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.

Regulation of steroid production and key genes in catfish Heteropneustes fossilis using recombinant gonadotropins.

The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHß, FSHß, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish.

Cloning of gonadotropin Gph-alpha, FSH-beta and LH-beta subunits and seasonal profiles of steroid hormones in wild-caught Nile perch, Lates niloticus.

The Nile perch (np; Lates niloticus) is a freshwater teleost species with a potential for aquaculture in freshwater surroundings. However, wild-caught breeders have persistently failed to spawn spontaneously in captivity. Cloning of the gonadotropin subunits and analysing seasonal variation in reproductive hormone levels for a 1-year period were done to gain knowledge on the physiological basis underlying the reproductive biology of np. The ß-follicle-stimulating hormone (FSH-ß) and ß-luteinizing hormone (LH-ß) subunits and their common α-glycoprotein (Gph-α) subunit were cloned using 3' and 5' RACE-PCR. The nucleotide sequences of the npgph-α, npfsh-ß, and nplh-ß subunits were 664, 580 and 675 nucleotides in length, encoding peptides of 124, 120 and 148 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with its counterparts in other teleost. Sequence analysis showed that npFSH-ß is more similar to higher vertebrate FSH-ßs than to higher vertebrate LH-ßs. Heterologous immunoassay was calibrated to analyse pituitary LH levels. While the LH immunoassay showed parallelism of npLH with that of tilapia (ta), no parallelism for FSH was found. Levels of pituitary LH were higher in females at gonadal stages of vitellogenic oocytes, mature secondary oocytes and mature tertiary oocytes with migrating nucleus than in pre-vitellogenic oocytes and early and late perinucleolus oocytes. Using competitive steroid ELISA, variations in the levels of the steroid hormones 11-ketotestosterone (11-KT) in males and E2 in females were characterized in relation to month and reproductive index of Nile perch. Our findings show that in females, gonadosomatic index and plasma E2 were highly correlated (R2 = 0.699, n = 172) and peaked from September to November while in males, the gonadosomatic index and plasma 11-KT peaked from October to November. In female fish, both steroid hormones were detected in the plasma but greatly varied in concentrations. E2 in particular, increased with the developmental stage of the gonads. The levels of steroid hormones, E2 and 11-KT in females and males respectively increased with fish size (total lengths) and suggest that females mature at a body length of 40-59 cm than their counter part males that mature at a total length of 60-70 cm. Taken together, we describe seasonal endocrine differences in wild-caught adult Nile perch which could potentially be exploited to manipulate the reproductive axis in cultured breeders.

Presence of GnRH3 in sturgeon and the roles of GnRH1 and GnRH2 on the regulation of LH/FSH in mature female sterlet Acipenser ruthenus in vitro.

Three forms of gonadotropin-releasing hormones (GnRHs), ArGnRH1, ArGnRH2, and ArGnRH3, were identified in sterlet. Compared with their orthologue, ArGnRH1 and ArGnRH2 have conserved core decapeptide but show low identity in the signal peptide and the rest of the sequences. The existence of the GnRH3 paralogue of sturgeon was predicted for the first time with TBLASTN by using the amino acid sequences of catshark and whale shark GnRH3 precursor as queries against the whole genome and transcript data of sterlet. The predicted ArGnRH3 cDNA sequence was composed of three exons containing all the elements of the GnRH family. The successful molecular cloning of GnRH3 from sterlets verified its expression in the brain of sturgeons. The analysis of the ArGnRH3 amino acid sequence revealed a completely conserved decapeptide sequence that shows 100% identity with the sequence of teleosts and differs in one amino acid with that of the cartilaginous fish (catshark and whale shark) at the 5th position. The structure of the phylogenetic tree showed that a total of 52 vertebrate GnRH sequences were clustered into three main clades corresponding to GnRH1, GnRH2, and GnRH3. The ArGnRH3 sequence is the oldest GnRH3 identified in teleosts. The tissue distribution analysis showed that ArGnRH1 was expressed in all the 13 examined tissues of females and in most of the tested tissues of male fish, with the highest expression in the pituitary and hypothalamus. ArGnRH2 is only expressed in the pituitary, hypothalamus, and gonads of both female and male sterlets. ArGnRH3 mRNA could be detected in the pituitary, hypothalamus, and gonad in both female and male fish. It is also present in the spleen, head kidney, and gill in female fish and in kidney and heart in male fish. However, the ArGnRH3 only showed weak expression in all the positive tissues. ArGnRH1 and ArGnRH2 active decapeptides were synthesized to investigate their roles on the regulation of LH/FSH using a mixed brain cell line from a sexually mature female sterlet. The results showed that ArGnRH1 and ArGnRH2 exerted different effects on the gene expression and release of gonadotropins. ArGnRH1 promoted the expression of fshß significantly around 48 h, and the expression was suppressed when the treatment time was extended to 72 h. ArGnRH1 had no significant effects on the level of either mRNA or secreted lh in any of the tested treatment length or concentrations. Moreover, ArGnRH1 did not stimulate the activity of gonadotropins in the maturation stage of female sturgeons. ArGnRH2 promoted the expression of fshß at 24 h and 48 h and increased mRNA level of lhß at 6 h and 48 h, accompanied by the significant secretion of LH at 72 h, although the high mRNA level of fsh did not correlate with the secretion of FSH in ArGnRH2-treated groups. In conclusion, ArGnRH2 plays an important role in the maturation stage of female sterlets. Therefore, ArGnRH2 has the potential to induce ovulation and spermiation in sturgeons.

