Arginine Vasotocin Directly Regulates Spermatogenesis in Adult Zebrafish (Danio rerio) Testes.

The neuropeptide vasopressin is known for its regulation of osmotic balance in mammals. Arginine vasotocin (AVT) is a non-mammalian homolog of this neuropeptide that is present in fish. Limited information suggested that vasopressin and its homologs may also influence reproductive function. In the present study, we investigated the direct effect of AVT on spermatogenesis, using zebrafish as a model organism. Results demonstrate that AVT and its receptors (avpr1aa, avpr2aa, avpr1ab, avpr2ab, and avpr2l) are expressed in the zebrafish brain and testes. The direct action of AVT on spermatogenesis was investigated using an ex vivo culture of mature zebrafish testes for 7 days. Using histological, morphometric, and biochemical approaches, we observed direct actions of AVT on zebrafish testicular function. AVT treatment directly increased the number of spermatozoa in an androgen-dependent manner, while reducing mitotic cells and the proliferation activity of type B spermatogonia. The observed stimulatory action of AVT on spermiogenesis was blocked by flutamide, an androgen receptor antagonist. The present results support the novel hypothesis that AVT stimulates short-term androgen-dependent spermiogenesis. However, its prolonged presence may lead to diminished spermatogenesis by reducing the proliferation of spermatogonia B, resulting in a diminished turnover of spermatogonia, spermatids, and spermatozoa. The overall findings offer an insight into the physiological significance of vasopressin and its homologs in vertebrates as a contributing factor in the multifactorial regulation of male reproduction.

Interaction between thyroid hormones and gonadotropin inhibitory hormone in ex vivo culture of zebrafish testis: An approach to study multifactorial control of spermatogenesis.

Reproduction is under multifactorial control of neurohormones, pituitary gonadotropins, as well as of local gonadal signaling systems including sex steroids, growth factors and non-coding RNAs. Among the factors, gonadotropin-inhibitory hormone (Gnih) is a novel RFamide neuropeptide which directly modulates gonadotropin synthesis and release from pituitary, and in the gonads, Gnih mediated inhibitory actions on gonadotropin response of zebrafish spermatogenesis. Thyroid hormones are peripheral hormones which are also known to interact with reproductive axis, in particular, regulating testicular development and function. This study investigated the interaction between Gnih and thyroid hormones in zebrafish spermatogenesis using in vivo and ex vivo approaches. Three experimental groups were established: "control" (non-treated fish), "methimazole" and "methimazole + T4". Fish were exposed to goitrogen methimazole for 3 weeks; T4 (100 µg/L) was added in the water from the second week only in the "reversal treatment" group. After exposure, testes were dissected out and immediately incubated in Leibovitz's L-15 culture medium containing hCG, Gnih or hCG + Gnih for 7 days. Germ cell cysts and haploid cell population were evaluated by histomorphometry and flow cytometry, respectively. Our results showed that hypothyroidism affected germ cell development in basal and gonadotropin-induced spermatogenesis, in particular, meiosis and spermiogenesis. Hypothyroid testes showed lower amount of spermatozoa, and decreased potency of hCG. We also showed that goitrogen treatment nullified the inhibitory actions of Gnih on the gonadotropin-induced spermatogenesis. This study provided evidences that thyroid hormones are important regulatory factors for hCG- and Gnih-mediated functions in zebrafish spermatogenesis.

Effects of gonadotropin-inhibitory hormone on early and late stages of spermatogenesis in ex-vivo culture of zebrafish testis.

