Lhb Expression Research Articles

Comparison of the single nucleotide polymorphisms and expression of FSHβ and LHβ genes between prolific and non-prolific goat breeds of Kerala

Attappady Black and Malabari breeds of goats are native to Kerala, a southern state of India. Attappady Black is a low prolific breed whereas Malabari breed is highly prolific. Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) are the gonadotropins which are considered to be the central hormones controlling prolificacy. The present study aimed to detect presence of SNPs in exonic regions of FSHβ and LHβ and the expression of these gonadotropins in both these breeds. Blood samples were collected from ten does five each from Attappady Black and Malabari goats and the genomic DNA was extracted. Multiple primers were designed based on Ensembl Genome Browser sequences to cover the entire exonic regions of both the genes for SNP detection. Pooled PCR products of FSHβ and LHβ separately from the two breeds were sequenced. Nucleotide sequencing showed synonymous single nucleotide polymorphism T → C, in FSHβ coding sequence, at position 413 (as per FSHβ ENSCHIG00000022859), in both Attappady Black and Malabari goats. Numerous other SNPs both novel and reported were detected in the non-coding regions of FSHβ. In LHβ of Attappady Black goat, an SNP was found at exonic position 455 (G → A) as per LHβ ENSCHIT00000012663.1. This transition resulted in replacement of amino acid arginine (R) with glutamine (Q). Upon individual sequencing of ten each of single birth and multiple birth Attappady Black goat samples, three animals with single birth and five animals with multiple birth showed G → A change in the LHβ transcript at position 455. Pituitary samples were collected from 6 animals each from both the breeds and expressions of both the genes were quantified using qRT-PCR technique. There was no significant difference in pituitary FSHβ expression between breeds. However, there was a significant difference (P < 0.01) in LHβ expression between the two breeds with an increase in expression of 2.61 folds in Attappady Black breed. This study forewarn a main role for LH in the genetic network controlling prolificacy in these goat breeds and this necessitates further investigation with larger sample size and also in other goat breeds.

Effects of LPS on the Secretion of Gonadotrophin Hormones and Expression of Genes in the Hypothalamus-Pituitary-Ovary (HPG) Axis in Laying Yangzhou Geese.

Simple SummaryLipopolysaccharide (LPS), an endotoxin from E. coli, has been proven to impair follicle development and steroidogenesis, secretion of pituitary and hypothalamus reproductive hormones in mammals. However, the effects of LPS on the avian reproductive axis remain elusive. Pathogenic bacterial infection due to the particular mating behavior on the water containing pathogens was reported to decrease the laying rate and cause economic loss in goose production. In this study, we showed that LPS infection disturbed the plasma pituitary gonadotrophin hormone concentrations and the gene expression of the reproductive axis in Yangzhou geese. Notably, for the first time we proved that both the expression of gonadotrophin-releasing hormone (GnRH) and gonadotropin-inhibiting hormone (GnIH), two important reproductive genes from the hypothalamus, were altered after LPS treatment in birds. Our results can explain the decreased laying rate in goose after bacterial infection, and also provide new insights into reproductive dysfunction caused by LPS and the immune challenge in birds.Lipopolysaccharide (LPS) from gram-negative bacteria was found to be involved in the decrease in laying performance in goose flocks with high stocking density during summer months. LPS injection delayed the increase in the laying rate and altered hierarchical follicle morphology. While there is evidence that LPS exerts suppressive effects on goose reproduction, the time course effects of LPS on the hypothalamus-pituitary-ovary (HPG) axis remain elusive. In this study, we investigated the expression of genes in the HPG axis and the plasma gonadotrophin hormone concentrations in breeding geese at 0, 6, 12, 24, and 36 h after intravenous injection with LPS. The results showed that LPS treatment enhanced and suppressed expression of hypothalamic gonadotropin-inhibiting hormone (GnIH) and gonadotrophin-releasing hormone (GnRH) mRNA, respectively, and similar effects were observed on the mRNA expression of their receptors, GnIHR and GnRHR, in the pituitary. LPS treatment transiently increased follicle FSHβ mRNA expression at 12 h and exerted no significant effect on LHβ mRNA expression in the pituitary. Regardless of the expression of FSHβ and LHβ, plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were significantly increased during 24–36 h after LPS treatment. In the ovary, StAR and Cyp11a1 were mainly expressed in the granulosa layer (GL) of hierarchical follicles, while Cyp17a1 and Cyp19a1 were mainly expressed in white follicles (WFs) and yellowish follicles (YFs), and to a lesser extent in the theca layer (TL). After LPS treatment, the mRNA levels of Cyp11a1 in the GLs, Cyp17a1 in the WFs and TL, and Cyp19a1 in the WFs, YFs, and TL were significantly decreased. However, LPS treatment transiently upregulated StAR expression at 12 h. These results indicate that the exposure of laying geese to LPS may impair the HPG axis and disturb ovarian steroidogenesis. Our research provides new insights into reproductive dysfunction caused by LPS and the immune challenge in birds.

