GTH Subunit mRNAs Research Articles

Masculinization in juvenile longtooth grouper, Epinephelus bruneus, with aromatase inhibitor: changes in GtH subunit mRNA expression and steroids hormone levels

We investigated the effects of fadrozol, an aromatase inhibitor (AI), and 17α-methyltestosterone (MT) on the induction of sex change in juvenile longtooth grouper Epinephelus bruneus, via histological observation of gonads. Changes in the mRNA expression of GtH subunits (FSH-β and LH-β) in the pituitary, and estradiol-17β (E2) and 11-ketotestosterone (11-KT) levels in the blood were also surveyed after AI and MT treatment. Juvenile longtooth groupers (113±17 g body weight; 16.2±1.2 cm body length) received intramuscular injections of AI at 3 (3-AI) and 5 (5-AI) mg/kg BW doses and MT at a 5 mg/kg BW (5-MT) dose. At week 7 post-injection, 3-AI and 5-MT oocytes were degenerated, and gonads of the 5-AI group initiated spermatogenesis. At week 21 post-injection, 3-AI- and 5-MT-treated gonads contained spermatogonia and spermatocytes, while 5-AI treatment induced advanced stages of spermatogenesis. The serum E2 level showed no significant differences throughout the experimental period, whereas that of 11-KT was significantly elevated in the 5-AI group at weeks 7 and 21 post-injection. A significant increase in the expression of FSH-β mRNA was evident in the 5-AI group at week 21 post-injection. In contrast, LH-β mRNA expression did not significantly differ among groups during the experimental period. These results imply that sex change has two stages in the longtooth grouper. In the first stage, oocytes are degenerated by the stimulation by 11-KT, and in the second stage spermatogenesis occurs, owing to the co-effects of 11-KT and FSH-β.

Expression of three gonadotropin subunits and gonadotropin receptor mRNA during male-to-female sex change in the cinnamon clownfish, Amphiprion melanopus

To quantify the sex-change progression from male to female in the cinnamon clownfish, Amphiprion melanopus, we divided gonadal development into three stages (I, mature male; II, male at 90days after removal of the female; and III, mature female), and the expression of GTH subunits and GTH receptors during each of these stages was investigated. The mRNA of the three GTH subunits and their receptors increased with progression from male to female. To understand the effect of gonadotropin-releasing hormone (GnRH) on this progression, we examined expression of genes encoding the GTH subunit mRNA in the pituitary and the GTH-receptor mRNA in the gonads in addition to investigating changes in plasma E2 levels after GnRH analogue (GnRHa) injection. GnRHa treatment increased mRNA expression levels of these genes, as well as plasma E2 levels, indicating that GnRH plays an important regulatory role in the brain-pituitary-gonad axis of immature cinnamon clownfish.

Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy, Acanthopagrus schlegeli: Effects of estradiol-17β on mRNA expression profiles

The cDNAs of three gonadotropin (GTH) subunits (GTHalpha, FSHbeta, and LHbeta) and two GTH receptors (FSHR and LHR) from pituitary and gonads of black porgy were cloned. The nucleotide sequences of the GTHalpha, FSHbeta, and LHbeta cDNA were 354, 363, and 414 base pairs (bps) in length with open reading frames (ORF) encoding peptides of 117, 120, and 137 amino acids, respectively. The FSHR and LHR cDNA was 2118 and 2076 bps in length with ORFs encoding peptides of 705 and 691 amino acids, respectively. To study the mechanism of the estradiol-17beta (E(2)) action, we examined the expression pattern of GTH subunit mRNAs in pituitary and GTH-receptor mRNAs in gonads, and the changes of plasma E(2) level when E(2) treatment was applied to immature black porgy. E(2) treatment increased mRNA expression levels of the genes and plasma E(2) levels, indicating that E(2) stimulated the increases in GTH subunit and GTH-receptor mRNAs. These data indicate that E(2) plays an important regulatory role in the brain-pituitary-gonad axis of immature black porgy. We provide the molecular characterization and expression of the GTH subunits and GTH receptors during sex change in the protandrous black porgy.

