Pathophysiology of The Hypothalamic-pituitary-adrenal & Gonadal Axes

我们已经证明了几种人类状态的特征是中枢应激系统的过度活动或活动不足，这不仅解释了情绪变化，也解释了患有此类疾病的患者发展、代谢、心血管或自身免疫并发症的倾向。中枢神经系统的这些变化还通过影响生殖系统的各个组成部分来影响生育能力。 我们先前已经报道，在中枢神经系统和外周产生昼夜节律的自振荡转录因子CLOCK/BMAL1节律性地抑制GR诱导的转录活性，表明CLOCK/BMAL1在靶组织中作为糖皮质激素作用的反向负调节因子发挥作用，可能是通过拮抗昼夜波动的循环糖皮质激素的生物作用。我们进行了一项人体研究，发现时钟对GR转录活动的这种负面调节在人类中也是有效的。作为这个昼夜节律项目的延伸，我们筛选了小鼠卵巢颗粒细胞中以昼夜节律方式调节的microRNA。MicroRNAs是一种短发夹状RNA，通过影响颗粒细胞的增殖和凋亡以及类固醇的合成，对生殖，特别是颗粒细胞表现出强大的生物学作用。另一方面，颗粒细胞是卵泡正常发育和类固醇激素产生所必需的成分。我们发现，从小鼠卵巢获得的原代颗粒细胞表现出几个时钟相关基因的昼夜振荡，如PER1/2和Cry1/2。我们发现，在我们筛选的600多个已知microRNA中，有10%的表达是昼夜节律的。我们进一步关注miR-196A，因为它显示了最显著的昼夜振荡。我们发现其靶基因HOX8B通过miR-196A呈现出昼夜表达，HOX8B是一种转录抑制因子，在细胞生长和凋亡调控中起重要作用。我们现在正在检验我们的发现在颗粒细胞生物学行为中的重要性。 衰老是降低妊娠成功率、最终发展为卵巢衰竭和绝经的重要因素，但这一生理过程背后的生物学机制尚未完全阐明。病理性不孕不育，如营养不良、压力和运动，也有可能与衰老相关性卵巢功能衰竭的部分机制相同。为了检测衰老对卵巢功能的影响，我们再次检测了从小鼠卵巢获得的初级颗粒细胞中microRNA的表达。我们获得了2岁小鼠的颗粒细胞，并以6-9周龄小鼠的细胞为对照，检测了600个microRNAs的表达。在这次筛选中，我们发现37个microRNAs在衰老细胞中受到显著调控。我们将重点放在miR-503和-322上，因为这些microRNAs在衰老的颗粒细胞中表现出最显著的减少(超过20倍)，并在X染色体上形成一个受共同启动子调控的基因复合体。在随后的蛋白质表达谱分析中，我们发现线粒体呼吸链中的ATP合成酶的几个亚基被这些microRNAs抑制而下调，这表明miR-503和-322是线粒体活动所必需的。最近的研究表明，这些miRNAs下调了Nix/Bnip3L、Ulk1、Gabarapl2、Sh3glb1、Atg12、Becn1和Bcl2l1等线粒体吞噬转录本的表达，从而通过抑制线粒体吞噬诱导基因的表达来维持线粒体的生物发生和能量产生。由于线粒体减少是老年卵巢生殖活性降低的主要特征，miR-503和-322很可能是老年卵巢线粒体功能障碍的介导者，进而可能是通过调节线粒体的生物发生/活性而增加老年不孕症发生率的潜在因素。 促性腺激素调节cAMP信号系统与类固醇激素受体，如孕激素(PR)、雌激素(ER)和糖皮质激素受体(GR)之间的信号串扰，对卵巢功能的调节是重要的。为了阐明它们在转录调控方面的相互作用，我们重点研究了CREB调节的转录辅助激活因子2(CRTC2)，这是一种已知的CREB(即cAMP信号)介导的转录调控的特异性辅助因子，并测试了它对PR或GR诱导的转录活性的影响。我们发现CRTC2作为GR和PR的共同调节因子，刺激前者的转录活性，而抑制后者的活性。我们进一步发现，在体内外，CRTC2与PR和GR的配体结合域通过其含有反式激活结构域的C末端部分发生物理作用。我们现在正在研究颗粒细胞中这种串扰的生物学重要性。

We have demonstrated that several human states are characterized by hyperactivity or hypoactivity of the central stress system, which explains not only mood changes but also the propensity of patients with such disorders to develop developmental, metabolic, cardiovascular or autoimmune complications. These changes in the central nervous system also influences fertility by affecting various components of the reproductive system. We have previously reported that CLOCK/BMAL1, the self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery, rhythmically repressed GR-induced transcriptional activity, indicating that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing the biologic actions of diurnally fluctuating circulating glucocorticoids. We performed one human study and revealed that this negative regulation on GR transcriptional activity by CLOCK was also functional in humans. As an extension of this circadian rhythm project, we screened microRNAs regulated in a circadian fashion in granulosa cells of mouse ovaries. MicroRNAs are short hairpin-like RNAs that demonstrate strong biological actions on reproduction, and specifically, granulosa cells by influencing proliferation and apoptosis, as well as steroidogenesis of these cells. Granulosa cells, on the other hand, are components of ovarian follicles required for their proper development and steroid hormone production. We have found that primary granulosa cells obtained from mouse ovaries showed circadian oscillation of several CLOCK-related genes, such as Per1/2 and Cry1/2. We found that expression of 10% of over 600 known microRNA we screened were in