and depression [78] can occur with PCOS in women. In this regard, and in addition to reproductive and metabolic dysfunction, early-to-mid gestation T-exposure reprograms (“organizes”) prepubertal and adult female behavior. T-exposed female monkeys exhibit increased male-typical infant vocalizations, diminished intimate social grooming of mothers and interest in infants, increased mounting of peers, and diminished, but not absent, engagement in female-typical sexual interactions with males [79], all independent of circulating sex hormone levels. Such behavioral reprogramming is difficult to reconcile with traditional female gender roles in human societies, potentially leading to sexual dysfunction and depression. Recent reports from in utero androgen excess rodent models clearly demonstrate anxiety-like behavior in female offspring accompanied by the upregulation of amygdala gene expression, including corticotropin-releasing hormone [80], an identical neural site and neuropeptide system implicated in the pathogenesis of anxious phenotype in monkeys and humans [81], leading to depression.
Mechanistic PCOS Insight from Non-Primate Animal Models of in utero Female Hyperandrogenism
Non-primate models of in utero androgen excess emulate many of the reproductive and metabolic traits found in PCOS women and T-exposed monkeys (Table 1). With their relative ease of manipulation, these models have generated a plethora of incisive pharmacological and molecular manipulations that provide additional insight into pathogenic mechanisms engaged by in utero androgen excess (recently reviewed by [16–18]). Differential gestational timing or duration of T exposure in female sheep (Table 1), illustrate the skew in gestational vulnerability to PCOS-like reprogramming of both reproductive and metabolic traits reported in female monkeys, and suggest that longer durations of T exposure commencing before mid-gestation induce a more pronounced PCOS-like phenotype. By late gestation, following cessation of maternal early-to-late T administration, increased ovarian theca cell expression of CYP17A1 and increased release of androstenedione are already present [82]. Postnatally, however, circulating androgen levels in such T-exposed sheep are not elevated, but ovarian androgen receptor expression is increased suggesting “functional hyperandrogenism” within an androgen target organ [83]. Early-to-late DHT exposure, while recapitulating T reprogramming of reproductive traits and insulin resistance, does not disrupt maturation of regular ovarian cycles or ovarian morphology (Table 1), suggesting limits to androgen receptor-mediated PCOS-like reprogramming in sheep. Maternal co-administration of the androgen receptor antagonist, flutamide, along with T during early-to-late gestation prevents early puberty, and likely LH hypersecretion, as well as onset of ovulatory dysfunction, PCOS-like ovarian morphology, and ovarian steroidogenic abnormalities [84]. Gestational flutamide co-treatment, however, fails to prevent metabolic phenotype, including insulin resistance, adipogenic constraint, hyperlipidemia, and fatty liver [16], again demonstrating limits to androgen receptor-mediated, PCOS-like reprogramming. In this regard, flutamide treatment of adult female mice previously exposed to fetal T reverses their acyclicity [14], and in some PCOS women, improves fertility, menstrual cyclicity, and LH levels [85], as well as normalizing progesterone negative feedback regulation of episodic GnRH/LH release [69].
Gestational co-administration of the peroxisome proliferator-activated receptor gamma or NR1C3, a nuclear transcription factor crucial for adipocyte maturation, along with T during early-to-late gestation, prevents insulin resistance and early puberty onset, and likely LH hypersecretion, in T-exposed female lambs, but does not prevent adipogenic dysfunction, hyperlipidemia and fatty liver [16]. In this regard, it is interesting that treatment of late gestation DHT-exposed female offspring as adults with the insulin sensitizer, metformin, restores normal cyclicity, as well as normalizes androgen and LH levels. Taken together, these findings suggest that while reprogramming of a variety of PCOS-like reproductive traits involves androgen receptor and/or insulin-mediated actions, adipogenic and lipogenic traits may involve additional reprogramming, perhaps engaging estrogenic T metabolites.
Mouse models have predominantly used the non-aromatizable androgen, DHT, to induce late gestation in utero androgen excess and PCOS-like reprogramming (e.g., [14, 17, 18]). Late gestation DHT administered to female GnRH-green fluorescent protein (GFP)-transgenic mouse dams (derived from CBB6/F1 strain) produces PCOS-like female offspring exhibiting hyperandrogenism, intermittent/absent cycles, aberrant ovarian follicle morphology, LH hypersecretion derived from accelerated episodic GnRH release, fatty liver, and enlarged adipocytes, without accompanying increased adiposity and insulin resistance (Table 1). The elegant use of genetic manipulation to globally delete androgen receptor (ARKO) protects fetal female mice (>98% C57BL/6J strain) from fetal DHT-induced, PCOS-like reprogramming, including absence of intermittent/absent cycles, aberrant follicle morphology, and enlarged adipocytes [86]. The wild type mice from which ARKO females are derived, nevertheless, do not exhibit fetal DHT-induced hyperandrogenism or LH hypersecretion, possibly reflecting GnRH-GFP and ARKO mouse strain differences in fetal female susceptibility to DHT fetal reprogramming. Neural androgen receptor expression may be particularly crucial for DHT-mediated, PCOS-like reprogramming since selective deletion of neuronal androgen receptor expression, NeuroARKO, provides the best protection against peri-pubertal onset, DHT induction of PCOS-like traits [87]. NeuroARKO mice, however, have not yet been challenged with late gestation DHT to ascertain if absence of neuronal androgen receptor abrogates in utero PCOS-like reprogramming. With less resemblance to in utero androgen excess in sheep, androgen receptors of in utero DHT-exposed female mice may mediate the majority of PCOS-like reprogramming.
GnRH-GFP transgenic mice have also enabled neuro-immunohistochemical delineation of hypothalamic changes that may underlie PCOS-like reprogramming of GnRH release and its negative feedback regulation. Late gestation DHT increases anatomical and functional gamma-aminobutyric acid (GABA) neuronal connectivity to GnRH neurons, generating increased firing rate of GnRH neurons, as well as LH hypersecretion, related to diminished progesterone negative feedback regulation [14, 88]. Such enhanced GABA excitatory connectivity, originating at least in part from the hypothalamic arcuate nucleus, is established before puberty, when circulating androgen levels are low [88]. Since GABA, but not GnRH, neurons express detectable levels of progesterone, estrogen, and androgen receptors, aberrant GABA excitatory connectivity may mediate diminished progesterone (and E2) negative feedback regulation demonstrated by in utero androgen excess female mice, and potentially rats, sheep and monkeys.
Unexpectedly, long-term administration