Differential expression of mRNA aromatase in ejaculated spermatozoa from infertile men in relation to either asthenozoospermia or teratozoospermia.

Oestrogen biosynthesis in ejaculated spermatozoa is an autonomous process, which may influence sperm functions. The purpose of this study was to evaluate the relationship between the expression of aromatase, sperm quality and seminal neutral α-glucosidase marker in semen of Tunisian infertile men: asthenozoospermia (A; n = 16), teratozoospermia (T; n = 12) and asthenoteratozoospermia (AT; n = 11) in comparison with 18 normozoospermic ones. Aromatase mRNA levels estimated by real-time PCR were reduced in groups T (52%) and AT (67%) compared to controls and inversely correlated with the percentage of normal forms. A higher coefficient of correlation was noted in presence of microcephaly or acrosome malformations (r = -0.64). The asthenozoospermic group was divided into two subgroups according to the relative amount of aromatase. The subgroup (A2) with higher aromatase transcript level was associated with an increased seminal pH, a decreased sperm viability, low sperm percentage motility and low neutral α-glucosidase semen levels. Our data highlight the involvement of aromatase in motility and morphology of spermatozoa. Thus, this enzyme could bring new insights about quality and fertilising capacity of human spermatozoa.

Estrogen, a female hormone involved in spermatogenesis.

In mammalian testes, aromatase irreversibly converts androgens (C19 steroid) into estrogens (C18) and is present in the endoplasmic reticulum of numerous tissues. In purified adult rat germ cells (pachytene spermatocytes and round spermatids) we have shown the presence of a functional aromatase (transcript, protein and biological activity) and the estrogen production is roughly identical to that of Leydig cells. In addition, transcripts of aromatase varied according to the germ cell type and the stages of seminiferous epithelium in an adult rat. In contrast with the androgen receptors mainly localized in somatic cells, estrogen receptors (ERs) are described in all testicular cells. Moreover, besides the presence of high affinity ERα and ERß a rapid membrane effects have been recently reported and we demonstrated that GPR30 (a transmembrane intracellular estrogen receptor) was expressed in adult rat pachytene spermatocytes and in round spermatids. Thus estrogens through both GPR30 and genomic effects are able to activate the rapid signaling cascade, which in turn triggers an apoptotic mitochondrial pathway (via an increase in Bax expression) and a concomitant decrease of cyclin A1 and B1 gene levels as well as in controlling apoptosis and maturation/differentiation of round spermatids. Hence, the role of estrogen (either intracrine, paracrine or autocrine) in spermatogenesis (proliferation, apoptosis, survival and maturation) is now obvious taking into account the simultaneous presence of a biologically active aromatase and the widespread distribution of estrogen receptors especially during the spermiogenesis steps.

Effect of methanol extract of Basella alba L. (Basellaceae) on the fecundity and testosterone level in male rats exposed to flutamide in utero.

We evaluated the effect of the methanol extract of Basella alba (MEBa) on testosterone level and fecundity/fertility in male rats exposed in utero to flutamide - an androgen receptor antagonist. For this purpose, 1.5- and 2.5 -month-old male rats exposed in utero to flutamide were treated with the MEBa (1 mg kg(-1) ) for 2 and 1 month respectively. Five days before the end of treatment, rats were housed with females to assess their fecundity/fertility. Thereafter, rats were sacrificed and blood collected for the quantification of testosterone. Flutamide-exposed male rats showed a decrease in their ano-genital distance (AGD, P < 0.05) and were infertile. In normal (methylcellulose-exposed) animals, MEBa provoked an increase in testosterone level in 1.5- (P < 0.008) and 2.5 -month-old rats (P < 0.01) concomitantly with the improvement in their fecundity by 25%. In flutamide-exposed male rats, MEBa increased testosterone level in 1.5 -month-old rats (P < 0.001) without any effect on their fecundity; while in 2.5- month-old rats, MEBa did not affect the testosterone level but improved fecundity (by 25%) and fertility (P < 0.001). This study demonstrated the positive effect of MEBa to enhance fecundity/fertility in normal male rats and in rats exposed to the antiandrogen flutamide during their foetal life.

Estrogen signaling in testicular cells.

