Follicle-stimulating Hormone Signaling Research Articles

OR24-05 Identifying Regulatory Elements Within a Novel Enhancer of FSHB Containing Two PCOS-Associated Single Nucleotide Polymorphisms

Polycystic ovary syndrome (PCOS) is the most common cause of female infertility, affecting approximately 10 percent of women by Rotterdam criteria, and is comorbid with obesity, type II diabetes, hypertension, and non-alcoholic fatty liver disease. As twin studies reveal that genetics account for approximately 70% of PCOS risk, genome-wide association studies (GWAS) can provide powerful insight into PCOS etiology. PCOS GWAS studies from several populations identified a risk locus containing the FSHB gene, which encodes the beta subunit of follicle-stimulating hormone (FSH). As FSH supplementation can restore ovulation in some PCOS patients, deficient FSH signaling could be a causative factor of anovulation and potentially other facets of PCOS. Two of the lead single nucleotide polymorphisms (SNPs) in association with PCOS, rs11031005 and rs11031006, fall within a highly conserved genomic region in mammals. We hypothesized that the conserved region (~450 base pairs) enhances FSHB transcription, and that one or both PCOS-related SNPs alter its function. We have shown that the conserved region from both human and mouse can act as an enhancer of FSHB in LβT2 cells, an immortalized, mouse-derived, mature pituitary gonadotrope cell line, and that its function is altered by the rs11031006 minor allele through modification of an SF1 consensus site. As elimination of the SF1 site reduced but did not completely abolish the function of the enhancer, we continued our investigation to identify additional regulatory sites. Transient transfection of LβT2 cells revealed a possible role for the rs11031005 SNP in FSHB regulation, with the minor allele decreasing enhancer-mediated FSHB transcription. This effect may be due to decreased binding of an unidentified transcription factor, as gel shift revealed that the rs11031005 minor allele reduced the intensity of a binding complex. Using truncations and sliding deletions, we identified three additional putative transcription factor binding sites with consensus sequences for ZEB1, PTX1, and SMAD. To support a role for the conserved region as an enhancer in native chromatin, we assessed the histone status in LβT2 chromatin. Compared to the proximal Fshb promoter, the enhancer-specific marker, H3K4me1, was enriched near the conserved region. Neither promoter/enhancer markers of active (H3K27Ac) or repressed (H3K27me3) chromatin were enriched near the conserved region, although levels of both modifications were consistent with the Fshb proximal promoter. Overall, our data support the role of this conserved region as a novel regulator of FSHB/Fshb transcription and reveal a possible mechanism to explain the contribution of PCOS-associated SNPs through FSHB regulation.

APPL1 as an important regulator of insulin and adiponectin-signaling pathways in the PCOS: A narrative review.

Adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) plays a central role as the main contributing factor in the adiponectin and insulin signaling. This review aims to discuss previous and recent findings concerning the role of APPL1 in the polycystic ovary syndrome (PCOS) patients with conclusions regarding more efficient therapeutic approaches. A literature review was performed in PubMed, Web of Science, ScienceDirect, Scopus, and Google Scholar from August 1999 to May 2020. This study reveals that APPL1 has a key role in adiponectin, insulin, and follicle-stimulating hormone signaling pathways occurring within the ovaries. Recent studies in mouse model systems have indicated that APPL1 can prevent diabetes, endothelial disorders, and insulin resistance. In contrast, APPL1 deficiency can lead to the metabolic and vascular disorders. APPL1 due to its potential roles in different signaling pathways might be suggested as a novel diagnostic and therapeutic option for prediction of ovarian dysfunctions and treatment of reproductive disorders, especially PCOS.

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.

The Role of FSH in Osteoporosis and Subsequent Oral Disease

Osteoporosis is an age‐related chronic disease in which osteoclasts degrade minerals of the bone faster than osteoblasts can replace them. In addition to increased risk of fracture, osteoporosis patients experience oral, dental and prosthodontic complications due to degradation of the alveolar and jaw bone. Elderly women are disproportionately impacted by this disease. One potential culprit for the gender‐based disparity is the increased levels of follicle stimulating hormone (FSH) during menopause. An isoform of the FSH receptor can be found on osteoclasts; when FSH binds to this receptor, osteoclast activity and differentiation are increased. Murine monocytes were differentiated into osteoclasts using RANK ligand, and FSH dependent signaling was measured through western blotting. Protein phosphorylation was detected in response to FSH in osteoclasts. A better understanding of the osteoclast FSH receptor could allow for pharmaceutical intervention to interrupt the pathway. Negative allosteric modulators (NAMs) that inhibit FSH signalling on granulosa cells could be used for inhibiting FSH signalling on osteoclasts.

