After cultivation of Leydig cells with spent Sertoli cell-cultured medium (SM) for the last 4 days of the 7-day culture period, LH binding to Leydig cells decreased to as low as 17.4% of that of the controls. For the controls, LH binding was measured in Leydig cells cultured in spent Leydig cell-cultured medium (LM). There was no difference between SM- and LM-cultures in the final survival rate or the percentage of cells showing histochemically demonstrated 3 beta-hydroxysteroid dehydrogenase activity. These data suggest that some factor or factors are secreted from the cultured Sertoli cells and inhibit the binding of LH to Leydig cells in culture.     

Regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured rat Sertoli and Leydig cells

New data are provided to show that (i) rat Sertoli cells produce two types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), and a plasminogen activator inhibitor type-1 (PAI-1); (ii) both tPA (but not uPA) and PAI-1 secretion in the culture are modified by FSH, forskolin, dbcAMP, GnRH, PMA and growth factors (EGF and FGF), but not by hCG and androstenedione (△4); (iii) in vitro secretion of tPA and PA-PAI-1 complexes of Sertoli cells are greatly enhanced by presence of Leydig cells which produce negligible tPA but measurable PAI-1 activity;(iv) combination culture of Sertoli and Leydig cells remarkably increases FSH-induced PAI-1 activity and decreases hCG- and forskolin-induced inhibitor activity as compared with that of two cell types cultured alone. These data suggest that rat Sertoli cells, similar to ovarian granulosa cells, are capable of secreting both tPA and uPA, as well as PAI-1. The interaction of Sertoli cells and Leydig cells is essential for the cells to response to     

Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures

Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.     

Paracrine regulation of testicular function

Data from several experimental approaches have been reviewed and the findings clearly indicate the existence of multiple interactions between testicular cells and the potential role of these interactions in the paracrine control of testicular functions. Both testicular interstitial fluid and spent media from cultured Sertoli cells had an acute steroidogenic effect on Leydig cells, and this effect is not species specific. The secretion of this steroidogenic factor(s), which is probably a protein, is enhanced by previous FSH treatment of Sertoli cells. Coculture for 2-3 days of pig Leydig cells with homologous or heterologous Sertoli cells enhances Leydig cell specific functions (hCG receptor number and hCG responsiveness) and induces Leydig cell hypertrophy. A similar but less pronounced trophic effect is seen when Leydig cells are cultured with spent media from Sertoli cells cultured in the presence of FSH and high concentrations of insulin, but the spent media from Sertoli cells cultured in the absence of these two hormones inhibits Leydig cell specific functions. Somatomedin-C might play an important role in the positive trophic effect of Sertoli cells on Leydig cells, since this peptide is secreted by Sertoli cells and it has trophic effects on the specific function of Leydig cells. Moreover, Sertoli cells, probably through a diffusible factor and cell-to-cell contacts, control the multiplication, meiotic reduction and maturation of germ cells. In turn, the activity of Sertoli cells is modulated by the stage of neighbouring germ cells. Thus, if a normal Sertoli cell function (which depends not only on FSH but also on Leydig and myoid cell secretory products) is an absolute requirement for germ cell multiplication and maturation, these cells, in turn, cyclically regulate Sertoli cell function and through these cells the size and probably the function of Leydig cells.     

Rat testis 17 beta-estradiol: identification by gas chromatography-mass spectrometry and age related cellular distribution

The aromatization of testosterone into 17 beta-estradiol (E2) was assessed in purified Leydig and Sertoli cells from rats aged 10-80 days. E2 was identified by gas chromatography-mass spectrometry (GC-MS) and measured both by radioimmunoassay (RIA) and GC-MS associated with stable isotope dilution. A potent competitive inhibitor of the aromatase activity, 4-hydroxyandrostenedione (4-OH-A) was used to test the enzymatic specificity. The basal aromatase activity was present in both cell types whatever the age of the animals. The basal E2 levels did not vary in Sertoli cells while a gradual increase was noted in Leydig cells until day 40, followed by a slight decrease in mature rats. In 10-day old animals, the aromatase activity was localized in Sertoli cells and highly stimulated by FSH; on day 20, both Sertoli and Leydig cells synthesized E2 although E2 from Sertoli cell origin was still predominant. Starting on day 20 until adulthood, the aromatase activity was under LH control in Leydig cells with a maximum around 40 days. The FSH and LH effects were mediated by cyclic AMP.     