Aromatase transforms irreversibly androgens into estrogens and is present in the endoplasmic reticulum of various tissues including the mammalian testis. In rat all testicular cells except peritubular cells express aromatase. Indeed in adult rat germ cells (pachytene spermatocytes and round spermatids) we have demonstrated the presence of a functional aromatase (transcript, protein and biological activity) and the estrogen output is equivalent to that of Leydig cells. In addition in the adult rat, transcripts of aromatase vary according to the germ cell type and to the stages of seminiferous epithelium. By contrast with the androgen receptors mainly localized in somatic cells, estrogen receptors (ERs) are described in most of the testicular cells including germ cells. Moreover, besides the presence of high affinity ERα and/or ERß, a rapid membrane effect has been recently reported and we demonstrated that GPR30 (a transmembrane intracellular estrogen receptor) is expressed in adult rat pachytene spermatocytes. Therefore estrogens through both GPR30 and ERα are able to activate the rapid EGFR/ERK/c-jun signaling cascade, which in turn triggers an apoptotic mitochondrial pathway involving an increase in Bax expression and a concomitant reduction of cyclin A1 and B1 gene levels. In another study in round spermatids of adult rat we have shown that the rapid membrane effect of estradiol is also efficient in controlling apoptosis and maturation / differentiation of these haploid germ cells. In man the presence of a biologically active aromatase and of estrogen receptors has been reported in Leydig cells, but also in immature germ cells and ejaculated spermatozoa. Thus the role of estrogen (intracrine, autocrine and / or paracrine) in spermatogenesis (proliferation, apoptosis, survival and maturation) and more generally, in male reproduction is now evidenced taking into account the simultaneous presence of a biologically active aromatase and the widespread distribution of estrogen receptors especially in haploid germ cells.

Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax.

Spermatogenesis is a precisely controlled and timed process, comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, maturation and differentiation of haploid spermatids giving rise to spermatozoa. It is well known that the maintenance of spermatogenesis is controlled by gonadotrophins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including oestrogens. However, it remains uncertain whether oestrogens are able to activate rapid signalling pathways directly in male germ cells. Classically, oestrogens act by binding to oestrogen receptors (ESRs) 1 and 2. Recently, it has been demonstrated that rapid oestrogen action can also be mediated by the G-protein-coupled oestrogen receptor 1 (Gper). The aim of the present study was to investigate ESRs and Gper expression in primary cultures of adult rat round spermatids (RS) and define if oestradiol (E2) is able to activate, through these receptors, pathways involved in the regulation of genes controlling rat RS apoptosis and/or maturation. In this study, we demonstrated that rat RS express ESR1, ESR2 and Gper. Short-time treatment of RS with E2, the selective Gper agonist G1 and the selective ESR1 and ERß agonists, 4,4',4"-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), respectively, determined activation of Extra-cellular signal-regulated kinase (ERK1/2) through the involvement of epidermal growth factor receptor transactivation. In addition, we investigated the effects of ESRs and Gper pathway activation on factors involved in RS maturation. Expression of cyclin B1 mRNA was downregulated by E2, G1 and PPT, but not by DPN. A concomitant and inverse regulation of the pro-apoptotic factor Bax mRNA expression was observed in the same conditions, with DPN being the only one determining an increase in this factor expression. Collectively, these data demonstrate that E2 activates, through ESRs and Gper, pathways involved in the regulation of genes controlling rat RS apoptosis and differentiation such as cyclin B1 and Bax.

Age- and cell-related gene expression of aromatase and estrogen receptors in the rat testis.

Spermatogenesis is a complex and coordinated process leading to the formation of spermatozoa. This event, which is under the control of numerous endocrine and paracrine factors, seems to also be controlled by estrogens which exert their effects via nuclear estrogen receptors (ESRs) ESR1 and ESR2. Estrogens are synthesized by aromatase which is biologically expressed in the rat testis. The objective of our study was to clarify the gene expression patterns of aromatase and ESRs according to age and in the two compartments of the adult rat testis. In the adult, transcripts of aromatase vary according to the germ cell type and to the stages of seminiferous epithelium, a maximum being observed at stage I. The ESR1 gene is highly expressed in the adult testis and in stages from VIIc-d to XIV. Moreover, both ESR mRNA levels are higher in purified round spermatids than in pachytene spermatocytes, suggesting a putative role of estrogens in the haploid steps of spermatogenesis. The variability of the results in the expression of both ESRs led us to explore the putative presence of variants in the rat testis. Concerning ESR1, we have shown the presence of the full-length form and of one isoform with exon 4 deleted. For ESR2, besides the wild type, three isoforms were observed: one with exon 3 deleted, another with an insertion of 54 nucleotides, and the last one with both modifications. Therefore, the stage-regulated expression of aromatase and ESR1 genes in the rat testis suggests a likely role of estrogens in spermatogenesis.