Gonadotropin-inhibitory hormone (Gnih) is known to play a role in the regulation of reproduction in vertebrates by influencing gonadotropin release and synthesis. While the endocrine actions of Gnih have been identified in several species, its paracrine/autocrine effects in the control of spermatogenesis are less defined. We have used ex vivo culture of zebrafish testis to investigate the role of gonadal zebrafish Gnih (zGnih) in the regulation of the spermatogenic process. We used FACScan cell cycle analysis, morphometric quantifications, BrdU incorporation and caspase-3 activity assays as well as measuring 11-Ketotestosterone (11-KT) level in the culture media. FACScan analysis and morphometric quantification results demonstrated direct action of zGnih on basal and gonadotropin (Lh and Fsh)-induced spermatogenesis. Treatment with zGnih (10 nM) significantly decreased the number of G0/G1 cells after 7-days of culture while no significant changes were found in the proportion area of spermatogonia cell types. Investigation of DNA synthesis using BrdU (5-Bromo-2'-Deoxyuridine) labeling showed that treatment with zGnih (10 nM) significantly decreased proliferative activity of type A spermatogonia, while increased the mitotic activity of type B spermatogonia. We also showed that treatment with zGnih (100 nM) completely eliminated 11-KT release induced by 100 ng/ml Fsh. Treatment with zGnih (10 and 100 nM) also inhibited both hCG and Fsh-induced spermatogenesis. These results, plus our previous findings, demonstrate that zGnih produced locally in the testis is a component of a complex multifactorial system that regulates testicular function in zebrafish.

Development of specific enzyme-linked immunosorbent assay for yellowtail kingfish (Seriola lalandi) follicle stimulating hormone using recombinant gonadotropins.

We developed a specific competitive enzyme-linked immunosorbent assay (ELISA) for yellowtail kingfish (Seriola lalandi) follicle stimulating hormone (FSH). We previously produced a full-length single chain recombinant yellowtail kingfish FSH using the Pichia pastoris expression system. We used the same method to produce the ß subunit of the hormone, against which polyclonal antibodies were raised in rabbits. We first confirmed immunoreactivity of the polyclonal antibodies with the recombinant full length FSH and FSHß as well as plasma and pituitary FSH of sexually immature and mature yellowtail kingfish by Western blot analysis. We then developed a precise and reproducible ELISA for yellowtail kingfish FSH and validated the assay in plasma and pituitary extracts. The intra- and inter-assay coefficients of variation was <2.2% and 10.2%, respectively. The sensitivity of the assay was 78 pg/ml. For further validation of the assay, we measured the plasma FSH in immature yellowtail kingfish treated with increasing doses (blank, 50, 100 and 150 µg/kg) of kisseptin2-10 peptide from a previous study. The dose response observed in treated females was not significant, however the increased plasma FSH levels coincided with the significantly higher estradiol levels we previously reported in the treated groups. We assessed the applicability of the assay in measuring circulating FSH in other species. We observed parallelism between the linearized FSH standard curve and displacement curves of serially diluted plasma from Atlantic bluefin tuna (Thunnus thynnus) and tilapia (Oreochromis niloticus). We also observed similar parallelism with full length recombinant giant grouper (Epinephelus lanceolatus) FSH. The ELISA we developed for yellowtail kingfish FSH will be useful in understanding the reproductive biology of the species as well as enhancing its aquaculture.

Effects of diisononyl phthalate on Danio rerio reproduction.

Di-isononyl phthalate (DiNP) is a high molecular weight phthalate commonly used as a plasticizer. It was introduced as a replacement for bis (2-ethylhexyl) phthalate (DEHP) which is used in the production of plasticized polyvinyl chloride (PVC). The purpose of this study was to investigate for the first time the effect of DiNP on female reproductive physiology in Danio rerio. Fish were exposed to five different doses of DiNP plus control (0 µg/L; 0.42 µg/L; 4.2 µg/L; 42 µg/L; 420 µg/L; 4200 µg/L) for a period of 21 days. We evaluated fish fecundity, oocyte growth, autophagic and apoptotic processes, as well as changes in morphological and biochemical composition of oocytes, using, qPCR analysis, histology and Fourier transform infrared imaging. The results demonstrate a non-monotonic dose response to DiNP. Greater differences were observed at the lowest (0.42 µg/L) and higher concentrations (420 µg/L; 4200 µg/L) of DiNP. The findings provide evidence that exposure to DiNP adversely affect oocytes growth and maturation, leading to abnormal gonadal development and reproduction in zebrafish.