In the last decade, the study of human sperm anatomy, at molecular level, has revealed the presence of several nuclear protein receptors. In this work, we examined the expression profile and the ultrastructural localization of liver receptor homolog-1 (LRH-1) in human spermatozoa. We evidenced the presence of the receptor by Western blotting and real time-RT-PCR. Furthermore, we used immunogold electron microscopy to investigate the sperm anatomical regions containing LRH-1. The receptor was mainly located in the sperm head, whereas its expression was reduced in the neck and across the tail. Interestingly, we observed the presence of LRH-1 in different stages of testicular germ cell development by immunohistochemistry. In somatic cells, it has been suggested that the LRH-1 pathway is tightly linked with estrogen signaling and the important role of estradiol has been widely studied in sperm cells. To assess the significance of LRH-1 in male gametes and to deepen understanding of the role of estrogens in these cells, we investigated important sperm features such as motility, survival and capacitation. Spermatozoa were treated with 10nm estradiol and the inhibition of LRH-1 reversed the estradiol stimulatory action. From our data, we discovered that human spermatozoa can be considered a new site of expression for LRH-1, evidencing its role in sperm motility, survival and cholesterol efflux. Furthermore, we may presume that in spermatozoa the LRH-1 effects are closely integrated with the estrogen signaling, supporting LRH-1 as a downstream effector of the estradiol pathway on some sperm functions.

The most common cause of male infertility is the testicular varicocele, a condition that impairs production and decreases quality of sperm. Male fertility also strictly depends on androgens acting through their own receptor. The enzyme 5α-reductase (SRD5A) is involved in the conversion of testosterone to 5α-dihydrotestosterone, both required for the development and maintenance of male reproductive function. Here, we evaluated, by western blotting analysis, the presence of SRD5A in human ejaculated spermatozoa and evidenced differences in sperm SRD5A content between healthy donors and varicocele-affected patients. Additionally, SRD5A sperm ultrastructural localization was also assessed by transmission electron microscopy and immunogold assay. We evidenced that SRD5A enzyme is present in the human spermatozoa and that its cellular content is lowered in sperm samples from varicocele patients compared to healthy subjects. The presence of SRD5A in human ejaculated spermatozoa highlights the potential role of this enzyme in sperm physiopathology suggesting that the decrease in its content, by affecting the conversion of testosterone into 5α-dihydrotestosterone, may be an important additional mechanism involved in the harmful effect of varicocele in male fertility.

The mechanisms through which sperm manage their energy metabolism are poorly understood. The present study provides biochemical and morphological evidence that mitochondrial citrate carrier (CIC) is present in ejaculated human sperm and is restricted to the midpiece. The inhibition of CIC with the specific substrate analog 1,2,3-benzenetricarboxylate resulted in the reduction of cholesterol efflux, protein tyrosine phosphorylation, phospho-AKT, phospho-p60src, hyperactivated motility and acrosome reaction, suggesting a role for this mitochondrial carrier in sperm physiology. Furthermore, inhibition of CIC by 1,2,3-benzenetricarboxylate resulted in a reduction of glucose-stimulated insulin secretion and autocrine insulin secretion by sperm. Remarkably, blocking CIC also reduced glucose-6-phosphate dehydrogenase activity, probably in accordance with its regulation on insulin secretion. Capacitation and glucose metabolism were stimulated by glucose as well as citrate, the specific substrate of CIC, implying a similar action because glucose and citrate both induced insulin secretion by sperm. In the present finding, we discovered a new site of action for CIC in the regulation of metabolism, and it may be assumed that CIC works with other factors in the regulation of sperm energy metabolism to sustain capacitation process and acrosome reaction.

We recently demonstrated that adrenocortical carcinoma cells express aromatase and estrogen receptors (ERs) and that 17β-estradiol enhances adrenocortical cell proliferation. To provide a clue to the role of estrogens in adrenal tumorigenesis, we investigated the expression profile of genes involved in sex steroid hormone production and activity in a large series of normal and neoplastic human adrenocortical tissues. Quantitative reverse transcriptase–polymerase chain reaction, Western blotting, and immunohistochemistry showed that ERα and ERβ, androgen receptor (AR), and aromatase were expressed in the adrenal cortex and in adrenocortical tumors. ERβ was the predominant ER subtype and was mainly expressed in the zona glomerulosa and fasciculata. Western blot analysis revealed the presence of a truncated form of AR in adrenocortical tissues. With respect to the normal adrenal cortex and adrenocortical adenomas, carcinomas were characterized by significantly lower ERβ levels, ERα upregulation, and aromatase overexpression. ER expression correlated with expression of nuclear hormone receptors, suggesting they could be involved in ER modulation. In agreement with our in vitro findings, the results of this study suggest that estrogens, locally produced by aromatase, could enhance adrenocortical cell proliferation though autocrine/paracrine mechanisms. This study opens new perspectives on the potential use of antiestrogens and aromatase inhibitors as therapeutic agents against ACC.

The newly synthesized 7-phenyl-3H-pyrrolo[3,2-f]quinolinones 16-26 and previously 27 and 28 were assayed for their in vitro antiproliferative activity on tumor cell lines, and the lead compound 16 in vivo on a singenic hepatocellular carcinoma in Balb/c mice. Results from FACS, immunofluorescence microscopy analysis, tubulin polymerization assay, and tritiated water release assay for the CYP19 activity confirmed the new compounds as potential anticancer agents acting by tubulin depolymerization, but devoid of aromatase activity unlike their geometric [2,3-h] isomers.

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.

The expression of estrogen receptor alpha (ERα) is generally associated with a less invasive and aggressive phenotype in breast carcinoma. In an attempt to understand the role of ERα in regulating breast cancer cells invasiveness, we have demonstrated that cell adhesion on fibronectin (Fn) and type IV Collagen (Col) induces ERα-mediated transcription and reduces cell migration in MCF-7 and in MDA-MB-231 cell lines expressing ERα. Analysis of deleted mutants of ERα indicates that the transcriptional activation function (AF)-1 is required for ERα-mediated transcription as well as for the inhibition of cell migration induced by cell adhesion on extracellular matrix (ECM) proteins. In addition, the nuclear localization signal region and some serine residues in the AF-1 of the ERα are both required for the regulation of cell invasiveness as we have observed in HeLa cells. It is worth noting that c-Src activation is coincident with adhesion of cells to ECM proteins and that the inhibition of c-Src activity by PP2 or the expression of a dominant-negative c-Src abolishes ERα-mediated transcription and partially reverts the inhibition of cell invasiveness in ERα-positive cancer cells. These findings address the integrated role of ECM proteins and ERα in influencing breast cancer cell motility through a mechanism that involves c-Src and seems not to be related to a specific cell type.