[In vitro capacitation]. / La capacitation in vitro.

Capacitation is a prerequisite for mammalian spermatozoa to fertilize oocytes. Lipids play a crucial role in the structural and functional organization of sperm plasma membrane. Lipid and membrane protein ordering changes dramatically during sperm capacitation but the resulting effects differ according to the regions of the sperm head. Lipids modifications are mainly characterized by a cholesterol efflux, dynamic cholesterol redistribution in particular in the apical zone of the head and also a phospholipids reorganization resulting to the scramblase activation. The existence of lipids ordered microdomains (lipid rafts) has been recently observed in sperm membranes. These lipid and membrane protein movements are believed to play a role in modulating signaling pathways mainly, the AMPc/PKA and ERK pathways. One of the early key events is the activation of adenylate cyclase by high levels of bicarbonate. All these pathways lead finally to the phosphorylation of Tyr-proteins. But capacitation seems to be more complex with the contribution of other kinases (from the PI3K/Akt pathway and phosphotyrosine kinases) towards the phosphorylation of other Ser/Thr and Tyr proteins. The reactive oxygen species (ROS) seem to be important in the control of mechanisms involved in capacitation.

Relationship between semen quality and seminal plasma components: alpha-glucosidase, fructose and citrate in infertile men compared with a normospermic population of Tunisian men.

The aim of this study was to evaluate the correlation between the secretory function of the male accessory glands and sperm parameters in normospermic controls and infertile patients. One hundred and fifty-nine men were investigated: they were composed of two groups: normospermic (n = 37) and infertile (n = 122) men with altered sperm characteristics. These infertile men were divided into the following groups: asthenozoospermia (n = 38), teratozoospermia (n = 40) and asthenoteratozoospermia (n = 44). The patients underwent semen analysis and measurements of fructose, neutral alpha-glucosidase and citric acid. The level of fructose was significantly decreased in asthenozoospermic and increased in asthenoteratozoospermic men. It was significantly correlated with semen volume, sperm count, motility and morphology. The seminal alpha-glucosidase levels were significantly correlated with semen volume and pH and citric acid was significantly correlated with pH. Thus, alpha-glucosidase and citric acid levels were associated with semen pH. The significant correlation between semen parameters, accessory glands and epididymal functions highlights the relationship between semen and normal genital tract function.

New insights about the evaluation of human sperm quality: the aromatase example.

Male contribution to the couple's infertility is at first evaluated by the routine examination of semen parameters upon optical microscopy providing valuable information for a rational initial diagnosis and for a clinical management of infertility. But the different forms of infertility defined according to the WHO criteria especially teratozoospermia are not always related to the chromatin structure or to the fertilization capacity. New investigations at the molecular level (transcript and protein) could be developed in order to understand the nature of sperm malformation responsible of human infertility and thus to evaluate the sperm quality. The profile analysis of spermatozoal transcripts could be considered as a fingerprint of the past spermatogenic events. The selection of representative transcripts of normal spermatozoa remains complex because a differential expression (increased, decreased or not modified levels) of specific transcripts has been revealed between immotile and motile sperm fractions issued from normozoospermic donors. Microarrays tests or real-time quantitative PCR could be helpful for the identification of factors involved in the male infertility. Differences in the expression of specific transcripts have been reported between normal and abnormal semen samples. With the aromatase example, we have noted a negative strong correlation between the amount of transcript and the percentage of abnormal forms especially in presence of head defects. Immunocytochemical procedures using fluorescent probes associated with either confocal microscopy or flow cytometry can be also helpful to proceed with further investigations about the localization of proteins in the compartmentalized spermatozoa or the acrosome reaction. The dual location of aromatase both in the equatorial segment, the mid-piece and the tail could explain the double role of this enzyme in acrosome reaction and motility.

Aromatase and estrogens in man reproduction: a review and latest advances.

