ABSTRACT
Anti-Müllerian hormone (AMH) is primarily produced by ovarian granulosa cells and contributes to follicle development. AMH is also produced in other tissues, including the brain and pituitary; however, its roles in these tissues are not well understood. In this study, we examined the effect of AMH on pituitary gonadotrophs. We detected AMH and AMH receptor type 2 expression in LßT2 cells. In these cells, the expression of FSHß- but not α- and LHß-subunits increased significantly as the concentration of AMH increased. LßT2 cells expressed Kiss-1 and Kiss-1R. AMH stimulation resulted in decreases in both Kiss-1 and Kiss-1R. The siRNA-mediated knockdown of Kiss-1 in LßT2 cells did not alter the basal expression levels of α-, LHß-, and FSHß-subunits. In LßT2 cells overexpressing Kiss-1R, exogenous kisspeptin stimulation significantly increased the expression of all three gonadotropin subunits. However, kisspeptin-induced increases in these subunits were almost completely eliminated in the presence of AMH. In contrast, GnRH-induced increases in the three gonadotropin subunits were not modulated by AMH. Our observations suggested that AMH acts on pituitary gonadotrophs and induces FSHß-subunit expression with concomitant decreases in Kiss-1 and Kiss-1R gene expression. Kisspeptin, but not GnRH-induced gonadotropin subunit expression, was inhibited by AMH, suggesting that it functions in association with the kisspeptin/Kiss-1R system in gonadotrophs.
Subject(s)
Anti-Mullerian Hormone/pharmacology , Gonadotrophs/metabolism , Gonadotropins, Pituitary/genetics , Kisspeptins/physiology , Receptors, Kisspeptin-1/physiology , Animals , Cell Line , Follicle Stimulating Hormone, beta Subunit/genetics , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Gonadotrophs/drug effects , Gonadotropin-Releasing Hormone/pharmacology , Kisspeptins/genetics , Luteinizing Hormone, beta Subunit/genetics , Mice , RNA, Small Interfering , Receptors, Kisspeptin-1/geneticsABSTRACT
Previous studies revealed an estradiol (E2)-dependent peak in brain activity, including neurosteroidogenesis and neurogenesis in the black porgy during the gonadal differentiation period. The brain-pituitary-gonadotropic axis is a key regulator of reproduction and may also be involved in gonadal differentiation, but its activity and potential role in black porgy during the gonadal differentiation period is still unknown. The present study analyzed the expression of regulatory factors involved in the gonadotropic axis at the time of gonadal differentiation (90, 120, 150â¯days after hatching [dah]) and subsequent testicular development (180, 210, 300 dah). In agreement with previous studies, expression of brain aromatase cyp19a1b peaked at 120 dah, and this was followed by a gradual increase during testicular development. The expression of gonadotropin subunits increased slightly but not significantly during gonadal differentiation and then increased significantly at 300 dah. In contrast, the expression of brain gnrh1 and pituitary gnrh receptor 1 (gnrhr1) exhibited a pattern with two peaks, the first at 120 dah, during the period of gonadal differentiation, and the second peak during testicular development. Gonad fshr and lhcgr increased during gonadal differentiation period with highest transcript level in prespawning season during testicular development. This suggests that the early activation of brain gnrh1, pituitary gnrhr1 and gths, and gonad gthrs might be involved in the control of gonadal differentiation. E2 treatment increased brain cyp19a1b expression at each sampling time, in agreement with previous studies in black porgy and other teleosts. E2 also significantly stimulated the expression of pituitary gonadotropin subunits at all sampling times, indicating potential E2-mediated steroid feedback. In contrast, no significant effect of E2 was observed on gnrh1. Moreover, treatment of AI or E2 had no statistically significant effect on brain gnrh1 transcription levels during gonadal differentiation. This indicated that the early peak of gnrh1 expression during the gonadal differentiation period is E2-independent and therefore not directly related to the E2-dependent peak in brain neurosteroidogenesis and neurogenesis also occurring during this period in black porgy. Both E2-independent and E2-dependent mechanisms are thus involved in the peak expression of various genes in the brain of black porgy at the time of gonadal differentiation.
