The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G 1 /S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was ϳ80 bp, and the MCM2-7: ORC ratio on this fragment was ϳ1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.In eukaryotic organisms, DNA replication initiates at many sites (1). In Saccharomyces cerevisiae, DNA replication initiates every ϳ40 kb at autonomously replicating sequences that recruit the origin recognition complex (ORC), 1 the sixsubunit initiator protein. In metazoans, initiation sites are less rigidly defined. In embryonic cells of Xenopus laevis, DNA replication initiates once every ϳ10 kb without sequence specificity (2). In somatic cells, initiation events are less frequent, occurring once every ϳ150 kb, and recent evidence indicates that initiations are controlled by genetic elements (1). At some loci, replication initiates at a precise location, whereas at other loci, initiation events are distributed throughout zones spanning up to 50 kb.The mechanism of DNA replication initiation is highly conserved among eukaryotic organisms (3, 4). A representative model system is Xenopus egg extracts (2, 4) where two factors, Cdc6 and Cdt1, bind to sites on chromatin that are marked by ORC. Subsequently, the hexameric MCM2-7 complex binds to the ORC-Cdc6-Cdt1 complex to establish the pre-replication complex (pre-RC). At the onset of DNA replication, the pre-RC is activated by the sequential action of the protein kinases Cdc7/Dbf4 and Cdk2/cyclin E (Cdk) (5, 6). Genetic and biochemical experiments suggest that the function of Cdc7/Dbf4 is the phosphorylation of the MCM2-7 complex (7). After MCM phosphorylation by Cdc7/Dbf4, Cdk2/cyclin E stimulates the association of Cdc45 with the pre-RC, likely via a direct interaction with the MCM2-7 complex (8, 9). The binding of Cdc45 coincides with activation of a chromatin-bound helicase that unwinds the DNA, allowing binding of the single-stranded...

Polycystic ovary syndrome (PCOS) is a complex hormonal disorder characterized by reproductive, endocrine, and metabolic abnormalities. As the origins of PCOS remain unknown, mechanismbased treatments are not feasible and current management relies on treatment of symptoms. Hyperandrogenism is the most consistent PCOS characteristic; however, it is unclear whether androgen excess, which is treatable, is a cause or a consequence of PCOS. As androgens mediate their actions via the androgen receptor (AR), we combined a mouse model of dihydrotestosterone (DHT)-induced PCOS with global and cell-specific AR-resistant (ARKO) mice to investigate the locus of androgen actions that mediate the development of the PCOS phenotype. Global loss of the AR reveals that AR signaling is required for all DHT-induced features of PCOS. Neuron-specific AR signaling was required for the development of dysfunctional ovulation, classic polycystic ovaries, reduced large antral follicle health, and several metabolic traits including obesity and dyslipidemia. In addition, ovariectomized ARKO hosts with wild-type ovary transplants displayed normal estrous cycles and corpora lutea, despite DHT treatment, implying extraovarian and not intraovarian AR actions are key loci of androgen action in generating the PCOS phenotype. These findings provide strong evidence that neuroendocrine genomic AR signaling is an important extraovarian mediator in the development of PCOS traits. Thus, targeting AR-driven mechanisms that initiate PCOS is a promising strategy for the development of novel treatments for PCOS.PCOS | androgen | animal model | neuroendocrine P olycystic ovary syndrome (PCOS) is the most frequent endocrine disorder of young women, with a prevalence of 6 to 15% (1), and accounts for more than 75% of anovulatory infertility (2). It is characterized by reproductive hormone dysregulation involving luteinizing hormone (LH) hypersecretion and hyperandrogenism (3), the consequences of which can be acne and hirsutism, as well as reduced fertility, due to aberrant follicular maturation, ovulatory disturbance, and miscarriage (3). Associated nonreproductive abnormalities, such as obesity, metabolic syndrome, hyperinsulinemia, insulin resistance, hepatic steatosis, and dyslipidemia predispose affected women to heightened risk of cardiovascular disease and type 2 diabetes (3, 4). However, despite the high prevalence and significant health impact, the pathogenesis of PCOS remains unclear so that mechanism-based treatments remain unattainable.Hyperandrogenism, the most consistent feature of PCOS (5), is implicated as a key mediator in the pathogenesis of PCOS. Supportive evidence includes that androgen excess from endogenous [congenital adrenal hyperplasia (6)] or exogenous [female-to-male transsexuals (7)] sources can produce polycystic ovaries. Furthermore, androgens induce reproductive, metabolic, and endocrine features of PCOS in rodent, sheep, and primate animal models of PCOS (8-10). As all androgen action is mediated via the androgen receptor (AR),...

