FSH Receptor mRNA Research Articles

Nesfatin-1-like peptide suppresses hypothalamo-pituitary-gonadal mRNAs, gonadal steroidogenesis, and oocyte maturation in fish†.

Nucleobindin (Nucb)-1 and Nucb2 are DNA and Ca2+ binding proteins with multiple functions in vertebrates. Prohormone convertase-mediated processing of Nucb2 results in the production of biologically active nesfatin-1. Nesfatin-1 is involved in the regulation of reproduction in many vertebrates, including fish. Our lab originally reported a nesfatin-1-like peptide (Nlp) encoded in Nucb1 that exhibits nesfatin-1-like metabolic effects. We hypothesized that Nlp has a suppressive role in the reproductive physiology of fish. In this research, whether Nlp regulates reproductive hormones and oocyte maturation in fish were determined. Single intraperitoneal (IP) injection of goldfish Nlp (50ng/g body weight) suppressed salmon and chicken gonadotropin-releasing hormone (sgnrh and cgnrh2), gonadotropin-inhibiting hormone (gnih) and its receptor (gnihr), and kisspeptin and brain aromatase mRNA expression in the hypothalamus of both male and female goldfish. In the pituitary, Nlp decreased mRNAs encoding lhb, fshb and kisspeptin and its receptor, while a significant increase in gnih and gnihr was observed. In the gonads, lh (only in male fish) and fsh receptor mRNAs were also significantly downregulated in Nlp-injected fish. Sex-specific modulation of gnih, gnihr, and kisspeptin system in the gonads was also observed. Nlp decreased sex steroidogenic enzyme encoding mRNAs and circulating levels of testosterone and estradiol. In addition, incubation of zebrafish ovarian follicles with Nlp resulted in a reduction in oocyte maturation. These results provide evidence for a robust role for Nlp in regulating reproductive hormones in goldfish and oocyte maturation in zebrafish, and these effects resemble that of nesfatin-1.

Evaluation of carvacrol on pituitary and sexual hormones and their receptors in the testicle of male diabetic rats.

Diabetes mellitus (DM) is a complex metabolic disease and it is also closely associated with a reduction in fertility in male patients. The purpose of the present study was to investigate the antidiabetic effect of carvacrol (CRV), as a potent antioxidant, on the numbers of germ cells and Sertoli cells in testicular tissue, and the messenger RNA (mRNA) and protein expression of some genes involved in spermatogenesis, including luteinizing hormone/choriogonadotropin receptor (LHCGR), follicle-stimulating hormone receptor (FSHR), and steroidogenic factor 1 (SF-1), as well as hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and insulin. Adult male Wistar rats (n = 32) were randomly divided into four groups (eight animals per group), including healthy control that received 0.2% Tween 80, diabetic control group, the diabetic group treated orally with CRV (75 mg/kg), and CRV group that received orally CRV (75 mg/kg). The duration of the treatment period lasted 8 weeks. In the diabetic group, the numbers of Sertoli cells and germ cells were significantly decreased, while the treatment with CRV prevented the degree of the damage to the cells mentioned earlier. CRV administration elevated the concentrations of insulin, T, FSH, and LH. Moreover, treatment with CRV significantly enhanced the levels of the mRNA and protein expression of SF-1, LHCGR, and FSHR. According to the obtained results, CRV administration could prevent the deleterious effects of DM on testicular germ cells, and it increases the levels of hormones and some essential genes, such as SF-1, LHCGR, and FSHR, involved in the process of spermatogenesis.

Receptor Binding Inhibitor Suppresses Carcinogenesis of Cervical Cancer by Depressing Levels of FSHR and ERβ in Mice.

FSH Receptor Binding Inhibitor (FRBI) blocked the binding of FSH to FSHR. Our initial study revealed FRBI reduced the maturation rate, enhanced the apoptosis of sheep Cumulus-Oocyte Complex (COCs). Little is known about whether FRBI modulates ERβ and FSHR levels in the normal uterine and cancerous tissues. The present study aimed to evaluate the FRBI effects on the expressions of Estrogen Receptor-beta (ERβ) and FSH receptor (FSHR) in the uteri. 150 mice were assigned to FRBI+FSH (COM), FSH and control groups (CG). Mice of COM-1, COM-2 and COM-3 groups were simultaneously intramuscularly injected with 500, 750 and 1000 µg FRBI with 10 IU FSH, respectively for five days. Western blotting and qPCR were utilized to determine the expression of ERβ and FSHR. In comparison with FSH group, uterine lumen and glands of COM groups became narrow. The uterine wall and endometrial epithelium were thinned, and uterine lumen became narrow. Epithelial cells were decreased. Uterine wall thicknesses of COM-1, COM-2 and COM-3 groups were reduced by 6.49%, 14.89% and 15.69% on day 30 as compared with FSH group. Uterine perimetrium thicknesses of COM-1, COM-2 and COM-3 groups were reduced by 16.17%, 17.93% and 19.92% on day 20 in comparison with FSH group. Levels of FSHR mRNAs and proteins of COM-1, COM-2 and COM-3 groups were less than FSH group on days 20 and 30 (P<0.05). ERβ protein of COM-3 group was less than FSH group. Serum estradiol (E2) and FSH concentrations of COM-2 and COM-3 were lower than that of FSH group on day 30. FRBI could decrease UWT and UPT, also block the uterine development, decline expression levels of ERβ and FSHR protein. Additionally, FRBI reduced the secretion of secretion of FSH and E2. Downregulating expression of FSHR and ERβ may be a potential treatment regimen for cervical cancer patients.

