Establishment of cell-lines stably expressing recombinant Japanese eel follicle-stimulating hormone and luteinizing hormone using CHO-DG44 cells: fully induced ovarian development at different modes.

The gonadotropins (Gth), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play central roles in gametogenesis in vertebrates. However, available information on their differential actions in teleost, especially in vivo, is insufficient. In this study, we established stable CHO-DG44 cell lines expressing long-lasting recombinant Japanese eel Fsh and Lh with extra O-glycosylation sites (Fsh-hCTP and Lh-hCTP), which were produced in abundance. Immature female eels received weekly intraperitoneal injections of Gths. Fsh-hCTP induced the entire ovarian development by 8 weeks from the beginning of injection; thus, the ovaries of most fish were at the migratory nucleus stage while the same stage was observed in eels after 4 weeks in the Lh-hCTP-treated group. In contrast, all pretreated and saline-injected eels were in the pre-vitellogenic stage. Gonadosomatic indices in the Fsh-hCTP-treated group were significantly higher than those in the Lh-hCTP group at the migratory nucleus stage because of the significantly higher frequency of advanced ovarian follicles. Ovarian mRNA levels of genes related to E2 production (cyp11a1, cyp17a1, cyp19a1, hsd3b, fshr, and lhr) were measured using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). All genes were induced by both Fsh-hCTP and Lh-hCTP, with a peak at either the mid- or late vitellogenic stages. Transcript abundance of cyp19a1 and fshr in the Lh-hCTP group were significantly higher than those in the Fsh-hCTP group, whereas no difference in the expression of other genes was observed between the groups. Fluctuations in serum levels of sex steroid hormones (estradiol-17ß, 11-ketotestosterone, and testosterone) in female eels were comparable in the Fsh-hCTP and Lh-hCTP groups, thus increasing toward the maturational phase. Furthermore, the fecundity of the eels induced to mature by Fsh-hCTP was significantly higher than that induced by Lh-hCTP. These findings indicate that Fsh and Lh can induce ovarian development in distinctively different modes in the Japanese eel.

Effect of GnRHa on plasma levels of Fsh and Lh in the female greater amberjack Seriola dumerili.

The effects of gonadotropin-releasing hormone agonist (GnRHa) on plasma levels of follicle-stimulating hormone (Fsh) and luteinising hormone (Lh) are reported for female greater amberjack Seriola dumerili with post-vitellogenic ovarian oocytes. Five females were implanted with pellets containing GnRHa (600 µg kg-1 body weight), while five other females were injected with saline. All females implanted with GnRHa-containing pellets ovulated 36-42 h post-implantation. The GnRHa implants elevated Lh, but not Fsh plasma levels within 42 h of GnRHa administration.

Photoperiodic regulation of plasma gonadotropin levels in previtellogenic greater amberjack (Seriola dumerili).

In Seriola species, exposure to a long photoperiod regime is known to induce ovarian development. This study examined photoperiodic effects on pituitary gene expression and plasma levels of follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) in previtellogenic greater amberjack (Seriola dumerili). The fish were exposed to short (8L:16D) or long (18L:6D) photoperiod. The water temperature was maintained at 22 °C. Compared with the short-photoperiod group, plasma Fsh levels were higher on days 10 and 30 in the long-photoperiod group, but plasma Lh levels did not significantly differ. On day 30, pituitary Fsh- and Lh-ß subunit gene expressions were also higher in the long-photoperiod group than the short-photoperiod group, whereas α-subunit gene expressions were higher on days 20 and 30. Throughout the experiment, average gonadosomatic index and plasma E2 levels did not significantly differ between the two groups. This study clearly demonstrated that a long photoperiod induced Fsh release in the previtellogenic fish followed by upregulation of pituitary Fsh and Lh subunit gene expressions. An increase in plasma Fsh levels may be a key factor that mediates the photoperiodic effect on the initiation of ovarian development.

Development of a homologous radioimmunoassay for red seabream follicle stimulating hormone and regulation of gonadotropins by GnRH in red seabream, Pagrus major.