Subject(s)
Brain/metabolism , Estradiol/pharmacology , Perciformes/physiology , Pituitary Gland/metabolism , Sex Differentiation , Testis/growth & development , Animals , Aromatase/genetics , Aromatase/metabolism , Aromatase Inhibitors/pharmacology , Female , Gene Expression Regulation, Developmental/drug effects , Gonadotropins, Pituitary/genetics , Gonadotropins, Pituitary/metabolism , Male , Perciformes/genetics , Perciformes/growth & development , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, LHRH/genetics , Receptors, LHRH/metabolism , Sex Differentiation/drug effects , Testis/drug effects , Testis/metabolismABSTRACT
In the present study, in vitro effects of synthetic vasotocin (VT), isotocin (4Ser, 8Ile- oxytocin; ITb) and the recently cloned IT gene paralog product (8Val-Isotocin, ITa) were studied on the expression of pituitary gonadotropin (GtH) subunit mRNA levels. In male pituitaries of early (preparatory phase) and late (prespawning phase) recrudescing catfish, Heteropneustes fossilis, VT (10â¯nM, 100â¯nM and 1000â¯nM) stimulated fshß expression dose-dependently. But in females, the dose-dependent effect was found only in the preparatory phase. In males, VT stimulated lhß expression only at higher doses. In females, VT produced a significant dose-dependent increase of the lhß expression only in the prespawning phase. VT stimulated the expression of gpα, dose-dependently in the preparatory phase in males and in the prespawning phase in females. The incubation of the pituitaries with ITb did not alter the fshß expression in either sex in both preparatory and prespawning phases. In males, ITb stimulated the expression of lhß and gpα only at the highest concentration (1000â¯nM) in both phases. In females, ITb stimulated both lhß and gpα expression only at 1000â¯nM in the preparatory phase and dose-dependently in the prespawning phase. The incubation of the pituitaries with ITa produced effects similar to ITb on the expression of fshß, lhß, and gpα. The results show that the basic peptide VT modulates both fshß and lhß expressions, which are influenced by the sex and reproductive stage. The neutral peptide ITA/ITb exerts an insignificant effect on the fshß expression regardless of sex or season. Both VT and ITa/ITb elicit a significant effect on the lhß expression in late recrudescent phase especially in females.
Subject(s)
Catfishes , Gonadotropins, Pituitary/genetics , Pituitary Hormones, Posterior/pharmacology , Reproduction/drug effects , Sexual Maturation/drug effects , Animals , Catfishes/genetics , Catfishes/growth & development , Catfishes/metabolism , Female , Follicle Stimulating Hormone, beta Subunit/genetics , Follicle Stimulating Hormone, beta Subunit/metabolism , Gene Expression Regulation, Developmental/drug effects , Gonadotropins, Pituitary/metabolism , In Vitro Techniques , Luteinizing Hormone, beta Subunit/genetics , Luteinizing Hormone, beta Subunit/metabolism , Male , Oxytocin/analogs & derivatives , Oxytocin/pharmacology , Pituitary Gland/drug effects , Pituitary Gland/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Reproduction/genetics , Seasons , Sex Characteristics , Sexual Maturation/genetics , Vasotocin/pharmacologyABSTRACT
Triclosan (TCS), a member of the class of compounds called pharmaceutical and personal care products (PPCPs), is a broad antibacterial and antifungal agent found in a lot of consumer products. However, TCS hormone effect mechanism in teleost female fish is not clear. Female Yellow River carp (Cyprinus carpio) were exposed to 1/20, 1/10 and 1/5 LC50 TCS (96h LC50 of TCS to carp) under semi-static conditions for 42days. Vitellogenin (Vtg), 17ß-estradiol (E2), testosterone(T), estrogen receptor (Er), gonadotropin (GtH), and gonadotropin-releasing hormone (GnRH) levels were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also examined the mRNA expressions of aromatase, GtHs-ß, GnRH, and Er by quantitative real-time PCR (qRT-PCR). The results indicated that 1/5 LC50 TCS induced Vtg in hepatopancreas of female carps by interference with the hypothalamic-pituitary-gonadal (HPG) axis at multiple potential loci through three mechanisms: (a) TCS exposure enhanced the mRNA expression of hypothalamus and gonadal aromatase which converts androgens into estrogens, subsequently increasing serum concentrations of E2 to induce Vtg in hepatopancreas; (b) TCS treatment increased GnRH and GtH-ß mRNA expression and secretion, causing the disturbance of reproductive endocrine and the increase of E2 to induce Vtg in hepatopancreas; (c) TCS exposure enhanced synthesis and secretion of Er, then it bound to Er to active Vtg synthesis. These mechanisms showed that TCS may induce Vtg production in female Yellow River carp by Er-mediated and non-Er-mediated pathways.
Subject(s)
Carps/metabolism , Endocrine Disruptors/toxicity , Estrogens/toxicity , Gonads/drug effects , Hormones/metabolism , Hypothalamo-Hypophyseal System/drug effects , Reproduction/drug effects , Triclosan/toxicity , Animals , Aromatase/genetics , Aromatase/metabolism , Carps/genetics , Estradiol/metabolism , Female , Fish Proteins/genetics , Fish Proteins/metabolism , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/metabolism , Gonadotropins, Pituitary/genetics , Gonadotropins, Pituitary/metabolism , Gonads/metabolism , Hepatopancreas/drug effects , Hepatopancreas/metabolism , Hormones/genetics , Hypothalamo-Hypophyseal System/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Testosterone/metabolism , Time Factors , Vitellogenins/metabolismABSTRACT
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.
