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Germ line activation of the luteinizing hormone receptor directly drives spermiogenesis in the flatfish Solea senegalensis

AuthorsChauvigné, François ; Zapater, Cinta ; Gasol, Josep M. ; Cerdà, Joan
Issue Date26-May-2014
Citation10th International Symposium on Reproductive Physiology of Fish, Expanding the khowledge base of reproductive success: from genes to the environment, Olhao, Portugal, 25-30 May 2014. Abstract book: 37 (2014)
AbstractIntroduction In mammals and teleosts, the differentiation of post-meiotic spermatids to spermatozoa (spermiogenesis) is thought to be controlled by the luteinizing hormone (LH) acting through the LH/choriogonadotropin receptor (LHCGR) to stimulate androgen secretion in the interstitial Leydig cells. However, a non-steroidal role of LH mediating the spermiogenic pathway remains unclear, in part because in mammals or amphibians complete spermatogenesis in vitro has never been obtained from isolated germ cells. In the flatfish Senegalese sole (Solea senegalensis), spermatogenesis occurs in spermatocysts which develop within seminiferous lobules (SLs), but germ cell development is semi-cystic, i.e. round spermatids are released from the supporting Sertoli cells into the SL lumen (SLL) where they elongate and transform into spermatozoa. Using this model, here we test the hypothesis that Lh can directly control spermiogenesis through the activation of the Lhcgrba in germ cells. Methods Cells from the sole SLL were extracted and characterized by flow cytometry or isolated by fluorescence-activated cell sorting (FACS). Immunofluorescence staining and Western blot on males treated or not with intramuscular injection of homologous recombinant Lh (rLh) (6 µg/kg) were carried out using a sole Lhcgrba-specific antibody, a piscine Lhβ antiserum or a 6xHis antibody. SLL cell extracts and FACS-purified spermatids were incubated in vitro with rLh in the presence or absence of different inhibitors, and the effect on spermatid differentiation assessed by quantitative flow cytometry. Changes in gene expression were determined by real-time qPCR. Results and Discussion Sole spermatids released into the SLL express the Lhcgrba which correlated with the detection of both native Lh and intramuscularly injected His-tagged rLh specifically bound to their cell membrane, thus demonstrating that circulating Lh can reach the SLL. In vitro incubation of spermatids isolated from the SLL with rLh specifically promoted their differentiation into spermatozoa, while homologous recombinant follicle-stimulating hormone and steroid hormones did not. The Lh-Lhcgrba induction of spermiogenesis in vitro was mediated through a cAMP/PKA signaling pathway which initiated the transcription of genes potentially involved in the function of spermatozoa. We further found that Lhcgrba expression in late spermatocytes and/or spermatids also occurs in distantly related fishes with cystic spermatogenesis, such as zebrafish and gilthead seabream, suggesting that this feature is likely conserved in teleosts regardless of the type of germ cell development. Conclusion These data reveal a novel role of Lh in vertebrate germ cells, whereby a Lhcgrba-activated signaling cascade in haploid spermatids directs gene expression and the progression of spermiogenesis.
Description10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 page
Appears in Collections:(ICM) Comunicaciones congresos
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