Naoki Kumakura

Correlation Between Messenger RNA Expression of Cytochrome P450 Aromatase and Its Enzyme Activity During Oocyte Development in the Red Seabream (Pagrus major)

Abstract In teleosts, estradiol-17β (E2) is an important hormone responsible for oocyte development. To elucidate the molecular mechanisms underlying E2 biosynthesis, we characterized the structure of red seabream (Pagrus major) cytochrome P450 aromatase (P450arom) that is directly involved in E2 biosynthesis and found changes in mRNA levels of P450arom during oocyte development induced by implantation of gonadotropin-releasing hormone analogue. A cDNA clone encoding P450arom is 1779 base pairs in length and encodes a protein of 519 amino acids in length, with a calculated molecular weight of 58.9 kDa. Northern blot analysis showed that P450arom mRNA levels increased gradually from Day 8, when oocytes reached the secondary yolk globule stage, and were maintained at high levels at the day of spawning (Day 15). The P450arom mRNA levels increased in association with an increase of the gonadosomatic index (gonad weight/body weight × 100%), serum E2, and P450arom enzyme activity (in vitro conversion of testosterone to E2 in the ovarian fragments). Furthermore, an increase in mRNA levels of the LHβ, but not FSHβ, correlated with increased P450arom mRNA levels during the course of ovarian development. In addition, the levels of P450arom mRNA increased in isolated ovarian follicles during the course of vitellogenic oocyte growth and became undetectable in follicles at the migratory nucleus and the mature stages. These findings, together with those of the previous studies, suggest that LH, not FSH, may regulate E2 biosynthesis via increased levels of P450arom mRNA during oocyte development of red seabream.

Effects of gonadotropin-releasing hormone agonist and dopamine antagonist on hypothalamus–pituitary–gonadal axis of pre-pubertal female red seabream (Pagrus major)

The effects of GnRH agonist (GnRHa) on the hypothalamus-pituitary-gonadal axis were studied in female pre-pubertal red seabream. Sexually immature 16-month-old fish were implanted intramuscularly with cholesterol pellets containing GnRHa or GnRHa in combination with domperidone, putative dopamine antagonist, and reared for 10-20 days. In both GnRHa and GnRHa+domperidone implanted groups, vitellogenesis was observed on Day 10 and ovulation was observed on Day 20, while ovarian development was not observed in the control fish throughout the experimental period. The levels of GnRH receptor mRNA were significantly higher in both GnRHa implanted groups than in the control. The expressions of all three gonadotropin subunit genes were up-regulated and serum luteinizing hormone levels were increased by the GnRHa implantation. Serum testosterone and estradiol-17beta levels were also increased on Day 10 and maintained high levels on Day 20. On the other hand, seabream (sb) GnRH mRNA levels in the brain were relatively low and unchanged in all experiment groups. The present study first shows that GnRH alone can induce precocious puberty in red seabream. These results indicate that the system of pituitary-gonadal axis has already been developed in 16-month-old fish and the commencement of sbGnRH secretion may be an important physiological event for the onset of puberty in the red seabream.

Physiological roles of FSH and LH in red seabream, Pagrus major

The duality of gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), has been confirmed in most teleost species, but very little is known about their biological functions. To elucidate the physiological roles of FSH and LH in fish reproduction, the expression profiles of GTH subunit genes during gonadal development were analyzed in both male and female red seabream. Furthermore, in vitro studies were carried out to examine the effects of GTHs on steroid hormone production and cytochrome P450 aromatase (P450arom) expression in red seabream gonads. In both sexes, LHβ mRNA was maintained at high levels from the early gametogenesis until spawning season, and declined with gonadal regression. Interestingly, FSHβ mRNA levels in males increased in parallel with testicular development, whereas those in female were remained low throughout oocyte development. From in vitro studies using purified red seabream FSH and LH, both GTHs had a similar potency in stimulating 11-ketotestosterone production by testicular slices, while the biological activity of FSH was much lower than that of LH in stimulating production of estradiol-17β by vitellogenic follicles. Moreover, expression of P450arom mRNA was induced by LH, but not FSH, in ovarian follicles in vitro. FSH was also ineffective in inducing maturational competence and final oocyte maturation. These results suggest that, unlike salmonids, FSH may play an important role during gametogenesis in male, but not female, red seabream, whereas LH may be involved in regulation of both early and late gametogenesis in both sexes.