Functional differences in the hypothalamic-pituitary-gonadal axis are associated with alternative reproductive tactics based on an inversion polymorphism.

The evolution of social behavior depends on genetic changes, yet, how genomic variation manifests itself in behavioral diversity is still largely unresolved. Chromosomal inversions can play a pivotal role in producing distinct behavioral phenotypes, in particular, when inversion genes are functionally associated with hormone synthesis and signaling. Male ruffs exhibit alternative reproductive tactics (ARTs) with an autosomal inversion determining two alternative morphs with clear behavioral and hormonal differences from the ancestral morph. We investigated hormonal and transcriptomic differences in the pituitary and gonads. Using a GnRH challenge, we found that the ability to synthesize testosterone in inversion carriers is severely constrained, whereas the synthesis of androstenedione, a testosterone precursor, is not. Inversion morphs were able to produce a transient increase in androstenedione following the GnRH injection, supporting the view that pituitary sensitivity to GnRH is comparable to that of the ancestral morph. We then performed gene expression analyses in a second set of untreated birds and found no evidence of alterations to pituitary sensitivity, gonadotropin production or gonad sensitivity to luteinizing hormone or follicle-stimulating hormone across morphs. Inversion morphs also showed reduced progesterone receptor expression in the pituitary. Strikingly, in the gonads, inversion morphs over-expressed STAR, a gene that is located outside of the inversion and responsible for providing the cholesterol substrate required for the synthesis of sex hormones. In conclusion, our results suggest that the gonads determine morph-specific differences in hormonal regulation.

Actions and Roles of FSH in Germinative Cells.

Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic-pituitary-gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and ß. The FSH ß-subunit (FSHß) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH ß-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.

Sensitivity of pituitary gonadotropes to hyperglycemia leads to epigenetic aberrations and reduced follicle-stimulating hormone levels.

The connection between metabolism and reproductive function is well recognized, and we hypothesized that the pituitary gonadotropes, which produce luteinizing hormone and follicle-stimulating hormone (FSH), mediate some of the effects directly via insulin-independent glucose transporters, which allow continued glucose metabolism during hyperglycemia. We found that glucose transporter 1 is the predominant glucose transporter in primary gonadotropes and a gonadotrope precursor-derived cell line, and both are responsive to culture in high glucose; moreover, metabolite levels were altered in the cell line. Several of the affected metabolites are cofactors for chromatin-modifying enzymes, and in the gonadotrope precursor-derived cell line, we recorded global changes in histone acetylation and methylation, decreased DNA methylation, and increased hydroxymethylation, some of which did not revert to basal levels after cells were returned to normal glucose. Despite this weakening of epigenetic-mediated repression seen in the model cell line, FSH ß-subunit ( Fshb) mRNA levels in primary gonadotropes were significantly reduced, apparently due in part to increased autocrine/paracrine effects of inhibin. However, unlike thioredoxin interacting protein and inhibin subunit α, Fshb mRNA levels did not recover after the return of cells to normal glucose. The effect on Fshb expression was also seen in 2 hyperglycemic mouse models, and levels of circulating FSH, required for follicle growth and development, were reduced. Thus, hyperglycemia seems to target the pituitary gonadotropes directly, and the likely extensive epigenetic changes are sensed acutely by Fshb. This scenario would explain clinical findings in which, even after restoration of optimal blood glucose levels, fertility often remains adversely affected. However, the relative accessibility of the pituitary provides a possible target for treatment, particularly crucial in the young in which hyperglycemia is increasingly common and fertility most relevant.-Feldman, A., Saleh, A., Pnueli, L., Qiao, S., Shlomi, T., Boehm, U., Melamed, P. Sensitivity of pituitary gonadotropes to hyperglycemia leads to epigenetic aberrations and reduced follicle-stimulating hormone levels.