FSHR ablation induces depression-like behaviors.

Alteration in reproductive hormones profile is associated with the increasing risk of menopausal depression in women. Serum follicle-stimulating hormone (FSH) level is changed during the menopause transition, while the effect of FSH on menopausal depression has remained undefined. In this study we investigated whether or how FSH affected menopausal depression in postmenopausal (ovariectomized) FSHR knockout mice (Fshr−/−). We found that Fshr−/− mice displayed aggravated depression-like behaviors, accompanied by severe oxidative stress in the whole brain, resulted from significantly reduced glutamate cysteine ligase modifier subunit (GCLm) in glutathione synthesis and glucose-6-phosphate dehydrogenase (G6PD) in NADP/NADPH transition. Importantly, administration of ROS scavenger N-acetyl cysteine (NAC, 150 mg · kg−1 · d−1, i.p. for 12 weeks) attenuated the depression-like behaviors of Fshr−/− mice. Consistent with these in vivo experiment results, we found that pretreatment with FSH (50, 100 ng/mL) dose-dependently increased protein levels of GCLm and G6PD, and decreased the ROS production in N2a mouse neuroblastoma cells. These findings demonstrate that FSH signaling is involved in pathogenesis of menopausal depression, and likely to maintain the redox-optimized ROS balance in neurons.

Neonatal Exposure to Agonists and Antagonists of Sex Steroid Receptors Affects AMH and FSH Plasma Level and Their Receptors Expression in the Adult Pig Ovary.

Simple SummaryThe ovarian development and the establishment of ovarian reserve during fetal and/or neonatal life is critical for future reproductive success. Many environmental chemicals are known to negatively affect development and physiology of human and animal ovaries by interfering with endocrine systems, resulting in aberrant reproductive functions. The present study shows the long-term impact of neonatal exposure to agonists and antagonists of sex steroid receptors on AMH and FSH signalling in the ovary of adult pigs. Our findings suggest alteration in ovarian follicle recruitment from ovarian reserve arising from neonatal disruption of androgen/estrogen signalling induced by environmental endocrine active compounds. Everyday use of many endocrine disruptors is already prohibited after their harmful impacts on normal physiology have become known. Nevertheless, market introduction of new chemicals with potential deleterious influence on reproductive physiology has continued. Our outcomes confirm that a neonatal window plays an essential role in the physiological programming of ovarian function in adult pigs. The influence of environmental chemicals on this critical neonatal window needs to be investigated in order to gain a comprehensive view of deleterious interactions between endocrine disrupting chemicals and ovarian function.In this study piglets were injected with testosterone propionate (TP, an androgen), flutamide (FLU, an antiandrogen), 4-tert-octylphenol (OP, an estrogenic compound), ICI 182,780 (ICI, an antiestrogen) or corn oil (controls) between postnatal days 1 and 10 (N = 5/group). Then plasma anti-Müllerian hormone (AMH) and follicle stimulating hormone (FSH) concentration and the expression of their receptors were examined in the adult pig ovary. TP and FLU decreased plasma AMH and FSH concentration. In preantral follicles, TP resulted in upregulation of AMHR2 and FSHR expression, but decreased AMH protein abundance. FLU upregulated AMHR2 expression, while OP increased FSHR mRNA. In small antral follicles, OP upregulated ACVR1 and BMPR1A expression, while FLU increased BMPR1A mRNA. FLU and ICI resulted in upregulation of AMHR2 expression. TP and FLU upregulated AMH expression, while it was downregulated in response to OP or ICI. Moreover, OP and ICI resulted in downregulation of FSHR expression, while FLU decreased FSHR protein abundance. In conclusion, neonatal exposure to either agonist or antagonist of androgen receptor affected AMH and FSH signalling systems in preantral follicles. In small antral follicles these systems were influenced by compounds with estrogenic, antiestrogenic, and antiandrogenic activity. Consequently, these hormonal agents may cause an accelerated recruitment of primordial follicles and affect the cycling recruitment of small antral follicles in pigs.