Regulation of 5 alpha-reductase activity in cultured immature leydig cells by human chorionic gonadotropin

The present studies examined the hormonal regulation of 5 alpha-reductase activity in cultured immature rat Leydig cells. Within the testis 5 alpha-reductase was concentrated in the interstitial cell compartment, and among interstitial cells, the enzyme was localized primarily in Band 3 of Percoll density gradients, which contains the majority of Leydig cells. Among various factors reported previously to stimulate testicular 5 alpha-reductase activity when administered in vivo to immature rats (LH/hCG, FSH, luteinizing hormone releasing hormone or prolactin), only LH/hCG directly stimulated 5 alpha-reductase activity of cultured immature Band 3 cells. Neither growth hormone which was reported previously to stimulate hepatic 5 alpha-reductase activity, nor insulin, insulin-like growth factor-I, or epidermal growth factor, which have been reported to modulate Leydig cell function, had any effect on 5 alpha-reductase activity of Band 3 cells. These studies suggest that the major factor directly stimulating 5 alpha-reductase activity in Leydig cells during early maturation is LH. However, it is possible that other factors acting indirectly may modulate the maturational rise in 5 alpha-reductase activity.     

Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1). whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2). whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1-100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.     

Interaction between Leydig and Sertoli cells in vitro

The interaction between Leydig and Sertoli cells grown in co-culture was studied. After 3 to 4 days in culture, Leydig and Sertoli cells formed monolayers. To distinguish functional Leydig cells from Sertoli cells, a histochemical test for delta 5,3 beta-HSD activity was performed, and cells which showed a positive reaction were defined as Leydig cells, in contrast to Sertoli cells which did not manifest enzyme activity. Testosterone and oestradiol levels in culture media were determined by radioimmunological assays. Sertoli cells in co-culture showed a tendency to organize themselves as in vivo, forming a kind of pseudo-wall of the tubule. This process becomes more evident with the time of culture. Co-cultures secreted more androgens than Leydig cells alone and more oestradiol than Sertoli cells alone. This influence was strengthened by the presence of follicle stimulating hormone (FSH) in the culture medium, which was not the case in cultures of Leydig and Sertoli cells cultured separately.     

LH contamination may explain FSH effects on rat Leydig cells

Treatment of immature, hypophysectomized male rats with 50 micrograms ovine FSH (NIH-FSH-S12) twice a day for 5 days stimulated the maximum quantity of 17 beta-hydroxyandrogen produced by isolated Leydig cells in response to hCG. Pretreatment of the FSH preparation with an LH antiserum in one study markedly reduced and in another study completely abolished this stimulatory effect of FSH, but only slightly impaired the capacity of the hormone to stimulate the Sertoli cell in vivo (epididymal androgen-binding protein). Administration of another highly potent FSH preparation (LER-1881) had no discernible effects on the dose-response characteristics of the Leydig cells but was superior to the NIH-FSH-S12 in its capacity for stimulating the Sertoli cell. When all hormone preparations were tested for their ability to stimulate steroid secretion from normal Leydig cells in vitro, a close correlation was obtained between their Leydig cell-stimulating activity (a measure of LH contamination) and their capacity to alter Leydig cell responsiveness after in-vivo treatment. FSH treatment had no effects on specific LH binding per 10(6) Leydig cells. It is concluded that the stimulatory influence of FSH on rat Leydig cells may to some extent be a result of the LH contaminating the hormone preparation.     

