The kisspeptin receptor, crucial for hypothalamic control of puberty and reproduction, is also present in the pituitary gland. Its role in the pituitary gland is not defined. Kisspeptin signaling via the Kiss1r could potentially regulate reproductive function at the level of pituitary gonadotrope. Using Cre/Lox technology, we deleted the Kiss1r gene in pituitary gonadotropes (PKiRKO). PKiRKO males have normal genital development (anogenital distance WT: 19.1 ± 0.4 vs. PKiRKO: 18.5 ± 0.4 mm), puberty onset, testes cell structure on gross histology, normal testes size, and fertility. PKiRKO males showed significantly decreased serum FSH levels compared to WT males (5.6 ± 1.9 vs. 10.2 ± 1.8 ng/ml) with comparable LH (1.1 ± 0.2 vs. 1.8 ± 0.4 ng/ml) and testosterone levels (351.8 ± 213.0 vs. 342.2 ± 183.0 ng/dl). PKiRKO females have normal puberty onset, cyclicity, LH and FSH levels and fertility. Overall, these findings indicate that absence of pituitary Kiss1r reduces FSH levels in male mice without affecting testis function. PKiRKO mice have normal reproductive function in both males and females.

The anterior pituitary secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate gonadal development, gametogenesis and the secretion of the gonadal steroid hormones. The gonadotroph is primarily regulated by hypothalamic secretion of gonadotropin-releasing hormone (GnRH) from neurons of the rostral hypothalamus and is mediated by GnRH receptor signaling. Kisspeptin (KISS1)/kisspeptin receptor (KISS1R) signaling in GnRH neurons plays an essential role in reproductive function. As the kisspeptin receptor is present in the pituitary, kisspeptin signaling via the Kiss1r may regulate reproductive function at the level of pituitary. Using Cre/Lox technology, we deleted the Kiss1r gene in pituitary gonadotropes (PKiRKO). PKiRKO male and females have normal genital development, puberty onset, and fertility. Females have normal LH, FSH and estradiol while males had significantly increased basal serum FSH levels with no differences in basal serum LH, or testosterone levels. Overall, these findings indicate that the pituitary KISS1R does not play a role in male reproduction.

Many women with hyperandrogenemia suffer from irregular menses and infertility. However, it is unknown whether androgens directly affect reproduction. Since animal models of hyperandrogenemia-induced infertility are associated with obesity, which may impact reproductive function, we have created a lean mouse model of elevated androgen using implantation of low dose dihydrotestosterone (DHT) pellets to separate the effects of elevated androgen from obesity. The hypothalamic-pituitary-gonadal axis controls reproduction. While we have demonstrated that androgen impairs ovarian function, androgen could also disrupt neuroendocrine function at the level of brain and/or pituitary to cause infertility. To understand how elevated androgens might act on pituitary gonadotropes to influence reproductive function, female mice with disruption of the androgen receptor (Ar) gene specifically in pituitary gonadotropes (PitARKO) were produced. DHT treated control mice with intact pituitary Ar (Con-DHT) exhibit disrupted estrous cyclicity and fertility with reduced pituitary responsiveness to GnRH at the level of both calcium signaling and LH secretion. These effects were ameliorated in DHT treated PitARKO mice. Calcium signaling controls GnRH regulation of LH vesicle exotocysis. Our data implicated upregulation of GEM (a voltage-dependent calcium channel inhibitor) in the pituitary as a potential mechanism for androgen's pathological effects. These results demonstrate that gonadotrope AR, as an extra-ovarian regulator, plays an important role in reproductive pathophysiology.