125 Transcriptomic changes in bovine ovarian cortex in response to FSH signaling

Preantral follicles serve as a reservoir of female gametes that could be used in assisted reproductive technologies in humans, livestock, and endangered animals. Invitro culture of ovarian cortex is a widely used method to grow preantral follicles. Follicle-stimulating hormone (FSH) is often added to the culture medium as a folliculogenesis-promoting factor. The roles of FSH in antral follicles is well known; however, the effects of FSH in preantral follicles and indirectly in the ovarian cortical cells is largely unknown. The aim of this study was to determine the transcriptomic responses of the ovarian cortex containing preantral follicles to FSH signalling over time. In 3 biological replicates, small strips of bovine ovarian cortex (10×5mm) were dissected from the medulla and evaluated under a stereomicroscope for removal of all visible antral follicles. Resulting cortical strips were cultured in defined medium with human-recombinant FSH or vehicle for 2 or 4 days at 38.5°C and 5% CO2. The RNA was isolated and subjected to cDNA library preparation and 3′-Tag RNA sequencing. Sequencing data analysis was performed using the edgeR and maSigPro packages (Bioconductor-R). Using a time-course analysis, genes up- or downregulated 2-fold or more and associated with an FDR<0.05 were considered differentially expressed (DEG) and were further analysed with NetworkAnalyst software. We found 252 DEG over time in response to FSH. In FSH-treated samples, significantly enriched biological functions from upregulated genes were associated with glycolysis, gluconeogenesis, carbon metabolism, and biosynthesis of amino acids. In contrast, significantly enriched biological functions from downregulated genes found in FSH-treated samples included phagosome and necroptosis. The germ cell markers BMP15, DAZL, DDX4, GDF9, and ZP2/ZP3 were expressed but unchanged by FSH, suggesting the presence of similar numbers of oocytes between samples. The gene B4GALT2, previously reported as a granulosa cell marker, was upregulated in FSH-treated samples at Day 4. The follicular marker RAB23 was expressed in all samples and not changed by FSH. One interesting finding was upregulation of MAPK signalling (represented by the genes MAPKAPK3, ELK4, MKNK2, and TGFB3) in response to FSH signalling, with no change in expression of the cAMP-response element responsive genes CYP19A1 and INHA. Together, these data indicate that FSH stimulates energy metabolism in ovarian cortical cells and represses negative cell function activity. We conclude that these responses are mostly mediated by granulosa cells, because the FSH receptor is not appreciably expressed in the ovarian cortex stroma. Moreover, the data suggest that FSH may utilise alternative signalling pathways, such as MAPK, in early follicles. This information enhances our understanding of FSH signalling pathways in the ovarian cortex, mediated by preantral follicles to create a positive environment for folliculogenesis.

Molecular Mechanisms of Action of FSH.

The glycoprotein follicle-stimulating hormone (FSH) acts on gonadal target cells, hence regulating gametogenesis. The transduction of the hormone-induced signal is mediated by the FSH-specific G protein-coupled receptor (FSHR), of which the action relies on the interaction with a number of intracellular effectors. The stimulatory Gαs protein is a long-time known transducer of FSH signaling, mainly leading to intracellular cAMP increase and protein kinase A (PKA) activation, the latter acting as a master regulator of cell metabolism and sex steroid production. While in vivo data clearly demonstrate the relevance of PKA activation in mediating gametogenesis by triggering proliferative signals, some in vitro data suggest that pro-apoptotic pathways may be awakened as a “dark side” of cAMP/PKA-dependent steroidogenesis, in certain conditions. P38 mitogen-activated protein kinases (MAPK) are players of death signals in steroidogenic cells, involving downstream p53 and caspases. Although it could be hypothesized that pro-apoptotic signals, if relevant, may be required for regulating atresia of non-dominant ovarian follicles, they should be transient and counterbalanced by mitogenic signals upon FSHR interaction with opposing transducers, such as Gαi proteins and β-arrestins. These molecules modulate the steroidogenic pathway via extracellular-regulated kinases (ERK1/2), phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3K)/protein kinase B (AKT), calcium signaling and other intracellular signaling effectors, resulting in a complex and dynamic signaling network characterizing sex- and stage-specific gamete maturation. Even if the FSH-mediated signaling network is not yet entirely deciphered, its full comprehension is of high physiological and clinical relevance due to the crucial role covered by the hormone in regulating human development and reproduction.