The reproductive regulation of LPXRFa and its receptor in the hypothalamo-pituitary-gonadal axis of the spotted scat (Scatophagus argus).

Gonadotropin-inhibitory hormone (GnIH) plays a critical role in regulating gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GtH), and steroidogenesis. The Lpxrfa (the piscine ortholog of GnIH) system has been found to regulate fish reproduction. To gain insight into the role of Lpxrfa in the regulation of spotted scat (Scatophagus argus) reproduction, spotted scat Lpxrfa (ssLpxrfa), and its receptor (ssLpxrfa-r) were cloned and analyzed. Tissue distribution and expression patterns at the hypothalamo-pituitary-gonadal axis (HPG axis) of sslpxrfa and sslpxrfa-r mRNA were also investigated during gonadal development of spotted scat. The open reading frame (ORF) of the sslpxrfa was 606bp encoding 201 amino acids and includes a putative signal peptide and two mature ssLpxrfa peptides with LPXRFamide motif at their C-terminus. The sslpxrfa-r ORF was 1449bp encoding 482 amino acids and contracted a seven-hydrophobic transmembrane (TM) domain structure. The tissue distribution showe d that the sslpxrfa was highly expressed in hypothalami, gill, and the gonads. In addition, sslpxrfa-r was highly expressed in hypothalami, pituitaries, and the gonads. Quantitative real-time polymerase chain reaction (qPCR) revealed that sslpxrfa had the highest expression in the hypothalami and pituitaries, and the lowest expression in the gonads in stage V. During gonadal development, the expression of sslpxrfa-r was gradually increased in the hypothalami but reduced in the gonads. However, no obvious trend was observed in the pituitaries. The expression of sslpxrfa and sslpxrfa-r decreased significantly after injection with 17β-estradiol (E2). However, the expression of both sslpxrfa and sslpxrfa-r was not changed after injection with 17α-methyltestosterone(17α-MT) in the hypothalami. In addition, no changes were observed in the expression of fshβ and lhβ in the pituitaries after injecting ssLpxrfa-1. However, ssLpxrfa-2 could downregulate the expression of sbgnrh and fshβ in the hypothalami and pituitaries, respectively. Taken together, these findings suggested that ssLpxrfa may participate in E2 feedback in reproduction and regulate the reproductive axis of spotted scat.

Loss of Inhibin Advances Follicle Activation and Female Puberty Onset but Blocks Oocyte Maturation in Zebrafish.

