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Expression of melatonin (MT1, MT2) and melatonin-related receptors in the adult rat testes and during development

It is well known that melatonin provokes reproductive alterations in response to changes in hours of daylight in seasonally breeding mammals, exerting a regulatory role at different levels of the hypothalamic-pituitary-gonadal axis. Although it has also been demonstrated that melatonin may affect testicular activity in vertebrates, until now, very few data support the hypothesis of a local action of melatonin in the male gonads. The aim of this study was to investigate whether MT1, MT2 melatonin receptors and the H9 melatonin-related receptor, are expressed in the adult rat testes and during development. A semi-quantitative RT-PCR method was used to analyse the expression of MT1, MT2 and H9 receptors mRNAs in several rat tissues, mainly focusing on testes during development and adult life. Our results provide molecular evidences of the presence of both MT1 and, for the first time, MT2 melatonin receptors as well as of the H9 melatonin-related receptor in the examined tissues, including adult testes. During development MT1 and MT2 transcripts are expressed at lower levels in testes of rats from 1 day to 1 week of age, lightly increased at 2 weeks of age and remained permanently expressed throughout development until 6 months. These data strongly support the hypothesis that melatonin acts directly in male vertebrate gonads suggesting that rat testes may be a suitable model to verify the role of indolamine in vertebrate testicular activity.

Recombinant Human FSH Reduces Sperm DNA Fragmentation in Men With Idiopathic Oligoasthenoteratozoospermia

A prospective randomized controlled study was designed to evaluate the effects of recombinant human follicle-stimulating hormone (rFSH) treatment on sperm DNA fragmentation in men with idiopathic oligoasthenoteratozoospermia (iOAT). One hundred twenty-nine men with sperm count less than 10 × 10(6) spermatozoa/mL and forward motility <25% were included; normal serum levels of FSH, luteinizing hormone (LH), and testosterone, and no other causes of infertility were enrolled. The patients were randomized into 2 groups: 65 men were treated on alternate days for 90 days with injections of 150 IU rFSH, and 64 subjects received nonantioxidant vitamin supplements. Main outcome measures were serum levels of FSH, LH, testosterone, and inhibin B and DNA fragmentation index (DFI) at baseline and after 90 days. No significant differences were observed between the 2 groups with regard to sperm parameters and hormone values. The DFI was similar between the 2 groups at the time of the enrollment but reduced significantly (P < .05) after rFSH therapy in study group, whereas no significant variation occurred in the control group. In the subgroup of patients with high basal DFI values (>15%), rFSH treatment significantly increased DFI (P < .01), whereas no significant variation occurred after 90 days of vitamin supplements. We conclude that rFSH administration improves sperm DNA integrity in iOAT men with increased DFI values. The degree of sperm DFI might be useful to identify those iOAT patients in which rFSH treatment can be advantageous.

Two Neuron Clusters in the Stem of Postembryonic Zebrafish Brain Specifically Express relaxin-3 Gene: First Evidence of Nucleus Incertus in Fish

We examined the spatial expression of the relaxin‐3 gene in the developing zebrafish brain, one of the vertebrate model systems in which this gene has been identified. Until the pharyngula stage, the gene is expressed diffusely in the brain, where, starting at about 40 hpf, the transcripts appear restricted in a midbrain cell cluster of the periaqueductal gray. Later, at 72 hpf, the transcripts are still evident in that cluster and distributed in a larger cell number; at this stage, the gene is also expressed posteriorly, in a smaller cell group that, as we report for the first time, could be homologous to mammalian nucleus incertus. The gene expression persists in both cell clusters at 96 hpf. This pattern indicates both conserved and divergent expression features of the relaxin‐3 gene among developing zebrafish and rat brains, where only scattered cells express the gene in the periaqueductal gray. Developmental Dynamics 237:3864–3869, 2008.

