Environmental influence on gametes and embryos

哺乳动物受精的一个普遍特征是，精子在卵中引起一系列重复的钙振荡，持续数小时，并以原核形成终止。小鼠中的这种钙振荡模式对于响应于受精的卵激活的早期事件和发生的足月子宫内发育都是必不可少的。 精子内负责诱导这些钙振荡的因子是睾丸特异性磷脂酶C，PLC zeta，其在精卵质膜融合后从精子头部释放，并且在迄今为止研究的所有哺乳动物精子中发现，包括人类。我们正在研究PLC zeta功能在人类精子样本中获得完成临床体外受精程序。到目前为止，我们已经招募了大约100对不孕夫妇参加这项研究，并从50多个程序中获得了精子样本，这是我们最终样本数量目标的1/3。我们假设缺乏足够的PLC zeta活性来诱导卵子激活所需的适当钙振荡可以解释不孕夫妇的失败，包括受精失败，植入前胚胎发育不良，甚至临床妊娠后流产。 钙的波动也受鸡蛋内因素的控制。 正在使用小鼠模型进行其他研究，以检查卵子内负责控制钙振荡行为和钙再吸收的分子，这些过程对于受精时钙振荡的持续至关重要。 我们正在产生磷脂酶C β 1基因的有条件敲除小鼠的过程中。该基因将从小鼠卵中遗传删除，以确定卵蛋白是否是产生功能性钙振荡所必需的。我们刚刚完成了一系列研究，证明受精卵周围环境中的钙进入对成功激活卵子至关重要。我们预计，通过更好地了解卵子激活过程中钙振荡行为的分子和细胞调节模式，我们可以了解早期胚胎发育如何被环境因素和疾病状态改变。 染色质重塑是原始卵母细胞在大腔卵泡内发育为完全生长的卵母细胞的关键过程，其能够在受精后发育成新的个体。转移相关蛋白3（MTA 3）是一种染色质重塑蛋白复合物的组成部分，在卵泡细胞和生长中的卵母细胞中高度表达。 我们使用慢病毒介导的RNA干扰技术来研究MTA 3在卵泡生长中的作用，发现它对支持卵母细胞生长和发育的颗粒细胞的增殖至关重要。 我们也正在培育在卵母细胞生长中缺乏MTA 3的小鼠，以确定这种蛋白质是否在有能力的卵母细胞的发育中起重要作用。 这些研究将揭示基本的表观遗传过程，这些过程可能会因暴露于环境化学品而中断，并可能影响人类生育能力。

A universal feature of fertilization in mammals is that the fertilizing sperm evokes a series of repetitive calcium oscillations in the egg that persist for several hours and terminate with pronucleus formation. This pattern of calcium oscillations in mice is essential for both early events of egg activation in response to fertilization and for full term intrauterine development to occur. The factor within sperm responsible for inducing these calcium oscillations is a testis-specific phospholipase C, PLC zeta, which is released from the sperm head after sperm-egg plasma membrane fusion, and is found in all mammalian sperm studied to date, including human. We are studying PLC zeta function in human sperm samples obtained at completion of clinical in vitro fertilization procedures. To date we have enrolled about 100 infertile couples in the study and obtained sperm samples from over 50 procedures, 1/3 of our final sample number goal. We hypothesize that a lack of sufficient PLC zeta activity to induce appropriate calcium oscillations required for egg activation could explain a spectrum of failure for the infertile couple, including failed fertilization, poor preimplantation embryo development, and even clinical pregnancy followed by miscarriage. Calcium oscillations are also controlled by factors within the egg. Additional studies are being performed using the mouse model to examine molecules within the egg that are responsible for controlling calcium oscillation behavior and calcium reuptake, processes that are essential for the continuation of calcium oscillations at fertilization. We are in the process of generating a conditional mouse knockout of the phospholipase C beta 1 gene. This gene will be genetically deleted from mouse eggs to determine if the egg protein is needed for production of functional calcium oscillations. We have just completed a set of studies documenting that calcium entry from the environment surrounding the fertilized egg is critical for successful egg activation. We anticipate that by achieving a better understanding of the molecular and cellular modes of regulation of calcium oscillatory behavior during egg activation, we can learn how early embryo development is altered by environmental factors and by disease states. Chromatin remodeling is a critical process during growth of a primordial oocyte to a fully-grown oocyte within a large antral follicle that it is competent to develop into a new individual after fertilization. Metastasis associated protein 3 (MTA3), a component of a chromatin remodeling protein complex, is very highly expressed in ovarian follicle cells and in growing oocytes. We used lentiviral-mediated RNA interference techniques to study the role of MTA3 in follicle growth and found that it is essential for proliferation of granulosa cells that support oocyte growth and development. We are also generating mice that lack MTA3 in growing oocytes to determine if this protein has an essential role in the development of a competent oocyte. These studies will shed light on basic epigenetic processes that can be disrupted by exposure to environmental chemicals and could impact on human fertility.