Cytochrome P450arom, androgen and estrogen receptors in pig sperm

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>This is the first report demonstrating that pig ejaculated spermatozoa express aromatase, estrogen and androgen receptors with a differential intra-cellular localization revealing a specie-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.</p

Day case parathyroidectomy: is this the right way for the patients?

Minimally-invasive video-assisted parathyroidectomy (MIVAP) can be considered as the primary treatment of choice for single parathyroid adenoma. Often, this technique is performed in a day surgery setting and is associated with regional anaesthesia (RA). Many studies have already reported the feasibility and safety of MIVAP in day surgery. Here our focus has been on the patient's personal experience with these procedures through an assessment of their recovery at home

Follicle-stimulating hormone receptor (FSHR) a promising novel target for cancer diagnosis in seminoma and embryonal carcinoma

Adult testicular germ cell tumors (TGCTs) are the most frequent malignant tumors in male patients aged 15–45 years, their incidence is increasing in recent years. There are two main subclasses of TGCTs: seminomas (SE) and non-seminomatous germ cell tumors (NSGCTs). SE have histological features of primordial germ cells, whereas NSGCTs have varying degrees of differentiation (i.e. embryonal carcinoma, EC), they present distinctive clinical features and differ for therapy and prognosis. NSGCTs tend to be metastatic at presentation, and have a worse prognosis than seminomas at an equivalent stage of disease. Despite general advances in the management of TGCTs, the molecular bases underlying their progression remain almost unknown. The effects of the Follicle-stimulating hormone (FSH), central hormone in mammalian reproductive biology, are mediated by FSHR, which was believed to be expressed primarily in ovary and testis. Recently, FSHR expression has been shown in the blood vessels of different solid tumors, including prostate, urothelial and breast carcinomas, suggesting a role in neoangiogenesis. The expression of FSHR at the periphery of tumors, also suggests that FSHR may be of relevance to the metastatic process. In normal human testis, estrogen physiological actions are mediated by estrogen receptor (ER) β and highly variable ERβ expression has been reported in the different TGCTs. ERβ loss is associated with advanced tumor stage in several cancers and previously, we showed a higher expression of ERβ1 in SE with respect EC. In this study, we evaluated the expression of FSHR in normal and neoplastic human testis tissues. Further, we compared FSHR expression with that of ERβ1 in the same samples. In normal testes, immunohistochemical studies showed the presence of FSHR prevalently in somatic testicular cells, while ERβ1 is expressed both in somatic and germinal testicular cells. Intriguingly, we discovered that FSHR was strongly expressed in EC and absent in SE. Conversely, immunostaining for ERβ1 revealed higher intensity in SE as compared to EC. These data suggest distinct physiopathological roles for the two receptors in TGCTs progression, being ERβ1 protective and FSHR harmful. Our data report for the first time the expression of FSHR in TGCTs, suggesting its possible involvement in testicular carcinogenesis. FSHR may be considered an useful molecular marker to distinguish seminoma from embryonal carcinoma, the most common TGCTs subtypes, and this could be informative in clinical decision making and patient counseling

The follicle-stimulating hormone receptor (FSHR) is expressed in human sperm and it may be considered as molecular marker of the detrimental effects related to the physiopathology of testicular varicocele

Localization of the follicle-stimulating hormone receptor (FSHR), has been always closely related to the testis and ovary. FSH/FSHR role in Sertoli cell, has been known, however, the sites of FSH action within the male reproductive system are not resolved yet. Few studies have raised the intriguing possibility that germ cells may exhibit FSHR, all the reports point to Sertoli cells as the exclusive FSH target cells in testis. Besides, the attention has been always paid on the FSHR several polymorphisms which affect receptor sensitivity and expression. The presence of FSHR in germinal cells from spermatogonia to spermatocytes, including round spermatids is controversial or excluded. The mechanisms by which testicular varicocele affects fertility remain undetermined. Recently, our studies showed that the disease causes damage in sperm at the molecular level opening a new chapter in the already multifaceted physiopathology of varicocele. Samples used in this study were from normozoospermic and from diagnosed varicocele of grade III on the left testis patients. To date four FSHR isoforms were discovered, FSHR1, FSHR2, FSHR3 and FSHR4. The activity of FSHR1 is mediated by G proteins, which activate adenylate cyclase. FSHR2 and FSHR3 also bind FSH, but this does not result in activation of adenylate cyclase. FSHR4 does not bind FSH. By western blot analysis, we showed that healthy sperm express FSHR1, FSHR2 and FSHR3 while FSHR4 is almost absent. Varicocele does not express FSHR2. Immunofluorescence assay evidences FSHR localization prevalently at the midpiece level, which was strongly reduced in varicocele sperm. Responses to different FSH concentrations on motility and survival were significantly reduced in varicocele respect to the normal sperm, probably due to the lower FSHR1 expression and FSHR2 absence. The FSHR significance in human male gamete also emerged from the acrosome reaction histochemical studies, during FSH treatment which significantly induced the process. Our data showed for the first time that human sperm express the FSHR and constrain the need of further studies on the molecular anatomy of human male gamete both in healthy and in pathological conditions related to the male genital apparatus, considering the high couple infertility linked to the male. The translation of these new researches in the clinic surgery of testicular varicocele needs to be taken into account since molecular alterations in sperm imply a decline in the acquisition of fertilizing ability, and to date controversies exist on the opportunity to intervene surgically

Statins reduce intratumor cholesterol affecting adrenocortical cancer growth

Mitotane causes hypercholesterolemia in ACC patients. We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to ER\u3b1 action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production and ER\u3b1 activity in vitro and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them INSIG, whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ER\u3b1 function. A mitochondrial target of ER\u3b1, the respiratory complex IV (COX IV) was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. In vivo experiments confirmed the ability of simvastatin to reduce tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ER\u3b1, COX IV expression and activity and increase TUNEL positive cells. Collectively these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production, inhibits mitochondrial respiratory chain inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth

Western blotting analysis of AR, P450arom, ERα and ERβ of pig sperm extracts

<p><b>Copyright information:</b></p><p>Taken from "Cytochrome P450arom, androgen and estrogen receptors in pig sperm"</p><p>http://www.rbej.com/content/5/1/23</p><p>Reproductive Biology and Endocrinology 2007;5():23-23.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1894639.</p><p></p> Individual sperm samples: 1–3. LNCaP extracts were used as positive controls for AR and ERβ (C+) while MCF7 extracts were used as positive controls for P450 arom and ERα (C+). No bands in negative controls (C-). β actin served as loading control. Molecular weights (KD) were indicated on the right of the blot

Immunofluorescence labelling of androgen and estrogen receptors in pig spermatozoa: A) AR red fluorescence in sperm proximal mid-piece

<p><b>Copyright information:</b></p><p>Taken from "Cytochrome P450arom, androgen and estrogen receptors in pig sperm"</p><p>http://www.rbej.com/content/5/1/23</p><p>Reproductive Biology and Endocrinology 2007;5():23-23.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1894639.</p><p></p> B) P450arom red brilliant light in the proximal tail of sperm with a diffuse labelling in the distal tail. C) ERα red fluorescence in the sperm mid-piece, together with a faint labelling in the tail. D) ERβ green intense light in the sperm acrosomal region. : immunonegative absorption controls. : 5 μm