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.

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.

Inhibition of activin signalling reduces the growth of LßT2 gonadotroph pituitary tumours in mouse.

Pituitary tumours are benign neoplasms that derive from hormone-producing cells of the pituitary gland. While medical treatments have emerged for most subtypes, gonadotroph tumours that express follicle-stimulating hormone (FSH) and/or luteinizing hormone still lack therapeutic options apart from surgery and radiotherapy. Activin ligands are physiological regulators of production and secretion of FSH by gonadotroph cells, but their role in gonadotroph tumourigenesis remains little explored. Using the LßT2 mouse gonadotroph cell line which produces FSH under activin stimulation, we first tested whether subcutaneous xenografts of LßT2 cells resulted in tumour formation in Rag2KO mice. Histological analysis confirmed the presence of LßT2 tumours with endothelial cells and macrophages in their microenvironment. FSH expression was found in a subset of clusters of LßT2 cells in the tumours. We subsequently addressed the consequences of targeting activin signalling via injection of a soluble activin decoy receptor (sActRIIB-Fc). sActRIIB-Fc treatment resulted in significantly decreased LßT2 tumour volume. Reduced Smad2 phosphorylation as well as inhibition of tumour-induced FSH production confirmed the efficient targeting of activin-downstream signalling in treated tumours. More interestingly, treated tumours showed significantly fewer endothelial cells associated with reduced Vegfa expression. In vitro treatment of LßT2 cells with sActRIIB-Fc had no effect on cell proliferation or apoptosis, but Vegfa expression was inhibited, pointing to a likely paracrine effect of LßT2 cells on endothelial cells through activin-mediated Vegfa regulation. Further in vitro and in vivo studies are now needed to pinpoint the exact roles of activin signalling in these processes prior to translating these observations to the clinic.

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.

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.

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.

HSP70 inhibits pig pituitary gonadotrophin synthesis and secretion by regulating the corticotropin-releasing hormone signaling pathway and targeting SMAD3.

High levels or long periods of stress have been shown to negatively impact cell homeostasis, including with respect to abnormalities in domestic animal reproduction, which are typically activated through the hypothalamus-pituitary-adrenal axis, in which corticotropin-releasing hormone (CRH) and heat shock protein 70 (HSP70) are involved. In addition, CRH has been reported to inhibit pituitary gonadotrophin synthesis, and HSP70 is expressed in the pituitary gland. The aim of this study was to determine whether HSP70 was involved in regulating gonadotrophin synthesis and secretion by mediating the CRH pathway in the porcine pituitary gland. Our results showed that HSP70 was highly expressed in the porcine pituitary gland, with over 90% of gonadotrophic cells testing HSP70 positive. The results of functional studies demonstrated that the HSP70 inducer decreased FSH and LH levels in cultured porcine primary pituitary cells, whereas an HSP70 inhibitor blocked the negative effect of CRH on gonadotrophin synthesis and secretion. Furthermore, our results demonstrated that HSP70 inhibited gonadotrophin synthesis and secretion by blocking GnRH-induced SMAD3 phosphorylation, which acts as the targeting molecule of HSP70, while CRH upregulated HSP70 expression through the PKC and ERK pathways. Collectively, these data demonstrate that HSP70 inhibits pituitary gonadotrophin synthesis and secretion by regulating the CRH signaling pathway and inhibiting SMAD3 phosphorylation, which are important for our understanding the mechanisms of the stress affects domestic animal reproductive functions.

FSHB Transcription is Regulated by a Novel 5' Distal Enhancer With a Fertility-Associated Single Nucleotide Polymorphism.

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone, signal the gonads to regulate male and female fertility. FSH is critical for female fertility as it regulates oocyte maturation, ovulation, and hormone synthesis. Multiple genome-wide association studies (GWAS) link a 130 Kb locus at 11p14.1, which encompasses the FSH beta-subunit (FSHB) gene, with fertility-related traits that include polycystic ovary syndrome, age of natural menopause, and dizygotic twinning. The most statistically significant single nucleotide polymorphism from several GWAS studies (rs11031006) resides within a highly conserved 450 bp region 26 Kb upstream of the human FSHB gene. Given that sequence conservation suggests an important biological function, we hypothesized that the region could regulate FSHB transcription. In luciferase assays, the conserved region enhanced FSHB transcription and gel shifts identified a binding site for Steroidogenic factor 1 (SF1) contributing to its function. Analysis of mouse pituitary single-cell ATAC-seq demonstrated open chromatin at the conserved region exclusive to a gonadotrope cell-type cluster. Additionally, enhancer-associated histone markers were identified by immunoprecipitation of chromatin from mouse whole pituitary and an immortalized mouse gonadotrope-derived LßT2 cell line at the conserved region. Furthermore, we found that the rs11031006 minor allele upregulated FSHB transcription via increased SF1 binding to the enhancer. All together, these results identify a novel upstream regulator of FSHB transcription and indicate that rs11031006 can modulate FSH levels.

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.

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 ßA (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.

Identification and in vivo validation of a 9-mer peptide derived from FSHß with FSHR antagonist activity.