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.

OR33-2 Blocking FSH Inhibits Hepatic Cholesterol Biosynthesis and Reduces Serum Cholesterol

Menopause is associated with dyslipidemia and an increased risk of cardio-cerebrovascular disease. The classic view assumes that the underlying mechanism of dyslipidemia is attributed to an insufficiency of estrogen. In addition to a decrease in estrogen, circulating follicle-stimulating hormone (FSH) levels become elevated at menopause. In this study, we found that blocking FSH reduced serum cholesterol via inhibiting hepatic cholesterol biosynthesis. First, the epidemiological results showed that the serum FSH levels were positively correlated with the serum total cholesterol levels, even after excluding serum estrogen effects. In addition, the prevalence of hypercholesterolemia was significantly higher in peri-menopausal women than that in pre-menopausal women. Furthermore, we generated an FSH-elevated mouse model by intraperitoneal injection of exogenous FSH in ovariectomized (OVX) mice, in which a normal level of estrogen (E2) was maintained by exogenous supplementation. Consistently, the results indicated that FSH, independent of estrogen, increased the serum cholesterol level in the FSH-elevated mouse model. Moreover, blocking FSH by FSHβ-antibody or ablating the FSH receptor (FSHR) gene could effectively prevent hypercholesterolemia induced by FSH injection or high-cholesterol diet feeding. Mechanistically, FSH, binding with hepatic FSHRs, activated the Gi2a/β-arrestin-2/Akt pathway and subsequently inhibited the binding of FoxO1 with the SREBP-2 promoter, thus preventing FoxO1 from repressing SREBP-2 gene transcription. This effect, in turn, resulted in the upregulation of SREBP-2, which drove HMGCR nascent transcription and de novo cholesterol biosynthesis, leading to the increase of cholesterol accumulation. This study uncovers a new opportunity for treating hypercholesterolemia during menopause by blocking FSH signaling, particularly for women in peri-menopause characterized by only FSH elevation.

Blocking FSH inhibits hepatic cholesterol biosynthesis and reduces serum cholesterol.

Menopause is associated with dyslipidemia and an increased risk of cardio-cerebrovascular disease. The classic view assumes that the underlying mechanism of dyslipidemia is attributed to an insufficiency of estrogen. In addition to a decrease in estrogen, circulating follicle-stimulating hormone (FSH) levels become elevated at menopause. In this study, we find that blocking FSH reduces serum cholesterol via inhibiting hepatic cholesterol biosynthesis. First, epidemiological results show that the serum FSH levels are positively correlated with the serum total cholesterol levels, even afteradjustment by considering the effects of serum estrogen. In addition, the prevalence of hypercholesterolemia is significantly higher in peri-menopausal women than that in pre-menopausal women. Furthermore, we generated a mouse model of FSH elevation by intraperitoneally injecting exogenous FSH into ovariectomized (OVX) mice, in which a normal level of estrogen (E2) was maintained by exogenous supplementation. Consistently, the results indicate that FSH, independent of estrogen, increases the serum cholesterol level in this mouse model. Moreover, blocking FSH signaling by anti-FSHβ antibody or ablating the FSH receptor (FSHR) gene could effectively prevent hypercholesterolemia induced by FSH injection or high-cholesterol diet feeding. Mechanistically, FSH, via binding to hepatic FSHRs, activates the Gi2α/β-arrestin-2/Akt pathway and subsequently inhibits the binding of FoxO1 with the SREBP-2 promoter, thus preventing FoxO1 from repressing SREBP-2 gene transcription. This effect, in turn, results in the upregulation of SREBP-2, which drives HMGCR nascent transcription and de novo cholesterol biosynthesis, leading to the increase of cholesterol accumulation. This study uncovers that blocking FSH signaling might be a new strategy for treating hypercholesterolemia during menopause, particularly for women in peri-menopause characterized by FSH elevation only.

MECHANISMS IN ENDOCRINOLOGY: Hormonal regulation of spermatogenesis: mutant mice challenging old paradigms.