The hypothalamic‐pituitary‐gonadal (HPG) axis controls the development and maintenance of reproductive function. The HPG axis is comprised of gonadotropin‐releasing hormone (GnRH) neurons in the hypothalamus that regulate the gonadotrophs in the anterior pituitary gland, stimulating the secretion of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) into the bloodstream. LH and FSH play essential roles in spermatogenesis in males, folliculogenesis and ovulation in females, and steroidogenesis in both sexes. Recently, kisspeptin (KISS1)/kisspeptin receptor (KISS1R) signaling in GnRH neurons has been shown by our group and others to play an essential role in HPG axis function. However, whether kisspeptin signaling via the Kiss1r affects reproductive function at the level of pituitary is not yet known. Using Cre/Lox technology, we knocked out the Kiss1r gene specifically in pituitary gonadotropes (PKiRKO) by crossing a αGSUCre mouse with a floxed Kiss1r mouse. Q‐RT‐PCR and immunohistochemistry were used to demonstrate a disruption in pituitary Kiss1r mRNA and KISS1R protein in PKiRKO mice relative to controls. Q‐RT‐PCR demonstrated a reduction in Kiss1r mRNA by 88% and 64% in the pituitary of male and female PKiRKO mice (n=8), respectively, compared with wild type (WT) mice (n=8). Immunostaining for KISS1R protein levels exhibited similar trends for protein knock down as observed for the relative mRNA levels. Our results revealed no difference in the age of puberty between WT and PKiRKO littermates, as assessed by the ages of vaginal opening, and first estrus for female mice, and preputial separation for male mice. Furthermore, we saw no difference in ovarian and testes weight respectively in female and male mice. While there were no differences in basal LH and FSH levels, upon performing a GnRH stimulation test in vivo, we observed a significant attenuation ( P <0.05) in stimulated luteinizing hormone (LH) levels in PKiRKO male mice compared with WT male mice, while stimulated LH and FSH levels were no different between WT and PKiRKO female mice. To directly assess the effects of KISS1 on pituitary gonadotroph function that are mediated via the KISS1R we sought to develop an in vitro primary culture system. To test the system, a GnRH dose response and time course study was performed on dispersed and adherent pituitary cells harvested from WT male and female mice. Groups of cells were treated with either 5nM, 30nM or 100nM GnRH, for a duration of 30 and 60 minutes. A significant increase in LH levels was observed in both male and female mice pituitary cells when treated with either 30nM or 100nM GnRH for 60 minutes. This culture system was then applied to calcium flux assays using the calcium indicator dye Fluo‐1. These calcium flux assays indicated that the WT male pituitaries were more responsive to GnRH (100nm) and kisspeptin (1nm) than PKiRKO males. Interestingly a combination of 1nM kp10 and 30nM GnRH potentiated the calcium response in WT males but not their PKiRKO littermates, suggesting that there is interplay of both KISS1 signaling and GnRH signaling at the level of the pituitary to augment pituitary action. These findings indicate overall that the pituitary Kiss1r may plan an important modulatory role and contribute to normal reproductive function. Support or Funding Information National Institutes of Health T32DK007751, JHU UMD Diabetes Research Center (P30 DK079637), RO1 DK101591 and R01HD068777. The American Physiological Society Porter Developmental and Minority Affairs Committee This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Androgen and its receptor (AR) play a critical role in reproductive function under both physiological and pathophysiological conditions. Female AR global knockout mice are subfertile due to both neuroendocrine and ovarian defects. Female offspring from prenatally androgenized heterozygous AR pregnant mice showed rescued estrous cyclicity and fertility. Ar is expressed in granulosa cells, theca interstitial cells, and oocytes in the ovary. We created mice with theca-specific deletion of Ar (ThARKO) by crossing Cyp17-iCre mice that express Cre recombinase under cytochrome P450 17A1 (Cyp17) promoter with Arfl/fl mice. ThARKO mice exhibited no significant differences in pubertal onset or fertility compared with control littermates, and neither estrogen or testosterone levels were different between these groups. Therefore, Ar expression in theca cells likely does not influence fertility nor androgen levels in female mice. We then tested the role of AR in theca cells under hyperandrogenemic condition. After treatment with a pathophysiological level of dihydrotestosterone (DHT), control mice (control-DHT) showed acyclicity and infertility. However, estrous cycles and fertility were altered to a significantly less degree in ThARKO-DHT mice than in control-DHT mice. Messenger RNA (mRNA) levels of Lhcgr (luteinizing hormone receptor) and Timp1 (tissue inhibitor of metalloproteinase 1, and inhibitor of matrix metalloproteinase) were significantly lower in control-DHT ovary compared with control-no DHT ovaries, whereas mRNA levels of Fshr (follicle-stimulating hormone receptor) were significantly higher. Timp1 gene expression was comparable in the ThARKO-DHT and the control-no DHT ovary. We speculate that the preserved level of Timp1 in ThARKO-DHT mice contributes to retained reproductive function.