Effect of high water temperature on brain-pituitary-gonad axis of the red seabream during its spawning season

In the present study, we examined the effect of high water temperature on brain-pituitary-gonad (B-P-G) axis of a perciform fish red seabream, Pagrus (Chrysophrys) major during its spawning season (April-May). High water temperature negatively affected all levels in B-P-G axis and turned out to be the termination factor of the spawning season of this species.

Effects of 11‐ketotestosterone and gonadotropin‐releasing hormone on follicle‐stimulating hormone and luteinizing hormone gene expression in castrated and sham‐operated male red seabream Pagrus major

In order to clarify the roles of androgen and gonadotropin-releasing hormone (GnRH) on gonadotropin (GTH; luteinizing hormone [LH] and follicle stimulating hormone [FSH]) synthesis, effects of castration and implantation of GnRH analog (GnRHa) or 11-ketotestosterone (11-KT) on expression of GTH subunit, α-glycoprotein subunit (αGSU), FSHβ, and LHβ genes, during the early spermatogenic stage in male red seabream Pagrus major were examined. Male red seabream underwent castration or sham-operation and were subsequently implanted with cholesterol pellets containing GnRHa, silicone capsules filled with 11-KT, or blank capsules (control). FSHβ mRNA levels increased due to castration, and it was reversed by treatment with 11-KT. 11-ketotestosterone treatment also decreased FSHβ mRNA levels in sham-operated fish. These results suggest that 11-KT acts on the pituitary to suppress FSH synthesis in male red seabream. On the other hand, neither castration nor replacement of 11-KT in castrated fish had effects on LHβ mRNA levels, whereas 11-KT treatment had slightly but significantly decreased LHβ mRNA in sham-operated fish, αGSU mRNA levels were not changed by castration or 11-KT treatment in both sham-operated and castrated fish. Meanwhile, treatment with GnRHa significantly decreased FSHβ mRNA levels in sham-operated fish, but not in castrated fish. This suggests that GnRHa may down-regulate expression of FSHβ mRNA through the production of 11-KT in testis. LHβ and αGSU mRNA levels in sham-operated fish, but not in castrated fish, were significantly elevated by treatment with GnRHa.

Regulation of GnRH and its receptor in a teleost, red seabream

Publisher Summary This chapter discusses the regulations of gonadotropin-releasing hormone (GnRH) and its receptor in a teleost, red seabream. The chapter aims to reveal the mechanisms underlying the regulation of the synthesis and release of GnRH to help solve several basic and practical problems in fish reproductive physiology and aquaculture, such as the control of puberty in fish. Red seabream, Pagrus (Chysophrys) major, belongs to the order Perciformes, the largest and evolutionally the most advanced fish group. It spawns almost every day even in captivity during its spawning season, and thus, red seabream is an excellent experimental model for the reproductive biology of fish. In addition, it is one of the most important species for aquaculture in Japan. The chapter describes the achievements in studies on GnRH and GnRH receptor (GnRH-R) in red seabream and proposes possible models for the regulatory mechanisms of the onset of puberty and the seasonal reproduction of this species.

Regulation of gonadotropin subunit genes expression by 11-ketotestosterone during early spermatogenesis in male red seabream, Pagrus major

To examine the roles of gonadal steroids in the regulation of expression of gonadotropin subunit genes, male red seabream were gonadectomized and a sub-group was treated with 11-ketotestosterone (11-KT). Castration of males during the early stage of spermatogenesis elicited a significant increase in FSHβ mRNA levels, which was prevented by 11-KT replacement. By contrast, LHβ mRNA levels were not changed by castration or 11-KT replacement. In addition, administration of 11-KT to sham-operated males suppressed the steady-state FSHβ and LHβ mRNA levels. These results indicate that 11-KT may function as a negative feedback regulator of FSHβ gene expression, and may act through the testis to down-regulate LHβ mRNA levels in male red seabream during this period.