Elevated temperature impairs gonadal development by suppressing the expression of the genes for kisspeptin, GnRH1 and GTH subunits in Nile tilapia Oreochromis niloticus.

Temperature is a preeminent factor in the regulation of fish reproduction and hinders gonadal development beyond a specific threshold. To comprehend the molecular mechanism responsible for reproductive suppression at different temperature, expression of the genes encoding kisspeptin (kiss2), gonadotropin-releasing hormone (gnrh1) and their receptors (gpr54, gnrh1r) in the brain, and the gonadotropin (GTH) subunits (fshb and lhb) in the pituitary were studied in juvenile Nile tilapia (Oreochromis niloticus) along with gonadal histology. Fish were acclimatized to three distinct temperatures, including 31 °C, 34 °C and 37 °C for 14 days. The mRNA levels of kiss2, gpr54, gnrh1, and gnrh1r were significantly decreased at 37 °C compared to 31 °C and 34 °C in the both sexes. In parallel, the expression level of fshb in the both sexes and lhb in the female were significantly lower at 37 °C in the pituitary. Histologically, the gonads of both sexes had normal growth of gametes at control temperature (31 °C), whereas the spermatogenesis and oocyte maturation were slowed down and atretic oocytes were found in the ovary at 37 °C acclimation temperature. Taken together, the results imply that elevated temperature beyond the specific threshold may have a negative impact on reproduction by suppressing the gene expressions of kisspeptin/GnRH1/GTH system and eventually restrains normal growth and maturation of gametes in the both sexes of Nile tilapia.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Lunar Age-Dependent Oscillations in Expression of the Genes for Kisspeptin, GnIH, and Their Receptors in the Grass Puffer during the Spawning Season.

Grass puffer is a semilunar-synchronized spawner: spawning occurs on beaches only for several days of spring tide around new moon (lunar age 0) and full moon (lunar age 15) every 2 weeks from spring to early summer. To investigate the role of kisspeptin and gonadotropin-inhibitory hormone (GnIH) in the semilunar-synchronized spawning, lunar age-dependent expression of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kissr2), GnIH (gnih), GnIH receptor (gnihr), gonadotropin-releasing hormone 1 (GnRH1) (gnrh1), and three gonadotropin (GTH) subunits (gpa, fshb, lhb) was examined in the male grass puffer, which was kept in an aquarium under natural light condition in a lunar month during the spawning period. In the brain, both kiss2 and kissr2 showed lunar variations with a peak at lunar age 10, while both gnih and gnihr showed semilunar variations with two peaks at lunar age 0 and 20. On the other hand, gnrh1 showed semilunar variation with two peaks at lunar age 0 and 15. In the pituitary, kiss2, kissr2, gnih, and gnihr showed similar variations to those shown in the brain. The fshb and lhb mRNA levels showed semilunar variations with two peaks at lunar age 0 and 15. The present study shows lunar and semilunar oscillations of kiss2/kissr2 and gnih/gnihr expressions, respectively, with their peaks around spring tide in the brain and pituitary along with the semilunar expressions of gnrh1 and the pituitary GTH subunit genes. These results suggest that the lunar age-dependent expressions of the kisspeptin, GnIH, and their receptor genes may be primarily important in the control of the precisely timed semilunar spawning of the grass puffer.

Simulation dataset of thermal and epithermal neutron self-shielding correction factors for 186W(n,γ)187W reaction rate experiments using tungsten foil targets.

In the experiments of neutron interaction with research samples, the incident neutron energy spectrum, distribution inside the irradiating sample volume, is affected by the unexpected neutron self-shielding effects. The nature of these effects is due to the formation and thickness of the irradiating sample, which significantly causes neutron self-absorption and multiple scattering inside the sample volume. The datasets presented in this article showed the thermal (Gth) and epithermal (Gepi) neutron self-shielding correction factors for the 186W(n,γ)187W neutron capture reaction rate in irradiating tungsten (W) foil samples with different thicknesses. The simulations were performed for three models of surface neutron source's geometries and relative orientations of the irradiating foil samples of isotropic cylinder surface neutron source with foil sample along to the center line, isotropic cylinder neutron source with foil sample flat to the center line, and isotropic spherical neutron source with foil sample placed at the center point. The range of sample thicknesses was from 10 µm to 2.5 mm. The uncertainties for each data point are also reported in the data table, making it more convenient for reuse in related experiments or evaluations.

