Male Pheromones Induce Ovulation in Female Honeycomb Groupers (Epinephelus merra): A Comprehensive Study of Spawning Aggregation Behavior and Ovarian Development.

This study characterizes the spawning phenomena of the honeycomb grouper (Epinephelus merra), which is a lunar-synchronized spawner that spawns a few days after full moon. To elucidate the aggregation characteristics of wild honeycomb groupers, the numbers of males and females at the spawning grounds were counted before and after the full moon. Approximately 20 males were consistently observed at the spawning grounds throughout the study period. Females appeared several days after full moon and rapidly increased in number, peaking four days after full moon (41 individuals). The maturation status of the females aggregating at the spawning grounds was investigated. The gonadosomatic index increased rapidly three days after full moon, and ovulation was confirmed. Individuals with ovulatory eggs were present for three days, after which the number of females at the spawning grounds decreased. Additionally, the role of males in final oocyte maturation (FOM) and ovulation in females during the spawning phase was investigated in captivity. FOM was induced in females reared in water with mature males, suggesting that male pheromones in the water induced FOM via activation of the hypothalamic-pituitary-gonadal axis. This suggests that spawning at the natural spawning grounds was the result of male-female interactions via pheromones.

Insulin-like growth factors 1 and 2 regulate gene expression and enzymatic activity of cyp17a1 in ovarian follicles of the yellowtail, Seriola quinqueradiata.

There is accumulating evidence that insulin-like growth factors (IGFs), primary mediators of somatic growth, play an important role in fish reproduction. Previously, we showed that IGF-1 and IGF-2 are expressed in the ovarian follicle cells of the yellowtail (Seriola quinqueradiata) during the vitellogenic phase, suggesting that IGFs may be involved in ovarian steroidogenesis. In this study, we examined the effects of IGF-1 and IGF-2 on gene expression and activity of steroidogenic enzymes in yellowtail ovary in vitro. IGF-1 and IGF-2 had no effect on mRNA levels of several steroidogenesis-related genes (star, cyp11a1, hsd3b, cyp17a2, and cyp19a1). However, both IGFs enhanced the transcription of cyp17a1 in vitellogenic ovaries, although such up-regulation was not found in the ovary at the pre-vitellogenic stage. The stage-dependent effects of IGFs were correlated with changes in ovarian cyp17a1 mRNA levels during the reproductive cycle: transcript abundances increased in conjunction with ovarian development. In addition, IGF-induced cyp17a1 gene expression was significantly inhibited by wortmannin, suggesting that PI3 kinase plays an essential role in IGF-mediated ovarian steroidogenesis. Furthermore, IGF-1 and IGF-2 promoted the conversion of both progesterone and 17α-hydroxyprogesterone to androstenedione in vitellogenic ovaries, suggesting that both IGFs stimulated 17α-hydroxylase and C17-20 lyase activities. Taken together, these findings suggest that IGF-1 and IGF-2 act directly on follicle cells to stimulate steroid production through an increase in gene expression and enzymatic activity of cyp17a1 via induction of PI3 kinase.

Changes in gene expression and cellular localization of insulin-like growth factors 1 and 2 in the ovaries during ovary development of the yellowtail, Seriola quinqueradiata.

A method of controlling the somatic growth and reproduction of yellowtail fish (Seriola quinqueradiata) is needed in order to establish methods for the efficient aquaculture production of the species. However, little information about the hormonal interactions between somatic growth and reproduction is available for marine teleosts. There is accumulating evidence that insulin-like growth factor (IGF), a major hormone related somatic growth, plays an important role in fish reproduction. As the first step toward understanding the physiological role of IGF in the development of yellowtail ovaries, we characterized the expression and cellular localization of IGF-1 and IGF-2 in the ovary during development. We histologically classified the maturity of two-year-old females with ovaries at various developmental stages into the perinucleolar (Pn), yolk vesicle (Yv), primary yolk (Py), secondary yolk and tertiary yolk (Ty) stages, according to the most advanced type of oocyte present. The IGF-1 gene expression showed constitutively high levels at the different developmental stages, although IGF-1 mRNA levels tended to increase from the Py to the Ty stage with vitellogenesis, reaching maximum levels during the Ty stage. The IGF-2 mRNA levels increased as ovarian development advanced. Using immunohistochemistry methods, immunoreactive IGF-1 was mainly detected in the theca cells of ovarian follicles during late secondary oocyte growth, and in part of the granulosa cells of Ty stage oocytes. IGF-2 immunoreactivity was observed in all granulosa cells in layer in Ty stage oocytes. These results indicate that follicular IGFs may be involved in yellowtail reproduction via autocrine/paracrine mechanisms.

Greater amberjack Fsh, Lh, and their receptors: Plasma and mRNA profiles during ovarian development.

To understand the endocrine regulation of ovarian development in a multiple spawning fish, the relationship between gonadotropins (Gths; follicle-stimulating hormone [Fsh] and luteinizing hormone [Lh]) and their receptors (Gthrs; Fshr and Lhr) were investigated in greater amberjack (Seriola dumerili). cDNAs encoding the Gth subunits (Fshß, Lhß, and glycoprotein α [Gpα]) and Gthrs were cloned. The in vitro reporter gene assay using recombinant hormones revealed that greater amberjack Fshr and Lhr responded strongly to their own ligands. Competitive enzyme-linked immunosorbent assays (ELISAs) were developed for measuring greater amberjack Fsh and Lh. Anti-Fsh and anti-Lh antibodies were raised against recombinant chimeric single-chain Gths consisting of greater amberjack Fshß (or Lhß) with rabbit GPα. The validation study showed that the ELISAs were precise (intra- and inter-assay coefficient of variation, <10%) and sensitive (detection limit of 0.2ng/ml for Fsh and 0.8ng/ml for Lh) with low cross-reactivity. A good parallelism between the standard curve and serial dilutions of greater amberjack plasma and pituitary extract were obtained. In female greater amberjack, pituitary fshb, ovarian fshr, and plasma E2 gradually increased during ovarian development, and plasma Fsh significantly increased during the post-spawning period. This suggests that Fsh plays a role throughout ovarian development and during the post-spawning period. Pituitary lhb, ovarian lhr, and plasma Lh were high during the spawning period, suggesting that the synthesis and secretion of Lh, and Lhr expression are upregulated to induce final oocyte maturation and ovulation.