Using a recombinant chimeric single-chain follicle stimulating hormone (FSH), we established a radioimmunoassay (RIA) for red seabream (Pagrus major) FSH (pmFSH) which became a powerful tool for studying reproductive physiology. We studied the profiles in plasma and pituitary concentrations of FSH and luteinizing hormone (LH) during sexual maturation. A pre-established RIA for red seabream LH was used for the LH measurements. The regulation of FSH and LH secretion from the pituitary was investigated using a gonadotropin-releasing hormone analog (GnRHa) in vivo and in vitro. Marked differences in plasma and pituitary FSH levels were observed between males and females; pituitary FSH content in males was much higher than that in females during all seasons, and plasma FSH levels in males were high during the spawning season, whereas those in females were unchanged. In contrast, plasma and pituitary levels of LH were elevated before and during the spawning season in males and females. Injecting or implanting (cholesterol pellet) a GnRHa into adult and juvenile red seabream resulted in significant increases in plasma LH concentrations; however, no significant change was observed in plasma FSH. Moreover, GnRHa stimulated only LH secretion in an in vitro experiment using dispersed pituitary cells. The discrete FSH and LH secretion profiles revealed suggest differential roles for the two gonadotropins during red seabream gametogenesis. In addition, the marked difference in pituitary FSH levels in males and females suggests the relative significance of FSH in male reproduction.

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.

High water temperature impairs ovarian activity and gene expression in the brain-pituitary-gonadal axis in female red seabream during the spawning season.

Temperature plays a pivotal role in the control of seasonal reproduction in temperate fish species. It is well known that temperatures that exceed a certain threshold impair gonadal development, maturation, and spawning. However, the endocrine mechanisms that underlie these effects are poorly understood. We evaluated the effect of high water temperature on the brain-pituitary-gonadal (B-P-G) axis of a perciform fish, red seabream, Pagrus (Chrysophrys) major during its spawning season (April-May). Fish were reared at either high (24 °C: H-group) or optimal/control (17 °C: C-group) temperatures for 5 or 10 d. After 5 d, the transcript abundance of gonadotropin-releasing hormone-1 (GnRH1) in the brain and GnRH receptor (GnRH-R) and FSH-ß in the pituitary were significantly lower in H-group than in C-group. Conversely, there was no difference in pituitary LH-ß mRNA levels, serum concentrations of estradiol-17ß (E2), or the gonadosomatic index (GSIs) between H- and C-groups on Day 5. After 10 d, the ovaries of all H-group fish had completely regressed and were filled with only perinucleolar stage primary oocytes and atretic oocytes. The brain GnRH1 expression, pituitary GnRH-R and pituitary LH-ß expression, serum E2 concentrations, and the GSI were significantly lower in the H-group on Day 10. Our results suggest that high water temperature is the proximate driver of the termination of the spawning season of female red seabream. The effect appears to be mediated by suppression of gene expression in the B-P-G axis.

Central distribution of kiss2 neurons and peri-pubertal changes in their expression in the brain of male and female red seabream Pagrus major.

kisspeptins that are encoded by kiss1 gene are now considered the key regulator of reproduction from a number of studies in mammals. In most vertebrates, a paralogue of kiss1, called kiss2, is also present, and the functional significance of kisspeptins is not known precisely. In the present study, we have cloned kiss2 from a perciform teleost, the red seabream Pagrus major. The amino acid sequence deduced from the red seabream kiss2 contained a highly conserved 10-amino-acid residue, Kiss2(10) or kp-10. A kiss1-like transcript was also identified, but it appears to be non-functional due to the presence of a "premature" stop codon. Neurons that express kiss2 mRNA were distributed in the dorsal (NRLd) and ventral (NRLv) parts of nucleus recessi lateralis in the hypothalamus. In some fish a few kiss2-expressing neurons were detected in the preoptic area and nucleus ventralis tuberis. The number of kiss2-expressing neurons in the NRLd was larger during the first spawning season in both males and females compared with fish in the post-spawning periods. In males the number of kiss2 neurons in the NRLd of maturing fish was also larger than those in the post-spawning periods. In males the number of kiss2 neurons in the NRLv showed a similar pattern of changes to that of NRLd, while significant changes were not detected for females. The numbers of gonadotropin-releasing hormone 1 (GnRH1)-immunoreactive neurons in the preoptic area showed a similar pattern of change as those of kiss2 cells of the NRLd in both males and females (and also the NRLv in males). These results are in good agreement with the hypothesis that kiss2 neurons are involved in pubertal processes via regulatory influences on GnRH1 neurons in red seabream.