circadian rhythms. We further focused on miR-196a, as it showed the most significant circadian oscillation. We found that its target gene HOX8B, a transcriptional repressor important for the regulation of cell growth and apoptosis, demonstrated diurnal expression through miR-196a. We are now examining importance of our finding in the biologic action of granulosa cells. Aging is an important factor for reducing the chance of successful pregnancy, eventually developing ovarian failure and menopause, but the biological mechanisms underlying this physiologic process have not completely been elucidated as yet. It is also possible that pathologic infertility, such as by malnutrition, stress and exercise, might share part of the mechanisms responsible for aging-dependent ovarian failure. To examine impact of aging on ovarian functions, we again examined microRNA expression in primary granulosa cells obtained from mouse ovaries. We obtained granulosa cells from 2-year old mice and tested expression of 600 microRNAs by employing the cells of young mice (6-9 week old) as controls. In this screening, we found that 37 microRNAs were significantly regulated in aged cells. We focused on miR-503 and -322, as these microRNAs showed the most significant reduction (over 20-fold) in aged granulosa cells and form a gene complex on chromosome X regulated by the shared promoter. We found in the subsequent protein expression profile analysis that several subunits of the ATP synthase, a component of the mitochondrial respiratory chain, were down-regulated by inhibition of these microRNAs, suggesting that miR-503 and -322 are required for mitochondrial activity. Recent publication indicated that these miRNAs down-regulate expression of mitophagy transcripts, such as Nix/Bnip3L, Ulk1, Gabarapl2, Sh3glb1, Atg12, Becn1, and Bcl2l1, thus these microRNAs maintain mitochondrial biogenesis and energy production by suppressing expression of mitophagy-inducing genes. Since reduction of mitochondria is a key feature of aged ovaries with reduced reproductive activity, it is likely that miR-503 and -322 are mediators of mitochondrial dysfunction observed in aged ovaries, and further, underlying factors for increased incidence of infertility in older subjects through modulation of mitochondrial biogenesis/activity. Crosstalk between the gonadotropin-regulating cAMP signaling system and that of the steroid hormone receptors, such as progesterone (PR), estrogen (ER) and glucocorticoid receptor (GR), are important for the regulation of ovarian functions. To elucidate their crosstalk at the transcriptional regulation, we focused on the CREB-regulated transcriptional coactivator 2 (CRTC2), a cofactor known to be specific for CREB (thus cAMP signaling)-mediated transcriptional regulation, and tested its effect on PR- or GR-induced transcriptional activity. We found that CRTC2 functions as a coregulator of GR and PR, stimulating former transcriptional activity, while repressing latter activity. We further found that CRTC2 physically interacts with the ligand-binding domain of PR and GR through its C-terminal portion harboring the transactivation domain in vitro and in vivo. We are now examining biologic importance of this crosstalk in granulosa cells.