Effects of sevoflurane on reproductive function of male rats and its main mechanism of action

Objective: To study the effects of sevoflurane on reproductive function and its main mechanism of action in male rats.Materials and methods: Forty adult male Sprague-Dawley rats were divided into 4 groups and exposed to 0, 50, 300 and 1800 ppm of sevoflurane, respectively. After 15 days, the serum levels of sex hormones and inflammatory factors were detected using enzyme-linked immunosorbent assay. Left testis was taken for conventional histopathological examination and TUNEL staining. Right testis was used for sperm production and daily sperm count were evaluated daily. Johnsen score was used to categorize the spermatogenesis. The expression of related genes in the hypothalamic-pituitary-gonadal axis were analyzed by quantitative real time polymerase chain reaction (qRT-PCR).Results: Exposure to sevoflurane increased the levels of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1), decreased the content of serum testosterone (T), reduced the concentration of testicular sperm, the production of daily sperm and Johnsen score, and damaged vas deferens in a dose dependent manner. In addition, chronic exposure to sevoflurane down-regulated transcription of gonadotropin-releasing hormone (GnRH) and kisspeptin (Kiss)-1 as well as its receptor GPR54 in hypothalamus, attenuated GnRH receptor and LH-β mRNA levels, but increased FSH-β mRNA in pituitary gland, and enhanced mRNA of LH receptor and FSH receptor, but decreased INH-α and INH-βA mRNA levels in testes.Discussion and conclusions: Sevoflurane induces disorders of spermatogenesis and causes testicular injury. The underlying mechanism may be related to the imbalance of sex hormones in the hypothalamic-pituitary-gonadal axis.

Cloning and expression analysis of the follicle-stimulating hormone receptor (FSHR) gene in the reproductive axis of female yaks (Bos grunniens)

Follicle-stimulating hormone receptor (FSHR) plays a central role in promoting follicle maturation through the follicle-stimulating hormone (FSH)-mediated cAMP pathway in animals. The objectives of the present study were to clone the FSHR gene of yaks (Bos grunniens) and compare differences in FSHR mRNA expression in the reproductive axis between yaks and cattle. Hypothalamus, anterior pituitary, oviduct, ovary, and uterus tissue samples were collected from adult female yaks (n = 5) and cattle (n = 5) during the follicular phase. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we found that the FSHR coding region of the yak is 2088 bp and encodes 695 amino acids. Its amino acid sequence showed 99.38%–72.22% similarity to the homologous genes of cattle, goats, sheep, cats, donkeys, horses, humans, chickens, monkeys, mice, rats, and wild boar. Real-time PCR analysis revealed that the FSHR gene was expressed in all tissues examined. Expression of the FSHR gene in the yak was higher in the uterus than other tissues (P < 0.05) but, in cattle, was higher in the ovary than other tissues (P < 0.05). The FSHR gene expression level in the cattle ovary was significantly higher than that in the yak ovary (P < 0.01). These results indicate that the FSHR gene is relatively conserved in the course of animal evolution. The variation in sequence and expression level of FSHR between the two species might be associated with the difference in their reproduction.

Molecular mechanism of FSHR expression induced by BMP15 in human granulosa cells.