FSH-FSHR interaction is critical for folliculogenesis, spermatogenesis and progression of several cancers. Therefore, FSHR is an attractive target for fertility regulation and cancer therapeutics. Based on homology and structural analysis of hFSH-FSHR(ECD) complex, a minimal continuous stretch within FSHß seat-belt loop (FSHß (89-97)) was identified to be crucial for FSHR interaction. The ability of FSHß (89-97) peptide to neutralize FSHR activity was evaluated by a panel of in vitro and in vivo experiments. The synthetic peptide significantly inhibited binding of [125I]-FSH to rat Fshr as well as FSH-induced cAMP production. In immature rats, FSHß (89-97) peptide administration reduced FSH-mediated increase in ovarian weight. The peptide inhibited transition of follicles from pre-antral to antral stage and hindered the cell cycle progression of granulosa cells beyond G0/G1 phase. In adult rats, administration of the peptide inhibited estradiol synthesis and significantly perturbed folliculogenesis.

Kisspeptin2 regulates hormone expression in female zebrafish (Danio rerio) pituitary.

Kisspeptin2 is a neuropeptide widely found in the brain and multiple peripheral tissues in the zebrafish. The pituitary is the center of synthesis and secretes various endocrine hormones. However, Kiss2 innervation in the zebrafish pituitary is unknown. In this study, the organization of Kiss2 cells and structures in the zebrafish pituitary by promoter-driving mCherry-labeling Kiss2 neurons were investigated. Kiss2 neurons in the hypothalamus do not project into the pituitary. Kiss2 cells are found in the female pituitary. Unidentified Kiss2 cells and extensions are located in the proximal pars distalis (PPD), similar to the distribution of Gnrh3 fibers. Kiss2 structures reside alongside Gnrh3 fibers. No Kiss2 structures are found in the male pituitary. The transcriptional expression of the kisspeptin receptor kiss1rb is detected in both female and male pituitaries. In situ hybridization shows that kiss1rb-positive cells are located in the PPD and pars intermedia (PI). In vitro Kiss2-10 treatment stimulates Akt and Erk phosphorylation and significantly induces lhß, fshß, and prl1 mRNA expression in the female pituitary. The results in this study suggest that Kiss2 and Kiss1rb may form an independent paracrine or autocrine system in the female zebrafish pituitary. Kiss2 and Kiss1rb signaling regulates the expression of pituitary hormones.

Human Follicle-Stimulating Hormone ß Subunit Expression Depends on FOXL2 and SMAD4.

Follicle-stimulating hormone (FSH), an essential regulator of mammalian fertility, is synthesized by pituitary gonadotrope cells in response to activins. In mice, activins signal via SMAD3, SMAD4, and FOXL2 to regulate transcription of the FSHß subunit (Fshb) gene. Gonadotrope-specific deletion of Foxl2, alone or in combination with Smad4, renders mice FSH-deficient. Whether human FSHB expression is similarly regulated is not known. Here, we used a combination of transgenic and conditional knockout mouse strains to assess the roles of activins, FOXL2, and SMAD4 in regulation of the human FSHB gene. First, we cultured pituitaries from mice harboring a human FSHB transgene (hFSHB mice) and measured both murine Fshb and human FSHB messenger ribonucleic acid (mRNA) expression in response to exogenous activins or two antagonists of endogenous activin-like signaling (follistatin-288 and SB431542). Both murine Fshb and human FSHB expression were stimulated by activins and reduced by the inhibitors. Next, we analyzed human FSHB expression in hFSHB mice carrying floxed Foxl2 and Smad4 alleles. Cre-mediated ablation of FOXL2 and SMAD4 strongly reduced basal and activin-stimulated murine Fshb and human FSHB expression in cultured pituitaries. Finally, the hFSHB transgene was previously shown to rescue FSH production and fertility in Fshb knockout mice. However, gonadotrope-specific Foxl2/Smad4 knockout females carrying the hFSHB transgene have significantly reduced murine Fshb and human FSHB pituitary mRNA levels and are hypogonadal. Collectively, these data suggest that similar to Fshb regulation in mice, FOXL2 and SMAD4 play essential roles in human FSHB expression.

A novel follicle-stimulating hormone vaccine for controlling fat accumulation.

Follicle-stimulating hormone (FSH) has been newly demonstrated to play a great role in promoting fat accumulation, providing a potential to target FSH for controlling fat accumulation and treating obesity. A short, 13-amino acid of FSHß (FSHß13AA) was indentified to be the FSH receptor-binding epitope in both humans and mice. By conservation analysis, we found such FSHß13AA is highly conserved across species. Accordingly, we designed a new FSH antigen by synthesizing a tandem of FSHß13AA (LVYKDPARPNIQK) and then conjugating it to ovalbumin (FSHß13AA-T-OVA). Then, we tested its efficacy in suppressing fat accumulation in both ovariectomized and intact mouse models. Vaccination with this novel antigen emulsified in mild adjuvant, Specol, was highly effective in preventing ovariectomy-induced body weight gain and fat accumulation in mice (P < 0.01). Mechanistically, FSH vaccination treatment inhibited lipid biosynthesis by inactivating PPARγ adipogenic signaling pathway and simultaneously enhanced adipocyte themogenesis via upregulating UCP1 expression in both visceral and subcutaneous adipose tissues. Moreover, injection of this novel FSH vaccine also substantially reduced (P < 0.05) fat accumulation in both intact male and female mice. These actions result from the specific binding of the generated antibody to the ß-subunit to block its action, rather than lowering the circulating levels of FSH, as evidenced by nearly no alterations in serum FSH levels in mice following FSH vaccination. Overall, we developed a novel FSH antigen and vaccine, and demonstrated it is highly efficacious in suppressing fat accumulation.

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.

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.

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.