The two pituitary gonadotrophins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and in particular LH-stimulated high intratesticular testosterone (ITT) concentration, are considered crucial for spermatogenesis. We have revisited these concepts in genetically modified mice, one being the LH receptor (R)-knockout mouse (LuRKO), the other a transgenic mouse expressing in Sertoli cells a highly constitutively active mutated Fshr (Fshr-CAM). It was found that full spermatogenesis was induced by exogenous testosterone treatment in LuRKO mice at doses that restored ITT concentration to a level corresponding to the normal circulating testosterone level in WT mice, ≈5 nmol/L, which is 1.4% of the normal high ITT concentration. When hypogonadal LuRKO and Fshr-CAM mice were crossed, the double-mutant mice with strong FSH signaling, but minimal testosterone production, showed near-normal spermatogenesis, even when their residual androgen action was blocked with the strong antiandrogen flutamide. In conclusion, our findings challenge two dogmas of the hormonal regulation of male fertility: (1) high ITT concentration is not necessary for spermatogenesis and (2) strong FSH stimulation can maintain spermatogenesis without testosterone. These findings have clinical relevance for the development of hormonal male contraception and for the treatment of idiopathic oligozoospermia.

Evolution of gonadotropin signaling on gonad development: insights from gene knockout studies in zebrafish.

Gonadal development is precisely regulated by the two gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Much progress on understanding the functions of LH and FSH signaling on gonad development has been achieved in the past decades, mostly from studies in mammals, especially genetic studies in both mouse and human. The functions of both LH and FSH signaling in nonmammalian species are still largely unknown. In recent years, using zebrafish, a teleost phylogenetically distant from mammals, we and others have genetically analyzed the functions of gonadotropins and their receptors through gene knockout studies. In this review, we will summarize the pertinent findings and discuss how the actions of gonadotropin signaling on gonad development have evolved during evolution from fish to mammals.

Estrogen Deficiency Promotes Hepatic Steatosis via a Glucocorticoid Receptor-Dependent Mechanism in Mice

SUMMARYGlucocorticoids (GCs) are master regulators of systemic metabolism. Intriguingly, Cushing’s syndrome, a disorder of excessive GCs, phenocopies several menopause-induced metabolic pathologies. Here, we show that the glucocorticoid receptor (GR) drives steatosis in hypogonadal female mice because hepatocyte-specific GR knockout mice are refractory to developing ovariectomy-induced steatosis. Intriguingly, transcriptional profiling revealed that ovariectomy elicits hepatic GC hypersensitivity globally. Hypogonadism-induced GC hypersensitivity results from a loss of systemic but not hepatic estrogen (E2) signaling, given that hepatocyte-specific E2 receptor deletion does not confer GC hypersensitivity. Mechanistically, enhanced chromatin recruitment and ligand-dependent hyperphosphorylation of GR underlie ovariectomy-induced glucocorticoid hypersensitivity. The dysregulated glucocorticoid-mediated signaling present in hypogonadal females is a product of increased follicle-stimulating hormone (FSH) production because FSH treatment in ovary-intact mice recapitulates glucocorticoid hypersensitivity similar to hypogonadal female mice. Our findings uncover a regulatory axis between estradiol, FSH, and hepatic glucocorticoid receptor signaling that, when disrupted, as in menopause, promotes hepatic steatosis.

Follicle-stimulating hormone enhances hepatic gluconeogenesis by GRK2-mediated AMPK hyperphosphorylation at Ser485 in mice.

