Gonadotropins in the Manchurian trout, Brachymystax lenok tsinlingensis

cDNA clones encoding gonadotropin (GTH) α, follicle-stimulating hormone (FSH) β and luteinzing hormone (LH) β were isolated from the pituitaries of maturing Manchurian trout (Brachymystax lenok tsinlingensis) and sequenced. The deduced amino acid sequences of GTH subunits showed high identities to masu salmon, Oncorhynchus masou: GTHα1 (95%), FSHβ (92%) and LHβ (97%), respectively. We are also attempting to produce recombinant FSH and LH using a eukaryotic expression system. In a pilot experiment, LH was secreted to the culture medium at 48 and 60 hrs after transfection. The results will be helpful to develop controlled reproduction of exterminating Manchurian trout.     

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 <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   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.     

Influence of estradiol-17β, testosterone, and 11-ketotestosterone on testicular development, serum steroid hormone, and gonadotropin secretion in male red sea bream Pagrus major

ABSTRACT:   In order to investigate the influence of estrogen and androgen on reproductive activities of male teleosts, male red sea bream were implanted with silicone capsules containing estradiol-17β (E2), testosterone (T) or 11-ketotestosterone (11-KT) in immature and early spermatogenic stages. One month after implantation of either E2 or T, the gonadosomatic index decreased in accordance with testicular regression in both stages. Implantation of E2 decreased circulating 11-KT levels but did not affect gonadotropin (GTH) subunits, follicle stimulating hormone-β (FSHβ), luteinizing hormone-β (LHβ), α glycoprotein subunit (αGSU) gene expression, and serum LH levels in both stages. Alternatively, T decreased serum 11-KT and LH levels, and FSHβ and LHβ mRNA levels in the early spermatogenic stage but not in the immature stage. These results suggest E2 may directly inhibit testicular development through the suppression of 11-KT production. Meanwhile, T may decrease serum 11-KT levels through the suppression of FSH and LH secretion, resulting to inhibition of testicular development in the early spermatogenic stage. Treatment with 11-KT did not affect the testis in either stage, whereas 11-KT increased LHβ and αGSU mRNA levels in immature, and decreased FSHβ mRNA levels in the early spermatogenic stage. These results suggest that 11-KT may have different effects on GTH subunit gene expression in each reproductive stage.     

Changes in the immunostaining intensities of follicle-stimulating hormone and luteinizing hormone during ovarian maturation in the female Japanese flounder

The role of gonadotropin (GTH) in the reproduction of the Japanese flounder, Paralichthys olivaceus, was studied by assessing the changes in the apparent activity of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary gland during gonadal maturation by immunohistochemical analyses. Corresponding changes in plasma levels of testosterone (T), estradiol-17β (E₂), and 17α,20β-dihydroxy-4–pregnen-3-one (DHP) were also studied. Reared fish at the early spawning to termination stages were sampled from May to August and wild fish at the previtellogenic to termination stages were caught at 3- to 4-week intervals between April and September offshore from the northern mainland of Japan by gill nets. The gonadosomatic index of the reared fish decreased from the early spawning stage to the termination stage, while that of the wild fish increased significantly from the previtellogenic stage to the early spawning stage and decreased thereafter. In the reared fish, the immunostaining intensities of FSH and LH were high during the spawning period, accompanied by high plasma levels of T, E₂, and DHP. In the wild fish, the immunostaining intensities of FSH and LH were low during the previtellogenic stage but increased during the maturing and spawning stages. These results indicate that both FSH and LH are likely associated with oocyte maturation in the Japanese flounder.     