Subject(s)
Gene Expression Regulation , Gonadotropins, Pituitary/metabolism , Kisspeptins/metabolism , Receptors, G-Protein-Coupled/metabolism , Seasons , Sexual Maturation/genetics , Tetraodontiformes/metabolism , Animals , Fish Proteins/genetics , Fish Proteins/metabolism , Gonadotropins, Pituitary/genetics , Kisspeptins/genetics , Pituitary Gland/metabolism , Receptors, G-Protein-Coupled/genetics , Reproduction/physiology , Temperature , Tetraodontiformes/genetics , Tetraodontiformes/growth & developmentABSTRACT
Differential effects of photoperiod and temperature on the temporal modulation of gonadotrophin subunit genes (glycoprotein α, gpα), follicle-stimulating hormone ß (fshß) and luteinizing hormone ß (lhß) expression were investigated in the stinging catfish Heteropneustes fossilis. Female H. fossilis were exposed to varying photoperiod and temperature conditions for 14 and 28 days in the early preparatory phase of the annual reproductive cycle. Gonadotrophin subunit gene expression, gonado-somatic index (IG ), ovarian histology and plasma steroid hormone levels were evaluated. The exposure of H. fossilis to long photoperiod (LP) of 16 h light or high temperature (HT) at 28 ± 2° C (mean ± s.e.), alone or in combination, resulted in significant increases in gpα, fshß and lhß messenger (m)RNA levels, IG , plasma oestradiol-17ß (E2 ), testosterone (T) and progesterone (P4 ) levels. The ovaries were filled with advanced yolky oocytes. On the other hand, the short photoperiod (SP) of 8 h light exposure decreased the transcript levels with higher inhibition in the normal temperature (NT) group at 18 ± 2° C (mean ± s.e.) than the HT group at 28 ± 2° C. Furthermore, the inhibition reached the highest level in total darkness (TD) of 24 h light deprivation under NT conditions at 18 ± 2° C. Consequently, the SP and TD treatments inhibited the IG , plasma E2 and T levels and ovarian development. The exposure to high temperature at 28 ± 2° C also modified the short photoperiod effect by elevating plasma E2 level. The plasma T level changed only mildly while the plasma P4 level showed the greatest fluctuations; the level reached the nadir in the SP + HT group but increased in the SP + NT group on day 28. A two-way ANOVA of the data showed differential effects of photoperiod and temperature; photoperiod produced a highly significant effect on fshß expression while temperature had a highly significant effect both on lhß and gpα levels. Thus, the differential expression of the gpα by the environmental variables ensures temporal synchronization of ovarian development and spawning.
Subject(s)
Catfishes/physiology , Gonadotropins, Pituitary/metabolism , Photoperiod , Temperature , Animals , Catfishes/genetics , Estradiol/blood , Female , Follicle Stimulating Hormone, beta Subunit/genetics , Follicle Stimulating Hormone, beta Subunit/metabolism , Gene Expression Regulation/radiation effects , Gonadotropins, Pituitary/genetics , Oocytes/metabolism , Oocytes/physiology , Ovary/metabolism , Progesterone/blood , RNA, Messenger/metabolism , Reproduction/radiation effects , Seasons , Testosterone/bloodABSTRACT
We determined the molecular mechanism underlying the environmental (photoperiodic) regulation of sexual maturation in fish, we examined the expression of sexual maturation-related hormones and vertebrate ancient long opsin (VAL-opsin) in goldfish (Carassius auratus) exposed to different light spectra (red and green light-emitting diodes). We further evaluated the effect of exogenous gonadotropin hormone (GTH) on the expression of VAL-opsin under different light conditions. Our results demonstrated that the expression of GTHs was higher in the fish exposed to green light, and VAL-opsin levels were increased in the fish receiving GTH injection. Therefore, we have uncovered a molecular mechanism underlying the environmental (light)-induced trigger for sexual maturation: VAL-opsin is activated by green light and GTH, which promotes the expression of sexual maturation genes.
