Nitric oxide involvement in the acrosome reaction triggered by leptin in pig sperm

<p>Abstract</p> <p>Background</p> <p>Nitric oxide (NO) is a signaling molecule produced by intracellular nitric oxide synthase (NOS) enzymes. This free radical appears to affect sperm capacitation, a maturation step preceding acrosome reaction. Recent studies have reported leptin ability to promote capacitation and acrosome reaction in pig male gametes.</p> <p>Methods</p> <p>This study has investigated nitric oxide production in leptin-treated pig spermatozoa by fluorescence-activated cell sorting, while the intracellular NOS isoforms were assessed by Western blot analysis. In addition, acrosome status of treated-spermatozoa was evaluated by FITC-PNA staining.</p> <p>Results</p> <p>Significant increases of nitric oxide levels and acrosome reaction extent were detected in leptin-treated spermatozoa, but both the effects were reversed in presence of <it>L</it>-NAME. Furthermore, the immunoblots of sperm extracts have evidenced three bands of ~160 Kd(bNOS), ~130 Kd (iNOS) and ~135 Kd (eNOS).</p> <p>Conclusions</p> <p>The identification of the three intracellular NOS isoforms suggests that pig spermatozoa could produce NO, while the augmented nitric oxide levels in leptin-treated male gametes indicates the capacity of the hormone to induce nitric oxide production. Furthermore, the inhibitory effect of <it>L</it>-NAME and of Ab-ObR on the promotion of acrosome reaction triggered by leptin suggests a possible involvement of NO in the hormone action.</p

Detection of estrogen receptors ER-alpha and ER-beta in human ejaculated immature spermatozoa with excess residual cytoplasm

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

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

Macrophage Autophagy and Oxidative Stress: An Ultrastructural and Immunoelectron Microscopical Study

The word autophagy broadly refers to the cellular catabolic processes that lead to the removal of damaged cytosolic proteins or cell organelles through lysosomes. Although autophagy is often observed during programmed cell death, it may also serve as a cell survival mechanism. Accumulation of reactive oxygen species within tissues and cells induces various defense mechanisms or programmed cell death. It has been shown that, besides inducing apoptosis, oxidative stress can also induce autophagy. To date, however, the regulation of autophagy in response to oxidative stress remains largely elusive and poorly understood. Therefore, the present study was designed to examine the ratio between oxidative stress and autophagy in macrophages after oxidant exposure (AAPH) and to investigate the ultrastructural localization of beclin-1, a protein essential for autophagy, under basal and stressful conditions. Our data provide evidence that oxidative stress induces autophagy in macrophages. We demonstrate, for the first time by immunoelectron microscopy, the subcellular localization of beclin-1 in autophagic cells

Cytochrome P450 aromatase expression in human seminoma

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 reduced content of estrogen and progesterone receptors in varicocele sperm may be indicative of the clinical surgery in the testicular varicocele

The enigma of testicular varicocele has always attracted the researcher’s attention as attested by the consistent literature, although conflicting data are reported (1). The detrimental role of varicocele in fertility is supported by the presence of an higher frequency of affected men in the infertile population (2). Varicocele influences male fertility in a variety of ways: spermatogenesis, semen quality and gamete biology. However, the mechanism/s by which the pathology impairs sperm production and activity, are not known yet. In spite of active interest, our knowledge about sperm molecular anatomy is very limited. Instead, it is important to fully elucidate the molecular sperm architecture, in order to clarify clinical cases of idiopathic infertility since not all the apparently normal sperm are able to fertilize. The presence of steroid/steroid receptor systems was demonstrated in human sperm, suggesting that both systemic and local steroids through sperm receptors, may influence male fertility. From our data, it emerged that varicocele causes a damage in the gamete at molecular level which includes a significant reduction of estrogen and progesterone receptors, opening a new chapter in the already multifaceted physiopathology of the disease. By the time of ovulation, estradiol and progesterone are almost everywhere in the egg microenvironment affecting ability of sperm to fertilize the oocyte. Therefore, the reduced responsiveness to these hormones as we observed in varicocele sperm, impedes their goal. Altogether, these studies constrain the need of further researches on the molecular anatomy of human male gamete both in healthy and in pathological conditions related 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 the reduction of steroid receptors in sperm implies a decline in the acquisition of fertilizing ability

Fibronectin and type IV collagen activate ERα AF-1 by c-Src pathway: effect on breast cancer cell motility

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Leptin and leptin receptor in pig spermatozoa: evidence of their involvement in sperm capacitation and survival

Several studies have recently investigated the role of leptin, the adipocyte-secreted hormone, in the growth and reproduction of rodents, humans, and domestic animals. The present study was designed to explore the expression of leptin and its receptor in pig spermatozoa. Successful Western blot evidenced a 16 kDa band for leptin and six isoforms, ranging from 120 to 40 kDa, for the leptin receptor. Both leptin and leptin receptor were interestingly located at sperm acrosomal level, suggesting their involvement in the oocyte fertilization events. In fact, both capacitation indexes and acrosin activity were enhanced by leptin, and these effects were reduced by the anti-leptin receptor antibody. Afterwards, we investigated the main transduction pathways regulated by the hormone. Our results showed that, in pig sperm, leptin can trigger the signal transducer and activator of transcription 3, a classical component of cytokine signal transduction pathways, whose expression has not been previously reported in male gamete; in addition it was found constitutively activated. Besides, leptin was able to induce the activation of phosphatidylinositol phosphate kinase 3 and MAP kinase pathways as well as of BCL2, a known antiapoptotic protein. These data address to a role of leptin and its receptor on pig sperm survival. The presence of leptin and its receptor in pig sperm suggests that they, through an autocrine short loop, may induce signal transduction and molecular changes associated with sperm capacitation and survival

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