Reproductive stage-related effects of salmon GnRH and sex steroid hormones on expression of genes encoding fushi tarazu factor 1 homolog and estrogen receptor α in masu salmon pituitary cells in vitro

Expression of genes encoding gonadotropin (GTH) subunits in the salmon pituitary was regulated by salmon gonadotropin-releasing hormone (sGnRH) and sex steroid hormones in a reproductive stage-dependent manner, probably through DNA-binding transcription factors. Direct effects of these hormones on expression of genes encoding salmon fushi tarazu factor 1 homolog (sFF1-I) and estrogen receptor α (ERα) were therefore examined by use of primary pituitary cell cultures of masu salmon at different reproductive stages. Pituitaries were collected in March (before initiation of gonadal maturation), in May (early maturing), in July (late maturing), and in September (spawning period). Amounts of sFF1-I and ERα mRNAs in the pituitary cells were determined by real-time polymerase chain reactions after a treatment with sGnRH, estradiol-17β (E2), testosterone (T) or 11-ketotestosterone (11KT). The amounts of sFF1-I mRNA were elevated by E2 in the males, and by sGnRH and T in the females before initiation of gonadal maturation and at the early maturing stage. The amounts of ERα mRNA in the early maturing females were elevated by sGnRH. Effects of sGnRH were not significant at the late maturing and spawning stages. The amounts of ERα mRNA in the spawning males were halved by 11KT and E2, and those of sFF1-I and ERα mRNAs in the late maturing females were decreased by T and 11KT. These results indicated that responsiveness of sFF1-I and ERα genes to sGnRH and sex steroid hormones is seasonally variable in relation to reproductive stages. Expression of sFF1 and ERα genes should be stimulated at the early stages of gonadal maturation prior to increases in the amounts of GTH subunit mRNAs, while attenuated after the late maturing period when stored amounts of GTH subunit mRNAs reached near the maximum.

Development of quantitative real-time PCR assays for fathead minnow ( Pimephales promelas) gonadotropin β subunit mRNAs to support endocrine disruptor research

Fathead minnows ( Pimephales promelas) are a widely-used small fish model for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). Quantitative real-time PCR assays for measuring fathead minnow gonadotropin (GtH) β subunit transcripts were developed and “baseline” transcript levels in pituitary tissue were examined over a range of age classes and spawning states. Among females, GtHβ transcripts did not vary significantly with gonadal–somatic index or gonad stage. However, in males, follicle-stimulating hormone β subunit transcripts decreased significantly with increasing gonad stage, while mean luteinizing hormone β subunit expression trended in the opposite direction. GtHβ transcript levels measured in pituitaries from fish that had spawned within the preceding 24 h were not significantly different from those from fish that were 2–3 days post-spawn. Exposure to the fungicide ketoconazole, a known steroidogenesis inhibitor, for 21 days significantly affected the abundance of GtHβ transcripts in pituitary tissue in males, but not females. This study provides critical data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression. Results of such experiments should facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain–pituitary–gonadal axis to stressors including EDCs.

Effects of insulin-like growth factor I on GnRH-induced gonadotropin subunit gene expressions in masu salmon pituitary cells at different stages of sexual maturation

Effects of insulin-like growth factor I (IGF-I) and salmon gonadotropin-releasing hormone (sGnRH) on expression of gonadotropin (GTH) subunit genes were examined using primary pituitary cell cultures of masu salmon ( Oncorhynchus masou). Fishes were assessed at three reproductive stages, i.e., in April (early maturation), in June (maturing), and in September (spawning). Amounts of GTH subunit mRNAs in pituitary cells were determined using real-time PCR after incubation with IGF-I and/or sGnRH. IGF-I alone had almost no effects on three GTH subunit mRNAs in both sexes, except for decrease in follicle-stimulating hormone (FSH) β mRNA in males in June. sGnRH alone was effective in stimulation of FSHβ and luteinizing hormone (LH) β gene expression in males in April. Thereafter it had no significant effects on GTH subunit mRNAs, although in September it tended to increase FSHβ and LHβ mRNAs in females. Co-administered IGF-I counteracted the sGnRH-induced expression of FSHβ and LHβ genes in males in April, but not in females in September. These results suggest that IGF-I is involved in direct regulation of GTH subunit genes during sexual maturation. In particular, IGF-I differently modulates sGnRH-induced GTH subunit gene expression, depending on reproductive stages.

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.

Molecular characterization of rockfish ( Sebastes schlegeli) gonadotropin subunits and their mRNA expression profiles during oogenesis

In an attempt to understand the reproductive regulation in viviparous teleosts, gonadotropin (GTH) subunit cDNAs were characterized and the expression levels of GTH subunit mRNAs in the pituitary glands of the rockfish, Sebastes schlegeli (Scorpaeniformes, Scorpaenidae), were examined by Northern blot analysis. The complete sequences of rockfish GTH subunits (GTHα, FSHβ, and LHβ) were determined by rapid amplification of cDNA ends (RACE) and nucleotide sequencing. Based on the RACE analysis, the cDNAs of GTHα, FSHβ, and LHβ consisted of 655, 540, and 529 nucleotides encoding peptides of 132, 130, and 143 amino acids, respectively. The mature peptides of rockfish FSHβ, LHβ, and common GTHα showed high sequence identities (FSHβ, 58–62%; LHβ, 86–94%; and GTHα, 87–88%) to those of other perciforme (e.g., orange spotted grouper, red seabream, stripped bass, and sea bass). In a sequence alignment of the mature peptides, rockfish FSHβ exhibited a unique feature, the lack of a conserved N-glycosylation site. This is in contrast to both LHβ and GTHα which contain one and two N-glycosylation sites, respectively, and is consistent with those of other teleosts. The mRNA levels of the GTHα subunit increased at the vitellogenic stage and remained steady from ovulation to post-parturition. FSHβ mRNA levels increased abruptly during the vitellogenic stages and then decreased during ovulation, embryonic development, and post-parturition. LHβ mRNA levels were observed to rapidly increase during the vitellogenic stage, reached its highest levels during ovulation and was then followed by a decrease at post-parturition. These results suggest that in the female rockfish FSH and LH syntheses are predominant during vitellogenesis and oocyte maturation, respectively.