Increased serum follicle-stimulating hormone (FSH) is correlated with fasting hyperglycaemia. However, the underlying mechanism remains unclear. Because excessive hepatic gluconeogenesis is a major cause of fasting hyperglycaemia the present study investigated whether FSH increases hepatic gluconeogenesis in mice. Ovariectomised mice supplemented with oestradiol (E2) to maintain normal levels of serum E2 (OVX+E2 mice) were injected with low or high doses of FSH. We knocked out Crtc2, a crucial factor in gluconeogenesis, and Fshr to discern their involvement in FSH signalling. To evaluate the role of the G-protein-coupled receptor (GPCR) kinase 2 (GRK2), which could affect glucose metabolism and interact directly with non-GPCR components, a specific GRK2 inhibitor was used. The pyruvate tolerance test (PTT), quantification of PEPCK and glucose-6-phosphatase (G6Pase), key enzymes of gluconeogenesis, GRK2 and phosphorylation of AMP-activated protein kinase (AMPK) were examined to evaluate the level of gluconeogenesis in the liver. A nonphosphorylatable mutant of AMPK Ser485 (AMPK S485A) was transfected into HepG2 cells to evaluate the role of AMPK Ser485 phosphorylation. FSH increased fasting glucose (OVX+E2+high-dose FSH 8.18 ± 0.60mmol/l vs OVX+E2 6.23 ± 1.33mmol/l), the PTT results, and the transcription of Pepck (also known as Pck1; 2.0-fold increase) and G6pase (also known as G6pc; 2.5-fold increase) in OVX+E2 mice. FSH also enhanced the promoter luciferase activities of the two enzymes in HepG2 cells. FSH promoted the membrane translocation of GRK2, which is associated with increased AMPK Ser485 and decreased AMPK Thr172 phosphorylation, and enhanced the nuclear translocation of cyclic AMP-regulated transcriptional coactivator 2 (CRTC2). GRK2 could bind with AMPK and induce Ser485 hyperphosphorylation. Furthermore, either the GRK2 inhibitor or AMPK S485A blocked FSH-regulated AMPK Thr172 dephosphorylation and gluconeogenesis. Additionally, the deletion of Crtc2 or Fshr abolished the function of FSH in OVX+E2 mice. The results indicate that FSH enhances CRTC2-mediated gluconeogenesis dependent on AMPK Ser485 phosphorylation via GRK2 in the liver, suggesting an essential role of FSH in the pathogenesis of fasting hyperglycaemia.

Effects of the insulin-like growth factor system on testicular differentiation and function: a review of the literature.

We recently described the occurrence of cryptorchidism, oligoasthenoteratozoospermia, and genital abnormalities in patients with distal 15q chromosome structural abnormalities. This observation brought us to hypothesize that insulin-like growth factor (IGF) receptor (IGF1R), mapping on the 15q 26.3 chromosomal band, may be involved in testicular function. To further evaluate this topic, we reviewed invitro and invivo studies exploring the role of the IGF system [IGF1, IGF2, IGF1R, insulin receptor substrates (IRS)] at the testicular level both in animals and in humans. In animals, IGF1/IGF1R has been found to be involved in testicular development during embryogenesis, in Sertoli cell (SC) proliferation, and in germ cell (GS) proliferation and differentiation. Interestingly, IGF1R seems to mediate follicle-stimulating hormone (FSH) effects through the PI3K/AKT pathway. In humans, IGF1 directly increases testicular volume. The molecular pathways responsible for testicular differentiation and IGF1/IGF1R signaling are highly conserved among species; therefore, the IGF system may be involved in FSH signaling also in humans. We suggest a possible molecular pathway occurring in human SCs, which involves both IGF1 and FSH through the PI3K/AKT pathway. The acknowledgment of an IGF1 mediation of the FSH-induced effects may open new ways for a targeted therapy in idiopathic non-FSH-responder oligoasthenoteratozoospermia.

Advanced glycation end products interfere in luteinizing hormone and follicle stimulating hormone signaling in human granulosa KGN cells

Advanced glycation end products accumulate in the ovarian granulosa-cell layer of women with polycystic ovarian syndrome. Taken that the MAPK/ERK-pathway is a key regulator of oocyte maturation and function, consisting the main pathway used by the gonadotrophic hormones (luteinizing hormone, follicle stimulating hormone) to control ovulation, the present study aims to assess advanced glycation end products' interference into luteinizing hormone-and follicle stimulating hormone-signaling via the MAPK/ERK-pathway in the human granulosa KGN cell line. KGN cells were treated with luteinizing hormone or follicle stimulating hormone in the absence or presence of human glycated albumin. The specific activation of the main components of the MAPK/ERK1/2-pathway (namely c-Raf, MEK and ERK1/2) was assessed. Treatment of KGN cells with an MEK1/2-inhibitor or a blocking anti-RAGE-antibody was also performed to shed further light on the mechanism of the involvement of advanced glycation end products in luteinizing hormone and/or follicle stimulating hormone-related signaling pathways. Luteinizing hormone treatment increased p-ERK1/2 levels in human granulosa cells, while the combined treatment of luteinizing hormone and human glycated albumin provoked a decrease of p-ERK1/2 levels. A similar reducing effect was also observed for the upstream molecule phospho-cRaf upon combined treatment, while treatment with an MEK-inhibitor confirmed that the phenomenon is MAPK/ERK-pathway-dependent. Similarly, follicle stimulating hormone treatment increased p-ERK1/2 and p-MEK1/2 levels, while the combined treatment of follicle stimulating hormone and human glycated albumin downregulated their levels. Advanced glycation end products reduce the luteinizing hormone- and follicle stimulating hormone-induced ERK1/2 activation that is critical for granulosa cell mitogenesis and proliferation. Inappropriate activation of ERK1/2 in granulosa cells may block the granulosa cell differentiation pathway and/or impair follicular responses to hormones, potentially leading to ovulation failure that characterizes polycystic ovarian syndrome.