Inhibin was first characterized in mammals as a gonadal dimeric protein that inhibited pituitary follicle-stimulating hormone (FSH) secretion. As in mammals, the inhibin-specific α subunit (INHA/Inha/inha) has also been characterized in teleosts; however, its functions and physiological importance in fish reproduction remain unknown. Using CRISPR/Cas9 method, we generated an inha-deficient zebrafish line and analyzed its reproductive performance. As expected, pituitary expression of fshb increased significantly in both the young and the adult inha mutant. The expression of lhb also increased in the mutant, but only in sexually mature adults. Interestingly, the expression of activin βA (inhbaa) increased significantly in both the ovary and the testis of inha mutant, and the expression of ovarian aromatase (cyp19a1a) also increased dramatically in the mutant ovary. The juvenile female mutant showed clear signs of early follicle activation or precocious puberty onset. However, the adult female mutant was infertile with follicles arrested at the full-grown stage without final oocyte maturation and ovulation. Although follicle growth was normal overall in the mutant, the size and distribution of yolk granules in oocytes were distinct and some follicles showed granulosa cell hypertrophy. In contrast to females, inha-null males showed normal spermatogenesis and fertility. As reported in mammals, we also found sporadic tumor formation in inha mutants. Taken together, our study not only confirmed some conserved roles of inhibin across vertebrates, such as inhibition of FSH biosynthesis and tumor formation, but also revealed novel aspects of inhibin functions such as disruption of folliculogenesis and female infertility but no obvious involvement in spermatogenesis in fish.

Effects of Kiss2 on the Expression of Gonadotropin Genes in the Pituitary of Nile Tilapia (Oreochromis niloticus).

Kisspeptin, expressed mainly in the hypothalamus, stimulates gonadotropin-releasing hormone neurons to facilitate reproduction. In some model animals, the kisspeptin is also expressed in the pituitary. Recently, a pathway has been suggested in which kisspeptin acts directly on the pituitary to secretion of gonadotropin in mammals. In the present study, pituitaries of the Nile tilapia (Oreochromis niloticus) were cultured at different concentrations of kisspeptin-10 (Kp-10, FNYNPLSLRF) for 3 hours to observe the effect of kisspeptin on the expression of follicle-stimulating hormone β subunit (fshβ) gene and luteinizing hormone β subunit (lhβ) gene. Pituitary tissues were cultured with 0.1 μM of Kp-10, luteinizing hormone releasing hormone (LHRH), or LHRH+Kp-10 for 3, 6, 12, and 24 hours to investigate changes in the expression of fshβ and lhβ mRNA. Pituitaries cultured with high concentration of Kp-10 more than 0.1 μM for 3 hours exhibited a significant increase of fshβ mRNA expression, but not lhβ mRNA. The expression of both fshβ and lhβ mRNA increased after 6 hours in 0.1 μM of Kp-10 medium in comparison with that in the control medium. Tissues cultured in the LHRH medium however exhibited increased expression of both genes not only at 6 but also 12 hours. There were no significant differences of fshβ and lhβ gene expression in tissues cultured with LHRH+KP-10 medium compared with the control. These results suggested that although kisspeptin plays an important role in fshβ and lhβ expression in the pituitary of Nile tilapia, its action is far more complicated than expected.

The microRNA-200 cluster on chromosome 23 is required for oocyte maturation and ovulation in zebrafish†.

The reproductive process is usually controlled by the hypothalamic-pituitary-gonad axis in vertebrates, while Kiss/gonadotropin-releasing hormone (GnRH) system in the hypothalamus is required for mammalian reproduction but dispensable for fish reproduction. The regulation of follicle stimulating hormone/luteinizing hormone (LH) expression in fish species is still unknown. Here, we identified miR-200s on chromosome 23 (chr23-miR-200s) as important regulators for female zebrafish reproduction. Knockout of chr23-miR-200s (chr23-miR-200s-KO) resulted in dysregulated expression of luteinizing hormone beta lhb (luteinizing hormone beta) and some hormone genes in the pituitary as revealed by comparative transcriptome profiling, leading to failure of oocyte maturation and ovulation as well as defects in reproductive duct development. Chr23-miR-200s mainly expressed in the pituitary and regulated lhb expression by targeting the transcription repressor wt1a. Injection of human chorionic gonadotropin (hCG) could rescue the defects of oocyte maturation in chr23-miR-200s-KO zebrafish, whereas GnRH or LHRH-A2 could not, suggesting that Chr23-miR-200s regulated lhb expression in a GnRH-independent pathway. It was remarkable that either injection of carp pituitary extraction, or co-injection of hCG with synthetic oxytocin and vasotocin could greatly rescue the defects of both oocyte maturation and ovulation in chr23-miR-200s-KO zebrafish. Altogether, our findings highlight an important function of chr23-miR-200s in controlling oocyte maturation by regulation LH expression, and oxytocin and vasotocin are potentially responsible for the ovulation in fish species.

