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-17β 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.
It is well accepted that the hypothalamus–pituitary–gonadal (HPG) axis mainly regulates the reproductive processes in teleosts as in other vertebrates. Gonadotropin-releasing hormone (GnRH) produced in the hypothalamus stimulates the production and release of gonadotropin (GTH) via GnRH receptor (GnRH-R) in the pituitary. Actually, the administration of GnRH by injection or cholesterol-based pellet has successfully induced oocyte maturation, ovulation, and spawning in Atlantic salmon, Salmo salar (Crim and Glebe, 1984), sea bass, Lates calcarifer (Harvey et al., 1985), and gilthead seabream, Sparus aurata (Barbaro et al., 1997). Fish used in these experiments had almost full-grown oocytes in their ovaries and the GnRH treatment induced the final oocyte maturation, and ovulation. However, in sexually immature fish, GnRH treatment alone (Crim and Evans, 1983; Dufour et al., 1991; Yaron et al., 1995) or in combination with testosterone (Crim and Evans, 1983; Holland et al., 1998), has often been shown to be unsuccessful in inducing vitellogenesis, oocyte maturation and ovulation. In juvenile teleosts, GnRH alone does not stimulate luteinizing hormone (LH) synthesis and release, resulting in a failure to induce sexual maturation (Crim and Evans, 1983; Dufour et al., 1991; Yaron et al., 1995). Moreover, if GnRH induced LH release from the pituitary of the fish treated with testosterone, LH release did not result in precocious puberty in female striped bass, Morone saxatilis (Holland et al., 1998). This suggests that other factor(s), such as follicle-stimulating hormone (FSH), are required for the initiation of ovarian development in this fish.
The red seabream is one of the most commercially important marine fish in Japan and has several advantages to investigate endocrine control of gametogenesis by GnRH–GTH system. This species attains puberty at 2–3 years of age for both males and females when they acquire full reproductive capacity. Our previous studies have shown that vitellogenesis and spawning can be induced by implantation of GnRH cholesterol pellet in sexually immature adult red seabream (Gen et al., 2001; Matsuyama et al., 1995). From these results, we expect that GnRH alone can also induce precocious puberty in the sexually immature juvenile red seabream.
Previous reports from our laboratory have shown that the biological activity of FSH is much lower than that of LH in in vitro production of estradiol-17β (E2) by vitellogenic follicles of red seabream (Tanaka et al., 1995). In addition, more recent studies have demonstrated that LH, but not FSH, induces the final maturation of oocytes of red seabream (Kagawa et al., 1998). Moreover, LHβ mRNA levels in the pituitary are maintained at high levels from the beginning of vitellogenesis to the spawning season. However, FSHβ mRNA remained low throughout sexual maturation in female red seabream (Gen et al., 2000). These results are different from those of other teleosts and suggest that LH, but not FSH, may be involved in regulation of early and late gametogenesis in female red seabream. Therefore, if GnRH treatment activates the pituitary–gonadal axis and stimulates LH release from the pituitary of juvenile red seabream, precocious puberty can be induced. However, the mechanisms of induced sexual maturation by GnRH implantation have not been elucidated yet in the red seabream. Moreover, GnRH-induced sexual maturation in red seabream is a good model to understand the regulatory mechanisms of puberty in fish.
It has been reported that dopamine has a direct effect on gonadotrophs to inhibit the LH release in several teleost species (see review Peter et al., 1991). The combined treatment of dopamine antagonist and GnRH agonist termed the Linpe method (Peter et al., 1988) has been proven highly successful in inducing oocyte maturation in a number of teleost species. In addition, a role for dopamine in the onset of puberty has been suggested in, for example, the juvenile spadefish Chaetodipterus faber, in which a decrease in dopaminergic activity in the hypothalamus was observed at the time of puberty (Marcano et al., 1995). Therefore, it may be possible that a reduction in dopaminergic inhibition of the gonadotropin release account for the onset of puberty. Although it has not been elucidated in red seabream whether dopamine act as an inhibitor of GTH release, domperidone was used as a putative dopamine antagonist in this study.
In the present study, we examined the effects of implantation of cholesterol pellet containing GnRHa and domperidone on sexual maturation in pre-pubertal female red seabream. Moreover, to understand the mechanisms of GnRH-induced sexual maturation, we investigated the changes in the expression of seabream (sb) GnRH, GnRH-R, GTH subunit genes, and changes in serum levels of LH, E2, and testosterone (T) during sexual maturation by implantation of GnRHa cholesterol pellet in juvenile female red seabream.
Immature 15-month-old red seabream (body weight=506.2±6.2 g), obtained from a commercial fish farm were kept in a sea pen (5×5×5 m) of the National Research Institute of Aquaculture, Nansei, Mie, Japan, in April 2000. In May (fish were 16-month-old), seven fish were sampled as an initial control and 60 fish were divided into three groups and the following treatments were performed: control group (GnRHa-free cholesterol pellet implanted), GnRHa cholesterol pellet implanted group, and GnRHa
Mean GSI and ovarian development of fish in each experimental group are shown in Table 1. In the initial control group, fish were immature and the mean GSI was below 0.7 and fish had ovaries containing oocytes at the perinucleolus and the oil droplet stage. On Day 10, the mean GSI in both treatment groups with GnRHa and GnRHa + domperidone had significantly increased (1.49±0.47 and 1.93±0.74, respectively) when compared with the control group (0.41±0.03, p<0.05). Histological examination revealed
The present study demonstrated that implantation of cholesterol pellet containing GnRHa induced vitellogenesis and ovulation in female juvenile red seabream. Our previous studies showed that spawning could be induced by GnRHa treatment in the post-pubertal female red seabream during the non-spawning season (Gen et al., 2001; Matsuyama et al., 1995). The 16-month-old fish used in the present study had completely immature gonads containing oocytes at the perinucleolus stage. Control fish without
This study was supported in part by a Bio-Design Program (BDP-02-IV-2-4) from the Ministry of Agriculture, Forestry, and Fisheries. We thank S. Yamaguchi, A. Mori, and Y. Nakamura for the technical assistance.
