Environmental influence on gametes and embryos

哺乳动物受精的一个普遍特征是，受精的精子会在卵子中引发一系列重复的钙振荡，这些振荡持续几个小时，最终形成原核。小鼠的这种钙振荡模式对于受精后卵子激活的早期事件和足月宫内发育都是必不可少的。精子内导致这些钙振荡的因素是一种睾丸特异性磷脂酶C，PLC Zeta，它在精子-卵子质膜融合后从精子头部释放出来，迄今在所有研究的哺乳动物精子中都发现了这种磷脂酶C，包括人类。我们正在研究PLC Zeta在完成临床体外受精程序时获得的人类精子样本中的功能。到目前为止，我们已经招募了大约100对不育夫妇参与研究，并从50多个手术中获得了精子样本，这是我们最终样本数量目标的三分之一。我们推测，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这对不育夫妇的一系列失败，包括受精失败、植入前胚胎发育不良，甚至临床妊娠后流产。 钙的振荡也受鸡蛋内部因素的控制。目前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。我们正在产生一种有条件的小鼠对磷脂酶Cβ1基因的敲除。这个基因将从小鼠的卵子中从基因上删除，以确定鸡蛋蛋白是否需要产生功能性的钙振荡。我们刚刚完成了一系列研究，证明从受精卵周围的环境中进入钙是成功激活卵子的关键。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控模式，我们可以了解环境因素和疾病状态是如何改变早期胚胎发育的。 染色质重塑是原始卵母细胞在大的有腔卵泡内发育为完全成熟卵母细胞的关键过程，受精后有能力发育成新的个体。转移相关蛋白3(MTA3)是染色质重塑蛋白复合体的组成部分，在卵泡细胞和发育中的卵母细胞中高表达。我们利用慢病毒介导的RNA干扰技术研究了MTA3在卵泡生长中的作用，发现MTA3对支持卵母细胞生长发育的颗粒细胞的增殖是必不可少的。我们还培育了在发育中的卵母细胞中缺乏MTA3的小鼠，以确定这种蛋白质是否在合格卵母细胞的发育中起到重要作用。这些研究将揭示基本的表观遗传过程，这些过程可能会被环境化学品的暴露破坏，并可能影响人类的生育能力。

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.