In silico insights into intra- and inter-species interactions of piscine gonadotropin hormones and receptor crosstalk.

In mammals, the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are macromolecules secreted during specific reproductive phases and display strict specificity towards their cognate receptors. However, fish gonadotropins (GTH) and their receptors (GTHR) display diverse species-specific expression patterns, secretion patterns, and intra- and interspecies cross-activation. To uncover the molecular basis of this diversity, we generated and analyzed 29 in-silico models of intra- and inter-species combinations of sturgeon, carp, tilapia, and human gonadotropins with piscine receptors and analyzed the resulting receptor activation and signal transduction of these GTHR-GTH complexes in-vitro. Our results suggest that unlike humans, the surface charge on piscine FSH/LH ß-seatbelt and N107huLHCGR/K104hFSHR homologs does not necessarily determine binding specificity. Instead, sequence and structural variations allow piscine GTHs significant conformational flexibility when binding to the receptor extracellular domain, thereby enabling cross-activation. The resulting diversity may support various reproductive strategies in different environmental niches.

Role of insulin-like growth factor 1 (IGF1) in the regulation of mitochondrial bioenergetics in zebrafish oocytes: lessons from in vivo and in vitro investigations.

Optimal mitochondrial functioning is indispensable for acquiring oocyte competence and meiotic maturation, whilst mitochondrial dysfunction may lead to diminished reproductive potential and impaired fertility. The role of the intra-ovarian IGF system in ovarian follicular dynamics has been implicated earlier. Although several studies have demonstrated the role of the IGF axis in facilitating mitochondrial function over a multitude of cell lines, its role in oocyte energy metabolism remains largely unexplored. Here using zebrafish, the relative importance of IGF1 in modulating oocyte mitochondrial bioenergetics has been investigated. A dramatic increase in ovarian lhcgr and igf1 expression accompanied heightened ATP levels and mitochondrial polarization in full-grown (FG) oocytes resuming meiotic maturation and ovulation in vivo. Concomitant with elevated igf1 expression and IGF1R phosphorylation, hCG (LH analog) stimulation of FG follicles in vitro prompted a sharp increase in NRF-1 and ATP levels, suggesting a positive influence of gonadotropin action on igf1 expression vis-à-vis oocyte bioenergetics. While recombinant IGF1 administration enhanced mitochondrial function, IGF1R immunodepletion or priming with PI3K inhibitor wortmannin could abrogate NRF-1 immunoreactivity, expression of respiratory chain subunits, ΔΨM, and ATP content. Mechanistically, activation of PI3K/Akt signaling in IGF1-treated follicles corroborated well with the rapid phosphorylation of GSK3ß at Ser9 (inactive) followed by PGC-1ß accumulation. While selective inhibition of GSK3ß promoted PGC-1ß, Akt inhibition could abrogate IGF1-induced p-GSK3ß (Ser9) and PGC-1ß immunoreactive protein indicating Akt-mediated GSK3ß inactivation and PGC-1ß stabilization. The IGF1-depleted follicles showed elevated superoxide anions, subdued steroidogenic potential, and attenuated G2-M1 transition. In summary, this study highlights the importance of IGF1 signaling in oocyte bioenergetics prior to resumption of meiosis.

Gonadotropins and Sex Steroid Hormones in Captive-Reared Small Yellow Croaker (Larimichthys polyactis) and Their Role in Female Reproductive Dysfunction.

The seed production of small yellow croaker (SYC) is constrained by reproductive dysfunction in captive-reared females. Reproductive dysfunction is closely linked to endocrine reproductive mechanisms. To better understand the reproductive dysfunction in captive broodstock, functional characterization of gonadotropins (GtHs: follicle stimulating hormone ß subunit, fshß; luteinizing hormone ß subunit, lhß; and glycoprotein α subunit, gpα) and sex steroids (17ß-estradiol, E2; testosterone, T; progesterone; P) was performed using qRT-PCR, ELISA, in vivo, and in-vitro assay. The pituitary GtHs and gonadal steroids levels were significantly higher in ripen fish of both sexes. However, changes in lhß and E2 levels in females were not significant in the developing and ripen stages. Furthermore, GtHs and steroids levels were lower in females compared to males throughout the reproductive cycle. In vivo administration of gonadotropin releasing hormone analogue (GnRHa) significantly increased the expression of GtHs in both dose- and time-related manners. The lower and higher doses of GnRHa led to successful spawning in male and female SYC, respectively. Sex steroids in vitro significantly inhibited the expression of lhß in female SYC. Overall, GtHs were shown to play a vital role in final gonadal maturation, while steroids promoted negative feedback in the regulation of pituitary GtHs. Lower levels of GtHs and steroids might be key components in the reproductive dysfunction of captive-reared female SYC.