Follicle-stimulating hormone receptor (FSHR) expression in granulosa cells is critical in enabling follicles to achieve accelerated growth. Although FSHR expression has been reported to be epigenetically regulated, the mechanism is unclear. Cooperation between oocytes and granulosa cells is also essential for normal follicular growth. Among oocyte-derived factors, bone morphogenetic protein 15 (BMP15) promotes follicular growth and is suggested to have epigenetic effects. We examined the role of BMP15 in the acquirement of FSHR in human granulosa cells. Immortalized non-luteinized human granulosa (HGrC1) cells were stimulated with trichostatin A (TSA) or BMP15 to analyze FSHR expression, histone modifications, and USF1/2 binding at the FSHR promoter region. Histone acetyl transferase (HAT) activity and phosphorylation of Smad 1/5/8 and p38 MAPK were examined with or without BMP15, SB203580, and LDN193189. CYP19A1 expression and estradiol production were also studied. TSA and BMP15 induced FSHR mRNA expression in a dose-dependent manner and histone modifications were observed with increased binding of USF1/2. BMP15 increased FSHR protein expression, which was suppressed by LDN193189. BMP15 increased phosphorylation of Smad 1/5/8 and significantly increased HAT activity, which was inhibited by LDN193189, but not by SB203580. BMP15 increased phosphorylation of p38 MAPK and USF1. LDN193189 suppressed BMP15-induced phosphorylation of both p38 MAPK and USF1, whereas SB203580 suppressed the phosphorylation of USF1. BMP15 increased CYP19A1 mRNA expression and estradiol production. BMP15 induced FSHR expression in human granulosa cells through Smad and non-Smad pathways. This mechanism of FSHR induction by BMP15 may be utilized for controlling follicular growth.

The IGF1 Receptor Is Involved in Follicle-Stimulating Hormone Signaling in Porcine Neonatal Sertoli Cells.

Experimental evidence has shown that the IGF1 receptor (IGF1R) is involved in testicular development during embryogenesis. More recently, data gathered from mice granulosa cells and zebrafish spermatogonia suggest that IGF1R has a role in Follicle-stimulating hormone (FSH) signaling. No evidence has been reported on this matter in Sertoli cells (SCs) so far. The aim of the study was to evaluate the role, if any, of the IGF1R in FSH signaling in SCs. The effects of FSH exposure on myosin-phosphatase 1 (MYPT1), ERK 1/2, AKT308, AKT473, c-Jun N-terminal kinase (JNK) phosphorylation and on anti-Müllerian hormone (AMH), inhibin B and FSH receptor (FSHR) mRNA levels were assessed with and without the IGF1R inhibitor NVP-AEW541 in purified and functional porcine neonatal SCs. Pre-treatment with NVP-AEW541 inhibited the FSH-induced MYPT1 and ERK 1/2 phosphorylation, decreased the FSH-dependent Protein kinase B (AKT)308 phosphorylation, but did not affect the FSH-induced AKT473 and JNK phosphorylation rate. It also interfered with the FSH-induced AMH and FSHR down-regulation. No influence was observed on the FSH-stimulated Inhibin B gene expression. Conclusion. These findings support the role of theIGF1R in FSH signaling in porcine SCs. The possible influence of IGF1 stimulation on the FSH-mediated effects on SCs should be further explored.

SUN-214 Neonatal Metformin Administration Exerts a Suppressive Effect on Sertoli Cell Proliferation in Rats

Sertoli cell (SC) proliferation, a factor defining SC population size, occurs during a restricted period of time. In rats, SCs start proliferating during fetal development and undergo mitosis for up to 15 days after birth. In humans, SCs proliferate during fetal and neonatal periods for up to 6 months after birth and there is a second stage of proliferation at the onset of puberty. Metformin (MET), a first line anti-hyperglycemic agent, is used in pediatric population at ages when SCs are still proliferating. In addition to its strong anti-diabetic properties, numerous studies have demonstrated that MET has anti-proliferative activity (1). In this context, it has been demonstrated in SC cultures obtained from 8-day old rats that MET decreases FSH-stimulated SC proliferation and that this decrease is accompanied by a reduction in Cyclin D1, D2, E1 and E2 expression and an increase in cell cycle inhibitor p21Cip expression (2). The aim of this study was to analyze the potential effect of in vivo neonatal administration of Metformin on rat Sertoli cell proliferation and its impact on the spermatogenic capacity in adult animals. Male Sprague Dawley rat pups were randomly assigned at postnatal day 3 (PND3) to the following groups: MET (receiving daily 200 mg/kg MET i.p., from day 3 to 7 inclusive) and control (receiving daily sterile saline solution i.p). At PND8, SCs were isolated from a set of animals to analyze the expression of cell cycle regulators and of SC maturation markers by RT-qPCR. Another set of animals were injected with BrdU (50 mg/kg) 90 min before sacrifice to evaluate cell proliferation. Our results showed that MET group exhibited a significant decrease in BrdU incorporation in SCs compared to controls (p<0.05; n=7 per group). Consistent with decreased SC proliferation, MET group showed a reduction in Cyclin D1 and E2 and an increase in cell cycle inhibitor p21Cip mRNA levels in isolated SC (p<0.05; n=6). In addition, MET group showed a decrease in androgen receptor mRNA levels (p<0.05; n=6) without changes in AMH or FSH receptor mRNA levels compared to controls. Given that a single SC can support a limited number of germ cells, we studied the impact of neonatal treatment with MET on sperm parameters in adult animals. Animals were treated as mentioned before and epididymal sperm parameters were evaluated at PND90. No differences in sperm morphology, motility or number between groups were observed. Altogether these results suggest that neonatal MET treatment exerted a suppressive effect on Sertoli cell proliferation with no further impact on spermatogenesis and sperm epididymal maturation. Reference: (1) Cai et al., Oncol Rep. 2013 Nov;30(5):2449-57. (2) Rindone et al., Reproduction. 2018 Aug; 156(2):93-101. Sources of Research Support: ANPCyT Grants, PICT2015-0228 and PICT 2014/945.