The sex-specific patterns of changes in hypothalamic-pituitary-gonadal axis during experimental autoimmune encephalomyelitis

Multiple sclerosis develops during reproductive years in a sex-specific manner. Various neuroendocrine changes have been described in this inflammatory, demyelinating, and debilitating disease. We here aimed to determine the extent and sex specificity of alterations in the hypothalamic-pituitary-gonadal axis in the rat model of multiple sclerosis named experimental autoimmune encephalomyelitis. During the disease course, the hypothalamic tissue showed transient upregulation of inflammatory marker genes Gfap, Cd68, Ccl2, and Il1b in both sexes, but accompanied by sex-specific downregulation of Kiss1 (in females only) and Gnrh1 (in males only) expression. In females, the expression of gonadotrope-specific genes Lhb, Cga, and Gnrhr was also inhibited, accompanied by decreased basal but not stimulated serum luteinizing hormone levels and a transient arrest of the estrous cycle. In contrast, Fshb expression and serum progesterone levels were transiently elevated, findings consistent with the maintenance of the corpora lutea, and elevated immunohistochemical labeling of ovarian StAR, a rate limiting protein in steroidogenic pathway. In males, downregulation of Gnrhr expression and basal and stimulated serum luteinizing hormone and testosterone levels were accompanied by inhibited testicular StAR protein expression. We propose that inflammation of hypothalamic tissue downregulates Kiss1 and Gnrh1 expression in females and males, respectively, leading to sex-specific changes downstream the axis.

Gonadotropin-releasing hormone-Cu complex (Cu-GnRH) transcriptional activity in vivo in the female rat anterior pituitary gland

Unlike gonadotropin-releasing hormone (GnRH) analogues characterized by amino acid replacement in decapeptide primary structure, Cu-GnRH molecule preserves the native sequence but contains a Cu2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of His2. Cu-GnRH can operate via cAMP/PKA signalling in anterior pituitary cells, suggesting that it may affect selected gonadotropic network gene transcription in vivo.We analysed pituitary mRNA expression of Egr-1, Nr5a1, and Lhb based on their role in luteinizing hormone (LH) synthesis; and Nos1, Adcyap1, and Prkaca due to their dependence on cAMP/PKA activity. In two independent experiments, ovariectomized rats received intracerebroventricular pulsatile (one pulse/h or two pulses/h over 5 h) microinjections of 2 nM Cu-GnRH; 2 nM antide (GnRH antagonist) + 2 nM Cu-GnRH; 100 nM PACAP6–38 (PACAP receptor antagonist) + 2 nM Cu-GnRH. Relative expression of selected mRNAs was determined by qRT-PCR. LH serum concentration was examined according to RIA.All examined genes responded to Cu-GnRH stimulation with increased transcriptional activity in a manner dependent on pulse frequency pattern. Increased expression of Nr5a1, Lhb, Nos1, Adcyap1, and Prkaca mRNA was observed solely in rats receiving the complex with frequency of two pulses/h over 5 h. Egr-1 transcription was up-regulated for both applied Cu-GnRH pulsatile patterns. The stimulatory effect of Cu-GnRH on gene transcription was dependent on both GnRH receptor and PAC-1 activation.In conclusion, obtained results indicate that Cu-GnRH complex is a GnRH analogue able to induce both IP3/PKC and cAMP/PKA-dependent gonadotrope network gene transcription in vivo.

Morphometric Evaluation of Spermatogenic Cells and Seminiferous Tubules and Exploration of Luteinizing Hormone Beta Polypeptide in Testis of Datong Yak.

