Endocrine control of aquaporin expression during gilthead seabream (Sparus aurata) spermatogenesis

Abstract

Introduction Recent studies in mammals have inferred that multiple classes of aquaporins may play different roles for water and solute transport during spermatogenesis and spermiogenesis. In teleosts, immunolocalization experiments in gilthead seabream have demonstrated that seven distinct aquaporins, Aqp0a, -1aa, -1ab, -7, -8b, -9b and -10b, are differentially expressed in the somatic and germ cell lineages of the mature (spermiating) testis. In this work, we used recombinant piscine gonadotropins and steroid hormones to investigate the in vivo and in vitro endocrine regulation of aquaporin expression during gilthead seabream spermatogenesis. Methods Males were sampled during the resting phase, at the onset of spermatogenesis and during the spermiating period. Plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) were measured by EIA. Testicular subsamples were used for immunofluorescence microscopy using paralog-specific antibodies, or incubated in vitro in the presence of European seabass recombinant follicle-stimulating hormone (rFsh) or luteinizing hormone (rLh), T, estradiol (E2), 11-KT or 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). Changes in aquaporin gene expression were determined by real-time qPCR. Results and Discussion During the resting phase, when plasma levels of T and 11-KT were low and the testis exclusively contained spermatogonia, only Aqp1ab was detected in the cytoplasm of spermatogonia, whereas Aqp9b and -10b were expressed in Leydig and Sertoli cells, respectively. At the onset of spermatogenesis and in the spermiating testis, androgen plasma levels progressively increased which correlated with the appearance of spermatocytes and spermatids, and few spermatozoa, in the seminiferous tubules. At both stages, Aqp0a and -9b were localized in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and spermatozoa. In vitro incubations of testis explants with rFsh and rLh indicated that both gonadotropins induced elevated expression levels of aqp0a, - 9b and - 10b at the onset of spermatogenesis, whereas aqp1aa and - 7 only increased in the presence of rLh. At the spermiating stage, gonadotropins only promoted the expression of aqp7 and - 8b. The response to the steroids was also different depending on the spermatogenic stage. Thus, at the onset of spermatogenesis aqp0a, - 1aa, - 7, - 9b and - 10b mRNA levels increased in the presence of 11-KT, and in the case of aqp9b also with E2 and 17,20β-P. In contrast, during spermiogenesis the mRNA levels of aqp0a, -8b, - 9b and - 10b were only stimulated with T, and 17,20β-P for aqp9b, whereas those of aqp7 were still responsive to 11-KT. Interestingly, aqp1ab expression was not regulated by gonadotropins or steroids at the onset of spermatogenesis or during spermiation, suggesting an earlier control of this channel. Conclusion These observations suggest a differential and complex gonadotropic and steroid regulation of aquaporin expression during gilthead seabream germ cell development