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.
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