Simple SummaryPrevious studies revealed that luteinizing hormone βeta polypeptide (LHB) plays an essential role in fertilization. Therefore, we aimed to confirm the importance of LHB in the testis of yak and to determine their association with male yak fertility. Histomorphological analysis of the testes is essential for predicting the fertilizing ability of the bull. To the best our knowledge, this is the first study to evaluate the micro anatomical changes and histometric alternation in testes of Datong yak. These findings could help to predict the sperm production capacity and to understand the specific molecular mechanisms of LHB during spermatogenesis.Histological examination of testes is essential for understanding infertility, sex development, and growth. Therefore, to understand the histomorphology of testes at different developmental stages, we performed hematoxylin and eosin staining of Yak testis. Our results revealed that the diameters of spermatogenic cells and their nuclei were significantly larger (p < 0.05) in the testis at six years compared to at six and 18 months. No significant difference was noted between 30 months and six years. The study was designed to compare the expression profile of LHB in Datong yak. The expression pattern of LHB was explored using quantitative PCR, semi-quantitative PCR, molecular bioinformatic, and Western blot analysis. Our observations indicated that expression of LHB was significantly higher (p < 0.05) in the testis of Datong yak. Western blotting indicated that the molecular mass of LHB protein was 16 kDa in yak. The protein encoded by yak LHB included conserved cysteine-knot domain regions. The high expression of LHB in testis indicated that LHB may be vital for the development of male gonads and the fertility of Datong yak.

Ovarian Androgens Maintain High GnRH Neuron Firing Rate in Adult Prenatally-Androgenized Female Mice.

Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive reproductive cycles. In polycystic ovary syndrome (PCOS), persistent high GnRH/luteinizing hormone (LH) frequency disrupts cycles and exacerbates hyperandrogenemia. Adult prenatally-androgenized (PNA) mice exhibit increased GnRH neuron firing rate, elevated ovarian androgens, and disrupted cycles, but before puberty, GnRH neuron activity is reduced in PNA mice compared with controls. We hypothesized that ovarian feedback mediates the age-dependent change in GnRH neuron firing rate in PNA vs control mice. Extracellular recordings of green fluorescent protein (GFP)-identified GnRH neurons were made 5 to 7 days after sham-surgery, ovariectomy (OVX), or, in adults, after OVX plus replacement of sub-male androgen levels with dihydrotestosterone implants (OVX + DHT). In 3-week-old mice, OVX did not affect GnRH neuron firing rate in either group. In adult controls, OVX increased GnRH neuron firing rate, which was further enhanced by DHT. In adult PNA mice, however, OVX decreased GnRH neuron firing rate, and DHT restored firing rate to sham-operated levels. In contrast to the differential effects of ovarian feedback on GnRH neuron firing rate, serum LH increased after OVX in both control and PNA mice and was not altered by DHT. Pituitary gene expression largely reflected changes expected with OVX, although in PNA but not control mice, DHT treatment increased Lhb expression. These results suggest prenatal androgen exposure programs marked changes in GnRH neuron regulation by homeostatic steroid feedback. PNA lowers GnRH neuron activity in low-steroid states (before puberty, OVX), and renders activity in adulthood dependent upon ongoing exposure to elevated ovarian androgens.

AMP-activated protein kinase activation reduces the transcriptional activity of the murine luteinizing hormone β-subunit gene.

Malnutrition is one of the factors that induces reproductive disorders. However, the underlying biological processes are unclear. AMP-activated protein kinase (AMPK) is an enzyme that plays crucial role as a cellular energy sensor. In the present study, we examined the effects of AMPK activation on the transcription of the murine gonadotropin subunit genes Cga, Lhb, and Fshb, and the gonadotropin-releasing hormone receptor Gnrh-r. Real-time PCR and transcription assay using LβT2 cells demonstrated that 5-amino-imidazole carboxamide riboside (AICAR), a cell-permeable AMP analog, repressed the expression of Lhb. Next, we examined deletion mutants of the upstream region of Lhb and found that the upstream regulatory region of Lhb (–2527 to –2198 b) was responsible for the repression by AICAR. Furthermore, putative transcription factors (SP1, STAT5a, and TEF) that might mediate transcriptional control of the Lhb repression induced by AICAR were identified. In addition, it was confirmed that both AICAR and a competitive inhibitor of glucose metabolism, 2-deoxy-D-glucose, induced AMPK phosphorylation in LβT2 cells. Therefore, the upstream region of Lhb is one of the target sites for glucoprivation inducing AMPK activation. In addition, AMPK plays a role in repressing Lhb expression through the distal –2527 to –2198 b region.