No abstract

As the mechanistic basis of polycystic ovary syndrome (PCOS) remains unknown, current management relies on symptomatic treatment. Hyperandrogenism is a major PCOS characteristic and evidence supports it playing a key role in PCOS pathogenesis. Classically, androgens can act directly through the androgen receptor (AR) or, indirectly, following aromatization, via the estrogen receptor (ER). We investigated the mechanism of androgenic actions driving PCOS by comparing the capacity of non-aromatizable dihydrotestosterone (DHT) and aromatizable testosterone to induce PCOS traits in WT and Ar-knockout (ARKO) mice. DHT and testosterone induced the reproductive PCOS-like features of acyclicity and anovulation in WT females. In ARKO mice, DHT did not cause reproductive dysfunction; however, testosterone treatment induced irregular cycles and ovulatory disruption. These findings indicate that direct AR actions and indirect, likely ER, actions of androgens are important mediators of PCOS reproductive traits. DHT, but not testosterone, induced an increase in body weight, body fat, serum cholesterol and adipocyte hypertrophy in WT mice, but neither androgen induced these metabolic features in ARKO mice. These data infer that direct AR-driven mechanisms are key in driving the development of PCOS metabolic traits. Overall, these findings demonstrate that differing PCOS traits can be mediated via different steroid signaling pathways and indicate that a phenotype-based treatment approach would ensure effective targeting of the underlying mechanisms.

Lifestyle, mainly dietary, interventions are first-line treatment for women with polycystic ovary syndrome (PCOS), but the optimal diet remains undefined. We combined a hyperandrogenized PCOS mouse model with a systematic macronutrient approach, to elucidate the impact of dietary macronutrients on the development of PCOS. We identify that an optimum dietary macronutrient balance of a low protein, medium carbohydrate and fat diet can ameliorate key PCOS reproductive traits. However, PCOS mice display a hindered ability for their metabolic system to respond to diet variations, and varying macronutrient balance did not have a beneficial effect on the development of metabolic PCOS traits. We reveal that PCOS traits in a hyperandrogenic PCOS mouse model are ameliorated selectively by diet, with reproductive traits displaying greater sensitivity than metabolic traits to dietary macronutrient balance. Hence, providing evidence to support the development of evidence-based dietary interventions as a promising strategy for the treatment of PCOS, especially reproductive traits.