Synergistic effects of salmon gonadotropin-releasing hormone and estradiol-17β on gonadotropin subunit gene expression and release in masu salmon pituitary cells in vitro

Effects of salmon gonadotropin-releasing hormone (sGnRH) and estradiol-17β (E2) on gene expression and release of gonadotropins (GTHs) were examined in masu salmon (Oncorhynchus masou) using primary pituitary cell cultures at three reproductive stages, initiation of sexual maturation in May, pre-spawning in July, and spawning in September. Amounts of GTH subunit mRNAs were determined by real-time polymerase chain reaction, and levels of GTH released in the medium were determined by RIA. In control cells, the amounts of three GTH subunit mRNAs (α2, FSHβ, and LHβ) peaked in July prior to spawning. FSH release spontaneously increased with gonadal maturation and peaked in September, whereas LH release remained low until July and extensively increased in September. Addition of E2 to the culture extensively increased the amounts of LHβ mRNA in May and July in both sexes. It also increased the α2 mRNA in July in the females. In contrast, sGnRH alone did not have any significant effects on the amounts of three GTH subunit mRNAs at all stages, except for the elevation of α2 and FSHβ mRNAs in July in the females. Nevertheless, synergistic effects by sGnRH and E2 were evident for all three GTH subunit mRNAs. In May, sGnRH in combination with E2 synergistically increased the amounts of LHβ mRNA in the males and α2 mRNA in the females. However, in July the combination suppressed the amounts of α2 and FSHβ mRNAs in the females. sGnRH alone stimulated LH release at all stages in both sexes, and the release was synergistically enhanced by E2. Synergistic stimulation of FSH release was also observed in May and July in both sexes. These results indicate that a functional interaction of sGnRH with E2 is differently involved in synthesis and release of GTH. The synergistic interaction modulates GTH synthesis differentially, depending on subunit, stage, and gender, whereas it potentiates the activity of GnRH to release GTH in any situation.

Molecular characterization of LH-β and FSH-β subunits and their regulation by estrogen in the goldfish pituitary

The gonadal steroids, along with gonadotropin-releasing hormone (GnRH) are involved in the regulation of gonadotropin (GtH) production in vertebrates. Goldfish have an annual reproductive cycle, characterized by seasonal fluctuations in the circulating levels of the reproductive hormones, including 17β-estradiol (E2). The purpose of the present study was to investigate the effect of E2 on basal and GnRH-induced GtH subunit (α, FSH-β and LH-β) gene expression in the goldfish pituitary. Northern analyses were performed to determine changes in steady state mRNA levels. Both in vivo and in vitro treatment with E2 resulted in a stimulation of all three GtH subunit mRNA levels, although a higher concentration was required for the stimulation of the FSH-β subunit mRNA levels. The effect of E2 on GnRH-induced GtH mRNA level was also investigated and demonstrated that E2 influences the GnRH-induced GtH subunit mRNA levels in a seasonally dependent manner. Overall, the present results indicate that E2 stimulates GtH subunit mRNA levels directly at the level of the pituitary in a seasonally dependent manner in goldfish.

Molecular cloning of the cDNA encoding the β subunit of thyrotropin and regulation of its gene expression by thyroid hormones in the goldfish, Carassius auratus

A cDNA encoding the β subunit of thyrotropin (TSH) was isolated from a goldfish (Carassius auratus) pituitary gland cDNA library. By comparing the sequence with other teleost TSHβs, a signal peptide of 19 amino acids and a mature hormone of 131 amino acids were predicted for goldfish TSHβ subunits. The resulting putative mature hormone of 131 amino acids had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 51–67% with TSHβs from other teleosts, 38–43% with tetrapod TSHβs, but only 27 and 29% with goldfish GTH-Iβ and -IIβ, respectively. We also examined the effects of thyroid hormones (TH) and thiourea (TU, an inhibitor of TH production) treatments on TSH and GTH subunit gene expressions in the goldfish pituitary gland. After thyroxine (T4) treatment, circulating T4 concentration increased and TSHβ mRNA level decreased. Supressing the amount of circulating T4 and triiodothyronine (T3) by TU treatment increased the TSHβ mRNA level. Moreover, T4 replacement therapy (simultaneous treatment of both TU and T4) caused a high level of circulating T4 and a low level of circulating T3, and a decrease in the TSHβ mRNA level. Thus, changing levels of circulating TH exert a negative feedback on the level of TSH β subunit mRNA in goldfish in vivo. On the other hand, GTH subunit mRNA levels were not affected by changes in the levels of circulating TH.