Activin effects on follicular growth in in vitro preantral follicle culture.

As the follicular environment transits from being activin dominant to inhibin dominant during folliculogenesis, it is assumed that activin plays an important role in the early stage of follicular growth. We examined the effects of activin on morphological, biochemical and molecular changes in isolated preantral follicles. Preantral follicles were mechanically isolated from 14-day old female C57BL/6 mice. Each follicle was cultured and observed for 14 days usingan in vitro follicle culture system containing FSH, FSH + activin A and FSH + inhibin in the culture medium. We subsequently examined FSH receptor (FSH-R) mRNA expression in isolated follicle cultures with or without activin on days 0 and 2. Activin was observed to significantly stimulate follicle enlargement on days 2, 4, 6 and 8, accelerate morphological changes and increase estradiollevels in culture medium on days 4, 12 and 14. In contrast, inhibin did not alter follicular growth. Additionally, activin stimulated the expression of FSH-R mRNA in isolated granulosa cells. It was demonstrated that activin stimulated the growth of preantral follicles, mainly during the early stage of folliculogenesis, by inducing FSH-R expression, in an isolated follicle culture system. J. Med. Invest. 66 : 165-171, February, 2019.

A polymorphism in the transcriptional regulatory region strongly influences ovine FSHR mRNA decay.

Follicle-stimulating hormone receptor (FSHR) is an important G protein-coupled receptor, which is required for steroidogenesis, follicular development and female infertility. Here, we report a novel polymorphism in the 3'-UTR that strongly influences ovine FSHR mRNA decay. The partial 3'-UTR sequence of Hu sheep FSHR gene was isolated and characterized, and a polymorphism (c.2327A>G) was identified. Luciferase assay and qRT-PCR showed that c.2327A>G polymorphism in the 3'-UTR exerts a strong regulatory role in FSHR transcription. This regulatory role is achieved by affecting FSHR mRNA decay. Furthermore, the c.2327A>G mutation in the 3'-UTR influences ARE (AU-rich element, a cis-acting element promoting mRNA decay)-mediated mRNA decay of Hu sheep FSHR gene. Together, our study identified a novel polymorphism and elucidated a new mechanism underlying transcriptional regulation of FSHR in mammals.

MiR-31 and miR-143 affect steroid hormone synthesis and inhibit cell apoptosis in bovine granulosa cells through FSHR

The regulatory role of microRNAs (miRNAs) has been explored in ovarian cells, and the effects of miRNAs on gonadal development, apoptosis, ovulation, and steroid production have been reported. In this study, we analyzed the effects of follicle stimulating hormone (FSH) on miR-31 and miR-143 expression levels in bovine granulosa cells (GCs). Our results demonstrated that the FSH receptor (FSHR) is a common target gene of miR-31 and miR-143 in bovine GCs. We further analyzed the roles of miR-31 and miR-143 in bovine GCs by transfecting miR-31 and miR-143 mimics and inhibitors. The Western blot and RT-PCR results showed that miR-31 and miR-143 reduced the mRNA and protein expression levels of FSHR. Moreover, miR-31 overexpression decreased the secretion of progesterone (P4), and miR-143 overexpression decreased both the synthesis of P4 and the secretion of estrogen (E2). In contrast, miR-31 inhibition increased the secretion of progesterone (P4), and miR-143 inhibition increased both the synthesis of P4 and the secretion of E2. Finally, we analyzed the possible effects of miR-31 and miR-143 on bovine GC apoptosis. The results showed that transfection with miR-31 and miR-143 mimics promoted GC apoptosis and that miR-143 and miR-31 inhibition reduced the rate of apoptosis in bovine GCs. Taken together, our results indicate that miR-31 and miR-143 decrease steroid hormone synthesis and inhibit bovine GC apoptosis by targeting FSHR.

