Diana Ferrara

GPR30 is overexpressed in post-puberal testicular germ cell tumors

GPR30 is a 7-transmembrane G protein-coupled estrogen receptor that functions alongside traditional estrogen receptors to regulate cellular responses to 17β-estradiol and environmental estrogens. In this study, we have evaluated by immunohistochemical analysis GPR30 expression in post-puberal testicular germ cell tumours (30 seminomas, 5 teratomas, 12 embryonal carcinomas, and 20 intratubular germ cell tumors). The GPR30 protein expression was detected at high level in all intratubular germ cell tumours, seminomas, and embryonal carcinomas, whereas in teratomas the expression was low. The immunohistochemical data were further confirmed by Western blot analysis. GPR30 protein expression has also been analyzed in GC1 and TCam-2 cell lines, respectively derived from immortalized type B murine spermatogonia and human seminoma. Our results indicate that GPR30 could be a potential therapeutic target; the design of a specific GPR30 inhibitors could be a useful molecular target to block neoplastic germ cells with a high proliferative rate for the treatment of TGCTs. See commentary: Role of GPR30 in testicular germ cell tumors: A potential new anticancer target

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.

Ethane 1,2-dimethane sulphonate is a useful tool for studying cell-to-cell interactions in the testis of the frog, Rana esculenta☆

Ethane 1,2-dimethane sulphonate (EDS), a toxin which specifically destroys Leydig cells (LC), has been used to study cellular interactions in the testis of the frog Rana esculenta. Animals received three consecutive EDS injections and were sacrificed on day 4, 8, and 28 from the first injection. No significant morphological differences were observed between present observation and that obtained, in a previous experiment, after four consecutive EDS injections. In fact, on day 4, in the germinal tubules adjacent to apparently normal LC, Sertoli cells surrounding primary spermatogonia (I SPG) show heterochromatic nuclei and loss of cellular adhesion. Interestingly, I SPG surrounded by the heterochromatic Sertoli cells present grossly swollen mitochondria with ballooned cristae. On day 8, sometimes in the interstitium many LC appear strongly damaged and the germinal tubules appear disorganized; the only cell type still distinguishable is the I SPG. On day 28 from the first EDS injection a new population of LC reappear in the interstitium and spermatogenesis normalizes. These data confirm the close relationship between the interstitial and the geminal compartments. Immunocytochemical data obtained using a polyclonal antibody anticonnexin-43 (Cx-43, the most abundant Cx found in mammalian testis) demonstrate the presence of Cx-43 in the frog testis. In particular, Cx-43 is present between LC in the interstitium, between Sertoli and germ cells in the cysts and between Sertoli cells and I SPG. Cx-43 immunopositivity sharply decreases on day 4 from the first EDS injection simultaneously with the loss of cellular adhesion between Sertoli and germ cells. On day 8 and 28 from the first EDS injection Cx-43, immunopositivity is restored and, this data is also supported by Western blot analysis. Our data provide, for the first time, evidence that Cx-43 protein is present in the frog testis and confirm that EDS is a useful tool for studying cellular communication at the paracrine pathway or through direct contact depending on the gap junctional pathway in R. esculenta testis

First evidence of prothymosin α in a non-mammalian vertebrate and its involvement in the spermatogenesis of the frog Rana esculenta ☆

A cDNA clone encoding for a Prothymosin alpha (Prot-alpha) has been isolated and characterized from the testis of the frog Rana esculenta. Frog Prothymosin alpha (fProt-alpha) predicted a 109 amino acid protein with a high homology to the mammalian Prot-alpha. fProt-alpha contains 28 aspartic and 25 glutamic acid residues and presents the typical basic KKQK amino acid sequence in the close carboxyl terminal region. Northern blot analysis revealed that fProt-alpha is highly expressed in the testis. A different expression of fProt-alpha transcript was found during the frog reproductive cycle with a peak in September/October in concomitance with germ cell maturation, strongly suggesting a role for this protein in the testicular activity. In situ hybridization evidenced that the only germ cells expressing fProt-alpha are the primary and secondary spermatocytes; in addition, the hybridization signal was stronger in the October testis. Taken together, our findings indicate that fProt-alpha might contribute to the efficiency of frog spermatogenesis with a role during the meiosis. This study is the first report on the isolation and characterization of a Prot-alpha in a non-mammalian vertebrate. In addition, our results indicate that the testis of the frog R. esculenta may be a useful model to increase the knowledge concerning the physiological role of Prot-alpha in vertebrates.

Differential Expression of Duplicated Genes for Prothymosin Alpha During Zebrafish Development

We show that ptma, a single copy gene found in all organisms investigated so far, is duplicated in zebrafish. The two genes, ptmaa and ptmab, are individually controlled as indicated by their different expression patterns during embryonic development. Only the ptmab transcript is observed at 4 and 8 hpf of development in all embryonic cells, whereas both genes are expressed at later stages as revealed by in situ hybridization studies. In most cases, the two genes are expressed in the same territories, but only the ptmaa transcript was found in the trigeminal ganglion and in endodermal pouches. In the eye, at 72 hpf, the ptmaa and ptmab transcripts were found in amacrine cells, whereas only the ptmab transcript appeared in horizontal cells. The existence of two prothymosin genes indicates that their function in cell proliferation and differentiation is more complex in fishes than in mammals. Developmental Dynamics 237:1112–1118, 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.

Testicular Activity of Mos in the Frog, Rana esculenta: A New Role in Spermatogonial Proliferation

Abstract Mos is a MAPK kinase kinase with an expression that is highly restricted to the gonads. Its function is mainly associated to the meiotic metaphase II arrest occurring during female gametogenesis, whereas to our knowledge, its role during spermatogenesis has not yet clarified. In the present paper, we report the isolation of c-mos cDNA and the identification of a 60-kDa Mos protein from the testis of the anuran amphibian, Rana esculenta. Both the transcript and the protein are always present at low levels in the testis during the frog annual sexual cycle, with single significant peaks of expression in March and May, respectively. Mos is mainly localized in the cytoplasm of primary and secondary spermatogonia (SPG). Therefore, we have used treatments with ethane-dimethane sulphonate (EDS), which blocks spermatogonial mitosis in frogs. Four days after a single EDS injection, Mos expression in SPG highly increases concomitantly with the temporary arrest of mitosis. From 8 to 28 days after the injection, the normal proliferative activity of SPG is restored, and Mos expression gradually decreases to control levels. These results strongly indicate that the c-mos proto-oncogene exerts a new role associated to the regulation of spermatogonial proliferation.

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.

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.