Subject(s)
Fish Proteins/metabolism , Goldfish/growth & development , Goldfish/metabolism , Gonadotropins, Pituitary/metabolism , Opsins/metabolism , Sexual Maturation/radiation effects , Animals , Fish Proteins/genetics , Gene Expression/radiation effects , Goldfish/genetics , Gonadotropins, Pituitary/genetics , Light , Opsins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sexual Maturation/genetics , Sexual Maturation/physiologyABSTRACT
In this study, three cDNA sequences encoding common glycoprotein α subunit (GTHα), follicle-stimulating hormone ß subunit (FSHß) and luteinizing hormone ß subunit (LHß) were isolated from Japanese sea bass (Lateolabrax japonicas). Comparison of the deduced amino acid sequences with other gonadotropic hormones (GTHs) indicated that their cysteine residues and potential N-linked glycosylation sites were highly conserved, and high homology with those of other perciformes was showed in phylogenetic analysis. GTHs transcripts were present highly in the pituitary and brain and weakly in testis and other tissues. During testicular development, GTHs transcriptional levels in pituitary and brain (expect FSHß subunit in brain) were significantly increased at spermiation period, stage V. Subsequently, the effects of hCG and GnRHa on the mRNA levels of GTHs subunits were examined. In brain, both hormones were detected to improve the expression of GTHα subunit mRNA. In pituitary, three GTHs subunits increased parallelly and abruptly in two hormone treatment groups. In testis, hCG was suggested to improve three GTHs subunits expression in Japanese sea bass for the first time. These results suggest that both gonadotropins are probably involved in the control of Japanese sea bass spermatogenesis and provide a framework for better understanding of the mechanisms of hormone-mediated reproduction control in Japanese sea bass and other teleosts.
Subject(s)
Bass/genetics , Gene Expression Regulation/physiology , Gonadotropins/genetics , Gonadotropins/metabolism , Phylogeny , Protein Subunits/genetics , Spermatogenesis/physiology , Amino Acid Sequence , Analysis of Variance , Animals , Base Sequence , Bass/physiology , Brain/metabolism , Chorionic Gonadotropin/pharmacology , Cloning, Molecular , Cluster Analysis , DNA Primers/genetics , Follicle Stimulating Hormone, beta Subunit/genetics , Gene Expression Regulation/drug effects , Gonadotropin-Releasing Hormone/analogs & derivatives , Gonadotropin-Releasing Hormone/pharmacology , Gonadotropins, Pituitary/genetics , Luteinizing Hormone, beta Subunit/genetics , Male , Molecular Sequence Data , Pituitary Gland/metabolism , Real-Time Polymerase Chain Reaction/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sequence Analysis, DNA/veterinary , Sequence Homology , Testis/metabolismABSTRACT
OBJECTIVE: To determine the effect of vaginal progesterone as luteal support on pregnancy outcomes in infertile patients who undergo ovulation induction with gonadotropins and intrauterine insemination (IUI). DESIGN: Prospective randomized trial. SETTING: Tertiary referral center. PATIENT(S): About 398 patients with primary infertility were treated during 893 ovarian stimulation and IUI cycles from February 2010 to September 2012. METHODS: All patients underwent ovarian stimulation with gonadotropins combined with IUI. Patients in the supported group received vaginal micronized progesterone capsules 200 mg once daily from the day after insemination until next menstruation or continuing for up to 8 weeks of pregnancy. Women allocated in the control group did not receive luteal phase support. MAIN OUTCOME MEASURE(S): Livebirth rate, clinical pregnancy rate and early miscarriage rate per cycle. RESULT(S): Of the 893 cycles, a total of 111 clinical pregnancies occurred. There were no significant differences between supported with progesterone and unsupported cycle in terms of livebirth rate (10.2% versus 8.3%, respectively, with a p value = 0.874) and clinical pregnancy rate (13.8% compared with 11.0% in unsupported cycle with a p value = 0.248). An early miscarriage rate of 3.6% was observed in the supported cycles and 2.7% in the unsupported cycles, with no significant differences between the groups (p value = 0.874). CONCLUSION(S): In infertile patients treated with mildly ovarian stimulation with recombinant gonadotropins and IUI, luteal phase support with vaginal progesterone is not associated with higher livebirth rate or clinical pregnancy rate compared with patients who did not receive any luteal phase support.
