Effects of follicle-stimulating hormone on energy balance and tissue metabolic health after loss of ovarian function.

Loss of ovarian function imparts increased susceptibility to obesity and metabolic disease. These effects are largely attributed to decreased estradiol (E2), but the role of increased follicle-stimulating hormone (FSH) in modulating energy balance has not been fully investigated. Previous work that blocked FSH binding to its receptor in mice suggested this hormone may play a part in modulating body weight and energy expenditure after ovariectomy (OVX). We used an alternate approach to isolate the individual and combined contributions of FSH and E2 in mediating energy imbalance and changes in tissue-level metabolic health. Female Wistar rats were ovariectomized and given the gonadotropin releasing hormone (GnRH) antagonist degarelix to suppress FSH production. E2 and FSH were then added back individually and in combination for a period of 3 wk. Energy balance, body mass composition, and transcriptomic profiles of individual tissues were obtained. In contrast to previous studies, suppression and replacement of FSH in our paradigm had no effect on body weight, body composition, food intake, or energy expenditure. We did, however, observe organ-specific effects of FSH that produced unique transcriptomic signatures of FSH in retroperitoneal white adipose tissue. These included reductions in biological processes related to lipogenesis and carbohydrate transport. In addition, rats administered FSH had reduced liver triglyceride concentration (P < 0.001), which correlated with FSH-induced changes at the transcriptomic level. Although not appearing to modulate energy balance after loss of ovarian function in rats, FSH may still impart tissue-specific effects in the liver and white adipose tissue that might affect the metabolic health of those organs.NEW & NOTEWORTHY We find no effect of follicle-stimulating hormone (FSH) on energy balance using a novel model in which rats are ovariectomized, subjected to gonadotropin-releasing hormone antagonism, and systematically given back FSH by osmotic pump. However, tissue-specific effects of FSH on adipose tissue and liver were observed in this study. These include unique transcriptomic signatures induced by the hormone and a stark reduction in hepatic triglyceride accumulation.

The effects of short-term and long-term phthalate exposures on ovarian follicle growth dynamics and hormone levels in female mice†.

Di(2-ethylhexyl) phthalate and diisononyl phthalate are widely used as plasticizers in polyvinyl chloride products. Short-term exposures to phthalates affect hormone levels, ovarian follicle populations, and ovarian gene expression. However, limited data exist regarding the effects of long-term exposure to phthalates on reproductive functions. Thus, this study tested the hypothesis that short-term and long-term exposure to di(2-ethylhexyl) phthalate or diisononyl phthalate disrupts follicle dynamics, ovarian and pituitary gene expression, and hormone levels in female mice. Adult CD-1 female mice were exposed to vehicle, di(2-ethylhexyl) phthalate, or diisononyl phthalate (0.15 ppm, 1.5 ppm, or 1500 ppm) via the chow for 1 or 6 months. Short-term exposure to di(2-ethylhexyl) phthalate (0.15 ppm) and diisononyl phthalate (1.5 ppm) decreased serum follicle-stimulating hormone levels compared to control. Long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate (1500 ppm) increased the percentage of primordial follicles and decreased the percentages of preantral and antral follicles compared to control. Both phthalates increased follicle-stimulating hormone levels (di(2-ethylhexyl) phthalate at 1500 ppm; diisononyl phthalate at 1.5 ppm) and decreased luteinizing hormone levels (di(2-ethylhexyl) phthalate at 0.15 and 1.5 ppm; diisononyl phthalate at 1.5 ppm and 1500 ppm) compared to control. Furthermore, both phthalates altered the expression of pituitary gonadotropin subunit genes (Cga, Fshb, and Lhb) and a transcription factor (Nr5a1) that regulates gonadotropin synthesis. These data indicate that long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate alters follicle growth dynamics in the ovary and the expression of gonadotropin subunit genes in the pituitary and consequently luteinizing hormone and follicle-stimulating hormone synthesis.

Dietary exposure to an environmentally relevant phthalate mixture alters follicle dynamics, hormone levels, ovarian gene expression, and pituitary gene expression in female mice.

