Successful embryonic development is dependent on the female gamete progressing correctly through meiosis. Assembly and positioning of the meiotic spindle is a crucial part of this process, with gene knockouts that impair these processes causing female infertility. Oocyte spindle organization and positioning is orchestrated by actin, involving actin-associated proteins in a cytoplasmic meshwork and in the oocyte cortex. Our research on actin-associated proteins in oocytes has identified nexilin as involved in these events, with data presented here showing that RNAi-mediated knockdown of nexilin results in meiotic arrest and aberrant organization of oocyte actin. We also have evidence that loss of nexilin affects the actin regulatory pathway involving the LIM-domain containing kinase (LIMK) and its substrate, the actin-depolymerization factor cofilin. The LIMK-cofilin pathway affects the depolymerization of F-actin filaments to monomeric G-actin, and thus this is a promising mechanism by which nexilin could impact actin-dependent processes. Nexilin is of broader relevance as well, due to its role in dilated and hypertropic cardiomyopathies (DCM and HCM, respectively). Thus, the impact of the research proposed here is wide-ranging, with relevance to reproduction, oocyte biology, muscle function, and cardiomyopathies. With onset of DCM typically being in one's 40s-60s, we hypothesize that a function-disrupting mutation in the NEXN gene could be a cause of female infertility during reproductive years, and then of heart disease later in life. Given that little is known about nexilin, our overall goal is to elucidate the functions of nexilin, its connection to the LIMK-cofilin pathway, and how nexilin dysfunction contributes to abnormalities in mammalian oocytes. We will achieve these goals with following Specific Aims.
In Aim 1, we will build on our data from RNAi-mediated knockdown nexilin in oocytes, and develop an oocyte-specific nexilin conditional knockout (cKO) model, to analyze the effects of loss of nexilin activity in oocytes, in vivo and in vitro.
Aim 2 will use state-of-the-art studies in cellular mechanics, live-cell imaging, and quantitative analyses to elucidate the mechanisms underlying the defects in spindle organization and translocation associated with nexilin deficiency.
This aim will test the hypotheses that aberrant spindle positioning associated with deficiencies in nexilin or the LIMK-cofilin pathway are attributed to (a) aberrant tension for cortical anchoring for spindle pulling to the oocyte periphery, or (b) defects in actin-based movement of the spindle in the oocyte cytoplasm.
Aim 3 will investigate how mutated forms of nexilin affect oocytes, eggs, and early embryos. This work will be an invaluable assessment of the severity of different disease-associated forms, and also provide answers to the question of if a woman has one of these NEXN mutations, what would the effects be on her oocytes? Overall, this project will shed light on a poorly understood but significant health-relevant protein by elucidating nexilin functions in oocytes and in general. In turn, this work will translate to understanding nexilin functions in cardiomyocytes and other cell types.
AND PUBLIC HEALTH RELEVANCE Successful embryo development is dependent on the egg progressing correctly through meiosis, and defects in these processes compromise the egg's ability to form a healthy embryo, and in turn are associated with female infertility or subfertility. The research proposed here will investigate the involvement of a novel molecular player in egg biology that also contributes to specific types of heart disease. This work will obtain answers to the question if a woman has a mutation in this heart disease-associated gene, what would be the effects on her eggs and her fertility?