Androgen excess in women is associated with metabolic dysfunction (e.g., obesity, hyperinsulinemia, insulin resistance, and increased risk of type 2 diabetes) and reproductive dysfunction (e.g., polycystic ovaries, amenorrhea, dysregulated gonadotropin release, and infertility). We sought to identify the effects of androgen excess on glucose metabolic dysfunction and the specific mechanisms of action by which androgens are inducing pathology. We developed a mouse model that displayed pathophysiological serum androgen levels with normal body mass/composition to ensure that the phenotypes were directly from androgens and not an indirect consequence of obesity. We performed reproductive tests, metabolic tests, and hormonal assays. Livers were isolated and examined via molecular, biochemical, and histological analysis. Additionally, a low-dose dihydrotestosterone (DHT) cell model using H2.35 mouse hepatocytes was developed to study androgen effects on hepatic insulin signaling. DHT mice demonstrated impaired estrous cyclicity; few corpora lutea in the ovaries; glucose, insulin, and pyruvate intolerance; and lowered hepatic insulin action. Mechanistically, DHT increased hepatic androgen-receptor binding to phosphoinositide-3-kinase (PI3K)-p85, resulting in dissociation of PI3K-p85 from PI3K-p110, leading to reduced PI3K activity and decreased p-AKT and, thus, lowered insulin action. DHT increased gluconeogenesis via direct transcriptional regulation of gluconeogenic enzymes and coactivators. The hepatocyte model recapitulated the in vivo findings. The DHT-induced hepatocyte insulin resistance was reversed by the androgen-receptor antagonist, flutamide. These findings present a phenotype (i.e., impaired glucose tolerance and disrupted glucose metabolism) in a lean hyperandrogenemia model (low-dose DHT) and data to support 2 molecular mechanisms that help drive androgen-induced impaired glucose metabolism.

BackgroundPolycystic ovarian syndrome (PCOS), the leading cause of female infertility, is characterized by hyperandrogenism, oligo‐or ammenorhea, and polycystic ovaries, and is associated with type 2 diabetes and insulin resistance. Our previous studies introduced a low dose dihydrotestosterone (DHT) mouse model that recapitulated features of PCOS and that displayed differential tissue specific responses to insulin action – insulin resistance in liver and adipose tissue but maintained insulin action in the pituitary and ovary.ObjectiveHere we seek to further characterize the low dose DHT mouse model and to determine molecular mechanisms involved in the differential tissue response to androgen/insulin action. We hypothesized that low dose DHT will result in hepatic and adipocyte dysfunction, differentially alter insulin‐stimulated glucose transport in energy storage compared to reproductive tissues, and augmented hepatic gluconeogenesis. Many DHT mouse models use concentrations 8‐fold that of normal female mice. We used a 4 mm DHT pellet which continuous release DHT and achieving serum DHT levels 1.7‐fold higher than controls, similar to the elevation of DHT seen in women with PCOS.MethodsReproductive tests (mouse estrous cyclicity, mating, and ovary histology), metabolic tests (PTT and GSIS) and blood sampling (hormonal assays: LH, FSH, insulin, leptin, Il‐6, and Tnfa) were performed. Radiolabeled ex vivo glucose transport assays, histological staining, quantitative real‐time PCR (qRT‐PCR), Luminex multiple ligand assay, and western blot immunostaining were performed on energy storage (liver and adipose tissue) and reproductive (pituitary and ovaries) tissues.ResultsLow dose DHT mice demonstrated unaltered LH and FSH serum levels, no weight gain, impaired pyruvate tolerance, hepatic steatosis and adipocyte hypertrophy compared to control mice. Mice implanted with DHT exhibited lower hepatic mRNA expression of enzymes used in glycolysis and glycogen synthesis compared to controls. Interestingly, gluconeogenic mRNA expression levels were unaltered but gluconeogenic protein levels were increased in DHT mice compared to controls. In addition, we assessed protein levels of intermediates in the insulin signaling pathways. DHT mice displayed lowered p‐AKT levels in the liver and WAT but in contrast maintained p‐AKT levels in the ovary and pituitary. However, insulin receptor protein levels were unaltered in all tissues compared to control mice.ConclusionThese findings suggest that low dose DHT serves as a mouse model of lean PCOS and that the molecular mechanism by which low dose DHT impairs glucose metabolism is partly due to increased hepatic gluconeogenic output and decreased adipocyte glucose uptake. In addition, the impairment in the insulin signaling pathway is downstream of the insulin receptor. Tissue specific targeting of AR via compounds such as SARMs may serve as therapeutic interventions for PCOS related glucose metabolism dysfunction.Support or Funding Information5T32DK007751‐19 Wolfe (PI) 09/12/1997 ‐06/30/17 NIH/NICHD Direct Costs: $300,812 Interdepartmental training program in cellular and molecular endocrinology The goal of the Interdepartmental Training Program in Cellular and Molecular Endocrinology at Johns Hopkins is to expand the pool of well‐trained and productive investigators in the biomedical sciences related to endocrinology. Role: TraineeR00HD068130 Wu (PI) 07/01/14‐06/30/17 NIH/NICHD Direct Costs: $226,790 Insulin and Androgen Interactions in the Infertility of Obesity We will attempt to define the complex interactions of insulin and androgens on the development of obesity induced infertility. We hope to uncover the mechanism that underlies the development of infertility in women with metabolic dysfunction such as frequently is observed in PCOS. Role: Trainee