Phthalates are chemicals ubiquitously used in industry. Individual phthalates have been found to adversely affect female reproduction; however, humans are exposed to a mixture of phthalates daily, primarily through ingestion. Previous studies show that exposure to an environmentally relevant mixture of phthalates (Mix) can affect female reproduction. Little research, however, has been conducted on the effects of short-term (1 month) and long-term (6 months) exposure to Mix on ovarian functions. Thus, this study tested the hypothesis that short-term and long-term exposure to Mix alters ovarian folliculogenesis, serum hormone concentrations, pituitary gene expression, and ovarian expression of genes involved in steroidogenesis, apoptosis, cell cycle regulation, and oxidative stress. Adult CD-1 female mice were exposed to vehicle control (corn oil) or Mix (0.15-1500 ppm) in the chow for 1 or 6 months. Exposure to Mix for 1 month increased the number of atretic follicles (0.15 ppm), altered ovarian gene expression (0.15 ppm, 1500 ppm), and decreased serum testosterone (1.5 ppm) compared to control. Exposure to Mix for 6 months increased serum follicle-stimulating hormone (FSH) (0.15 ppm), decreased serum luteinizing hormone (LH) (0.15 ppm, 1.5 ppm, and 1500 ppm), decreased serum estradiol (1500 ppm), altered pituitary gene expression (1500 ppm), increased the number (1500 ppm) and percentage (1.5 ppm and 1500 ppm) of primordial follicles, and decreased the percentage of preantral (1500 ppm) and antral (1.5 ppm and 1500 ppm) follicles compared to control. These data indicate that exposure to Mix can alter folliculogenesis, steroidogenesis, and gene expression in female mice.

Oocyte quality is enhanced by hypoglycosylated FSH through increased cell-to-cell interaction during mouse follicle development.

Macroheterogeneity in follicle-stimulating hormone (FSH) ß-subunit N-glycosylation results in distinct FSH glycoforms. Hypoglycosylated FSH21 is the abundant and more bioactive form in pituitaries of females under 35 years of age, whereas fully glycosylated FSH24 is less bioactive and increases with age. To investigate whether the shift in FSH glycoform abundance contributes to the age-dependent decline in oocyte quality, the direct effects of FSH glycoforms on folliculogenesis and oocyte quality were determined using an encapsulated in vitro mouse follicle growth system. Long-term culture (10-12 days) with FSH21 (10 ng/ml) enhanced follicle growth, estradiol secretion and oocyte quality compared with FSH24 (10 ng/ml) treatment. FSH21 enhanced establishment of transzonal projections, gap junctions and cell-to-cell communication within 24 h in culture. Transient inhibition of FSH21-mediated bidirectional communication abrogated the positive effects of FSH21 on follicle growth, estradiol secretion and oocyte quality. Our data indicate that FSH21 promotes folliculogenesis and oocyte quality in vitro by increasing cell-to-cell communication early in folliculogenesis, and that the shift in in vivo abundance from FSH21 to FSH24 with reproductive aging may contribute to the age-dependent decline in oocyte quality.

RNA-seq analysis identifies age-dependent changes in expression of mRNAs - encoding N-glycosylation pathway enzymes in mouse gonadotropes.

Follicle-stimulating hormone (FSH) is a glycoprotein that is assembled as a heterodimer of α/ß subunits in gonadotropes. Each subunit contains two N-glycan chains. Our previous in vivo genetic studies identified that at least one N-glycan chain must be present on the FSHß subunit for efficient FSH dimer assembly and secretion. Moreover, macroheterogeneity observed uniquely on human FSHß results in ratiometric changes in age-specific FSH glycoforms, particularly during menopausal transition. Despite the recognition of many prominent roles of sugars on FSH including dimer assembly and secretion, serum half-life, receptor binding and signal transduction, the N-glycosylation machinery in gonadotropes has never been defined. Here, we used a mouse model in which gonadotropes are GFP-labeled in vivo and achieved rapid purification of GFP+ gonadotropes from pituitaries of female mice at reproductively young, middle, and old ages. We identified by RNA-seq analysis 52 mRNAs encoding N-glycosylation pathway enzymes expressed in 3- and 8-10-month-old mouse gonadotropes. We hierarchically mapped and localized the enzymes to distinct subcellular organelles within the N-glycosylation biosynthetic pathway. Of the 52 mRNAs, we found 27 mRNAs are differentially expressed between the 3- and 8-10-month old mice. We subsequently selected 8 mRNAs which showed varying changes in expression for confirmation of abundance in vivo via qPCR analysis, using more expanded aging time points with distinct 8-month and 14-month age groups. Real time qPCR analysis indicated dynamic changes in expression of N-glycosylation pathway enzyme-encoding mRNAs across the life span. Notably, computational analysis predicted the promoters of genes encoding these 8 mRNAs contain multiple high probability binding sites for estrogen receptor-1 and progesterone receptor. Collectively, our studies define the N-glycome and identify age-specific dynamic changes in mRNAs encoding N-glycosylation pathway enzymes in mouse gonadotropes. Our studies suggest the age-related decline in ovarian steroids may regulate expression of N-glycosylation enzymes in mouse gonadotropes and explain the age-related N-glycosylation shift previously observed on human FSHß subunit in pituitaries of women.