Evidence for the involvement of prothymosin α in the spermatogenesis of the frog Rana esculenta

Prothymosin alpha (PTMA) is a small acidic protein abundantly and ubiquitously expressed in mammals and involved in different biological activities. Until now, its specific function in spermatogenesis has never been properly investigated. Recently, the isolation of a cDNA encoding for PTMA from the testis of the frog Rana esculenta has been reported: ptma transcript is highly expressed throughout the frog reproductive cycle, peaking in September/October, in concomitance with the germ cell maturation; it is specifically localized in the cytoplasm of primary and secondary spermatocytes and, at a lower level, in the interstitial compartment, in Leydig cells.In this article we support the involvement of PTMA in the meiotic phases of frog spermatogenesis. The expression of ptma mRNA increases in the testis of frogs treated with the antiandrogen cyproterone acetate, which blocks the II meiotic division and induces an increase in SPC cysts; on the contrary, it highly decreases in the testis of animals kept at 4 degrees C and treated with human corionic gonadotropin, in concomitance with the induced block of spermatogenesis and the disappearance of meiotic cells in the tubules. Furthermore, for the first time we have also evidenced by immunohistochemistry the expression of PTMA in the nuclei of secondary spermatocytes, spermatids, and spermatozoa, as well as in the cytoplasm of interstitial Leydig cells. Taken together our data suggest for an important role of PTMA in germ cell maturation and/or differentiation during R. esculenta spermatogenesis.

Inhibition of the increased 17beta-estradiol-induced mast cell number by melatonin in the testis of the frog Rana esculenta, in vivo and in vitro.

SUMMARY In the present study, we have utilized 17β-estradiol to induce the increase of mast cell number in order to verify the melatonin effect on mast cell accumulation in the frog testicular interstitium. Data obtained from in vivo experiments confirm that 17β-estradiol increases the mast cell number and indicate a melatonin-inhibitory role in their accumulation in the frog testis. In addition, melatonin interferes with the effects of estradiol on the increase of mast cell number in short-term cultured testes, and this result has also been obtained in a dose-response experiment at physiological concentration. The data suggest that melatonin acts on mast cell number directly via its local action in the frog gonads. In conclusion, our study shows, for the first time, that melatonin may interfere, probably via estrogen receptors, with the differentiation and/or proliferation of mast cells induced by estradiol treatment either in vivo or in vitro in the testis of the frog Rana esculenta.

Connexin 43 expression in the testis of the frog Rana esculenta.

Testicular cell-to-cell interactions play a key role in the regulation of spermatogenesis. In the testis, cell contacts are mediated through several mechanisms, including paracrine and direct contacts depending on gap junctional pathways. Gap junctions require connexin (Cx) channels and connexin-43 (Cx43) represent the most abundant Cx found in mammalian testis. Little is known about Cx expression in non-mammalian testis. Here we report the partial cloning of a Cx43 transcript of 381 bp from Rana esculenta testis. We also demonstrate that, in the frog testis, Cx43 transcript and protein show a parallel temporal and spatial pattern of expression throughout the reproductive annual cycle, with higher levels from September to January (when spermatogenesis is at a maximum level). In situ hybridization, carried out on testis collected in October, indicated that Leydig cells (LC) and Sertoli cells express Cx43 transcript, while the hybridization signal was less intense in germ cells. To obtain more information on Cx43 expression in the frog testis, we have used ethane-dimethane sulphonate (EDS), a toxin that specifically destroys LC. RT-PCR analysis shows a progressive decrease in Cx43 expression in EDS-treated testis from day 1 to day 4 after the injection, associated with LC destruction. Moreover, Cx43 expression returns to normal on day 28, when a new population of LC reappear in the interstitium, indicating that Cx43 is mainly expressed by LC. Taken together our data provide evidence that Cx43 is present in the frog testis with an important role in spermatogenesis.

The expression level of frog relaxin mRNA (fRLX), in the testis of Rana esculenta, is influenced by testosterone.