Colocalization of GPR120 and anterior pituitary hormone-producing cells in female Japanese Black cattle.

Negative energy balance in domestic animals suppresses their reproductive function. These animals commonly use long-chain fatty acids (LCFAs) from adipocytes as an energy source under states of malnutrition. The G-protein coupled receptor, GPR120, is a specific receptor for LCFAs, but its role in reproductive function remains unknown in domestic animals. The purpose of this study was to examine whether GPR120 is involved in the reproductive system of cattle. GPR120 mRNA expression was evaluated in brain, pituitary, and ovarian tissue samples by RT-PCR. GPR120 gene expression was detected with high intensity only in the anterior pituitary sample, and GPR120-immunoreactive cells were found in the anterior pituitary gland. Double immunohistochemistry of GPR120 in the anterior pituitary hormone-producing cells, such as gonadotropes, thyrotropes, lactotropes, somatotropes, and corticotropes, was performed to clarify the distribution of GPR120 in the anterior pituitary gland of ovariectomized heifers. Luteinizing hormone ß subunit (LHß)- and follicle-stimulating hormone ß subunit (FSHß)-immunoreactive cells demonstrated GPR120 immunoreactivity at 80.7% and 85.9%, respectively. Thyrotropes, lactotropes, somatotropes, and corticotropes coexpressed GPR120 at 21.1%, 5.4%, 13.6%, and 14.5%, respectively. In conclusion, the present study suggests that GPR120 in the anterior pituitary gland might mediate LCFA signaling to regulate gonadotrope functions, such as hormone secretion or production, in cattle.

Short-term but not long-term high-fat diet induces an increase in gene expression of gonadotropic hormones and GPR120 in the male mouse pituitary gland.

High-fat diet (HFD) is associated with the regulation of reproductive functions. This study aimed to investigate the effects of short-term HFD on the mRNA expression levels of follicle-stimulating hormone ß subunit (FSHß), luteinizing hormone ß subunit (LHß), gonadotropin-releasing hormone receptor, and long-chain fatty acid receptor, GPR120, in the matured male mouse pituitary gland. Adult male mice were fed either control chow or HFD for 1, 2, 5, 10, 30 and 150 days. Fshb and Gpr120 mRNA expression levels in the pituitary glands were significantly increased during 2 to 30 days of HFD feeding. Gnrh-r mRNA in the 30 days HFD fed group and body weight in the 30 and 150 days HFD fed groups were higher than control. However, there were no significant differences in plasma non-esterified fatty acids or glucose levels during the 150 days of HFD feeding. These results suggest that male mice feeding a short-term HFD induces FSHß synthesis and GPR120 expression in their pituitary gonadotropes.

AMP-activated protein kinase activation reduces the transcriptional activity of the murine luteinizing hormone ß-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 LßT2 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 LßT2 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.

Gpr120 mRNA expression in gonadotropes in the mouse pituitary gland is regulated by free fatty acids.