Effects of pulsatile intravenous follicle-stimulating hormone treatment on ovarian function in women with obesity.

OBJECTIVE: To establish conditions for effective hypothalamic suppression in women with normal and high body mass index (BMI) and test the hypothesis that intravenous (IV) administration of pulsatile recombinant follicle-stimulating hormone (rFSH) can overcome the clinically evident dysfunctional pituitary-ovarian axis in women with obesity. DESIGN: Prospective interventional study. SETTING: Academic medical center. PATIENT(S): Twenty-seven normal-weight women and 27 women with obesity, who were eumenorrheic and aged 21-39 years. INTERVENTION(S): Two-day frequent blood sampling study, in early follicular phase, before and after cetrorelix suppression of gonadotropins and exogenous pulsatile IV rFSH administration. MAIN OUTCOME MEASURE(S): Serum inhibin B and estradiol (E2) levels (basal and rFSH stimulated). RESULT(S): A modified gonadotropin-releasing hormone antagonism protocol effectively suppressed production of endogenous gonadotropins in women with normal and high BMIs, providing a model to address the functional role of FSH in the hypothalamic-pituitary-ovarian axis. The IV rFSH treatment resulted in equivalent serum levels and pharmacodynamics in normal-weight women and those with obesity. However, women with obesity exhibited reduced basal levels of inhibin B and E2 and a significantly decreased response to FSH stimulation. The BMI was inversely correlated with serum inhibin B and E2. In spite of this observed deficit in ovarian function, pulsatile IV rFSH treatment in women with obesity resulted in E2 and inhibin B levels comparable with those in normal-weight women, in the absence of exogenous FSH stimulation. CONCLUSION(S): Despite normalization of FSH levels and pulsatility by exogenous IV administration, women with obesity demonstrate ovarian dysfunction with respect to E2 and inhibin B secretion. Pulsatile FSH can partially correct the relative hypogonadotropic hypogonadism of obesity, thereby providing a potential treatment strategy to mitigate some of the adverse effects of high BMI on fertility, assisted reproduction, and pregnancy outcomes. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov #NCT02478775.

Identification of follicle-stimulating hormone-responsive genes in Sertoli cells during early postnatal mouse testis development.

BACKGROUND: In the mouse testis, Sertoli cells rapidly divide during a narrow window of time pre-pubertally and differentiate thereafter. The number of Sertoli cells determines the testis size and germ cell-carrying capacity. Follicle-stimulating hormone (FSH) binds its cognate FSH-receptors expressed on Sertoli cells and acts as a mitogen to regulate their proliferation. Fshb-/- mutant adult male mice have reduced Sertoli cell number and testis size and reduced sperm number and motility. However, FSH-responsive genes in early postnatal mouse Sertoli cells are unknown. OBJECTIVES: To identify FSH-responsive genes in early postnatal mouse Sertoli cells. MATERIALS AND METHODS: A fluorescence-activated cell sorting method was developed to rapidly purify Sertoli cells from control and Fshb-/- mice carrying a Sox9 GfpKI allele. These pure Sertoli cells were used for large-scale gene expression analyses. RESULTS: We show that mouse Sertoli cells rarely divide beyond postnatal day 7. Our in vivo BrdU labeling studies indicate loss of FSH results in a 30% reduction in Sertoli cell proliferation in mice at 5 days of age. Flowsorted GFP+ Sertoli cells with maximal Fshr expression were 97%-98% pure and mostly devoid of Leydig and germ cells as assessed by Taqman qPCR quantification of gene expression and immunolabeling of the corresponding cell-specific markers. Large-scale gene expression analysis identified several differentially regulated genes in flow-sorted GFP+ Sertoli cells obtained from testis of control and Fshb-/- mice at 5 days of age. The top 25 networks identified by pathway analysis include those related to the cell cycle, cell survival and most importantly, carbohydrate and lipid metabolism and molecular transport. DISCUSSION: Several of the FSH-responsive genes identified in this study could serve as useful markers for Sertoli cell proliferation in normal physiology, toxicant-induced Sertoli cell/testis injury, and other pathological conditions. CONCLUSION: Our studies reveal that FSH-regulates macromolecular metabolism and molecular transport networks of genes in early postnatal Sertoli cells most likely in preparation for establishment of functional associations with germ cells to successfully coordinate spermatogenesis.