Crosstalk between FSH and relaxin at the end of the proliferative stage of rat Sertoli cells.

Follicle-stimulating hormone (FSH) stimulates the proliferation of immature Sertoli cells through the activation of PI3K/AKT/mTORC1 and MEK/ERK1/2 pathways. Mature Sertoli cells stop proliferating and respond to FSH by stimulating cAMP production. To gain insight into possible mechanisms involved in this switch as well as the impact of paracrine factors that stimulate cell proliferation, we analyzed the effects of FSH and relaxin on intracellular signaling pathways involved with proliferation and differentiation in Sertoli cells from 15-day-old rats, which are close to the transition between the two stages. FSH stimulated 3H-thymidine incorporation and cyclin D1 expression, changes associated with proliferation. In contrast, FSH inhibited AKT and ERK1/2 phosphorylation, activated cAMP production and induced changes in several cell cycle genes that were compatible with differentiation. Relaxin also stimulated 3H-thymidine incorporation but increased phosphorylation of ERK1/2 and AKT. When both hormones were added simultaneously, relaxin attenuated FSH-mediated inhibition of ERK1/2 and AKT phosphorylation and FSH-mediated activation of cAMP production. FSH but not relaxin increased CREB phosphorylation, and relaxin but not FSH shifted NF-κB expression from the cytoplasm to the nucleus. Relaxin did not inhibit the effects of FSH on inhibin α and Bcl2 expression. We propose that at this time of Sertoli cell development, FSH starts to direct cells to differentiation through activation of cAMP/CREB and inhibition of ERK1/2 and AKT pathways. Relaxin counteracts FSH signaling through the inhibition of cAMP and activation of ERK1/2, AKT and NF-κB, but does not block the differentiation process triggered by FSH.

Insulin signalling and glucose transport in the ovary and ovarian function during the ovarian cycle.

Data derived principally from peripheral tissues (fat, muscle and liver) show that insulin signals via diverse interconnecting intracellular pathways and that some of the major intersecting points (known as critical nodes) are the IRSs (insulin receptor substrates), PI3K (phosphoinositide kinase)/Akt and MAPK (mitogen-activated protein kinase). Most of these insulin pathways are probably also active in the ovary and their ability to interact with each other and also with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) signalling pathways enables insulin to exert direct modulating influences on ovarian function. The present paper reviews the intracellular actions of insulin and the uptake of glucose by ovarian tissues (granulosa, theca and oocyte) during the oestrous/menstrual cycle of some rodent, primate and ruminant species. Insulin signals through diverse pathways and these are discussed with specific reference to follicular cell types (granulosa, theca and oocyte). The signalling pathways for FSH in granulosa cells and LH in granulosa and theca cells are summarized. The roles of glucose and of insulin-mediated uptake of glucose in folliculogenesis are discussed. It is suggested that glucose in addition to its well-established role of providing energy for cellular function may also have insulin-mediated signalling functions in ovarian cells, involving AMPK (AMP-dependent protein kinase) and/or hexosamine. Potential interactions of insulin signalling with FSH or LH signalling at critical nodes are identified and the available evidence for such interactions in ovarian cells is discussed. Finally the action of the insulin-sensitizing drugs metformin and the thiazolidinedione rosiglitazone on follicular cells is reviewed.

Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation.

Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs.