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Leydig cell tumors (LCTs) of the testis are steroid-secreting tumors associated with various steroid biosynthetic abnormalities and endocrine dysfunctions. Despite their overall rarity, LCTs are still of substantial interest owing to the paucity of information regarding their exact nature and malignant potential. In the present study, we disclose the ability of androgens to inhibit Leydig tumor cell proliferation by opposing to self-sufficient in situ estrogen production. In rat Leydig tumor cells, R2C, androgen treatment significantly decreases the expression and the enzymatic activity of cytocrome P450 aromatase, responsible for the local conversion of androgens into estrogens. This inhibitory effect relies on androgen receptor (AR) activation and involves negative regulation of the CYP19 gene transcriptional activity through the nuclear orphan receptor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1). Ligand-activated AR up-regulates the expression of DAX-1 and promotes its increased recruitment within the steroidogenic factor-1 site-containing region of the aromatase proximal promoter II in association with the nuclear receptor corepressor. The biological relevance in LCTs of the newly highlighted functional interplay between AR, DAX-1, and aromatase is underlined by our in vivo observations, revealing a marked down-regulation of AR and DAX-1 expression and a strong increase in aromatase levels in testes tissues from old Fischer rats with spontaneously developed Leydig cell neoplasia, compared with normal testes tissues from younger animals. In elucidating a mechanism by which androgens modulate the growth of Leydig tumor cells, our finding support the hypothesis that maintaining the adequate balance between androgen and estrogens may represent the key for blocking estrogen-secreting Leydigioma development, opening new prospects for therapeutic intervention.

Background: Androgens and estrogens are crucial for mammalian sperm differentiation but their role in biology of mature male gamete is not still defined. The expression of proteins involved in the biosynthesis and action of these steroid hormones has been demonstrated in human spermatozoa, but very few data have been reported in mature sperm from non human species. The purpose of the current study was to investigate the expression of aromatase (P450arom), estrogen (ERalpha/ERbeta) and androgen (AR) receptors in ejaculated spermatozoa of pig., Methods: The immunfluorescence experiments were carried out treating pig sperm with anti-P450arom, anti-ERalpha, anti-ERbeta and anti-AR as primary antibodies, while Texas-Red/FITC conjugated IgG were applied as secondary antibodies. Furthermore, Western blot analysis was performed on sperm lysates., Results: Aromatase was immunolocalized in the sperm tail, ERalpha and AR were localised in the sperm midpiece, while ERbeta was confined in the acrosomal region of the male gamete. Immunoblots detected a ~52 kDa aromatase band, a ~110 kDa AR band, a ~67 kDa ERalpha and two ERbeta bands, at ~50 kDa and ~59 kDa., Conclusion: This is the first report demonstrating that pig ejaculated spermatozoa express aromatase, estrogen and androgen receptors with a differential intra-cellular localization revealing a species-specific expression pattern. Therefore, pig sperm could be considered as a potential estrogen source while the different hormone cellular sites suggest distinct roles of androgens and estrogens in pig sperm physiology.

Background: The enzyme cytochrome P450 aromatase, catalysing the conversion of androgens into estrogens, has been detected in normal human testicular cells suggesting a physiological role of local estrogen biosynthesis on spermatogenesis control. Estrogens, regulating cell growth and apoptosis, can also be involved in tumorigenesis process, but the possible link between estrogens and testicular neoplastic process is, up to now, scarcely known. This study examined aromatase expression in human seminoma, which is the most common germ cell tumour of the testis., Methods: The tumour-bearing testes were obtained from 20 patients with classic seminoma undergoing to therapeutic orchidectomy. Paraffin embedded tissues were processed for immunohistochemistry using a mouse monoclonal antibody generated against human placental cytochrome P450 arom, as primary antibody, and a biotinylated goat-anti-mouse IgG, as secondary antibody. Furthermore, Western blot analysis of seminoma extracts was carried out., Results: Intense P450 arom immunoreactivity was observed in the seminoma cells and Western blot analysis confirmed the immunodetection. A strong immunostaining was also detected in cells of intratubular germ cell neoplasia (IGCN), adjacent to seminoma., Conclusion: The present study demonstrated, for the first time in human, aromatase expression in neoplastic cells of seminoma suggesting a relation between local estrogen biosynthesis and germ cell tumorigenesis. The P450 arom immunolocalization in the cells of IGCN, representing the common precursor of most germ cell tumors, seems to support these findings.