Rerouting of follicle-stimulating hormone secretion and gonadal function.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are synthesized in the same pituitary cell, i.e., gonadotrope. They both consist of a common α-subunit that is noncovalently assembled with a hormone-specific ß-subunit in gonadotropes. The heterodimers exit gonadotropes through distinct modes of trafficking and secretion. The FSH is constitutively secreted, whereas LH is secreted in pulses through the regulated pathway that involves dense core granules. Based on several in vitro mutagenesis studies, the carboxy terminus heptapeptide of human LH-ß subunit is identified as a gonadotrope sorting determinant. When heptapeptide is genetically fused to human FSH-ß subunit and the mutant transgene expressed on a Fshb null genetic background, the rerouted FSH mutant dimer enters the LH secretory pathway, stored in dense core granules, coreleased with LH on gonadotropin releasing hormone stimulation and rescues Fshb null mice as efficiently as the constitutively secreted wild-type FSH. The rerouted FSH markedly suppresses follicle atresia and significantly enhances ovulations per cycle and prolongs the female reproductive life span. Gonadotropin rerouting is emerging as a novel paradigm to treat ovarian dysfunction in women, and may explain the origins of ovarian cyclicity as well as provide clues to understand gene and protein networks that maintain optimal ovarian function throughout the female reproductive life span.

Lhb-/-Lhr-/- Double Mutant Mice Phenocopy Lhb-/- or Lhr-/- Single Mutants and Display Defects in Leydig Cells and Steroidogenesis.

In the mouse, two distinct populations of Leydig cells arise during testis development. Fetal Leydig cells arise from a stem cell population and produce T required for masculinization. It is debated whether they persist in the adult testis. A second adult Leydig stem cell population gives rise to progenitor-immature-mature adult type Leydig cells that produce T in response to LH to maintain spermatogenesis. In testis of adult null male mice lacking either only LH (Lhb-/-) or LHR (Lhr-/-), mature Leydig cells are absent but fetal Leydig cells persist. Thus, it is not clear whether other ligands signal via LHRs in Lhb null mice or LH signals via other receptors in the absence of LHR in Lhr null mice. Moreover, it is not clear whether truncated LHR isoforms generated from the same Lhr gene promoter encode functionally relevant LH receptors. To determine the in vivo roles of LH-LHR signaling pathway in the Leydig cell lineage, we generated double null mutant mice lacking both LH Ligand and all forms of LHR. Phenotypic analysis indicated testis morpho-histological characteristics are identical among double null and single mutants which all showed poorly developed interstitium with a reduction in Leydig cell number and absence of late stage spermatids. Gene expression analyses confirmed that the majority of the T biosynthesis pathway enzyme-encoding mRNAs expressed in Leydig cells were all suppressed. Expression of thrombospondin-2, a fetal Leydig cell marker gene was upregulated in single and double null mutants indicating that fetal Leydig cells originate and develop independent of LH-LHR signaling pathway in vivo. Serum and intratesticular T levels were similarly suppressed in single and double mutants. Consequently, expression of AR-regulated genes in Sertoli and germ cells were similarly affected in single and double mutants without any evidence of any additive effect in the combined absence of both LH and LHR. Our studies unequivocally provide genetic evidence that in the mouse testis, fetal Leydig cells do not require LH-LHR signaling pathway and a one-to-one LH ligand-LHR signaling pathway exists in vivo to regulate adult Leydig cell lineage and spermatogenesis.

The solute carrier family 7 member 11 (SLC7A11) is regulated by LH/androgen and required for cystine/glutathione homeostasis in mouse Sertoli cells.