The research of the progesterone and estrogen secretion level and the FSHR, LHR gene quantitative in granular cells cultured in vitro in different time of laying hens

To research the hormone secretion levels of progesterone and estrogen and the gene expression levels of two go-nadotropin receptors follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in granular cells of laying hen, and the effect of culture time on the levels of hormone secretion and expression of related receptor gene in granulosa cells was inferred. The experiment using the method of cells culture in vitro, the granular cells supernatants of hens were collected at 0 h, 24 h, 48 h, 72 h, 96 h, the progesterone and estrogen concentrations in cell supernatants were determined by ELISA kits, and detected the expression of FSHR and LHR gene in granular cells by real-time fluorescent quantitative PCR. The results showed that the progesterone and estrogen secretion reduced in the early culture of 0 h~48 h(P < 0.05), with the culture time increases to 72 h, the secretion of two hormones began to in-crease, and reaching the level of the initial level of culture. When the cells were cultured to 96 h, the rogesterone and estrogen secretion was reduced again. The lower levels of FSHR and LHR mRNA expression in granular cells appeared with the increase of culture time, compared with the group of cell culture to 0 h, the mRNA expression levels of each groups reduced obviously(P < 0.05). The amount of progesterone and estrogen in the cultured follicular granulosa cells decreased with the increase of in vitro culture time, and then increased. This might be related to the growth state of cells cultured in vitro. But on the whole, with the extension of the training time, the secretion of proges-terone and estrogen in the cells decreased. This may be related to the decreased expression of the FSHR and LHR genes in the two go-nadotropin receptors.

FSH receptor binding inhibitor restrains follicular development and possibly attenuates carcinogenesis of ovarian cancer through down-regulating expression levels of FSHR and ERβ in normal ovarian tissues

ObjectivesThe current study aimed to investigate FSH receptor binding inhibitor (FRBI) effects in the expressions of FSH receptor (FSHR) and estrogen receptor-beta (ERβ) in the mice ovaries at the gene and protein levels, also to find the potential efficacy of FRBI on suppressing ovarian cancer through down-regulating over-expression of FSHR and ERβ in the normal ovarian tissues. Methods180 female mice were randomized into six groups (n = 30). Mice of FRBI-1, FRBI-2 and FRBI-3, FRBI-4 were intramuscularly injected with FRBI of 10, 20, 30 and 40 mg/kg, respectively, for five consecutive days. The qPCR and Western blotting were used to determine expression levels of FSHR and ERβ mRNAs and proteins in mouse ovaries. ResultsThe ovarian cortex thickness (OCT) of the FRBI-4 group were less than that FSH group on day 30 (P < 0.05). The numbers of secondary follicles (SF) and the maximum transverse diameters (MTD) of secondary follicles of FRBI-3 and FRBI-4 groups were decreased as compared to FSH group (P < 0.05 or P < 0.01) by 24.11% and 27.47% on day 20 based on the control group (CG) levels. On day 15, the reductions of FSHR mRNA levels in FRBI-2, FRBI-3 and FRBI-4 were 27.78%, 29.37% and 43.65% (P < 0.05 or P < 0.01), respectively in comparison with CG. ERβ and FSHR protein levels of FRBI-treated mice were gradually decreased as compared to and CG and FSH group. ERβ protein level of FRBI-4 was less than that of CG on day 20 (P < 0.05). On days 15 and 20, estradiol (E2) concentrations of FRBI-2, FRBI-3 and FRBI-4 groups were lower than those of the CG and FSH group (P < 0.05 or P < 0.01). ConclusionsFRBI could reduce OCT and follicle numbers. A high dose of FRBI (30 mg/kg to 40 mg/kg) could suppress ovarian and follicular development, and attenuate expression levels of ERβ and FSHR mRNAs and proteins in the ovaries, additionally inhibit E2 production. Therefore, FRBI will possibly be utilized to restrain the carcinogenesis of ovarian cancer by down-regulating overexpression of FSHR and ERβ in the ovaries.

Multigenerational obesity-induced perturbations in oocyte-secreted factor signalling can be ameliorated by exercise and nicotinamide mononucleotide.