Background: Advances in the definition of the function and the mechanism of estrogen action in different tissues have come from human and animal models of estrogen insufficiency. Recently we have demonstrated that aromatase is present and biologically active in human ejaculated sperm, suggesting that autonomous estradiol sperm production may influence sperm functions. In the present study we investigate a possible physiological role for enzymatically active P450 aromatase in human ejaculated sperm., Methods and Results: To confirm the presence of mRNA coding for P450 aromatase, total RNA isolated from human sperm underwent RT-PCR and then Southern blot analysis. In non-capacitating medium, we observed that only estradiol and aromatizable steroids were able to increase sperm motility/migration; concomitantly they enhanced protein tyrosine phosphorylation and increased p-44/42 extracellular signal-regulated kinase activity. When we tested acrosin activity, it emerged that estradiol and aromatizable androgens were also able to induce the acrosome reaction evaluated by two different cytological staining techniques (triple-stain and fluorescein isothiocyanate-Pisum sativum agglutinin). All these events were enhanced by the 2'-O-dibutyryladenosine-3',5'-cyclic monophosphate and inhibited in the presence of the specific aromatase inhibitor, letrozole., Conclusions: From this study, it appears that a link exists between the locally produced estradiol (from ejaculated sperm), sperm capacitation and the acrosome reaction. The induction of both events by aromatizable androgens in the absence of exogenous mediators suggests that estrogen biosynthesis in ejaculated sperm is a process that may influence the intrinsic sperm fertilizing capability.

The generation of cytochrome P450 aromatase (P450arom) and estrogen receptor (ER) knockout mice has raised new interest in the physiological role of estrogens in male reproduction. Testicular expression of P450arom, the enzyme that converts androgens into estrogens, has been shown in both somatic and germ cell types in several species, whereas in humans, testicular expression is confined to the somatic cells. The aim of this study was to determine whether P450arom is present in human ejaculated spermatozoa. Using RT-PCR and specific primers, we amplified the highly conserved helical, aromatic, and heme-binding sequences of the conventional human P450arom from RNA isolated from human spermatozoa. Employing a rabbit polyclonal antiserum directed against human placental P450arom, immunoblotting analysis demonstrated aromatase protein expression, which was localized primarily to the tail and midpiece of spermatozoa. Measurement of enzymatic activity using a sensitive (3)H(2)O aromatase assay revealed that activity was enhanced by the 2'-O-dibutyryl cAMP and completely inhibited in the presence of the specific aromatase inhibitor, letrozole. These results represent the first demonstration that human spermatozoa are a potential site of estrogen biosynthesis. The physiological relevance of estrogen synthesis in spermatozoa remains to be elucidated and opens a new area of investigation in male fertility.

The mechanisms by which varicocele affects fertility remain undetermined. Estrogens play a key role in the human male reproduction and human sperm expresses the estrogen receptors (ERs) and aromatase. In this study, by Western blotting we evidenced the ERs content concomitantly in healthy sperm and in oligoastenoteratozoospermic (OAT) samples without and with varicocele. In varicocele a strong reduction of the ERβ was observed, while the ERα was almost absent. Besides, transmission electron microscopy (TEM) confirmed the reduction of ERs expression in 'varicocele' sperm, indicating that varicocele has a detrimental effect on sperm structure at molecular level. To further define the estrogen significance in male gamete and the pathophysiology of varicocele we investigated both the expression of ERα and ERβ in normal and pathologic sperm samples as well as we evaluated estradiol (E2) action on lipid and glucose sperm metabolism. Responses to E2 treatments on cholesterol efflux, protein tyrosine phosphorylations, motility, and acrosin activity in varicocele sperm were reduced or absent. The evaluation of the triglycerides content, lipase and acyl-CoA dehydrogenase activities, suggest that E2 exerts a lipolytic effect on human sperm metabolism. Concerning glucose metabolism, it appears that E2 induces G6PDH activity concomitantly to the insulin secretion. In 'varicocele' sperm, the E2 did not induce energy expenditure. OAT sperm had E2-responsiveness but in a lesser extent with respect healthy sperm. This study discovered a novel role for E2/ERs in human sperm physiology, since they modulate sperm metabolism and new detrimental effects related to the pathophysiology of the varicocele condition. J. Cell. Physiol. 226: 3403-3412, 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Background: A key role of estrogens in human sperm biology has been recently suggested by aromatase and estrogen receptor detection in human testicular germ cells and ejaculated spermatozoa. However, the involvement of these hormones in the sperm maturation process is still not defined. The aim of this work was to investigate the expression of estrogen receptors, ER-alpha and ER-beta, in human ejaculated immature spermatozoa with excess residual cytoplasm. Methods: Immunofluorescence labelling has been used to localize ER-alpha and ER-beta proteins in immature spermatozoa isolated by Percoll gradient, while Western blot analysis was carried out on sperm protein extracts. Results: Both estrogen receptors were localized in excess residual cytoplasm of immature sperm, while sperm tails showed only ER-beta. Furthermore, in the same cells, immunoblots detected the presence of the full-length (~67 kDa) ER-alpha and (~59 kDa) ER-beta proteins, together with a ~50 kDa ER-beta species, lacking in mature sperm. Conclusion: The present investigation demonstrated ER-alpha and ER-beta presence in excess residual cytoplasm of human abnormal sperm cells, suggesting the hypothesis that both the 'classical' ERs could be able to mediate estrogen action in spermatogenetic cells. Furthermore, the presence of the short ER-beta form in abnormal germ cells and its disappearance in mature sperm, support estrogen modulation via different ER forms during sperm maturation. [ABSTRACT FROM AUTHOR]