Functional Characterization of Three GnRH Isoforms in Small Yellow Croaker Larimichthys polyactis Maintained in Captivity: Special Emphasis on Reproductive Dysfunction.

Fish reproduction is regulated by the brain-pituitary-gonad (BPG) axis where the gonadotropin-releasing hormone (GnRH) plays a central role. Seed production of small yellow croaker (Larimichthys polyactis) is performed using captive-reared broodstock known to undergo reproductive dysfunction, which is connected to endocrinological dysfunction. To determine the endocrinological mechanism of GnRHs in the BPG axis of small yellow croaker, full-length sequences of three GnRH isoforms encoding sbGnRH (GnRH1), cGnRH-II (GnRH2), and sGnRH (GnRH3) were cloned and characterized from brain tissue. qRT-PCR, in vivo, and in vitro experiments were performed for functional characterization. The mRNA expression of GnRH1 in the brain and gonadotropin subunits (GPα, FSHß, and LHß) in the pituitary were significantly higher at the ripen stage during gonadal development and GnRH1 at spawning stage during spawning events. Expression of both GnRH1 and GtH subunits was significantly lower in females than males. GtH subunits were induced at higher concentrations of GnRH1 in vivo and in vitro. Sex-steroids significantly inhibited the GnRH1 expression in vitro in a dose-dependent manner. Taken together, results indicated that GnRH1 plays a key role in gonadal maturation and sex-steroids induced negative feedback in the regulation of GnRH. A lower level of GnRH1 and GtHs might be responsible for reproductive dysfunction in a female small yellow croaker.

Response of Pituitary Cells and Tissues to Neurokinin B and F in the Nile tilapia.

Neurokinin B (NKB) is a neuropeptide involved in the regulation of reproductive endocrine system of vertebrate animals, including fish. However, the pathway of NKB action in fish has not been clearly elucidated. In order to clarify the effect of NKB and NKF (neurokinin F) on gonadotropic hormone (GTH) gene expression in the pituitary, we studied the changes of LHß and FSHß gene expressions by using two different pituitary culture methods (whole pituitary culture or dispersed pituitary cell culture). Pituitaries were removed from mature female and male Nile tilapia. Changes of LHß and FSHß gene expressions were measured and compared after the treatment with NKB or NKF peptides at concentrations 0 to 1,000 nM. Expression of GTH genes in the whole pituitary cultures treated with NKB or NKF peptides did not show significant difference except in female at one concentration when treated with NKF. On the contrary, there were significant changes of GTH gene expressions in the dispersed pituitary cell cultures when treated with NKB and NKF peptides. These results suggest that dispersed pituitary cell culture is more relevant than whole pituitary culture in studying the function of pituitary, and that NKB and NKF could act directly on the pituitary to regulate the expression of GTH genes.

Glyceroltriheptanoate (GTH) occurrence in animal by-products: a monitoring study to minimise safety-related risk of misuse.

Early in this century, the crisis connected to the spread of bovine spongiform encephalopathy caused a great concern related to the use of animal by-products (ABPs). According to the Commission Regulation (EU) No 1069/2009, these materials are classified in three categories according to their related risk. In 2011 Commission Regulation (EU) No 142/2011 established that meat and bone meal (MBM) and fat deriving from ABPs not intended for human consumption (category 1 and 2) are required to be permanently marked with glyceroltriheptanoate (GTH), at a minimum concentration of 250 mg kg-1 of fat, while category 3 processed animal proteins (PAPs) must not contain this compound. PAPs are bio resources, which could be used in a renewable and regenerative way in a circular economy model for a conscious usage of raw materials. The aim of this study was to provide information on GTH occurrence in MBM and, if any, in PAPs. Samples were collected from 2017 to 2021 and analysed by GC-MS. Detected non-compliant samples were exclusively of MBM category 1 and 2, probably due to the addition of an inadequate amount of GTH during the manufacturing processes. These results highlighted the importance of National Monitoring Programs as a useful tool to minimise safety related risk due to the misuse of GTH. Thus, investigating the critical points in feed supply-chain and sharing the information on its occurrence may help to improve animal and human wellness and safety.