The mammalian testis is a complex organ which produces spermatozoa and synthesizes steroids. The transformation of androgens into estrogens is catalyzed by aromatase, an enzymatic complex encoded by a single copy-gene (cyp19) which contains 18 exons, 9 of them being translated. In man besides Leydig cells, we have demonstrated the existence of a biologically active aromatase in immature germ cells and in ejaculated spermatozoa. In addition the presence of estrogen receptors (ERalpha and ERss) in immature germ cells and in spermatozoa has been reported. Concerning aromatase, a 30% decrease of the amount of mRNA is observed in immotile compared to motile sperm fraction from the same sample. In asthenoteratozoospermic, teratozoospermic and asthenozoospermic patients, the aromatase gene expression is decreased respectively by 67%, 52% and 44%, when compared to normospermic controls. Statistical analyses between the sperm morphology and the aromatase/GAPDH ratio have revealed a high degree of correlation (r=-0.64) between that ratio and the percentage of abnormal spermatozoa (especially microcephaly). In men genetically deficient in aromatase diminutions of sperm number and motility have been published. Therefore besides gonadotrophins and testosterone, estrogens are likely playing a relevant role in spermiogenesis and human male gamete maturation.

Impact of dietary restriction on peroxidative effects of nickel chloride in wistar rats.

ABSTRACT The purpose of this study, carried out in Wistar rats, was to evaluate the protective effect of dietary restriction (performed by intermittent fasting) against oxidative stress induced by a low concentration of nickel chloride in kidney, liver, uterus, and ovary. Lipid peroxidation (TBARS), catalase activity, and the levels of vitamins E and A in the blood were investigated in rats feed for 1 month either daily (N) or 1 day over two (intermittent fasting, IF) and then injected (NNi, IFNi) or not with nickel chloride (30 mumoles/kg body weight/day) for 10 days. Ni induced a significant increase of TBARS in organs of N rats. Intermittent fasting alone or associated to nickel treatment did not result in TBARS change in IF and IFNi rats. Catalase activity levels were found to be similar in N and IF rats. In Ni-treated rats a transient increase of catalase activity appeared at day 1 in the kidney and days 1 and 3 in the liver. Then, catalase activity was found to be inhibited until day 10. In the uterus and ovary, catalase activity was always found to be inhibited. In IFNi rats, no significant increase of catalase activity was observed as compared to IF rats. Vitamin E was inhibited from the 1st to the 10th day in Ni rats, whereas no significant changes were noted in IFNi rats. A moderate decrease of vitamin A was only found at days 1 and 3 in Ni rats. In conclusion, intermittent fasting is able to protect from oxidative stress induced by low concentration of Ni, but catalase and Vitamins E and A do not seem to be involved.

Inhibitory effects of 1alpha, 25dihydroxyvitamin D3 and Ajuga iva extract on oxidative stress, toxicity and hypo-fertility in diabetic rat testes.

The aim of the current study is to investigate the therapeutic and preventive effects of 1alpha, 25dihydroxyvitaminD3 (1,25 (OH)2 D3) and Afuga iva (AI) extract on diabetes toxicity in rats testes. Thus diabetic rats were treated with 1alpha, 25dihydroxyvitaminD3 or Ajuga iva extract as both therapeutic and preventive treatments on diabetes toxicity in rats testes. Our results showed that diabetes induced a decrease in testosterone and 17beta-estradiol levels in testes and plasma. Besides, a fall in testicular antioxidant capacity appeared by a decrease in both antioxidant (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities) and nonenzymatic antioxidant (copper (Cu), magnesium (Mg) and iron (Fe) levels). All theses changes enhanced testicular toxicity (increase in testicular aspartate amino transaminase (AST), alanine amino transaminase (ALT), lactate dehydrogenase (LDH) activities and the lipid peroxidation and triglyceride (TG) levels). In addition, a decrease in testicular total cholesterol (TCh) level was observed in diabetic rats testes. All the changes lead to a decrease in the total number and mobility of epididymal spermatozoa. The administration of 1alpha,25dihydroxyvitaminD3 and Ajuga iva extract three weeks before and after diabetes induction interfered and prevented diabetes toxicity in the reproductive system. 1,25 (OH)2 D3 and Ajuga iva extract blunted all changes observed in diabetic rats. To sum up, the data suggested that 1,25 (OH)2 D3 and Ajuga iva extract have a protective effect on alloxan-induced damage in reproductive system by enhancing the testosterone and 17beta-estradiol levels, consequently protecting from oxidative stress, cellular toxicity and maintaining the number and motility of spermatozoids.