The androgen receptor (AR) is expressed throughout the hypothalamic-pituitary-gonadal (HPG) axis, and findings from female global AR knockout mice confirm that AR-mediated androgen actions play important roles in regulating female reproductive function. We generated neuron-specific AR knockout mice (NeurARKO) to investigate the functional role of neuronal AR-mediated androgen action in regulating the female HPG axis and fertility. Relative to control females, NeurARKO females exhibited elevated luteinizing hormone (LH) levels at diestrus (p < 0.05) and a compromised serum LH response to ovariectomy and E2 priming (p < 0.01). Furthermore, NeurARKO females displayed reduced Kiss1 mRNA expression in the anteroventral periventricular nucleus at diestrus (p < 0.05) and proestrus (p < 0.05), but elevated Kiss1 (p < 0.05) and neurokinin B (Tac2, p < 0.05) mRNA expression in the arcuate nucleus at proestrus compared to WT controls. Ovarian follicle dynamics were also altered in NeurARKO ovaries at 3 months of age, with a significant reduction in large antral follicle numbers at the proestrus stage compared to control WT ovaries (p < 0.05). Increased follicular atresia was evident in NeurARKO ovaries with a 4-fold increase in unhealthy large preantral follicles (p < 0.01). Despite the findings of aberrant neuroendocrine and ovarian characteristics in the NeurARKO females, estrous cyclicity and overall fertility were comparable between NeurARKO and WT females. In conclusion, our findings revealed that selective loss of neuronal AR actions impacts the kisspeptin/GnRH/LH cascade leading to compromised ovarian follicle dynamics.

Polycystic ovary syndrome (PCOS) is a complex disorder characterized by endocrine, reproductive, and metabolic abnormalities. Despite PCOS being the most common endocrinopathy affecting women of reproductive age, the etiology of PCOS is poorly understood, so there is no cure and symptomatic treatment is suboptimal. Hyperandrogenism is the most consistent feature observed in PCOS patients, and recently aberrant neuroendocrine signaling and adipose tissue function have been proposed as playing a role in the development of PCOS. To investigate the role of adipose tissue and the brain as key sites for androgen receptor (AR)-mediated development of PCOS, we combined a white and brown adipose and brain-specific AR knockout (AdBARKO) mouse model with a dihydrotestosterone (DHT)-induced mouse model of PCOS. As expected, in wildtype (WT) control females, DHT exposure induced the reproductive PCOS traits of cycle irregularity, ovulatory dysfunction, and reduced follicle health, whereas in AdBARKO females, DHT did not produce the reproductive features of PCOS. The metabolic PCOS characteristics of increased adiposity, adipocyte hypertrophy, and hepatic steatosis induced by DHT in WT females were not evident in DHT-treated AdBARKO females, which displayed normal white adipose tissue weight and no adipocyte hypertrophy or liver steatosis. Dihydrotestosterone treatment induced increased fasting glucose levels in both WT and AdBARKO females. These findings demonstrate that adipose tissue and the brain are key loci of androgen-mediated actions involved in the developmental origins of PCOS. These data support targeting adipocyte and neuroendocrine AR-driven pathways in the future development of novel therapeutic strategies for PCOS.

Polycystic ovary syndrome (PCOS) is a common heterogeneous disorder, however the etiology and pathogenesis of PCOS are poorly understood and current management is symptom based. Defining the pathogenesis of PCOS traits is important for developing early PCOS detection markers and new treatment strategies. Hyperandrogenism is a defining characteristic of PCOS and studies support a role for androgen driven actions in the development of PCOS. Therefore, we aimed to determine the temporal pattern of development of PCOS features in a well characterized dihydrotestosterone (DHT)-induced PCOS mouse model after 2, 4 and 8 weeks of DHT exposure. Following 2 weeks of treatment, DHT induced the key PCOS reproductive features of acyclicity, anovulation and multi-follicular ovaries as well as a decrease in large antral follicle health. DHT treated mice displayed the metabolic PCOS characteristics of increased body weight and exhibited increased visceral adiposity after 8 weeks of DHT treatment. DHT treatment also led to an increase in circulating cholesterol after 2 weeks exposure and had an overall effect on fasting glucose levels, but not triglycerides, aspartate transaminase (AST) and alanine transaminase (ALT) levels or hepatic steatosis. These data reveal that in this experimental PCOS mouse model, acyclicity, anovulation and increased body weight are early features of a developing PCOS phenotype whereas adiposity, impaired glucose tolerance, dyslipidemia and hepatic steatosis are later developing features of PCOS. These findings provide insights into the likely sequence of PCOS trait development and support the addition of body weight criteria to the early diagnosis of PCOS.