STUDY QUESTIONCan maternal and offspring high-fat diet (HFD)-induced changes in mRNA expression levels in mice be ameliorated by interventions in female offspring?SUMMARY ANSWEROur results indicate that exercise and nicotinamide mononucleotide (NMN) can ameliorate the negative effects of maternal and post-weaning HFD in female offspring.WHAT IS KNOWN ALREADYMaternal and post-weaning HFD can perturb offspring developmental trajectories. As rates of maternal obesity are rising globally, there is a need for effective treatments in offspring to ameliorate the negative effects from a maternal obesogenic environment. Modulation of the nicotinamide adenine dinucleotide (NAD+) pathway by exercise and the NAD+ precursor NMN has previously been shown to reduce the effects of obesity.STUDY DESIGN, SIZE, DURATIONThis study consisted of a multigenerational study using C57Bl6 mice. Mice were fed a control (chow) or HFD ad libitum throughout mating, pregnancy and lactation (n = 13–25). Female offspring (n = 72) were then also supplied either a chow or HFD post-weaning. At 9 weeks of age offspring from HFD dams were subjected to exercise on a treadmill for 9 weeks or at 16 weeks of age administered NMN (i.p.) for 2.5 weeks. At 18.5 weeks mice were euthanized and ovaries and cumulus–oocyte complexes (COC) were collected to examine the possibility of ameliorating the negative effects of maternal and post-weaning HFD.PARTICIPANTS/MATERIALS, SETTING, METHODSOvary and COC mRNA expression was analysed using RT-qPCR. An initial screen of candidate genes was developed to test which molecular pathways may be involved in generating adverse reproductive system effects. For histological analysis, ovarian tissue was fixed in paraformaldehyde and embedded in paraffin and stained with haematoxylin and eosin. The numbers of primordial, primary, secondary and antral follicles were counted.MAIN RESULTS AND THE ROLE OF CHANCEIn the offspring’s COC, maternal obesity increased both growth differentiation factor 9 (Gdf9: 2-fold; P < 0.05, HFD versus chow) and bone morphogenetic protein 15 (Bmp15: 4-fold; P < 0.05, HFD versus chow) mRNA expression levels while exercise and NMN interventions did not regulate Gdf9 and Bmp15 in the same manner. In whole ovary, maternal diet programmed a 25–50% reduction in FSH receptor and sirtuin-3 mRNA expression levels in daughter ovaries (P < 0.05, HFD versus chow). There was a significant interaction between HFD and intervention on the proportion of large preantral and preovulatory follicles (P < 0.05). However, the increase in preovulatory follicles did not translate to increased oocyte yield. NMN administration resulted in reduced body weight in HFD-fed individuals.LIMITATIONS, REASONS FOR CAUTIONIt is unclear if the changes in oocyte mRNA expression levels reported here will impact oocyte quality and fertility in offspring. Offspring ovulation rate or fecundity could not be studied here and fertility trials are required to determine if the changes in gene expression do reduce fertility.WIDER IMPLICATIONS OF THE FINDINGSOur results demonstrate that maternal and offspring HFD perturbs key signalling pathways that are known to regulate fertility in mice, highlighting the importance of interventions in helping to prevent the declining rates of fertility in the context of the current obesity epidemic.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by grants and fellowships from the National Health and Medical Research Council to R.B.G. (APP1023210, APP1062762, APP1117538) and to M.J.M. and D.A.S. (APP1044295). DAS is a consultant to and inventor on patents licenced to Ovascience, Metrobiotech and GlaxoSmithKline. The other authors declare that there is no conflict of interest.

FSH receptor binding inhibitor impacts K-Ras and c-Myc of ovarian cancer and signal pathway.

The present study aimed to investigate FSHreceptor binding inhibitor (FRBI) effects on relative factors (K-Ras, c-Myc and Vascular endothelial growth factor (VEGF)) to ovarian cancer, and expression levels of FSH receptor (FSHR) mRNAs and proteins in the cumulus-oocyte complex (COCs), to determine changes of protein kinase A (PKA) in sheep granulosa cells, further to elucidate signaling pathway of FRBI action. COCs were cultured in vitro for 24h under supplementation of varying concentrations of FRBI (0, 10, 20, 30 and 40μg/mL) or FSH (10IU/mL). Concentrations of K-Ras, c-Myc, VEGF, cAMP and FSH were detected in IVM media fluids, respectively. The results showed that the concentrations of c-Myc, K-Ras and FSH of FRBI groups were gradually reduced with the increase of FRBI doses. VEGF level of the FRBI-4 group was significantly greater than control group (CG). Expression levels FSHR mRNA and protein and PKA of FRBI-3 and FRBI-4 groups were less than that of CG or FSH group (P<0.05 or P<0.01). Inositol trisphosphate (IP3) concentrations of FRBI-3 and FRBI-4 groups were less than FSH group (P<0.05). FRBI administration doses had significant negative correlations to levels or concentrations of K-Ras, c-Myc, VEGF, FSHR mRNA and protein and PKA protein. K-Ras had significant positive correlations with FSHR mRNA and protein and PKA protein. In conclusion, FRBI could promote the production of VEGF of sheep COCs. Higher doses of FRBI (30 and 40μg/mL) suppressed the production of c-Myc and K-Ras, and declined FSH concentrations in the IVM medium fluid, and decreased the expressions of FSHR at the gene and protein levels, additionally attenuated expression of PKA protein in the granulosa cells.