Divergent expression patterns of pituitary gonadotropin subunit and GnRH receptor genes to continuous GnRH in vitro and in vivo

Continuous, as opposed to pulsatile, delivery of hypothalamic gonadotropin-releasing hormone (GnRH) leads to a marked decrease in secretion of pituitary gonadotropins LH and FSH and impairment of reproductive function. Here we studied the expression profile of gonadotropin subunit and GnRH receptor genes in rat pituitary in vitro and in vivo to clarify their expression profiles in the absence and continuous presence of GnRH. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb expression. These data suggest that blockade of Fshb expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment.

Changes in expression of reproduction-related hormones in the brain and pituitary during early ovarian differentiation and development in the red spotted grouper Epinephelus akaara, with emphasis on FSHβ and LHβ

The brain-pituitary-gonadal (BPG) axis, an important endocrine system, is closely related to gonadal development in teleosts. This study aimed to investigate the regulation of reproduction-related hormones in the BPG axis during early ovarian differentiation and development from 30 to 160 days post hatching (dph) in the red spotted grouper. The immune signals of FSHβ and LHβ were detected by immunohistochemistry. kiss1, kiss2, gpr54, gnrh1, fshβ, and lhβ gene expression was analyzed using qRT-PCR. 17β-estradiol (E2) levels were analyzed by ELISA. The immune signals of FSHβ and LHβ were detected in the central, ventral, or marginal part of the proximal pars distalis area at 55, 80, 100, 120, and 160 dph, along with a few signals detected in the external border of the pars intermedia area at 55, 120, and 160 dph. As a result of histological observation, we determined that ovarian differentiation began at 80 dph and finished at 120 dph. At 30 dph before ovarian differentiation, only the FSHβ signal was detected but with weak reaction, suggesting the earlier appearance of FSHβ than LHβ after hatching. Comparison of the signal intensity and gene expression of FSHβ and LHβ indicated that LH had stronger signals and higher transcript levels at 80 dph while E2 levels also increased, suggesting that LH may play a more active role in the onset of ovarian differentiation. gnrh1 and the kisspeptin genes showed no clear changes, but gpr54 (kisspeptin receptor) significantly increased during the ovarian differentiation phase. These results suggest that the kisspeptin receptor GPR54 may be involved in ovarian differentiation in the red spotted grouper.

Nr5a1b promotes and Nr5a2 inhibits transcription of lhb in the orange-spotted grouper, Epinephelus coioides†.

Nr5a1 (Sf-1) up-regulates lhb expression across vertebrates; however, its regulatory roles on fshb remain to be defined. Moreover, the involvement of Nr5a2 in the regulation of gonadotropin expression is not clear either. In the present study, the involvement of Nr5a1b (a homologue of Nr5a1) and Nr5a2 in the regulation of lhb and fshb expression in the orange-spotted grouper was examined. Dual fluorescent immunohistochemistry using homologous antisera showed that in the pituitary of orange-spotted groupers, Lh cells contain both immunoreactive Nr5a1b and Nr5a2 signals, whereas Fsh cells contain neither of them. In LβT2 cells, Nr5a1b up-regulated basal activities of lhb and fshb promoters possibly via Nr5a sites, and synergistically (on lhb promoter) or additively (on fshb promoter) with forskolin. Surprisingly, Nr5a2 inhibited basal activities of lhb promoter possibly via Nr5a sites and attenuated the stimulatory effects of both forskolin and Nr5a1b. In contrast, Nr5a2 had no effects on fshb promoter. Chromatin immunoprecipitation analysis showed that both Nr5a1b and Nr5a2 bound to lhb promoter, but not fshb promoter in the pituitary of the orange-spotted grouper. The abundance of Nr5a1b bound to lhb promoter was significantly higher at the vitellogenic stage than the pre-vitellogenic stage, whereas that of Nr5a2 exhibited an opposite trend. Taken together, data of the present study demonstrated antagonistic effects of Nr5a1b and Nr5a2 on lhb transcription in the orange-spotted grouper and revealed novel regulatory mechanisms of differential expression of lhb and fshb genes through Nr5a homologues in vertebrates.