Novel expression of importin alpha homologue in marine teleost, Pagrus major.

Importin alpha proteins are critical modulators of the classical nuclear protein import pathway. Although the physiological roles of importin alpha have been extensively studied in invertebrates and mammals, very little is known about their counterparts in lower vertebrates. In this study, to elucidate the roles of importin alpha in a teleost species, we isolated and characterized red seabream (Pagrus major) importin alpha cDNA derived from ovary and found changes in the mRNA levels of importin alpha in male and female red seabream during sexual maturation. The 1846-bp cDNA encodes a 520 amino acid protein that includes the importin beta-binding domain, a short acidic domain, and an armadillo (arm) repeat domain. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) showed transcription of red seabream importin alpha in testis and ovary but not in the other tissues. The importin alpha mRNA levels in males increase in association with testicular development, whereas those in females remain high throughout sexual maturation. These findings suggest that red seabream ovary-derived importin alpha may be controlled in a tissue-specific manner and may perform unique functions in the gonad in addition to its involvement in nuclear transport.

Effects of gonadotropin-releasing hormone on pituitary-ovarian axis of one-year old pre-pubertal red seabream.

To identify the pubertal development of the brain-pituitary-gonad (BPG) axis in female red seabream (Pagrus major), we investigated the effects of gonadotropin-releasing hormone agonist (GnRHa) on seabream (sb) GnRH mRNA levels in the brain, gonadotropin subunit mRNA levels in the pituitary, and serum concentrations of luteinizing hormone (LH), testosterone (T) and estradiol-17beta (E2) in pre-pubertal fish. Sexually immature 12-month-old fish were implanted with a cholesterol pellet containing GnRHa and maintained for 10-20 days. In the brain, GnRHa had no effect on sbGnRH mRNA levels. In the pituitary, although no marked changes were observed in follicle-stimulating hormone (FSH) beta subunit mRNA levels, the expression of glycoprotein (GP) alpha, and LHbeta subunit genes in the pituitary was drastically up-regulated (approximately 4- and 5-fold, respectively) and serum LH levels were also increased (approximately 3-fold) by GnRHa implantation. However, ovaries of GnRHa treated fish contained only oocytes at the peri-nucleolus stage, and oocyte development such as vitellogenesis and oocyte maturation did not occur throughout the experimental period. In these fish, even though LH was released, only slight increases in serum concentrations of T and E2 were observed. These results indicate that the pituitary gonadotropin cells of pre-pubertal 12-month-old fish were already receptive to GnRH stimulus, and acquired the ability to synthesize and release of LH as in the case of adult fish. Deficient factors for the onset of puberty by GnRHa treatment will be discussed.

Disturbance of plasma melatonin profile by high dose melatonin administration inhibits testicular maturation of precocious male masu salmon.

We have previously shown that the testicular development of underyearling male masu salmon Oncorhynchus masou reared under a long photoperiod was accelerated by oral melatonin treatment (0.5 mg melatonin/kg body weight/day), suggesting that melatonin mediates photoperiodic signaling. In this study, we further examined the effects of a disturbance in the plasma melatonin profile on gonadal development in underyearling male masu salmon by administering a higher dose of melatonin. Fish randomly selected in June were divided into two groups. They were reared under a light:dark (LD) cycle of 16:8 (lights on 04:00-20:00 hr) and fed with pellets sprayed with melatonin or vehicle twice a day at 08:30 and at 15:30 hr (7.5 mg melatonin/kg body weight/day) until October. Fish were sampled on Day 0, 25, 60, 90 and 120. The plasma melatonin levels were high in the dark phase and low in the light phase in the control group, while they were constantly high with no significant change in the melatonin-treated group. Melatonin treatment had inhibitory effects on the gonadosomatic index and plasma testosterone levels. Pituitary salmon gonadotropin-releasing hormone content and luteinizing hormone content were significantly lower in the melatonin-treated group on Day 60 and 90, respectively. These results indicate that the plasma melatonin profile is important for mediating photoperiodic signals that regulate brain-pituitary-gonadal axis in underyearling precocious male masu salmon.