Insulin Sensitizers Modulate GnRH Receptor Expression in PCOS Rats

Insulin sensitizers like metformin and pioglitazone are clinically used since last decades for the treatment of PCOS, but their efficacy and possible role in PCOS patients remains questionable. Also, the mechanism by which these insulin sensitizers show effect is not clear. To evaluate the effect of metformin and pioglitazone on leutinizing hormone and follicle stimulating hormone receptor mRNA expression, hyperandrogenism and insulin resistance in high fat diet induced and letrozole induced PCOS in rats. Pre-pubertal female rats were divided into four groups: group I received normal pellet diet and group II, III and IV received high fat diet. After 105d of dietary manipulation, metformin and pioglitazone treatment was given to group III and group IV animals respectively for 21d. Similarly, adult female rats were divided into four groups: group I received 1% carboxymethyl cellulose (CMC) and group II, III, IV received letrozole for 21d. After 21d of letrozole administration, metformin and pioglitazone treatment was given to group III and group IV animals respectively for 21d. Oral glucose tolerance test, lipid profile, fasting glucose, insulin, estrus cycle, hormonal profile, ovary weight, leutinizing hormone receptor and follicle stimulating hormone receptor mRNA expression was measured. Polycystic ovarian morphology was assessed through histopathological changes of ovary. Metformin and pioglitazone treatment improve both metabolic and reproductive parameters of PCOS including hyperinsulinemia and hyperandrogenism. LH receptor and FSH receptor mRNA expression were altered by pioglitazone and metformin treatment.

Effects of liver-soothing therapy on the luteotropic hormone receptor and follicle stimulating hormone receptor of perimenopausal syndrome rats with liver qi stagnation

Objective To explore the mechanism of Liver-soothing therapy on the Luteotropic hormone receptor (LHR) and Follicle stimulating hormone receptor (FSHR) in Perimenopausal Syndrome (PMS). Methods A total of 30 nature aging rats (13-month-old) were randomly assigned into three groups; PMS Liver-qi stagnation model (n=8), PMS Liver-qi stagnation model (n=8) treated with Chaihu-Shugan powder (4.0 g/kg•d) and PMS Liver-qi stagnation model (n=8) treated with Danzhi-Xiaoyao powder (4.0 g/kg•d). The PMS Liver-qi stagnation syndrome rat model were established by immobilizing the nature aging rats. Twelve-week-old female rats (n=8) were used as normal controls. Water decoctions of Chaihu-Shugan powder or Danzhi-Xiaoyao powder were administered respectively for 3 weeks while the rat models established. The serum E2, FSH, LH level were measured by radioimmunoassay. The LHR, FSHR in ovary were measured by quantitative real-time PCR. Results Compared with Liver-qi stagnation syndrome rat model, FSH in the Chaihu-Shugan powder group (4.32 ± 0.33 mIU/ml vs. 5.24 ± 0.45 mIU/ml) decreased (P<0.01), and LH in the Danzhi-Xiaoyao powder group (6.76 ± 0.52 mIU/ml vs. 8.08 ± 0.59 mIU/ml) decreased (P<0.01). Compared with normal controls, LHR mRNA, FSHR mRNA level increased in PMS Liver-qi stagnation model. Compared with Liver-qi stagnation syndrome rat model, LHR mRNA, FSHR mRNA level decreased (7.42 ± 2.54, 4.91 ± 1.76 vs. 3.80 ± 1.36) in the ovary of Danzhi-Xiaoyao powder group (P<0.01), but there was no remarkable FHR, LHR expression changes in Chaihu-Shugan powder group. Conclusions The mechanism of Liver-soothing therapy may be related to the regulation of endocrine and decrease of LHR, FSHR. Key words: Perimenopause; Stagnation of liver-qi; Models, animal; Soothing Liver; Luteotropic hormone receptor; Rats

Expression of follicle-stimulating hormone receptor (FSHR), protein kinase B-2 (AKT2) and adapter protein with PH domain, PTB domain, and leucine zipper (APPL1) in pig ovaries.