Gonadotropins, gonadotropin receptors and their expressions during sexual maturation in yellowtail, a carangid fish

To study the physiological roles of gonadotropins (GtHs) in the yellowtail, the cDNAs encoding each GtH subunit (GPHα, FSHβ and LHβ) and their receptors (FSHR and LHR) were isolated from the pituitary gland and gonads using the polymerase chain reaction (PCR). In addition, thyrotropin (TSH) and its receptor (TSHR) cDNAs, were isolated from the pituitary gland, ovary and testis. The changes in the mRNA levels of each subunit were determined at different stages of maturation. The isolated cDNAs of GPHα, FSHβ, LHβ and TSHβ were 662, 545, 595 and 879 bp long, respectively. The amino acid sequence identity of the yellowtail GPHα, FSHβ, LHβ and TSHβ subunits was 85–63, 68–33, 93–65 and 74–46%, respectively, as compared with other fish species. Northern blot analysis showed that GPHα and FSHβ were strongly expressed in pituitary at the early vitellogenic stage and during spermatogenesis, whereas LHβ was expressed significantly in the late vitellogenic stage, and in both spermatogenesis and spermiation. Full-length cDNAs encoding FSHR, LHR, and TSHR were obtained from the testes and ovaries. The FSHR, LHR and TSHR cDNA encoded a protein of 680, 702 and 778 amino acids, and showed the highest identity with tilapia FSHR (76%), tilapia LHR (84%) and striped bass TSHR (94%), respectively. Northern blot analyses indicated that all of these receptors are expressed differently at different stages in the ovaries and testes.     

Molecular cloning of the three gonadotropin subunits and early expression of FSHβ during sex differentiation in the nile tilapia, Oreochromis niloticus

Gonadotropin (GTH)α, FSHβ and LHβ cDNAs were cloned from the Nile tilapia. Northern blot analysis detected a single band for each subunit. Preliminary studies indicate that FSHβ is expressed as early as 0 days after hatching (dah) in the fish pituitaries.     

Immunohistochemical changes in production of pituitary hormones during artificial maturation of female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   Specific antibodies against follicle-stimulating hormone β subunit (FSHβ), prolactin (PRL), and somatolactin (SL) of the Japanese eel Anguilla japonica were produced. These antibodies, as well as antibodies against luteinizing hormone β subunit (LHβ) and growth hormone (GH) produced previously, were used to examine changes in the production of pituitary hormones in female eels during maturation induced by salmon pituitary homogenate (SPH) injection. Immunohistochemical observations showed a decrease in FSH production after SPH injection, suggesting that SPH inhibits FSH production. In contrast, LH production increased markedly with maturation. The number of GH producing cells decreased gradually during maturation, possibly because of inhibition by exogenous GH present in the SPH and/or endogenous insulin-like growth factor-I produced by the stimulation of salmon GH. Although changes in the number of PRL producing cells with maturation were not evident, the number of SL producing cells showed a peak at the late vitellogenic stages, and thereafter decreased to the migratory nucleus stage. These results suggest that GH and SL are involved in sexual maturation in SPH injected eels, as in other fishes.     

Modulation of glycoprotein hormone α- and gonadotropin IIβ-subunit mRNA levels in the pituitary gland of mature male African catfish, Clarias gariepinus

The cDNAs encoding the glycoprotein hormone -subunit (GP) and the gonadotropin II-subunit (GTH II) were cloned from the pituitary gland of the African catfish, Clarias gariepinus. Using RNase protection analysis, we studied the steady-state mRNA levels of GP as well as GTH II in the pituitary gland of adult male catfish. Castration of adult male catfish resulted in a significant decrease of GTH II mRNA levels, whereas there was no change in the GP mRNA levels. Treatment of intact males with a single dose of 11-ketotestosterone (11-KT) resulted in dose-dependent increases in mRNA levels of both GP and GTH II. We conclude that 11-KT, a prominent, non-aromatizable teleost androgen, has a stimulatory effect on the pituitary mRNA levels of GP and GTH II of adult male fish.     