SUMMARY Frog relaxin (fRLX) belongs to the relaxin/insulin gene family present in the testis of Rana esculenta and is specifically expressed by Leydig cells. Since the expression of fRLX transcript changes during the reproductive cycle and is more abundant when circulating levels of androgens are relatively high, we investigated the effect(s) of testosterone and its antagonist (cyproterone acetate, CPA) on its expression pattern, in the testis of the frog Rana esculenta. Results from in vivo and in vitro experiments demonstrate that testosterone strongly induces a significant increase of fRLX mRNA expression in frog testes and, this effect is counteracted by CPA, supporting the existence of intratesticular (autocrine/paracrine) mechanisms of action. Interestingly, in both the control and testosterone-treated testes, fRLX mRNA expression was markedly decreased 24 h post-treatment, as compared to that measured at 2 h and 8 h post-treatment, suggesting that factor(s), other than testosterone, may act(s) in controlling its expression. In addition, RT-PCR analysis and in situ hybridization performed on frog testis injected with CPA for 15 days, on alternate days, showed a strong decrease of fRLX expression, suggesting that CPA counteracts the effect of testosterone on fRLX expression. Taken together our results strongly indicate that changes in the production, by the Leydig cells, of both testosterone and fRLX may represent a marker for the study of Leydig cell activity in the testis of the frog Rana esculenta.

Effects of melatonin treatment on Leydig cell activity in the testis of the frog Rana esculenta.

This study was conducted to verify the effect(s) of melatonin treatment on frog Leydig cells. Morphological observation after melatonin treatment indicates that many frog Leydig cells show degenerative changes (i.e. heterochromatic nuclei, loss of cellular adhesion) while in adjacent germinal tubules several Sertoli cells show heterochromatic nuclei, confirming the presence of a paracrine effect between interstitial and germinal compartments. The effect of melatonin on frog Leydig cell steroidogenesis was investigated in in vitro experiments; after 6 h of incubation melatonin severely inhibits both control and GnRH-induced testosterone secretion. In addition, in order to verify the effect of indolamine on frog Leydig cell activity, we investigated, by in situ hybridization, the presence of frog relaxin (fRLX, a transcript specifically expressed by these cells) in the testes of melatonin-injected animals after 48 h. fRLX signal completely disappeared from the testis of melatonin- injected frogs. The results of the present study indicate that melatonin treatment provokes Leydig cell morphological changes, blocks GnRH-antagonist-induced testosterone secretion and decreases fRLX expression. Taken together these results strongly indicate that melatonin acts on Leydig cells in the testis of the frog Rana esculenta.

Expression of prothymosin alpha in meiotic and post-meiotic germ cells during the first wave of rat spermatogenesis.

Prothymosin alpha (PTMA) is a highly acidic small polypeptide, that is, widely distributed and conserved among mammals. Its possible involvement in male gametogenesis has been mentioned but not clarified yet; in particular, it has been suggested that, in non‐mammalian vertebrates, it could play a role during GC meiosis and differentiation. In the present work we investigated the possible association between PTMA and meiotic and post‐meiotic phases of mammalian spermatogenesis. Three different time points during postnatal development of rat testis were analyzed, that is, 27 dpp (completed meiosis), 35 dpp (occurring spermiogenesis), and 60 dpp (first wave of spermatogenesis definitely ended). RT‐PCR and Western blot analyses showed that the expression levels of both Ptma mRNA and corresponding protein decrease in total extracts from 27 to 60 dpp. The in situ hybridization localized the transcript in interstitial Leydig cells, peritubular myoid cells and, inside the tubules, in germ cells from pachytene spermatocytes to newly formed haploid spermatids. The immunohistochemistry analysis localized the protein in the same cell types at 27 dpp, while at 35 and 60 dpp the haploid cells remain the only germ cells that still express it. In particular, PTMA specific localization in the heads of spermatids and epididymal spermatozoa, associated with the acrosome system, supports for the first time the hypothesis of a direct function in male germ cells. J. Cell. Physiol. 224: 362–368, 2010.

Endometrial LGR7 expression during menstrual cycle

In a prospective observational study, 50 healthy patients aged 18-39 years, with regular ovulatory cycle and normal hormone levels, underwent endometrial biopsy in the proliferative and secretory phase of the menstrual cycle for semiquantitative reverse-transcription polymerase chain reaction analysis of mRNA for LGR7, the classic relaxin receptor. LGR7 is constitutively expressed in human endometrium, and an increased LGR7 immunostaining is demonstrated in the secretory phase, confirming the involvement of relaxin in the physiology of endometrium and suggesting its role in implantation.