Follicle-stimulating hormone (FSH) regulates oogenesis and spermatogenesis by binding to its receptor (FSHR) on target cells in the ovary and testis, respectively. The signaling cascades activated after ligand binding are extremely complex and have been shown to include protein kinase A and phosphatidylinositol 3-kinase/protein kinase. The adapter protein APPL1 (adapter protein with PH domain, PTB domain, and leucine zipper), which is an assortment of other signaling proteins, was previously identified to interact with the FSH receptor (FSHR) and the protein kinase B (AKT) pathway. APPL1 plays an important role in promoting cell survival within the preovulatory follicle granulosa layer. Here, we aimed to evaluate the FSHR, AKT2, and APPL1 gene and protein expression levels in the ovaries of different prolific porcine breeds (Wannan Black [WB] and Large White [LW] pigs) using immunohistochemistry and qRT-PCR, respectively. Our results showed that FSHR, AKT2, and APPL1 mRNA levels were significantly higher (P < 0.05) in the ovaries of WB pigs than in the ovaries of LW pigs. Additionally, the FSHR, AKT2, and APPL1 proteins were mainly found distributed in the granulosa cells and oocytes. This study showed that high levels of FSHR, AKT2, and APPL1 were expressed in the ovaries of high prolific breed pigs.

Transcriptome Analysis on Single Small Yellow Follicles Reveals That Wnt4 Is Involved in Chicken Follicle Selection.

Ovarian follicle selection is an important process impacting the laying performance and fecundity of hens, and is regulated by follicle-stimulating hormone (FSH) through binding to its receptor [follicle-stimulating hormone receptor (FSHR)]. In laying hens, the small yellow follicle (6–8 mm in diameter) with the highest expression of FSHR will be recruited into the preovulatory hierarchy during ovarian follicle development. The study of molecular mechanism of chicken follicle selection is helpful for the identification of genes underlying egg-laying traits in chicken and other poultry species. Herein, the transcriptomes of chicken small yellow follicles differing in the mRNA expression of FSHR were compared, and a total of 17,993 genes were identified in 3 pairs of small yellow follicles. The Wnt signaling pathway was significantly enriched in the follicles with the greatest fold change in FSHR expression. In this pathway, the expression level of Wnt4 mRNA was significantly upregulated with a log2(fold change) of 2.12. We further investigated the expression, function, and regulation of Wnt4 during chicken follicle selection and found that Wnt4 mRNA reached its peak in small yellow follicles; Wnt4 stimulated the proliferation of follicular granulosa cells (GCs), increased the expression of StAR and CYP11A1 mRNA in prehierarchical and hierarchical follicles, increased the expression of FSHR mRNA, and decreased the expression of anti-Müllerian hormone and OCLN mRNA. Treatment with FSH significantly increased Wnt4 expression in GCs. Moreover, Wnt4 facilitated the effects of FSH on the production of progesterone (P4) and the mRNA expression of steroidogenic enzyme genes in the GCs of hierarchical follicles, but inhibited the effects of FSH in the GCs of prehierarchical follicles. Collectively, these data suggest that Wnt4 plays an important role in chicken follicle selection by stimulating GC proliferation and steroidogenesis. This study provides a theoretical basis for improving the egg-laying performance of chicken and a reference for the elucidation of the molecular mechanism of follicular selection in mammals.

Expression of FSHR in chondrocytes and the effect of FSH on chondrocytes

ObjectiveChondrocytes express many kinds of hormone receptors. The function of Follicle stimulating hormone (FSH) in the ovary is mediated by FSH receptor (FSHR). FSH receptor (FSHR) is found in many non-ovarian tissues, however it has been unclear if chondrocytes express FSHR. The purpose of this study is to determine it. MethodsMouse primary chondrocytes and human articular cartilage tissues were examined. The expression and sequence of FSHR mRNA by reverse transcription polymerase chain reaction (RT-PCR) and sequenced, respectively, and its protein expression was tested using western blotting and location was observed under immunofluorescence microscopy. Ovarian tissue was as a positive control. After FSH stimulated mouse chondrocytes, intracellular cAMP levels were assessed by ELISA, and gene expression relative to Mouse WNT Signaling Pathway was tested by RT2 Profiler PCR Arrays. ResultsFSHR was detected at the transcriptional level and confirmed to have the same sequence as that of ovary-derived mRNA of FSHR. FSHR proteins presented at the same line as the positive proteins of ovary, in mouse chondrocytes and human cartilage tissue, respectively. FSHR proteins were located at the cell membrane. Intracellular cAMP contents were significantly elevated up to 7-fold in mouse chondrocytes by forskolin (100 mM) (P < 0.001); however, different doses of FSH did not change the cAMP contents in mouse primary chondrocytes. RT2 Profiler PCR Arrays demonstrated that FSH could cause changes in gene expression among the 84 preordained genes, such as Fosl1, Rhou, and Dkk1, in mouse chondrocytes relative to the control. ConclusionMouse chondrocytes and human articular cartilage express functional FSHR. Moreover, FSH can act on chondrocytes and cause genetic changes.