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Androgen and its receptor (AR) play a critical role in reproductive function, under both normal physiological and clinically pathophysiological conditions. Many women with hyperandrogenemia suffer irregular menses, oligo-anovulation and infertility. Whether high androgen levels directly or secondarily affect reproduction is unknown. Further, most animal models of hyperandrogenemia induced infertility are associated with obesity which adds a confounding variable to analysis. Therefore, we created a lean elevated androgen model by implantation of a low dose dihydrotestosterone (DHT) that separates the effects of obesity from elevated androgen. Reproduction is tightly controlled by the hypothalamus-pituitary-gonadal axis, androgen could theoretically alter neuroendocrine function at the level of brain and/or pituitary. The goal of this study is to understand how high androgen signaling via the androgen receptor (AR) in pituitary gonadotropes affects reproductive function. Mice with disruption of the Ar gene specifically in pituitary gonadotropes (PitARKO) were produced to explore the role of elevated androgen on the development of reproductive dysfunction in female mice. PitARKO-DHT mice showed significantly improved estrous cyclicity and fertility compared to DHT treated control littermates with intact pituitary Ar (Con-DHT). During 90 days of mating, PitARKO-DHT mice had a significantly increased number of litters (2.3±0.6 vs 0.5±0.3) and number of pups (5.3±2.8 vs 17.0±4.5) compared to Con-DHT mice. The improved reproductive function in PitARKO-DHT mice is partially due to improved pituitary responsiveness to GnRH stimulation. Ex vivo pituitary primary culture confirmed that DHT significantly reduced both basal and GnRH stimulated LH secretion. In vivo, LH secretion is preserved in PitARKO-DHT mice after GnRH stimulation compared to CON-DHT mice. GnRH induces the release of gonadotropins via an increase in cytosolic Ca2+ concentration, and reduced cytoplasmic Ca2+ concentrations may interfere with exocytosis of granules of LH. GEM, a calcium binding protein, inhibits voltage-gated Ca2+ channel activity and reduces Ca2+ influx in gonadotropes, which can affect gonadotropin secretion. We observed that Gem mRNA levels were significantly increased in Con-DHT treated mice (6 fold higher) compared to PitARKO-DHT mice. Further, the intracellular calcium influx signals were dramatically attenuated in Con-DHT mice compared to Con-no DHT mice, and were preserved in PitARKO-DHT mice after GnRH stimulation. These findings highlight gonadotrope AR as an extra ovarian regulator playing an important role in reproductive physiology and that high androgens may impact function through AR regulated Gem expression.

Women with elevated androgen often exhibit hyperinsulinemia and insulin resistance. Many models have been created to produce metabolic dysfunction by manipulating androgen levels. Previously we reported that hyperandrogenemia produced by implantation of a DHT pellet may induce whole body insulin resistance through hepatic androgen receptor (AR) in vitro. Therefore, we conditionally disrupted the hepatic AR both developmentally (by breeding the albumin-CRE mouse (alb-CRE; liver-specific Cre expression) with ARfl/fl mice (LivARKO)) and acutely (by tail vein injection in ARfl/fl mice of adeno-associated virus with albumin promoter driven Cre vector (adLivARKO)). Impaired glucose tolerance, pyruvate tolerance, and insulin tolerance were prevented in both LivARKO-DHT and adLivARKO-DHT mice compared to control mice treated with DHT (ARfl/fl; cre-; Con-DHT) mice. Further, the glucose infusion rate is significantly reduced in LivARKO-DHT mice compared to Con-DHT mice as measured during hyperinsulinemia-euglycemia clamp suggesting improved insulin-induced glucose disposal. We observed that primary hepatocytes treated with DHT from both women and female mice showed reduced PI3K-pAKT and pFOXO1 activation by insulin, and increased ex vivo glucose production. LivARKO primary hepatocytes were resistant to DHT induced attenuated insulin signaling and increased gluconeogenesis (ex vivo) and this was confirmed in LivARKO in vivo. These data support a paradigm in which in females, elevated androgen disrupts metabolic function via hepatic AR.

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.

Elimination of estrogen receptorα (ERα) from kisspeptin (Kiss1) neurons results in premature LH release and pubertal onset, implicating these receptors in 17β-estradiol (E2)-mediated negative feedback regulation of GnRH release during the prepubertal period. Here, we tested the dependency of prepubertal negative feedback on ERα in Kiss1 neurons. Prepubertal (postnatal d 14) and peripubertal (postnatal d 34) wild-type (WT) and Kiss1 cell-specific ERα knockout (KERαKO) female mice were sham operated or ovariectomized and treated with either vehicle- or E2-containing capsules. Plasma and tissues were collected 2 days after surgery for analysis. Ovariectomy increased LH and FSH levels, and E2 treatments completely prevented these increases in WT mice of both ages. However, in prepubertal KERαKO mice, basal LH levels were elevated vs WT, and both LH and FSH levels were not further increased by ovariectomy or affected by E2 treatment. Similarly, Kiss1 mRNA levels in the medial basal hypothalamus, which includes the arcuate nucleus, were suppressed with E2 treatment in ovariectomized prepubertal WT mice but remained unaffected by any treatment in KERαKO mice. In peripubertal KERαKO mice, basal LH and FSH levels were not elevated vs WT and were unaffected by ovariectomy or E2. In contrast to our previous findings in adult animals, these results demonstrate that suppression of gonadotropins and Kiss1 mRNA by E2 in prepubertal animals depends upon ERα activation in Kiss1 neurons. Our observations are consistent with the hypothesis that these receptors play a critical role in restraining GnRH release before the onset and completion of puberty.

Kisspeptin, encoded by the Kiss1 gene, binds to a specific G protein-coupled receptor (kisspeptin1 receptor) to regulate the central reproductive axis. Kisspeptin has also been reported to be expressed in peripheral tissues, including the testes. However, factors regulating testicular kisspeptin and its role in reproduction are unknown. Our objective herein was to begin to address kisspeptin function in the testis. In particular, we sought to determine the level of kisspeptin in the testis in comparison with the brain and other tissues, how these levels change from the prepubertal period through sexual maturation, and the factors involved in kisspeptin regulation in the testis. Immunohistochemical analysis of testis sections using a validated kisspeptin antibody localized kisspeptin to the Leydig cells. Kisspeptin was not detected in germ cells or Sertoli cells within the seminiferous tubules at any developmental time period studied, from prepuberty to sexual maturation. A developmental time course of testicular kisspeptin revealed that its mRNA and protein levels increased during development, reaching robust levels at postnatal day 28, correlating with pubertal onset. In vitro studies of primary mouse Leydig cells, as well as in vivo studies, indicated clearly that LH is involved in regulating levels of Leydig cell kisspeptin. Interestingly, gonadectomy resulted in elevated LH but reduced serum kisspeptin levels, suggesting that testicular kisspeptin may be secreted. These data document kisspeptin expression in mouse Leydig cells, its secretion into peripheral serum, and its regulation by changes in reproductive neuroendocrine function.