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.

Involvement of melatonin in transducing moon-related signals into the reproductive network of the female honeycomb grouper Epinephelus merra.

Most groupers (genus Epinephelus) inhabiting tropical and subtropical waters exhibit lunar-related reproductive cycles. Their gametes develop synchronously toward and are released around the species-selected moon phase. Periodical changes in cues from the moon are likely used as zeitgeber, and the hypothalamic-pituitary-gonadal (HPG) axis may be activated after cues are perceived by the sensory organ and transduced as internal signals. The objective of this study was to examine weekly changes in mRNA expression profiles of gonadotropin-releasing hormones (gnrh1 and gnrh2) and the ß-subunit of gonadotropins (fshß and lhß) during the spawning season (May to June) of the female honeycomb grouper Epinephelus merra, which spawns around the full moon period. When mature females were collected based on the lunar cycle, the gonadosomatic index peaked around the full moon. Ovarian histology revealed that oocytes laden with yolk developed toward the full moon and, subsequently, ovulatory follicles appeared around the last quarter moon, confirming lunar-related spawning with a full moon preference. Real-time quantitative polymerase chain reaction analyses revealed high abundances of fshß and lhß toward the first quarter moon, whereas concentrations of gnrh1 and gnrh2 increased around the last quarter moon and the first quarter moon, respectively, suggesting that transcription levels of these hormones fluctuate with the lunar cycle. The measurement of melatonin in the eye around the new moon and the full moon revealed that the ocular melatonin content was higher around the new moon than around the full moon, suggesting that the honeycomb grouper can perceive changes in moonlight. In addition, implantation of an osmotic pump containing melatonin into the body cavity of E. merra reduced the transcription levels of gonadotropins, suggesting that melatonin negatively affects hormonal synthesis at the HPG axis. We concluded that melatonin plays an essential role in transducing periodical changes in moonlight and that decreases in melatonin levels from the new moon to the full moon activate the HPG axis for entrainment of gonadal development and spawning.

Localization of three forms of gonadotropin-releasing hormone in the brain and pituitary of the self-fertilizing fish, Kryptolebias marmoratus.

The localization of gonadotropin-releasing hormone (GnRH) in the brain and pituitary of the self-fertilizing mangrove killifish Kryptolebias marmoratus was examined by immunohistochemistry and in situ hybridization to understand its neuroendocrine system. The genome assembly of K. marmoratus did not have any sequence encoding GnRH1, but sequences encoding GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH) were found. Therefore, GnRH1 was identified by in silico cloning. The deduced amino acid sequence of the K. marmoratus GnRH1 (mature peptide) was identical to that of the medaka GnRH. GnRH1 neurons were detected in the ventral part of the preoptic nucleus by immunohistochemistry and in situ hybridization, and GnRH1-immunoreactive (ir) fibers were observed throughout the brain. GnRH1-ir fibers were in close contact with luteinizing hormone (LH)-ir cells in the pituitary using double immunohistochemistry. GnRH2 neurons were detected in the midbrain tegmentum by immunohistochemistry and in situ hybridization. Although GnRH2-ir fibers were observed throughout the brain, they were not detected in the pituitary. GnRH3 neurons were detected in the lateral part of the ventral telencephalic area by both methods. GnRH3-ir fibers were observed throughout the brain, and a few GnRH3-ir fibers were in close contact with LH-ir cells in the pituitary. These results indicate that GnRH1 and possibly GnRH3 are responsible for gonadal maturation through LH secretion and that all three forms of GnRH function as neurotransmitters or neuromodulators in the brain of K. marmoratus.

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.

Androstenedione and 17α-methyltestosterone induce early ovary development of Anguilla japonica.