Studies with purified immature porcine Leydig cells in primary culture

The steroidogenic capacity of purified immature porcine Leydig cells in culture was studied over several days. The cells were obtained by fractionating crude testicular interstitial cell suspensions on a discontinuous Percoll gradient (d = 1.037, 1.042, 1.052, 1.098 g/ml), and characterized by specific binding of 125I-human chorionic gonadotropin (hCG), testosterone (T) and cyclic adenosine 3':5'-monophosphate (cAMP) production in response to hCG, and the enzymatic determination of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The Leydig cells were recovered in a density band between 1.052-1.068 g/ml and grown in a chemically defined medium (Mather et al., 1981). In the absence of hCG, T production was low throughout the 6 days of culture. However, in response to hCG (10 mIU/ml), the cultured Leydig cells showed a progressive increase in T synthesis, which reached a maximum at Days 3-4. 8-Br-cAMP (1 mM) induced a comparable rise in T production to that obtained with hCG throughout the culture period. In contrast, 8-Br-cAMP induced a near maximal increase in dehydroepiandrosterone (DHEA) production from Day 1. This paper demonstrates that purified immature porcine Leydig cells in primary culture are a valuable model to study the ontogeny of Leydig cell function.     

Location of enzymes of androgen and estrogen biosynthesis in the testis of the ground squirrel (Citellus lateralis)

The intratesticular localization of enzymes of androgen and estrogen biosynthesis was studied in the ground squirrel (Citellus lateralis). In mature animals, interstitium and tubules were isolated by manual dissection. Microsomes were prepared and enzymes assayed by analysis of product formation after incubation with appropriate 3H-labeled substrates. In the immature testis, tubules and interstitium are not readily separable; thus, distribution was inferred after analysis of whole testicular microsomes from control, follicle-stimulating hormone (FSH)-treated, and luteinizing hormone (LH)-treated animals. To verify the cellular composition of tissues and the status of steroidogenic organelles in Leydig and Sertoli cells, samples were also analyzed by light and electron microscopy. In mature squirrels, enzymes of androgen biosynthesis were concentrated in the interstitium; however, levels present in the tubules were sufficient to account for a substantial fraction of whole testicular activity (1/3 to 1/5). By contrast, virtually all of the testicular aromatase was accounted for by that in the seminiferous tubules. The purity of these fractions was checked by light microscopy; they showed little cross-contamination. In whole testicular microsomes of immature squirrels, androgen biosynthetic enzymes had a much lower specific activity than in mature animals; however, the opposite was true for aromatase, its activity being approximately 5-fold higher in prepubertal animals. Luteinizing hormone treatment markedly stimulated hydroxylase and lyase but not aromatase. Luteinizing hormone also induced an increase in Leydig cell size and a dramatic proliferation of smooth endoplasmic reticulum. These changes were correlated with increased serum testosterone. As shown previously in rats, 3 beta-hydroxysteroid dehydrogenase was independent of LH control. Follicle-stimulating hormone had no effect on any of the enzymes studied, but induced some increase of agranular reticulum in Sertoli cells. Results from immature squirrels thus corroborate data from mature animals, showing a predominant interstitial location of androgen biosynthetic enzymes. While we cannot explain the absence of FSH stimulation of aromatase activity, the data do not refute the findings in mature animals showing a predominant tubular location of this enzyme. We conclude that the distribution of steroidogenic enzymes in the testis of squirrels differs in several important respects from rats, although both are members of the order Rodentia.     

Spent media from immature seminiferous tubules and Sertoli cells inhibit adult rat Leydig cell aromatase activity

Adult rat Leydig cell aromatase activity is stimulated 2.5 fold by LH or dbcAMP. Spent media prepared from seminiferous tubules or Sertoli cells of immature rats depress both the basal and the LH stimulated estradiol syntheses (25 and 20% decreases, respectively). These inhibitory effects are further enhanced when FSH is added to the culture medium of seminiferous tubules or Sertoli cells. Rat serum as well as culture media from other cell lines are ineffective while seminiferous tubule media from other immature animals (mouse, guinea-pig, calf) inhibit the aromatase activity. This Sertoli cell factor is a heat stable protein (molecular weight greater than 10 kDa), different from the LHRH-like Sertoli cell compound, which acts on the aromatase activity at a step beyond the adenylate cyclase.     