Immunoreactive changes in pituitary FSH and LH cells during seasonal reproductive and spawning cycles of female chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis>

The physiological functions of pituitary gonadotropins (GtHs) are well established in higher vertebrates, whereas those in teleosts are still poorly understood. To describe the role of GtHs during gonadal development of female chub mackerel Scomber japonicus, changes in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were investigated immunohistochemically during the seasonal reproductive and spawning cycles. FSH and LH cells were identified in the different cell types of the proximal pars distalis (PPD); FSH cells were located in the central PPD, whereas LH cells were localized along the border of the pars intermedia. To examine changes in FSH and LH cells, the percentage of FSH or LH cell-occupying area in the PPD was evaluated and represented as FSHβ-immunoreactive (ir) or LHβ-ir levels, respectively. FSHβ-ir levels increased significantly from immature to the completion of vitellogenesis, whereas LHβ-ir levels were maintained at high levels from early vitellogenesis to post-spawning. During the spawning cycle, which consisted of four stages from just after spawning to the next oocyte maturation, both FSHβ-ir and LHβ-ir levels showed no significant changes among different stages; however, LHβ-ir levels remained relatively high, and FSHβ-ir levels were constantly low. These results suggest that both FSH and LH may be involved in vitellogenesis and LH may act at final oocyte maturation in female chub mackerel, although the role of FSH during the spawning cycle is still unclear.     

Production of biologically-active recombinant goldfish gonadotropins in transgenic rainbow trout

The feasibility of using rainbow trout Oncorhynchus mykiss embryos as an expression system for proteins was investigated. For model proteins, we selected two goldfish gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). To produce single-chain goldfish FSH (scgfFSH) and LH (scgfLH), cDNAs encoding glycoprotein hormone (GP) α and FSHβ were fused in tandem, and cDNAs encoding GPα and LHβ were fused in tandem. The fused cDNAs were ligated with β-actin promoter, and microinjected into fertilized rainbow trout eggs. After 4-days incubation, the embryos were subjected to western blotting and in vitro bioassays. The recombinant proteins produced by the embryos were immunoreactive to antisera against goldfish GPα, N-glycosylated, and biologically active. We conclude that scgfFSH and scgfLH were successfully produced in transgenic rainbow trout.     

The duality of teleost gonadotropins

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.     

Gonadotropin-releasing hormone and gonadotropin in goldfish and masu salmon

Reproductive activities in vertebrates are regulated by an endocrine system, consisting of the brain-pituitary-gonad axis. In teleosts, gonadotropin-releasing hormone (GnRH) in the brain stimulates gonadotropin (GTH) release in the pituitary gland, but because of lack of the portal vessel, it is not known when and how much GnRH is released for the regulation of GTH release. There are multiple molecular types of GnRH in teleosts and several distinct populations of GnRH neurons in the brain. However, we do not know which types and populations of GnRH neurons regulate reproductive activities. Here we summarize our recent studies on GnRH and GTH in masu salmon Oncorhynchus masou and goldfish Carassius auratus. Immunocytochemistry showed the location and molecular types of GnRH neurons. Salmon (sGnRH) and chicken-II GnRH (cGnRH-II) neuronal fibers were widely distributed in the brain of both masu salmon and goldfish. Only sGnRH fibers were observed in the pituitary of masu salmon, whereas both sGnRH and cGnRH-II fibers were observed in the goldfish pituitary, indicating that species specific GnRH profiles are involved in the regulation of pituitary function in teleosts. A series of experiments in masu salmon and goldfish suggest that among GnRH neuron populations GnRH neurons in the ventral telencephalon and the hypothalamus regulate GTH release, and that GnRH of the terminal nerve origin is not essential to gonadal maturation and ovulation. The biological function of other GnRH neurons remains unkown. Two GTHs appear to be characteristic of teleost; however, regulation of reproduction by these GTHs is a question that remains to be elucidated. In salmonid species, it is proposed that GTH I stimulates early gonadal development, whereas GTH II acts in later stages. When GTH expression was examined in goldfish, both GTH I and II mRNA levels in the pituitary gland showed increases in accordance with gonadal development, unlike the sequential expression of GTH subunits in salmonids. The expression of these GTH subunit mRNAs were affected by water temperature, starvation, and steroid hormones in goldfish, but in what manner these two GTHs regulate gonadal development remains to be clarified.     