Subject(s)
Corpus Luteum Maintenance/drug effects , Infertility, Female/therapy , Infertility, Male/therapy , Insemination, Artificial, Heterologous , Ovulation Induction , Progesterone/pharmacology , Progestins/pharmacology , Administration, Intravaginal , Adult , Birth Rate , Capsules , Drug Compounding , Female , Gonadotropins, Pituitary/administration & dosage , Gonadotropins, Pituitary/genetics , Gonadotropins, Pituitary/pharmacology , Humans , Luteal Phase/drug effects , Male , Pregnancy , Pregnancy Rate , Progesterone/administration & dosage , Progesterone/chemistry , Progestins/administration & dosage , Progestins/chemistry , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Spain/epidemiologyABSTRACT
OBJECTIVE: We examined how the sex steroids influence the synthesis of gonadotropins. MATERIALS AND METHODS: The effects of sex steroids estradiol (E2), progesterone (P4), and dihydrotestosterone (DHT) in pituitary gonadotroph cell model (LßT2 cells) in vitro and ovary-intact rats in vivo were examined. The effects of sex steroids on Kiss1 gene expression in the hypothalamus were also examined in ovary-intact rats. RESULTS: In LßT2 cells, E2 increased common glycoprotein alpha (Cga) and luteinizing hormone beta (Lhb) subunit promoter activity as well as their mRNA expression. Although gonadotropin subunit promoter activity was not modulated by P4, Cga and Lhb mRNA expression was increased by P4. DHT inhibited Cga and Lhb mRNA expression with a concomitant decrease in their promoter activity. During the 2-week administration of exogenous E2 to ovary-intact rats, the estrous cycle determined by vaginal smears was disrupted. P4 or DHT administration completely eliminated the estrous cycle. Protein expression of all three gonadotropin subunits within the pituitary gland was inhibited by E2 or P4 treatment in vivo; however, DHT reduced Cga expression but did not modulate Lhb or follicle-stimulating hormone beta subunit expression. E2 administration significantly repressed Kiss1 mRNA expression in a posterior hypothalamic region that included the arcuate nucleus. P4 and DHT did not modulate Kiss1 mRNA expression in this region. In contrast, P4 administration significantly inhibited Kiss1 mRNA expression in the anterior region of the hypothalamus that included the anteroventral periventricular nucleus. The expression of gonadotropin-releasing hormone (Gnrh) mRNA in the anterior hypothalamic region, where the preoptic area is located, appeared to be decreased by treatment with E2 and P4. CONCLUSION: Our findings suggest that sex steroids have different effects in the hypothalamus and pituitary gland.
Subject(s)
Kisspeptins , Ovary , Rats , Female , Animals , Kisspeptins/genetics , Kisspeptins/metabolism , Hypothalamus/metabolism , Gonadotropins, Pituitary/genetics , Gonadotropins, Pituitary/metabolism , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/metabolism , Estradiol/pharmacology , RNA, Messenger/metabolism , Dihydrotestosterone/pharmacology , Gene ExpressionABSTRACT
Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth and maturation, but no clear differences between breeds with different ovulation rates have been found. Therefore, this study investigated mRNA expression of FSHß, LHß, FSH receptor (FSHR), LH receptor (LHR), and estrogen receptor-ß (ERß) genes in prolific Lezhi black (LB) goats and nonprolific Tibetan (TB) goats by real-time PCR. Follicles and pituitaries were recovered from goats at 12-24 h after onset of estrus. Real-time PCR analysis revealed that the expression levels of FSHß and LHß mRNA were significantly higher (p < 0.01) in pituitary of LB than in TB does, but the expression levels of FSHR and LHR mRNA in follicle of TB were greater (p < 0.05). Expression level of follicular ER ß was not different between the two breeds. Data provide evidence that the greater ovulation rate in the LB goat as compared to the TB breed is associated with a greater gonadotropin expression during follicular phase.
Subject(s)
Estrogen Receptor beta/analysis , Goats/metabolism , Gonadotropins, Pituitary/analysis , RNA, Messenger/analysis , Receptors, Gonadotropin/analysis , Animals , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Female , Gene Expression Profiling , Goats/genetics , Gonadotropins, Pituitary/genetics , Gonadotropins, Pituitary/metabolism , Ovarian Follicle/chemistry , Ovarian Follicle/metabolism , Ovulation/genetics , Ovulation/physiology , Pituitary Gland/chemistry , Pituitary Gland/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Receptors, Gonadotropin/genetics , Receptors, Gonadotropin/metabolismABSTRACT
The cDNAs of the α-subunit of the pituitary gonadotrophic hormones (GTHα) of fish of the order Osteoglossiformes or the superorder Osteoglossomorpha have never been sequenced. For a better understanding the phylogenetic diversity and evolution of PGHα in fish and for future biotechnological synthesis of the gonadotrophic hormones (ag-FSH and ag-LH), of Arapaima gigas, one of the largest freshwater fishes of the world, its GTHα cDNA was synthesized by reverse transcriptase and the polymerase chain reaction starting from total pituitary RNA. The ag-GTHα-subunit was found to be encoded by 348 bp, corresponding to a protein of 115 amino acids, with a putative signal peptide of 24 amino acids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for forming 5 disulfide linkages, 2 putative N-linked glycosylation sites and 3 proline residues, were found to be conserved on the basis of the known sequences of vertebrate gonadotrophic hormones. Phylogenetic analysis, based on the amino acid sequences of 38 GTHα-subunits, revealed the highest identity of A. gigas with members of the Acipenseriformes, Anguilliformes, Siluriformes and Cypriniformes (87.1-89.5 %) and the lowest with Gadiformes and Cyprinodontiformes (55.0 %). The obtained phylogenetic tree agrees with previous analysis of teleostei, since A. gigas, of the order of Osteoglossiformes, appears as the sister group of Clupeocephala, while Elopomorpha forms the most basal group of all other teleosts.