GPR120 is a long-chain fatty acid (LCFA) receptor that is specifically expressed in gonadotropes in the anterior pituitary gland in mice. The aim of this study was to investigate whether GPR120 is activated by free fatty acids in the pituitary of mice and mouse immortalized gonadotrope LßT2 cells. First, the effects of palmitate on GPR120, gonadotropic hormone b-subunits, and GnRH-receptor expression in gonadotropes were investigated in vitro. We observed palmitate-induced an increase in Gpr120 mRNA expression and a decrease in follicle-stimulating hormone b-subunit (Fshb) expression in LßT2 cells. Furthermore, palmitate exposure caused the phosphorylation of ERK1/2 in LßT2 cells, but no significant changes were observed in the expression levels of luteinizing hormone b-subunit (Lhb) and gonadotropin releasing hormone-receptor (Gnrh-r) mRNA and number of GPR120 immunoreactive cells. Next, diurnal variation in Gpr120 mRNA expression in the male mouse pituitary gland was investigated using ad libitum and night-time restricted feeding (active phase from 1900 to 0700 h) treatments. In ad libitum feeding group mice, Gpr120 mRNA expression at 1700 h was transiently higher than that measured at other times, and the peak blood non-esterified fatty acid (NEFA) levels were observed from 1300 to 1500 h. These results were not observed in night-time-restricted feeding group mice. These results suggest that GPR120 is activated by LCFAs to regulate follicle stimulating hormone (FSH) synthesis in the mouse gonadotropes.

Differentially expressed lncRNA-m433s1 regulates FSH secretion by functioning as a miRNA sponge in male rat anterior pituitary cells†.

Long noncoding RNAs (lncRNAs) are important regulators that have multiple functions in a variety of biological processes. However, the contributions of lncRNAs to follicle-stimulating hormone (FSH) secretion remain largely unknown. In this study, we first identified a novel lncRNA, lncRNA-m433s1, as an intergenic lncRNA located in the cytoplasm. We next used MS2-RIP assays to demonstrate that lncRNA-m433s1 interacted with miR-433. Furthermore, we detected the levels of lncRNA-m433s1, miR-433, and Fshß expression, FSH concentrations, and apoptosis upon overexpression and knockdown of lncRNA-m433s1, revealing that lncRNA-m433s1 upregulated Fshß expression. Globally, lncRNA-m433s1 reduced the inhibitory effect of miR-433 on Fshß and further regulated FSH secretion as a competing endogenous RNA (ceRNA) by sponging miR-433. This ceRNA model will provide novel insight into the regulatory mechanisms of lncRNAs associated with rat reproduction.

Thermal manipulation and GnRHa therapy applied to the reproduction of lambari-do-rabo-amarelo, Astyanax altiparanae females (Characiformes: Characidae) during the non-breeding season.

Lambari-do-rabo-amarelo Astyanax altiparanae in the wild reproduce during spring and summer, but females undergo vitellogenesis throughout the year, including the non-spawning winter period when water temperatures are low. The present study investigated the physiological role of temperature modulation on the hypothalamus-pituitary-gonads axis of lambari during winter, as well as the effects of gonadotropin releasing hormone agonist (GnRHa) therapy. Captive females were exposed to two different temperatures (20 °C and 27 °C) and were injected weekly with GnRHa for 21 days during winter (Control, CTR; Low dose; LD and high dose of GnRHa, HD). At the end of the 21-days period gonadosomatic index (GSI), oocyte stage of development and theoretical fecundity were evaluated, together with plasma levels of 17ß-estradiol (E2). Gene expression of the two pituitary gonadotropins follicle-stimulating hormone (fshß) and luteinizing hormone (lhß), as well as hepatic vitellogenin-A (vtgA) expression were also analyzed. At the end of the experimental period, females from the six different experimental conditions were induced to spawn using human chorionic gonadotropin (hCG). Spawning performance parameters and plasma levels of the maturation inducing steroid (MIS) were analyzed. Gene expression of fshß did not change with temperature manipulation, but females exposed to 27 °C and supplemented with a HD of GnRHa exhibited an increased fshß gene expression, associated with higher E2 levels. The higher water temperature alone was able to increase E2 levels. At both water temperatures GnRHa injections induced a decrease in E2 levels. GnRHa injected females had a lower vtgA gene expression levels at 20 °C. Even with differences in the gene expression of gonadotropins among the various temperature/GnRHa treatments, GSI and oocyte diameter did not change, but GnRHa enhanced the number of vitellogenic oocytes at 20 °C. The reproductive performance of lambari induced to spawn with hCG was better after the combined treatment with GnRHa and summer temperature.