Effects of recombinant vertebrate ancient long opsin on reproduction in goldfish, <Emphasis Type="Italic">Carassius auratus</Emphasis>: profiling green-wavelength light

This study was conducted to identify the possible effect of recombinant vertebrate ancient long (VAL) opsin as a non-visual “photoreceptor” in the deep brain of goldfish, Carassius auratus. In addition, we investigated the effects of green-wavelength light on the predictable reproductive function of VAL-opsin as a green-sensitive pigment in the deep brain. To determine this, we quantified changes in gonadotropin hormone (GTH) [GTHα, follicle stimulating hormone (FSH) and luteinizing hormone (LH)] and estrogen receptor (ER; ERα and ERβ) mRNA expression levels associated with goldfish reproduction as well as changes in plasma FSH, LH, and 17β-estradiol (E₂) activities after injection of recombinant VAL-opsin protein in two concentrations (0.1 or 0.5 μg/g body mass) for 4 weeks (injection once weekly) and examined the possible impact of green-wavelength light (500, 520, and 540 nm) on the function of VAL-opsin. As a result, all parameters associated with reproduction significantly increased with time and light-emitting diode (LED) exposure. Based on these results, we suggested that VAL-opsin in the deep brain is involved in goldfish maturation, and it is possible that green-wavelength light improves the ability of VAL-opsin to promote maturation by increasing VAL-opsin expression.     

Effects of gonadotropin-releasing hormone agonist and testosterone on gonadotropin subunit gene expression in female red seabream (Pagrus major)

We examined the effects of gonadotropin-releasing hormone agonist (GnRHa) and testosterone (T) on the level of gonadotropin subunit mRNAs in the pituitary of ovariectomized or intact female red seabream. Ovariectomy induced increase of seabream (sb) GnRH, glycoprotein (GP) α and luteinizing hormone (LH) β mRNA levels. GnRHa treatment also stimulated GPα and LHβ mRNA levels. T treatment reduced GPα and LHβ mRNA expression probably via negative feedback action on sbGnRH. Both GnRHa and T treatment had no effect on follicle-stimulating hormone (FSH) β mRNA levels. These results suggest that the regulatory mechanisms of GPα and LHβ gene expression differ from those of FSHβ gene.     

Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder

ABSTRACT:   To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus , localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic–hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.     

Production of recombinant goldfish gonadotropins by baculovirus in silkworm larvae

Recombinant gonadotropins (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of goldfish Carassius auratus were produced by baculovirus in silkworm larvae. Hemolymph containing recombinant FSH (rFSH) or LH (rLH) was collected from silkworm larvae, and its biological activity was examined in vivo using male goldfish and female bitterling Rhodeus ocellatus ocellatus. Injection of hemolymph containing rFSH or rLH induced milt production in male goldfish, whereas only rLH induced ovulation in female bitterling. These results suggest, that biologically active goldfish recombinant GTH could be applied for the induction of gonadal development in aquaculture fishes as a substitute of pituitary extract, if a method of large scale production is established.     

Molecular cloning and expression of salmon pituitary hormones

A cDNA library was prepared from chinook salmon pituitaries. Growth hormone (GH), prolactin (PRL) and the β subunit of gonadotropin (GTH) genes were screened using synthetic oligonucleotides as probes. Full size cDNA clones coding for these polypeptide hormones were isolated and characterized. The cDNA sequences for PRL and βGTH have been reported earlier from our laboratories. The cDNA clone for GH contains 1148 bp and codes for a preGH of 210 amino acids. The chinook salmon GH, reported in the present investigation, differs from chum salmon GH in only 1 amino acid, and from coho salmon GH in 5 amino acids. Plasmids containing modified nucleotide sequences coding for GH, PRL and βGTH were constructed individually into an expression vector using the heat-inducible λ pL promotor. Mature PRL, GH and unglycosylated βGTH were expressed in the bacteria at elevated temperature.