Cortisol differentially affects cell viability and reproduction-related gene expression in Atlantic cod pituitary cultures dependent on stage of sexual maturation

Through the action of cortisol, stress can affect reproductive biology with behavioural and physiological alterations. Using mixed sex primary pituitary cultures from Atlantic cod (Gadus morhua), the present study aimed to investigate potential direct effects of basal and stress level cortisol on the pituitary in terms of cell viability and reproduction-related gene expression at different stages of sexual maturity. Stress level of cortisol stimulated cell viability in cells derived from sexually maturing and mature fish. In cells from spent fish, high cortisol levels did not affect cell viability in terms of metabolic activity, but did stimulate viability in terms of membrane integrity. Basal cortisol levels did not affect cell viability. Ethanol, used as solvent for cortisol, decreased cell viability at all maturity stages, but did generally not affect gene expression. Genes investigated were fshb, lhb and two Gnrh receptors expressed in cod gonadotropes (gnrhr1b and gnrhr2a). Cortisol had dual effects on fshb expression; stimulating expression in cells from mature fish at stress dose, while inhibiting expression in cells from spent fish at both doses. In contrast, cortisol had no direct effect on lhb expression. While gnrhr2a transcript levels largely increased following cortisol treatment, gnrhr1b expression decreased in cells from spent fish and was unaffected at other maturity stages. These findings demonstrate that cortisol can act directly and differentially at the pituitary level in Atlantic cod and that factors facilitating these actions are dose-dependently activated and vary with level of sexual maturity.

SAT-420 Gonadotropin-Releasing Hormone Stimulates Luteinizing Hormone Exocytosis without Elevating Lhb Expression

Continuous but not pulsatile delivery of GnRH leads to marked down regulation of luteinizing hormone (LH) secretion and blockade of reproductive functions. However, the relationship between LH beta subunit gene (Lhb) expression and LH secretion has not been studied in detail. Here we studied the relationship between Lhb expression, LH release and intracellular LH content under different experimental paradigms. Experiments were performed in vivo and in vitro using female and male rats and in cultured rat pituitary cells. In vitro experiments included evaluation of Lhb expression during the prolonged absence of GnRH, influence of culture medium on gene expression, continuous vs repetitive GnRH application, and ELISA vs Western blot analysis of LH content. Experiments showed that Lhb expression declined progressively after pituitary removal and cell dispersion, indicating the loss of regulated gene expression. Addition of GnRH receptor agonists did not rescue Lhb expression, suggesting that other factor(s) account(s) for regulated gene expression. On the other hand, GnRH stimulated LH secretion in vitro, leading to gradual depletion of the secretory pool during continuous receptor activation. Inhibition of transcription by actinomycin D had only a minor effect on in vitro LH secretion, in contrast to cycloheximide, a protein synthesis inhibitor. In vivo administration of a GnRH receptor agonist was accompanied with a rapid increase in serum LH levels and a progressive depletion of the intrapituitary LH levels, without affecting Lhb expression. These results indicate that GnRH controls LH synthesis and exocytosis post-transcriptionally and that pulsatile GnRH release protects gonadotrophs from depletion of the LH secretory pool.