GnRH Analog Stimulates Gonadotropin II Gene Expression in Maturing Sockeye Salmon

Our previous study suggested that, in the pituitaries of pre-spawning chum salmon, salmon gonadotropin-releasing hormone (sGnRH) stimulates expression of genes for gonadotropin (GTH) IIβ but not for Iβ, since the levels of mRNAs encoding sGnRH and GTH II but not I were increased during the final stages of spawning migration. In the present study, a capsule of GnRH analog (GnRHa) was implanted into the dorsal muscle of maturing sockeye salmon to clarify function of GnRH on expression of GTH subunit genes in pre-spawning homing salmonids. The amounts of GTH subunit mRNAs in the individual pituitaries were analyzed by a quantitative dot blot analysis using single-stranded sense DNA as the standard. The levels of GTH α and IIβ mRNAs in the GnRHa-implanted fish were significantly higher than those in the control fish in both the males and females, whereas the levels of GTH Iβ mRNA did not show any significant differences in both sexes. These results indicate that GnRH elevates expression of GTH subunit genes which encode the components of GTH II, α and IIβ chains, in the pituitary of maturing sockeye salmon, and then accelerates final maturation.

Changes in the Levels of Gonadotropin Subunit mRNAs in the Pituitary of Pre-Spawning Chum Salmon

We only have little information on expression of gonadotropin (GTH) subunit genes during spawning migration in salmonids. Changes in the levels of mRNAs for GTH subunits (GTH α2, Iβ and IIβ) were therefore analyzed in the pituitaries of chum salmon (Oncorhynchus keta) during the final stages of spawning migration to the Ishikari river. The fish were caught at Atsuta, a fisherman's village facing the Ishikari bay, and at Chitose, a tributary of the Ishikari river, in 1993 and 1994. The former is referred to as seawater (SW) fish, and the latter as freshwater (FW) fish. The levels of GTH subunit mRNAs in the pituitaries were determined by a quantitative dot blot analysis, using single-stranded sense DNA as the standard. The sense DNAs have the same nucleic acid sequences of mRNAs. The level of GTH α2 mRNA in the FW males was higher than that in the SW ones. Similar tendency was seen in the females. No significant changes were observed in the levels of GTH Iβ mRNA in both the males and females. Whereas, the level of GTH IIβ mRNA in the FW fish was higher than that in the SW fish regardless of sexes in 1994. Although not statistically significant in the males, similar tendency was seen in the 1993 fish. The present study thus showed that the level of GTH IIβ mRNA was increased concomitantly with that of GTH a2 mRNA during the final stages of spawning migration.

Gonadotropin-I and -II subunit gene expression of male striped bass (Morone saxatilis) after gonadotropin-releasing hormone analogue injection: quantitation using an optimized ribonuclease protection assay.

In fish, both gonadotropin (GtH)-I and -II are involved in the spermatogenic process, but the differential regulation of these hormones by GnRH is still poorly understood. To gain further insight into the GnRH regulation of GtH-I and -II gene expression in the male striped bass, we have developed and optimized a ribonuclease protection assay for the simultaneous measurement of all GtH subunit mRNAs in a single pituitary gland. The RNA extraction protocol enables the determination of GtH protein content in the same sample, thus enhancing the power of the method. Maturing striped bass males were injected intramuscularly with [D-Ala6,Pro9Net]-LHRH (GnRHa) and sampled at 6 and 24 h postinjection. The mRNA levels of the alpha subunit and GtH-IIbeta increased after 6 h (4- and 6-fold, respectively), while the GtH-Ibeta mRNA levels increased only 2-fold after 24 h. Interestingly, GnRHa stimulation caused a significant increase in beta-actin mRNA levels. GnRHa treatment also resulted in a 2-fold decrease in pituitary GtH-II content, associated with a dramatic increase of plasma GtH-II levels from undetectable levels (< 0.2 ng/ml) to 13+/-2 ng/ml after 6 h. These results demonstrate that both GtH-Ibeta and -Ilbeta are expressed during striped bass spermatogenesis and that the two genes are subjected to differential regulation by GnRHa.

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 β and II β 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.