Subject(s)
Fishes/genetics , Gonadotropins, Pituitary/genetics , Phylogeny , Pituitary Gland/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cluster Analysis , Conserved Sequence/genetics , DNA Primers/genetics , Fishes/classification , Fishes/metabolism , Gonadotropins, Pituitary/isolation & purification , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
The aim of this study was to clarify the roles of 2 gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on sex differentiation in the protogynous Malabar grouper, Epinephelus malabaricus. To do this, the mRNA expression patterns of GTH subunits (cga, fshb, and lhb) in the fish pituitary throughout gonadal sex differentiation were investigated. Real-time reverse transcriptase (RT)-PCR showed that cga and fshb were present in the undifferentiated and ovarian differentiation stages, and that the expression levels significantly increased after ovarian differentiation (AOD). However, lhb was not expressed before ovarian differentiation (BOD) and was first detected AOD. Next, to investigate the differentiation and distribution of Fshb and Lhb-producing cells in the pituitary of fish throughout gonadal sex differentiation, immunohistochemical analysis was used to detect teleost GTH subunits. Positive immunoreactivity against Fshb and Lhb was not detected in the pituitary BOD; Fshb and Lhb-positive cells first appeared in the pituitary AOD. It therefore seems unlikely that pituitary gonadotropins play a major role in the control of gonadal sex differentiation in the Malabar grouper.
Subject(s)
Bass/metabolism , Gonadotropins, Pituitary/metabolism , Sex Differentiation/physiology , Animals , Bass/genetics , Female , Follicle Stimulating Hormone/genetics , Follicle Stimulating Hormone/metabolism , Gonadotropins, Pituitary/genetics , Immunohistochemistry , Luteinizing Hormone/genetics , Luteinizing Hormone/metabolism , Male , Ovary/cytology , Ovary/metabolism , Real-Time Polymerase Chain Reaction , Sex Differentiation/geneticsABSTRACT
The endocrine regulation of reproduction in a multiple spawning fish with an asynchronous-type ovary remains largely unknown. The objectives of this study were to monitor changes in the mRNA expression of three gonadotropin (GtH) subunits (GPα, FSHß, and LHß) during the reproductive cycle of the female chub mackerel Scomber japonicus. Cloning and subsequent sequence analysis revealed that the cDNAs of chub mackerel GPα, FSHß, and LHß were 658, 535, and 599 nucleotides in length and encoded 117, 115, and 147 amino acids, respectively. We applied a quantitative real-time PCR assay to quantify the mRNA expression levels of these GtH subunits. During the seasonal reproductive cycle, FSHß mRNA levels remained high during the vitellogenic stages, while GPα and LHß mRNA levels peaked at the end of vitellogenesis. The expression of all three GtH subunits decreased during the post-spawning period. These results suggest that follicle-stimulating hormone (FSH) is involved in vitellogenesis, while luteinizing hormone (LH) functions during final oocyte maturation (FOM). Both GPα and FSHß mRNA levels remained high during the FOM stages of the spawning cycle and increased further just after spawning. Thus, FSH synthesis may be strongly activated just after spawning to accelerate vitellogenesis in preparation for the next spawning. Alternatively, LHß mRNA levels declined during hydration and then increased after ovulation. This study demonstrates that chub mackerel are a good model for investigating GtH functions in multiple spawning fish.
Subject(s)
Gonadotropins, Pituitary/genetics , Perciformes/genetics , Perciformes/physiology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , Female , Follicle Stimulating Hormone, beta Subunit/genetics , Gene Expression Regulation , Glycoprotein Hormones, alpha Subunit/genetics , Gonadotropins, Pituitary/chemistry , Luteinizing Hormone, beta Subunit/genetics , Male , Molecular Sequence Data , Ovary/physiology , Perciformes/anatomy & histology , Phylogeny , Protein Subunits/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reproduction/genetics , Reproduction/physiology , Vitellogenesis/genetics , Vitellogenesis/physiologyABSTRACT
Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.