Reproductive stage- and sex-dependant effects of neurohypophyseal nonapeptides on gonadotropin subunit mRNA expression in the catfish Heteropneustes fossilis: An in vitro study.

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 (10 nM, 100 nM and 1000 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 (1000 nM) in both phases. In females, ITb stimulated both lhß and gpα expression only at 1000 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.

Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells.

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.

Sex steroids differentially regulate fshb, lhb and gnrhr expression in Atlantic cod (Gadus morhua).

Depending on the stage of gonad maturation, as well as other factors, gonadal steroids can exert either a positive or negative feedback at the brain and pituitary level. While this has been demonstrated in many teleost species, little is known about the nature of steroid feedback in Gadiform fish. Using an optimized in vitro model system of the Atlantic cod pituitary, the present study investigated the potential effects of two physiologically relevant doses of estradiol, testosterone (TS) or dihydrotestosterone (DHTS) on cell viability and gene expression of gonadotropin subunits (fshb/lhb) and two suggested reproduction-relevant gonadotropin-releasing hormone receptors (gnrhr1b/gnrhr2a) during three stages of sexual maturity. In general, all steroids stimulated cell viability in terms of metabolic activity and membrane integrity. Furthermore, all steroids affected fshb expression, with the effect depending on both the specific steroid, dose and maturity status. Conversely, only DHTS exposure affected lhb levels, and this occurred only during the spawning season. Using single-cell qPCR, co-transcription of gnrhr1b and gnrhr2a was confirmed to both fshb- and lhb- expressing gonadotropes, with gnrhr2a being the most prominently expressed isoform. While steroid exposure had no effect on gnrhr1b expression, all steroids affected gnrhr2a transcript levels in at least one maturity stage. These and previous results from our group point to Gnrhr2a as the main modulator of gonadotropin regulation in cod and that regulation of its gene expression level might function as a direct mechanism for steroid feedback at the pituitary level.

Production of biologically active recombinant goose FSH in a single chain form with a CTP linker sequence.

FSH is a glycoprotein hormone secreted by the pituitary gland that is essential for gonadal development and reproductive function. In avian reproduction study, especially in avian reproduction hormone study, it is hindered by the lack of biologically active FSH. In order to overcome this shortcoming, we prepared recombinant goose FSH as a single chain molecule and tested its biological activities in the present study. Coding sequences for mature peptides of goose FSH α and ß subunits were amplified from goose pituitary cDNA. A chimeric gene containing α and ß subunit sequences linked by the hCG carboxyl terminal peptide coding sequence was constructed. The recombinant gene was inserted into the pcDNA3.1-Fc eukaryotic expression vector to form pcDNA-Fc-gFSHß-CTP-α and then transfected into 293-F cells. A recombinant, single chain goose FSH was expressed and verified by SDS-PAGE and western blot analysis, and was purified using Protein A agarose affinity and gel filtration chromatography. Biological activity analysis results showed that the recombinant, chimeric goose FSH possesses the function of stimulating estradiol secretion and cell proliferation, in cultured chicken granulosa cells. These results indicated that bioactive, recombinant goose FSH has been successfully prepared in vitro. The recombinant goose FSH will have the potential of being used as a research tool for studying avian reproductive activities, and as a standard for developing avian FSH bioassays.

Delayed onset of puberty in male offspring from bisphenol A-treated dams is followed by the modulation of gene expression in the hypothalamic-pituitary-testis axis in adulthood.