Estrogens and male reproduction: a new concept.

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them estrogens are the end products obtained from the irreversible transformation of androgens by a microsomal enzymatic complex named aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rats, the aromatase activity is mainly located in Sertoli cells of immature rats and then in Leydig cells of adult rats. We have demonstrated that germ cells represent an important source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes compared to gonocytes or round spermatids; conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank voles, bears, and monkeys express aromatase. In humans, we have shown the presence of a biologically active aromatase and of estrogen receptors (alpha and ss) in ejaculated spermatozoa and in immature germ cells in addition to Leydig cells. Moreover, we have demonstrated that the amount of P450arom transcripts is 30% lower in immotile than in motile spermatozoa. Alterations of spermatogenesis in terms of number and motility of spermatozoa have been described in men genetically deficient in aromatase. These last observations, together with our data showing a significant decrease of aromatase in immotile spermatozoa, suggest that aromatase could be involved in the acquisition of sperm motility. Thus, taking into account the widespread localization of aromatase and estrogen receptors in testicular cells, it is obvious that, besides gonadotrophins and androgens, estrogens produced locally should be considered to be physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis.

Estrogens and male reproduction: a new concept

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them estrogens are the end products obtained from the irreversible transformation of androgens by a microsomal enzymatic complex named aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rats, the aromatase activity is mainly located in Sertoli cells of immature rats and then in Leydig cells of adult rats. We have demonstrated that germ cells represent an important source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes compared to gonocytes or round spermatids; conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank voles, bears, and monkeys express aromatase. In humans, we have shown the presence of a biologically active aromatase and of estrogen receptors (alpha and ß) in ejaculated spermatozoa and in immature germ cells in addition to Leydig cells. Moreover, we have demonstrated that the amount of P450arom transcripts is 30 percent lower in immotile than in motile spermatozoa. Alterations of spermatogenesis in terms of number and motility of spermatozoa have been described in men genetically deficient in aromatase. These last observations, together with our data showing a significant decrease of aromatase in immotile spermatozoa, suggest that aromatase could be involved in the acquisition of sperm motility. Thus, taking into account the widespread localization of aromatase and estrogen receptors in testicular cells, it is obvious that, besides gonadotrophins and androgens, estrogens produced locally should be considered to be physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis.

RNA dynamics of fertile and infertile spermatozoa.

The presence of a complex population of mRNAs in human mature spermatozoa is well documented; among them, transcripts of aromatase and ERs (oestrogen receptors) have been described but their significance is not clear. Therefore, to clarify the role of this complex population of mRNAs in human ejaculated sperm, we have isolated on discontinuous density gradients two main fractions from the same sample: high- and low-motile spermatozoa. The levels of different transcripts coding for molecules involved in nuclear condensation [Prm-1 (protamine 1) and Prm-2], capacitation [eNOS (endothelial nitric oxide synthase), nNOS (neuronal nitric oxide synthase), c-myc], motility and sperm survival (aromatase) have been assessed using semi-quantitative RT (reverse transcriptase)-PCR. The viability of sperm as well as the percentage of apoptosis were identical in high- and low-motile fractions. No significant change in the c-myc/Prm-2 ratio between the two populations of spermatozoa was observed. Conversely the amount of Prm-1 mRNA was significantly higher in low-motile than in high-motile fraction; in most of the high-motile sperm samples analysed, eNOS and nNOS transcripts were undetectable, whereas they were observed in low-motile sperm. Moreover, a partial or complete disappearance of c-myc transcripts was observed after capacitation. As to the aromatase expression, a significant decrease in the amount of transcripts in immotile sperm fraction was recorded in all samples studied. To conclude, analysing mRNA profiles in humans could be helpful either as a diagnostic tool to evaluate male fertility, since they reflect spermatogenesis gene expression, and/or a prognosis value for fertilization, since these RNAs are delivered to oocytes.

Protective effect of 17beta-estradiol on oxidative stress and liver dysfunction in aged male rats.