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.

Seasonal variation of the three native gonadotropin-releasing hormone messenger ribonucleic acids levels in the brain of female red seabream.

We studied the seasonal variation of the expression of genes encoding the three native gonadotropin-releasing hormones (GnRHs), namely salmon(s) GnRH, chicken(c) GnRH-II, and seabream(sb) GnRH in red seabream, Pagrus (Chrysophrys) major, in order to better understand the regulatory mechanisms of GnRH gene expression by environmental and endocrine factors. Female red seabream, reared under natural conditions, were collected monthly or bimonthly from October to June, and the levels of the three distinct GnRH messenger ribonucleic acids (mRNAs) in the brains of those fish (n = 4-6) were determined by ribonuclease (RNase) protection analysis. The levels of sbGnRH mRNA correlated well with the observed ovarian histology; the levels of sbGnRH mRNA of immature fish in October and December were low, and increased in February and March in conjunction with active vitellogenesis. The sbGnRH mRNA levels reached a maximum level in April (spawning season), after which they rapidly decreased together with the observed ovarian regression in June. In contrast, the levels of sGnRH mRNA showed no variation, while those of cGnRH-II mRNA were elevated only slightly in March and April. The increase in sbGnRH mRNA levels correlates with the increase in day length, water temperature and serum steroids levels, suggesting that these factors are candidates for regulators of sbGnRH synthesis.

Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream, Pagrus major.

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.

Ontogenic origin of salmon GnRH neurons in the ventral telencephalon and the preoptic area in masu salmon.

During the ontogeny of masu salmon Oncorhynchus masou, neurons producing the salmon type of gonadotropin-releasing hormone (sGnRH) were first detected in the olfactory epithelium of the eyed egg and, subsequently, in the brain, suggesting a migration of these cells. Among sGnRH neurons distributed from the olfactory nerve (ON) through the preoptic area (POA), those in the ventral telencephalon (VT) and the POA are indicated to regulate gonadotropin secretion. Thus, it is of interest to know whether all the sGnRH neurons originate from the olfactory epithelium. In the present study, we examined by in situ hybridization whether sGnRH neurons are present in the VT-POA of fish, whose olfactory epithelia including sGnRH clusters were cauterized just after hatching (44 days after fertilization). Fish were sampled in June (212 days after the operation). Neurons expressing sGnRH mRNA were detected in the VT-POA as well as in the ON, ventral olfactory bulb, and transitional area between the olfactory bulb and telencephalon (which is considered to correspond to the terminal nerve ganglion) in the control group. In contrast, neurons expressing sGnRH mRNA were not detected in the VT-POA in the olfactory epithelium lesioned (OEL) group. Furthermore, pituitary sGnRH content in the OEL group was just above the detectable limit and was significantly lower than that in the corresponding control group in both sexes. These results indicate that sGnRH neurons in the VT-POA are derived from the olfactory epithelium in masu salmon, although the possibility cannot be ruled out that sGnRH neurons in the VT-POA arise from the VT-POA, but were delayed in expressing sGnRH because of the trauma of cauterization.

Biosynthesis of steroids in ovarian follicles of red seabream, Pagrus major (Sparidae, Teleostei) during final oocyte maturation and the relative effectiveness of steroid metabolites for germinal vesicle breakdown in vitro.