FSH regulates cultured Leydig cell function via Sertoli cell proteins: an in vitro study

The effects of follicular stimulating hormone (FSH) on testicular steroidogenic activity has been studied by testing the capacity of conditioned medium (CM) by both unstimulated (control) Sertoli cells (C-CM) and FSH stimulated Sertoli cells (FSH-CM) to influence porcine cultured Leydig cell activity. Leydig cells cultured in FSH-CM for 48 hrs, as compared to C-CM, show a significant (P less than 0.05) increase in [125I]-hCG binding (150% +/- 4) and hCG-stimulated testosterone (T) secretion (266% +/- 42). In addition, the stimulating effect of FSH-CM on Leydig cell function as compared to C-CM, is trypsin sensitive, non dialyzable, heat stable, acid resistant and is chromatographed following gel filtration (Sephadex G 100) into two different peaks of activity. These data suggest that FSH regulates Leydig cell function via (at least two types of) Sertoli cell secreted proteins.     

Activité aromatase dans les cellules germinales mâles: quelle signification fonctionnelle?

The ability of the male gonad to convert androgens into estrogens is well known. According to age, aromatase activity has been already measured in immature and mature rat Leydig cells as well as in Sertoli cells. Recently, in different studies, a cytochrome P450_arom has even been immunolocalized not only in Leydig cells but also in germ cells of mouse, brown bear and rooster whereas in pig, ram and human the aromatase is mainly present in Leydig cells. Our purpose was to investigate the testicular cell distribution of cytochrome P450_arom mRNA in adult rat using RT-PCR. With 2 highly specific primers located on exons 8 and 9, we have been able to amplify a 289 bp aromatase fragment not only in Leydig cells and Sertoli cells but more importantly in highlyenriched preparations of pachytene spermatocytes, round spermatids and testicular spermatozoa. These amplified products showed 100% homology with the corresponding fragment of the rat ovary cDNA. In parallel, using an anti-human cytochrome P450_arom antibody we have demonstrated the presence of a 55 kDa protein in seminiferous tubules and crude germ cell (pachytene spermatocytes and round spermatids) preparation of the mature rat. After incubation with tritiated androstenedione, the aromatase activities in the microsomal fractions were 3.12±0.19 pmoles/mg/h in the testis, 1.25±0.13 in the seminiferous tubules and 1.53±0.15 in the crude germ cells. In purified testicular spermatozoa the aromatase activity was 2.96±0.69 pmoles/mg/h and found to be 5-fold higher when compared to that of either purified pachytene spermatocytes or round spermatids. Using a quantitative RT-PCR method with a standard cDNA 29 bp shorter, we have compared the amount of cytochrome P450_arom mRNA in mature rat Leydig cells and Sertoli cells. In purified Leydig cells from 90 day-old rats the P450_arom mRNA level was: 36.2±3.4×10^?3 amoles/μg RNA whereas in Sertoli cells the mRNA level was 10 fold lower. In pachytene spermatocytes, round spermatids and testicular spermatozoa the P450_arom mRNA levels were re pectively 367.2±76.6, 117.6±22.0 and <1×10^?3 amole/μg RNA. In conclusion we have demonstrated that the P450 aromatase is present not only in Sertoli cells and Leydig cells from mature rat testis but a biologically active aromatase exists also in germ cells (pachytene spermatocytes, round spermatids and spermatozoa). The existence of an additional source of estrogens within the genital tract of the male is now well documented and that suggests a putative role for these hormones during the male germ cell development.