In aging liver oxidative stress increases due to the decrease in antioxidant bio-molecules such as estrogens which can be modified by hormonal replacement therapy (HRT). With this in mind, we hypothesized that age-related decline in steroidogenesis may be associated with the impairment of the antioxidant defense cells in liver, the increase in lipid peroxidation, hepatic dysfunction and histological changes; estrogens prevent all these changes induced by aging. 17beta-estradiol treatment was initiated in 12 month-old Wistar rats, and continued until 18 months of age. Our results showed that 17beta-estradiol (E2) level in the serum of the aged untreated rats was reduced by -32% in 18 month-old rats compared to the young animals (4-month-old). The superoxide dismutase (SOD), catalase (CAT), and gluthatione peroxidase (GPX) activities were reduced by -47, -46, and -29% respectively in old rat liver. In addition, the TBARs in liver and hepatic dysfunction parameters in plasma such as gamma-glutamyl transferase (GGT), phosphatase alkalin (PAL) as well as bilirubin level increased significantly in old rats, and histological changes were investigated. In E2-treated rats, protective effects were observed. Indeed, 17beta-estradiol attenuates all changes induced by aging. The 17beta-estradiol level was higher in old E2-treated rats compared to the control rats. Moreover, the SOD, CAT and GPX activities were higher by +28, +15, and +11% respectively. This anti-aging effect of estrogens was clarified by a lower level of lipid peroxidation and liver dysfunction parameters as well as by histological observation.

Estrogen-induced growth inhibition of human seminoma cells expressing estrogen receptor beta and aromatase.

It is now well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. Studying a human testicular seminoma cell line, JKT-1, we show here that 17beta-estradiol (10(-12) to 10(-6) M) induced in vitro a significant dose-dependent decrease of cell growth. This antiproliferative effect was maximum after 4 days of exposure at a physiologically intratesticular concentration of 10(-9) M, close to the K(d) of ER, and reversed by ICI 182780, an ER antagonist, suggesting an ER-mediated pathway. By RT-PCR and Western blot we were able to confirm that JKT-1, like tumoral seminoma cells and normal human testicular basal germ cells, expresses estrogen receptor beta (ERbeta), including ERbeta1 and ERbeta2, a dominant negative variant, but not ERalpha. Using immunofluorescence and confocal microscopy, ERbeta was observed as perinuclear intracytoplasmic spots in JKT-1 and tumoral seminoma cells without significant translocation of ERbeta into the nucleus, under 17beta-estradiol exposure. Double staining observed by confocal microscopy revealed that ERbeta colocalized in JKT-1 cells with cytochrome C, a mitochondrial marker. We report for the first time the expression of a functional aromatase complex in seminoma cells as assessed by RT-PCR, Western blot and enzymatic assay. Seminoma cells are able to respond to estrogens through a possible autocrine or paracrine loop. These preliminary results support estrogen-dependency of human testicular seminoma, the most frequent tumor of young men, and suggest potential pharmacological use. Whether this estrogen control, however, involves an ERbeta-mediated stimulation of cell apoptosis and/or an ERbeta-mediated inhibition of cell proliferation, remains to be further determined.

Aromatase in testis: expression and role in male reproduction.