Endocrine effects as 11-ketotestosterone (11-KT), an unaromatizable androgen, regulating the follicles growth in the previtellogenic stage of eel reproduction have been widely elucidated. However, the influence of aromatizable androgens on the brain-pituitary-gonad axis during oogenesis in A. japonica has not been clearly elaborated. In the study, androstenedione (AD) and 17α-methyltestosterone (MT) were employed together to induce ovary development of seven-year-old female Anguilla japonica through feeding or exposure in the migration season. After female A. japonica had been fed with commercial diet containing 5 mg AD and MT kg d-1 body weight respectively for 45 d in fresh water (Trial I), the development of oocytes still remained at the oil droplet stage, but the GSI and follicle diameter increased significantly. The serum 11-KT level and expression of liver vitellogenin mRNA were significantly elevated. After female fish had been exposed to seawater containing 50 µg L-1 AD and MT respectively for 45 d (Trial II), the ovaries of A. japonica almost reached midvitellogenic stage and the GSI and follicle diameter increased significantly. Yolk granular layer was observed in the peripheral ooplasm. The serum 11-KT level maintained consistently low, and the serum E2 level declined significantly to a relatively low level. The expression levels of ovarian arα and cyp19a1, brain (with pituitary together) mGnRH and lhß increased significantly. The results showed that A. japonica in Trial II appeared a higher ovarian development than those in Trial I. These findings indicated that AD and MT increased the oil droplet and enlarged follicle diameter in previtellogenic stage, while the vitellogenesis and gonadotropin release did not occur in Trial I. In Trial II, AD and MT promoted vitellogenesis by stimulating the ovary expression of arα and by up-regulating brain mGnRH and pituitary lhß expression.

Temperature affects sexual maturation through the control of kisspeptin, kisspeptin receptor, GnRH and GTH subunit gene expression in the grass puffer during the spawning season.

Water temperature is an environmental factor of primary importance that influences reproductive function in fish. To understand the molecular and physiological mechanisms underlying the regulation of reproduction by temperature, we examined changes in expression of genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r) and three gonadotropin-releasing hormones (gnrh1, gnrh2 and gnrh3) in the brain and genes encoding gonadotropin (GTH) subunits (gpa, fshb and lhb) in the pituitary of grass puffer exposed to a low temperature (14°C), normal temperature (21°C) and high temperature (28°C) for 7days. In addition, the plasma levels of cortisol were examined after exposed to three temperature conditions. The gonadosomatic index was significantly decreased in both low and high temperature conditions. The levels of kiss2 and kiss2r mRNAs were significantly decreased at both low and high temperature conditions compared to normal temperature (control) condition. gnrh1 but not gnrh2 were significantly decreased in both temperature conditions, while gnrh3 showed a decreasing tendency in low temperature. Consequently, the levels of fshb and lhb mRNAs were significantly decreased in both low and high temperature conditions. Interestingly, the plasma levels of cortisol were significantly increased in low temperature but remain unchanged in high temperature, suggesting that the fish were under stress in the low temperature conditions but not in the high temperature conditions. Taken together, the present results indicate that anomalous temperature have an inhibitory effect on reproductive function through suppressing kiss2/kiss2r/gnrh1/fshb and lhb expression and these changes may occur in a normal physiological response as well as in a malfunctional stress response.

Acetylshikonin from Zicao exerts antifertility effects at high dose in rats by suppressing the secretion of GTH.

Zicao is being highlighted as a promising Chinese medicine due to all the beneficial effects that have been associated with its use. Unfortunately, studies on the toxicity of Zicao in different species are still missing and should be carried out. In this study, we investigated whether Acetylshikonin (AS) from Zicao has an anti-fertility effect through mating experiments and explored its underling mechanism. Sprague-Dawley rats received no treatment or were treated with 120, 360 or 1080 mg/kg AS extract by intragastric administration for 2 weeks. The rat pregnancy rate of the 1080 mg/kg dose group was significantly decreased relative to control group, while it recovered after a month of drug withdrawal, which indicated that the effect of antifertility is reversible. Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels in rat were significantly decreased by AS. The secretion of FSH in rat anterior pituitary cells was decreased but the synthesis was not affected. AS reduced the number of developing follicle and mature follicle in rat ovarian cortical. Maybe all of these resulted from AS decreased the expression of synaptotagmin-1 and SNAP-25 which were the critical proteins of exocytosis. Our data suggested that AS at high dose can suppress the ability of pregnancy of the rats through decreasing serum FSH and LH levels by affecting exocytosis process of gonadotropic hormone (GTH).

GnIH plays a negative role in regulating GtH expression in the common carp, Cyprinus carpio L.