The steroid synthesis pathway in the ovarian follicles of the red seabream during final oocyte maturation (FOM) was investigated by incubating intact follicles with different radioactively labeled steroid precursors. During FOM, the steroidogenic shift from estradiol-17beta to 20 beta-hydroxylated progestin production occurred mainly due to a combination of inactivation of C 1720-lyase and activation of 20 beta-hydroxysteroid dehydrogenase. Of the steroids produced, 1720 beta-dihydroxy-4-pregnen-3-one (1720 beta-P) and 1720 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) exhibited the greatest effect on germinal vesicle breakdown (GVBD) in vitro. 1720 beta-P was further converted to its 5 beta-reduced form, 1720 beta-dihydroxy-5 beta-pregnan-3-one (1720 beta-P-5 beta), which had lower GVBD activity, suggesting that 5 beta-reduction plays a role in the inactivation of the maturation-inducing ability of 1720 beta-P. In contrast, no 5 beta-reduced metabolite of 20 beta-S was found. Serum levels of 1720 beta-P and 20 beta-S, measured by ELISA, showed that circulating levels of both progestins increased during FOM, and 20 beta-S levels were approximately twice as high as 1720 beta-P levels. This study clarified the complete steroidogenesis pathway during FOM in red seabream ovarian follicles and showed that two 20 beta-hydroxylated progestins, 1720 beta-P and 20 beta-S, act as maturation-inducing hormones in this species. The catabolites of these two progestins and their physiological roles in reproduction are also discussed.

Three GnRH systems in the brain and pituitary of a pleuronectiform fish, the barfin flounder Verasper moseri.

To clarify the possible function of gonadotropin-releasing hormone (GnRH) in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri, the distribution of three forms of GnRH in various areas of the brain was examined by radioimmunoassay, and the localization of GnRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary was determined by immunocytochemistry. The dominant form in the pituitary was seabream GnRH (sbGnRH), levels of which were much higher than those of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In contrast, sbGnRH levels were extremely low in all other brain areas examined. Levels of sGnRH and cGnRH-II were high in the anterior and posterior part of the brain, respectively. sbGnRH-ir cell bodies were located in the preoptic area, whereas sbGnRH-ir fibers were localized mainly in the preoptic area-hypothalamus-pituitary and formed a distinctive bundle of axons projecting to the pituitary. sGnRH-ir cell bodies were located in the ventromedial part of the rostral olfactory bulbs and in the terminal nerve ganglion (the transitional area between the olfactory bulb and the telencephalon). cGnRH-II-ir cell bodies were localized to the midbrain tegmentum. sGnRH-ir and cGnRH-II-ir fibers were observed throughout the brain except in the pituitary gland. These results indicate that sbGnRH is responsible for the neural control of the reproductive endocrinology of the barfin flounder (hypothalamo-hypophysial system), and that sGnRH and cGnRH-II function as neurotransmitters or neuromodulators in the brain.

Maturational changes in brain contents of salmon GnRH in rainbow trout as measured by a newly developed time-resolved fluoroimmunoassay.

A newly developed time-resolved fluoroimmunoassay (TR-FIA) for salmon gonadotropin-releasing hormone (sGnRH) was applied to investigate changes in sGnRH content in discrete brain areas at three different gonadal stages in the rainbow trout, Oncorhynchus mykiss. The sensitivity (6.8 pg/well), specificity, intraassay coefficients of variation (<7.4%), and interassay coefficients of variation (<10.3%) of the assay system were almost the same as those for the radioimmunoassay. Displacement curves of serially diluted brain extracts of nine teleost fish (freshwater fish and seawater fish) including rainbow trout paralleled that of the sGnRH standard, indicating that the sGnRH TR-FIA is widely applicable to the measurement of the brain sGnRH contents of various fishes. The sGnRH content in female hypothalamus decreased during final gonad maturation, whereas the sGnRH levels in pituitary were highest at the time of spermiating in males or ovulating in females, decreasing significantly thereafter. In contrast, there were no changes in the sGnRH contents of olfactory bulbs, telencephalon, optic tectum + thalamus, and cerebellum + medulla oblongata during final maturation, except for olfactory bulbs of males. Changes in sGnRH contents in the hypothalamus and the pituitary indicate that sGnRH is involved in final maturation (ovulation or spermiation) in the rainbow trout.