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids, among them estrogens are the end products obtained from the irreversible transformation of androgens by aromatase (P450arom). In the rat the pattern of P450arom expression differs among the testicular somatic cell types according to age; in addition, we have shown that gonocytes, spermatogonia, spermatocytes (preleptotene, pachytene), spermatids and spermatozoa, represent an important source of estrogens; the expression of aromatase is three-fold higher in pachytene spermatocyte (PS) compared to gonocytes. In man both Leydig cells and immature germ cells (PS and round spermatids, RS) as well as ejaculated spermatozoa expressed a biologically active aromatase revealed as a single band of 49 kDa on western blots. Up today P450arom has been demonstrated in male germ cells of all mammals so far studied (mice, bank vole, bear and monkey). The aromatase gene is highly conserved and is unique in humans; its expression is regulated in a cell-specific manner via the alternative use of various promoters located in the first exon. Nevertheless, data concerning the regulation of P450arom especially in germ cells are scarce. We have demonstrated that TGFbeta inhibits the expression of Cyp19 in PS and RS via the SMAD pathway although TNFalpha exerts a stimulatory role in PS, which is amplified in presence of dexamethasone. It is noteworthy that dexamethasone alone exerts a positive effect on Cyp19 expression in PS and a negative one in RS. Cyclic AMP is also a positive regulator of P450arom gene expression in germ cells. In addition, we have shown that androgens and estrogens modulate Cyp19 gene expression, whatever the testicular cell type studied, which favored the presence of androgens and estrogens responsive elements on the Cyp19 gene promoter(s). Moreover, in presence of seminiferous tubules conditioned media, the amount of aromatase transcripts is increased in Leydig cells, therefore, suggesting that other locally produced modulators are involved in the regulation of the aromatase gene expression and among them the liver receptor homolog-1 (LRH-1) from germ cells origin is concerned. Using RACE-PCR we have confirmed that promoter II directs the expression of aromatase gene, whatever the testicular cell type studied in the rat but the involvement of another promoter, such as PI.4 is suggested. Finally, the aromatase gene is constitutively expressed both in somatic and germ cells of the testis and the identification of the promoter(s) concerned as well as their detailed regions which direct(s) the expression of Cyp19 gene is obviously very important but largely unknown especially according to the ontogeny of the male gonad.

Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors: relationship to sperm motility and capacitation.

The existence of a complex population of mRNA in human sperm is well documented but their role is not yet elucidated. Using discontinuous density gradients, we have isolated high and low motile sperm from the same semen sample. The levels of different transcripts coding for molecules either involved in nuclear condensation (protamines 1 and 2) or in capacitation [endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and c-myc] were then assessed in the two populations using semi-quantitative RT-PCR. Sperm viability was estimated by eosin-nigrosin staining and by hypo-osmotic swelling test; apoptosis percentage was measured by the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling technique. The contamination by somatic and germ cells was assessed by looking for specific molecular markers of these cells, respectively CD-45 and E-cadherin for somatic cells and c-kit for germ cells. The viability of sperm was unchanged in high and low motile fractions, as well as DNA fragmentation percentage. The amount of Prm-1 mRNA was significantly higher in low density motile than in the high motile fraction. In most of high motile sperm samples eNOS and nNOS transcripts were undetectable whereas they were present in the low motile sperm. In contrast, no significant variation was found in the c-myc/Prm-2 mRNA ratio between the two populations. Moreover, a partial or complete disappearance of c-myc transcripts was observed after capacitation. Thus analysing mRNA profiles could be helpful as a diagnostic tool and prognosis value for fertilization.

Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors.

It is now well established that oestrogens play a part in germ cell function. These hormones are synthesised by the cytochrome P450 aromatase (P450 arom) and act via two kinds of receptor (ERalpha and ERbeta). Although the presence of aromatase and oestrogen receptors in mammalian testis is now well documented, the localisation of these proteins in human germ cells is not yet clear. The primary purpose of the current study was to look for the expression of aromatase and oestrogen receptors in human germ cells. Human immature germ cells were collected from semen samples with an excess of rounds cells (>20%) and purified spermatozoa were obtained after sedimentation on a discontinuous PureSperm gradient. Expression of aromatase and oestrogen receptors was determined by RT-PCR with specific primers, and by Western blot using monoclonal antibodies. RT-PCR products for aromatase, ERalpha and ERbeta were amplified from total RNA isolated from human germ cells and spermatozoa. We identified an ERalpha isoform variant that lacks exon 4 in human germ cells and visualised P450 arom as a single band of 49 kDa in germ cells, as we have already reported for human ejaculated spermatozoa. By Western blot, we identified two proteins for ERbeta at approximately 50 and approximately 60 kDa, which could correspond to the long and short forms of ERbeta formed from the use of alternative start sites. In human ejaculated spermatozoa, ERbeta protein was not detected, even though we could amplify mRNA. Using Western blot analysis and a monoclonal antibody specific for ERalpha, we detected two proteins in human immature germ cells: one of the expected size (66 kDa) and a second one of 46 kDa. In mature spermatozoa, only the 46 kDa band was observed and we speculate it may be related to the ERalpha isoform lacking exon I. In conclusion, we have identified P450 arom and ER proteins (full-length and variant) in human germ cells. Further studies are now required to elucidate the mechanism of action of oestrogens on human male germ cells, in terms of both genomic and 'non-genomic' pathways.