Luteinizing hormone (LH) stimulates testosterone production from Leydig cells. Both LH and testosterone play important roles in spermatogenesis and male fertility. To identify LH - and testosterone - responsive transporter genes that play key roles in spermatogenesis, we performed large-scale gene expression analyses on testes obtained from adult control and Lhb knockout mice. We found a significant reduction in cystine/glutamate transporter encoding Slc7a11 mRNA in testes of Lhb null mice. We observed that Slc7a11/SLC7A11 expression was initiated pre-pubertally and developmentally regulated in mouse testis. Immunolocalization studies confirmed that SLC7A11 was mostly expressed in Sertoli cells in testes of control and germ cell-deficient mice. Western blot analyses indicated that SLC7A11 was significantly reduced in testes of mutant mice lacking either LH or androgen receptor selectively in Sertoli cells. Genetic and pharmacological rescue of Lhb knockout mice restored the testicular expression of Slc7a11 comparable to that observed in controls. Additionally, Slc7a11 mRNA was significantly suppressed upon Sertoli cell/testicular damage induced in mice by cadmium treatment. Knockdown of Slc7a11 in vitro in TM4 Sertoli cells or treatment of mice with sulfasalazine, a SLC7A11 inhibitor caused a significant reduction in intracellular cysteine and glutathione levels but glutamate content remained unchanged as determined by metabolomic analysis. Knockdown of Slc7a11 resulted in compensatory upregulation of other glutamate transporters belonging to the Slc1a family presumably to maintain intracellular glutamate levels. Collectively, our studies identified that SLC7A11 is an LH/testosterone-regulated transporter that is required for cysteine/glutathione but not glutamate homeostasis in mouse Sertoli cells.

First-in-class humanized FSH blocking antibody targets bone and fat.

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a KD of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHß subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.

The Capacity of the Ovarian Cancer Tumor Microenvironment to Integrate Inflammation Signaling Conveys a Shorter Disease-free Interval.

PURPOSE: Ovarian cancer has one of the highest deaths to incidence ratios across all cancers. Initial chemotherapy is effective, but most patients develop chemoresistant disease. Mechanisms driving clinical chemo-response or -resistance are not well-understood. However, achieving optimal surgical cytoreduction improves survival, and cytoreduction is improved by neoadjuvant chemotherapy (NACT). NACT offers a window to profile pre- versus post-NACT tumors, which we used to identify chemotherapy-induced changes to the tumor microenvironment. EXPERIMENTAL DESIGN: We obtained matched pre- and post-NACT archival tumor tissues from patients with high-grade serous ovarian cancer (patient, n = 6). We measured mRNA levels of 770 genes (756 genes/14 housekeeping genes, NanoString Technologies), and performed reverse phase protein array (RPPA) on a subset of matched tumors. We examined cytokine levels in pre-NACT ascites samples (n = 39) by ELISAs. A tissue microarray with 128 annotated ovarian tumors expanded the transcriptional, RPPA, and cytokine data by multispectral IHC. RESULTS: The most upregulated gene post-NACT was IL6 (16.79-fold). RPPA data were concordant with mRNA, consistent with elevated immune infiltration. Elevated IL6 in pre-NACT ascites specimens correlated with a shorter time to recurrence. Integrating NanoString (n = 12), RPPA (n = 4), and cytokine (n = 39) studies identified an activated inflammatory signaling network and induced IL6 and IER3 (immediate early response 3) post-NACT, associated with poor chemo-response and time to recurrence. CONCLUSIONS: Multiomics profiling of ovarian tumor samples pre- and post-NACT provides unique insight into chemo-induced changes to the tumor microenvironment. We identified a novel IL6/IER3 signaling axis that may drive chemoresistance and disease recurrence.

Sex Differences Across the Lifespan: A Focus on Cardiometabolism.

Women's health and sex differences research remain understudied. In 2016, to address the topic of sex differences, the Center for Women' s Health Research (CWHR) at the University of Colorado (cwhr@ucdenver.edu) held its inaugural National Conference, "Sex Differences Across the Lifespan: A Focus on Metabolism" and published a report summarizing the presentations. Two years later, in 2018, CWHR organized the 2nd National Conference. The research presentations and discussions from the 2018 conference also addressed sex differences across the lifespan with a focus on cardiometabolism and expanded the focus by including circadian physiology and effects of sleep on cardiometabolic health. Over 100 participants, including basic scientists, clinicians, policymakers, advocacy group leaders, and federal agency leadership participated. The meeting proceedings reveal that although exciting advances in the area of sex differences have taken place, significant questions and gaps remain about women's health and sex differences in critical areas of health. Identifying these gaps and the subsequent research that will result may lead to important breakthroughs.

In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†.

Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980-2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

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.