Subject(s)
Salmo salar , Animals , Gene Expression , Gonadotropins/metabolism , Gonadotropins/pharmacology , Gonadotropins, Pituitary/genetics , Male , Salmo salar/genetics , Salmo salar/metabolism , Sexual Maturation/geneticsABSTRACT
The specific role of gonadotropin-releasing hormone (GnRH) on brain sexual differentiation remains unclear. To investigate whether gonadotropin and, in turn, testosterone (T) secretion is regulated by GnRH during the critical period for brain differentiation in sheep fetuses, we attempted to selectively suppress pituitary-testicular activation during midgestation with the long-acting GnRH antagonist degarelix. Fetuses received subcutaneous injections of the antagonist or vehicle on day 62 of gestation. After 2 to 3 weeks we examined consequences of the intervention on baseline and GnRH-stimulated plasma luteinizing hormone (LH) and T levels. In addition, we measured the effect of degarelix-treatment on messenger RNA (mRNA) expression for the pituitary gonadotropins and key gonadal steroidogenic enzymes. Baseline and GnRH-stimulated plasma LH levels were significantly suppressed in degarelix-treated male and female fetuses compared to control values. Similarly, T concentrations were suppressed in degarelix-treated males. The percentage of LHß-immunoreactive cells colocalizing c-fos was significantly reduced by degarelix treatment indicating that pituitary sensitivity was inhibited. Degarelix treatment also led to the significant suppression of mRNA expression coding for the pituitary gonadotropin subunits and for the gonadal enzymes involved in androgen synthesis. These findings demonstrate that pharmacologic inhibition of GnRH early in gestation results in suppression of LH secretion and deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T exposure during the critical period of sheep gestation when the brain is masculinized. Thus, disturbance to gonadotropin secretion during this phase of gestation could have long-term consequence on adult sexual behaviors and fertility.
Subject(s)
Gestational Age , Gonadotropin-Releasing Hormone/antagonists & inhibitors , Gonadotropins, Pituitary/metabolism , Oligopeptides/administration & dosage , Pituitary Gland, Anterior/embryology , Sheep/embryology , Animals , Brain/embryology , Female , Fetal Blood/chemistry , Gonadotropin-Releasing Hormone/administration & dosage , Gonadotropin-Releasing Hormone/physiology , Gonadotropins, Pituitary/genetics , Injections, Subcutaneous/veterinary , Luteinizing Hormone/blood , Male , Ovary/chemistry , Ovary/embryology , Pituitary Gland, Anterior/chemistry , Pituitary Gland, Anterior/drug effects , Pregnancy , RNA, Messenger/analysis , Sex Differentiation/physiology , Testis/chemistry , Testis/embryology , Testosterone/bloodABSTRACT
We examined the expression of dual specificity phosphatase 1 (DUSP1) by gonadotropin-releasing hormone (GnRH) stimulation and investigated the role of DUSP1 on gonadotropin gene expression using LbetaT2 gonadotroph cell line. DUSP1 expression was markedly increased 60 min after GnRH stimulation, and mitogen-activated protein kinase 3/1 (MAPK3/1) activation was gradually decreased after 60 min. GnRH-induced MAPK3/1 activation was completely inhibited by U0126, a MEK inhibitor, whereas GnRH-induced DUSP1 expression was partially inhibited by U0126. GnRH-induced DUSP1 induction was inhibited by triptolide, a diterpenoid triepoxide. In contrast, this compound potentiated MAPK3/1 activation. U0126 prevented GnRH-stimulated gonadotropin subunit promoter activation dose dependently, and 10 muM of U0126 reduced the effects of GnRH on the Lhb and Fshb promoters to 79.15% and 55.66%, respectively. GnRH-stimulated activation of Lhb and Fshb promoters as well as serum response factor (Srf) promoters were almost completely inhibited by triptolide, suggesting that this component had a nonspecific effect to the cells. Dusp1 siRNA reduced the expression of DUSP1 and augmented MAPK3/1 phosphorylation, but it did not increase of gonadotropin promoters. By overexpression of DUSP1, both GnRH-stimulated Lhb and Fshb promoters were significantly reduced. We have previously shown that insulin-like growth factor 1 (IGF1) increases MAPK3/1 but does not activate gonadotropin subunit promoters. IGF1 failed to induce DUSP1 expression. In addition, under pulsatile GnRH stimulation, DUSP1 expression was observed following high-frequency GnRH pulses but not following low-frequency pulses. Our study demonstrated that DUSP1, induced by GnRH, functions not only as an MAPK3/1-inactivating phosphatase but also as an important mediator in gonadotropin subunit gene expression regulation.