Bisphenol A (BPA) is a synthetic endocrine-disrupting chemical of high prevalence in the environment, which may affect the function of the hypothalamic-pituitary-testis (HPT) axis in adult rats. The aim of the present study was to evaluate whether exposure to BPA during hypothalamic sexual differentiation at doses below the reproductive no observable adverse effect level of the World Health Organization causes changes in the regulation of the HPT axis. For this, 0.5 or 5mgkg-1 BPA was injected subcutaneously to the mothers from gestational day 18 to postnatal day (PND) 5. In adulthood (PND90), the mRNA expression of genes related to HPT axis was evaluated in hypothalamus, pituitary and testis. Hypothalamic expression of gonadotrophin-releasing hormone (Gnrh) and estrogen receptor 2 (Esr2) mRNA was increased in both BPA-treated groups compared to control group. In the pituitary, follicle stimulating hormone beta subunit (Fshb) and androgen receptor (Ar) mRNA expression was increased compared to control group in rats treated with 0.5mgkg-1 of BPA, whereas estrogen receptor 1 (Esr1) mRNA expression was only increased in the group treated with 5mgkg-1of BPA, compared to control group. In the testis, there was increased expression of FSH receptor (Fshr) and inhibin beta B subunit (Inhbb) transcripts only in rats treated with 0.5mgkg-1 of BPA. Serum testosterone and LH concentrations were increased in the group treated with 5mgkg-1of BPA. The results of the present study demonstrate for the first time that perinatal exposure to low doses of BPA during the critical period of hypothalamic sexual differentiation modifies the activity of the HPT axis in the offspring, with consequences for later life in adult rats.

Gonadotropin subunits of the characiform Astyanax altiparanae: Molecular characterization, spatiotemporal expression and their possible role on female reproductive dysfunction in captivity.

To better understand the endocrine control of reproduction in Characiformes and the reproductive dysfunctions that commonly occur in migratory fish of this order when kept in captivity, we chose Astyanax altiparanae, which has asynchronous ovarian development and multiple spawning events, as model species. From A. altiparanae pituitary total RNA, we cloned the full-length cDNAs coding for the follicle-stimulating hormone ß subunit (fshb), the luteinizing hormone ß subunit (lhb), and the common gonadotropin α subunit (gpha). All three sequences showed the highest degree of amino acid identity with other homologous sequences from Siluriformes and Cypriniformes. Real-time, quantitative PCR analysis showed that gpha, fshb and lhb mRNAs were restricted to the pituitary gland. In situ hybridization and immunofluorescence, using specific-developed and characterized polyclonal antibodies, revealed that both gonadotropin ß subunits mRNAs/proteins are expressed by distinct populations of gonadotropic cells in the proximal pars distalis. No marked variations for lhb transcripts levels were detected during the reproductive cycle, and 17α,20ß-dihydroxy-4-pregnen-3-one plasma levels were also constant, suggesting that the reproductive dysfunction seen in A. altiparanae females in captivity are probably due to a lack of increase of Lh synthesis during spawning season. In contrast, fshb transcripts changed significantly during the reproductive cycle, although estradiol-17ß (E2) levels remained constant during the experiment, possibly due to a differential regulation of E2 synthesis. Taken together, these data demonstrate the putative involvement of gonadotropin signaling on the impairment of the reproductive function in a migratory species when kept in captivity. Future experimental studies must be carried to clarify this hypothesis. All these data open the possibility for further basic and applied studies related to reproduction in this fish model.

Retinoic acid and retinaldehyde dehydrogenase are not involved in the specific induction of the follicle-stimulating hormone ß subunit by trichostatin A, a selective inhibitor of histone deacetylase.

The selective histone deacetylase inhibitor, trichostatin A (TSA), increases follicle-stimulating hormone ß subunit (FSHß) mRNA expression but not α- and luteinizing hormone ß (LHß)-subunits in both the pituitary gonadotrophic cell line LßT2 and primary cultures of rat anterior pituitary cells. TSA increased histone acetylation in whole cell lysates in both cells. In addition, retinaldehyde dehydrogenases (RALDHs), which are retinoic acid (RA)-synthesizing enzymes, were induced by TSA in these cells. Anacardic acid, a histone acetyltransferase inhibitor that prevents histone acetylation, significantly inhibited TSA-induced FSHß mRNA expression as well as TSA-induced RALDH2 and RALDH3 mRNA expression. Similar to the effect of TSA, gonadotropin-releasing hormone stimulated RALDH expression in LßT2 cells. RA directly applied to the pituitary cells stimulated the transcriptional activity of the FSHß promoter. In addition, α- and LHß-subunit promoters were also activated by RA. Our results suggest that TSA specifically increases FSHß expression with a concomitant increase in RALDHs; however, RALDH and RA are not directly involved in the specific regulation of FSHß by TSA.