应用卵巢损伤模型研究原始卵泡起始募集的分子调控机制

An ovarian cyst is a common discovery in women and affects up to 10% of women of reproductive age. Surgical management by laparoscopic cystectomy is often the rule. However, as shown by sharply decrease of AFC (antral follicle counts) and serum AMH (anti-Müllerian hormone) levels, the treatment can result in a nontrivial reduction in ovarian reserve. Until now, there are seldom reports about the effects of ovarian surgery on the pool of primordial follicles, the real ovarian reserve in the ovary. To study if ovarian surgery has any effect on the activation of primordial follicles, we set up a mouse surgical model with cutting 1/3 of one lateral ovary in mice. The other lateral ovary of the mice will keep untouched as control. Ovaries were collected shortly after the surgery to detect changes of MAPK, PI3K, mTOR signaling pathways. Markers of primordial follicle activation were also checked at the same time. Follicular development was then followed by follicle counts 3 weeks after the surgery. Our preliminary data has shown that ovarian injury can activate primordial follicles near the surgical site. Future works will focus on which signaling pathway function after ovarian injury by using specific inhibitors of the related pathways. RNA-seq will be also used to find key regulators after ovarian surgery. The results will demonstrate the mechanism on how the initial recruitment of primordial follicles be regulated by stromal signals. The study will also provide patients with ovarian cyst a better way to protect their ovarian reserve and fertility after ovarian surgery.

卵巢囊肿，包括子宫内膜异位症和畸胎瘤，是一类临床常发的妇科良性肿瘤，育龄女性发病率约为10%。临床上通常采用外科手术方法摘除囊肿，然而大量研究表明，卵巢手术比囊肿本身对卵巢储备的损害还要大。上述卵巢储备在临床上主要指卵巢有腔卵泡计数，并不是严格意义上由原始卵泡组成的卵巢储备，而关于卵巢手术对于原始卵泡的影响目前尚无报道。本研究以小鼠手术模型为基础，通过卵巢手术后短时间内信号通路的变化以及对于卵泡发育的长期影响，研究卵巢损伤是否激活原始卵泡；并通过相关信号通路抑制剂特异性阻断作用进行机制的探讨；最后通过RNA-seq筛选特定信号通路参与调节原始卵泡激活的关键分子，进而建立原始卵泡激活的卵泡外调节网络。本项目的意义在于通过卵巢手术模型，将动态的原始卵泡初始募集过程同步化，有利于研究原始卵泡激活的外源性调节机制；也能够为卵巢手术中保护卵巢储备提供新的理论依据并对临床具有一定的指导意义。

在哺乳动物的卵巢中，原始卵泡数目作为卵巢储备的评价标准，是所有发育卵泡的基础。防止育龄期女性原始卵泡的过度消耗，可以通过抑制部分原始卵泡的激活，使绝大多数原始卵泡仍处于休眠状态。卵巢疾病中相关的干预措施，例如卵巢囊肿切除术、卵巢楔形切除、激光治疗多囊卵巢综合征（PCOS）等，均已被报道会改变卵泡的发育状态，但其机制仍旧不明。.基于以上认知，我们建立了小鼠单侧卵巢部分切除的模型，手术后发现手术激活了卵巢中mTOR信号通路，从而加快了原始卵泡的激活和生长。此外，我们发现原始卵泡的激活呈现区域性，其中绝大多数出现在切口附近，切口附近磷酸化rps6的信号从间质过渡到原始卵泡包质，呈现动态变化。通过对手术侧和非手术侧卵巢总RNA和核糖体RNA的测序结果发现，神经营养因子家族中的神经生长因子（NGF）在手术侧卵巢中无论是总RNA还是核糖体RNA均较之非手术侧有明显的变化，同时免疫组化结果也显示，NGF在切口附近呈现局域性升高。体外培养实验中，重组NGF因子处理后的新生乳鼠卵巢，其原始卵泡激活比例会明显增加。小鼠腹腔注射NGF受体抑制剂k252a，发现该抑制剂可以有效抑制因手术导致的原始卵泡激活。在过去的研究中认为，排卵的过程是一个导致卵巢周期性出现微小损伤的过程，并且发现排卵后NGF的升高会参与原始卵泡的激活，因此我们猜测可能是排卵过程引起的损伤导致了卵巢局部NGF的表达升高，从而使得局部原始卵泡激活进入下一轮生长。