Subject(s)
Dual Specificity Phosphatase 1/biosynthesis , Gene Expression/drug effects , Gonadotropin-Releasing Hormone/pharmacology , Gonadotropins, Pituitary/genetics , Pituitary Gland/enzymology , Animals , Butadienes/pharmacology , Cell Line , Diterpenes/pharmacology , Dual Specificity Phosphatase 1/genetics , Enzyme Activation/drug effects , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Epoxy Compounds/pharmacology , Follicle Stimulating Hormone, beta Subunit/genetics , Kinetics , Luteinizing Hormone, beta Subunit/genetics , Mice , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Nitriles/pharmacology , Phenanthrenes/pharmacology , Promoter Regions, Genetic/geneticsABSTRACT
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 90 days 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 E(2) levels after GnRH analogue (GnRHa) injection. GnRHa treatment increased mRNA expression levels of these genes, as well as plasma E(2) levels, indicating that GnRH plays an important regulatory role in the brain-pituitary-gonad axis of immature cinnamon clownfish.
Subject(s)
Gonadotropins, Pituitary/genetics , Hermaphroditic Organisms/genetics , Perciformes/growth & development , Perciformes/genetics , Receptors, Gonadotropin/genetics , Sex Determination Processes/genetics , Amino Acid Sequence , Animals , DNA, Complementary/genetics , Estradiol/blood , Female , Gene Expression Profiling , Gonadotropins, Pituitary/chemistry , Gonadotropins, Pituitary/metabolism , Gonads/metabolism , Male , Molecular Sequence Data , Phylogeny , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Gonadotropin/metabolism , Sex Determination Processes/physiology , Species SpecificityABSTRACT
Female reproduction is under multifactorial control of brain-pituitary-peripheral origin. The present study provides information on seasonal changes in circulating LH and GH concentrations, as well as transcript levels for a number of genes involved in the regulation of reproduction and growth in female goldfish. We also provide information on the effects of treatments with GnRH and/or GnIH, and their interaction with T3, at three stages of gonadal recrudescence. Maximum basal concentration of LH was observed at late recrudescence (Spring) while no seasonal changes in basal serum GH levels was detected. Serum LH and GH levels were stimulated by GnRH as expected, depending on the season. GnIH stimulated basal GH concentrations in gonadally regressed fish. GnIH inhibitory action on GnRH-induced LH response was observed in late, but not in mid recrudescence. T3 actions on basal and GnRH- or GnIH-induced GH secretion were generally inhibitory, depending on season. Administration of T3 attenuated GnRH-induced LH responses in mid and late stages of gonadal recrudescence, and the presence of GnIH abolished inhibitory actions of T3 in fish at mid recrudescence. Our results also demonstrated seasonal patterns in basal and GnRH- and/or GnIH-induced transcript levels for ERα, ERßI, FSHR, aromatase, TRαI, TRß, IGF-I, and Vtg in the liver and ovary. However, there were no clear correlations between changes in transcript levels and circulating levels of LH and GH. The results support the hypothesis that GnRH, GnIH, and T3 are contributing factors in complex reciprocal control of reproduction and growth in goldfish.
Subject(s)
Goldfish/physiology , Gonadotropins, Pituitary/genetics , Growth Hormone/genetics , Neuropeptides/pharmacology , Thyroid Hormones/pharmacology , Animals , Female , Goldfish/growth & development , Gonadotropins, Pituitary/metabolism , Gonadotropins, Pituitary/pharmacology , Growth Hormone/blood , Growth Hormone/metabolism , Luteinizing Hormone/blood , Luteinizing Hormone/genetics , Neuropeptides/physiology , Reproduction/physiology , Seasons , Thyroid Hormones/physiologyABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are a class of environmental pollutants with suspected toxic effects on reproductive and developmental processes. This study investigated a hypothesis that maternal exposure to TCDD damages gonadotropin-regulated steroidogenesis in fetal gonads to imprint defects in sexual behavior as well as the maturation of gonadal tissues. Oral administration of 1 microg/kg TCDD to pregnant Wistar rats at gestational day (GD) 15 attenuated the expression of luteinizing hormone (LH), a regulator of gonadal steroidogenesis, in the pituitaries of male and female fetuses at GD20. TCDD treatment also reduced the fetal expression of testicular and ovarian steroidogenic proteins, including steroidogenic acute-regulatory protein. These changes in pituitary and gonadal proteins were fetus-specific, and this seems not to be because of the greater delivery of TCDD to the brain of fetuses than adults. This is because a reduction in LH production was not reproduced even although TCDD was administered intraventricularly to adult rats. Direct supplementation of equine chorionic gonadotropin (eCG), an LH-mimicking hormone, to TCDD-exposed fetuses at GD17 restored the reduced expression of gonadal steroidogenic proteins. Maternal exposure to TCDD delayed the development of gonadal tissues in male and female pups and impaired their sexual behavior. However, eCG treatment at the fetal stage again restored not only tissue maturation but also many of the behavioral defects that occurred at adulthood. These results demonstrate that TCDD disrupts steroidogenesis in fetuses by targeting pituitary gonadotropin production and imprints demasculinization in males and defeminization in females in terms of their copulatory behavior.