Molecular characterization of gonadotropin-inhibitory hormone (GnIH) gene and effect of intramuscular injection of GnIH peptide on the reproductive axis in Catla catla

Gonadotropin-inhibitory hormone (GnIH) plays an important role in reproduction by inhibiting the expression of gonadotropins in birds and mammals, but in fishes, it is ambiguous. In this study, we cloned 606 bp long cDNA of GnIH from Catla catla brain (cGnIH). The encoded preproGnIH peptide generated three putative peptides (cGnIH-I, -II, -III) of different size. Phylogenetic analysis of GnIH showed clustering of different peptide sequence with its orthologs in separate clades. The real-time PCR analysis showed the expression of cGnIH in brain, gonads, intestine, stomach, heart, gill and liver with the highest expression in the brain and gonads of both sexes. The basal GnIH mRNA expression was higher in spawning and spent phase of the male brain and spawning phase of the female brain. In testis, the expression was highest in spent phase, while in ovary the expression did not change significantly during reproductive phases. The in vivo experiment of cGnIH-III peptide exhibited the higher expression of HPG axis genes, lhb, fshb, cgnrh, kiss2 and kiss1r and serum hormone level of LH and FSH as soon as 3 h after the intramuscular delivery. These results suggest that the GnIH is positively involved in regulation of reproduction in HPG axis of C. catla.

Medaka follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression levels

The two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are of particular importance within the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates. In the current study, we demonstrate the production and validation of Japanese medaka (Oryzias latipes) recombinant (md) gonadotropins Fshβ (mdFshβ), Lhβ (mdLhβ), Fshβα (mdFshβα), and Lhβα (mdLhβα) by Pichia pastoris, the generation of specific rabbit antibodies against their respective β subunits, and their use within the development and validation of competitive enzyme-linked immunosorbent assays (ELISAs) for quantification of medaka Fsh and Lh. mdFsh and mdLh were produced as single-chain polypeptides by linking the α subunit with mdFshβ or mdLhβ mature protein coding sequences to produce a “tethered” polypeptide with the β-chain at the N-terminal and the α-chain at the C-terminal. The specificity of the antibodies raised against mdFshβ and mdLhβ was determined by immunofluorescence (IF) for Fshβ and Lhβ on medaka pituitary tissue, while comparison with fluorescence in situ hybridization (FISH) for fshb and lhb mRNA was used for validation. Competitive ELISAs were developed using antibodies against mdFshβ or mdLhβ, and the tethered proteins mdFshβα or mdLhβα for standard curves. The standard curve for the Fsh ELISA ranged from 97.6 pg/ml to 50 ng/ml, and for the Lh ELISA from 12.21 pg/ml to 6.25 ng/ml. The sensitivity of the assays for Fsh and Lh was 44.7 and 70.8 pg/ml, respectively. A profile of pituitary protein levels of medaka Fsh and Lh comparing juveniles with adults showed significant increase of protein amount from juvenile group (body length from 12 mm to 16.5 mm) to adult group (body length from 21 mm to 26.5 mm) for both hormones in male medaka. Comparing these data to a developmental profile of pituitary mRNA expression of medaka fshb and lhb, the mRNA expression of lhb also increased during male maturation and a linear regression analysis revealed a significant increase of lhb expression with increased body length that proposes a linear model. However, fshb mRNA expression did not change significantly during male development and therefore was not correlated with body length. In summary, we have developed and validated homologous ELISA assays for medaka Fsh and Lh based on proteins produced in P. pastoris, assays that will be used to study the functions and regulations of Fsh and Lh in more detail.

Altered GnIH precursor gene expression during early development of Nile tilapia, Oreochromis niloticus

ABSTRACT This study investigated the expression patterns and relationships among gonadotropin inhibiting hormone (GnIH), gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) mRNA during early developmental phases of Nile tilapia. GnIH mRNA could be detected immediately after hatching and was maintained until 11 weeks post-hatching (wph), at which time presumptive puberty occurs in this species. Subsequently, levels remained low for up to 24 wph. GnIH expression was relatively high until the onset puberty, and started to decrease when the expression of GnRH1, LH-β, and FSH-β mRNA increased. Expression of GnRH1 was low until 11 wph, but increased significantly at 12 wph. Markedly high of expression occurred at 12 and 24 wph. Results from this study suggest that GnIH is involved in the inhibition of sexual maturation during early development periods until the onset of puberty in this species.

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 lhβ 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 lhβ expression.