In birds and mammals, the gonadotrophin-inhibitory hormone (GnIH) effectively inhibits the expression of gonadotrophin (GtH). In teleosts, the effects of GnIH are still unclear and under much debate. The aim of this study is to evaluate the functions of GnIH/receptors of gonadotrophin-inhibitory hormone (GnIHRs) system during reproduction in the common carp, Cyprinus carpio L. We cloned the full cDNA sequences of GnIH /GnIHRs. Real-time PCR results showed that GnIH/GnIHRs were distributed extensively across the whole hypothalamus-pituitary-gonad (HPG) axis. We also examined the changes of GnIH/GnIHRs in the HPG axis during reproduction. GnIH mRNA expression was decreased to the minimum value in Apr, the spawning month, and increased immediately after the completion of reproduction. Expression pattern of GnIH during reproduction was the opposite to those of Gonadotrophin release hormone 3 (GnRH3) and luteinizing hormone (LH). Expression patterns of GnIHRs were similar to that of GnIH in the hypothalamus. In the pituitary, GnIHR2/3 peaked in March before spawning. In the ovaries, the GnIHR1 decreased to the minimum value in April, but GnIHR2/3 increased. By injection and incubation with synthesized GnIH-III peptide, we confirmed the negative influence of GnIH on mRNAs of the follicle-stimulating hormone-ß and LH-ß subunits in the common carp. These results show that the GnIH/GnIHRs system is involved in the negative regulation of reproduction in HPG axis of the common carp.

Molecular identification of GnIH/GnIHR signal and its reproductive function in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides).

Gonadotropin-inhibitory hormone (GnIH) and its receptor (GnIHR) play an important role in reproduction regulation in birds, mammals and some teleost species. In protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides), the GnIH/GnIHR signaling pathway and its reproductive function have not been addressed yet. In this study, GnIH and GnIHR in orange-spotted grouper were characterized. gGnIH possessed three putative peptides (gGnIH-I, -II, -III), while gGnIHR showed the characteristics of G protein-coupled receptor and was clustered with GPR147. Functional assays demonstrated that three synthetic gGnIH peptides significantly decreased the forskolin-induced CRE promoter activity, but only gGnIH-I could significantly decrease SRE promoter activity in COS-7 cells transfected with gGnIHR. During the process of ovarian differentiation and development, gGnIH mRNA level in hypothalamus was low at the gonadal primordium stage with gonia, then increased significantly at the early differentiated gonad with primary growth oocytes, while decreased significantly at the developing gonads with cortical-alveolus and vitellogenic stage oocytes. During MT-induced sex reversal, gGnIH mRNA level in hypothalamus increased significantly when the fish completely reversed from female to male. However, gGnIHR mRNA level in pituitary decreased significantly in intersex and completely reversed male fish. Intraperitoneal injection (i.p.) of three gGnIH peptides significantly decreased GnRH1 mRNA levels in hypothalamus, and gGnIH-II significantly inhibited synthesis of LHß in pituitary. In summary, we firstly identified the GnIH/GnIHR signal in protogynous orange-spotted grouper, which might be involved in the regulation of the reproductive function of sex differentiation, gonadal development and sex reversal via regulating the synthesis of both GnRH and GtH.

Endocrine regulation of testosterone production by Leydig cells in the catfish, Clarias batrachus: probable mediators of growth hormone.

Growth hormone (GH), in the recent past, has been recognized as a potent steroid stimulating hormone independent of gonadotropin (GtH). However, the mode and mechanism of its steroidogenic action in the testis is not yet elucidated, particularly in fish. The present study was designed to understand the mode and mechanism of steroidogenic action of growth hormone in testis of the catfish, Clarias batrachus through in vivo and in vitro Leydig cell culture studies using the signaling molecule inhibitors. Exogenous administration of GtH, GH and insulin to the male catfish increased testicular and circulating testosterone level. In vitro treatment of Leydig cells with these hormones also increased testosterone production. The steroidogenic action of GH appeared to be indirect and mediated through Leydig cell produced insulin-like growth factor I (IGF-I), as the treatments with actinomycin D, cycloheximide and anti-IGF-I abolished the GH-induced testosterone production by Leydig cells. The GH-induced stimulation in IGF-I production by the isolated Leydig cells further substantiates this notion. GH appears to employ cAMP/PKA and tyrosine kinase signaling pathways to induce IGF-I production, as the adenylyl cyclase inhibitor (SQ 22,536), cAMP-dependent protein kinase (PKA) blocker (H-89) and tyrosine kinase inhibitor (lavendustin A) abolished the GH-induced IGF-I production and in turn testosterone by the Leydig cells. This study suggests that GH exerts independent androgenic effect in the catfish testis indirectly through augmenting the Leydig cell production of IGF-I.