Nexilin: An Actin-Binding Protein as a Potential Common Molecular Mediator Between Cardiomyocyte and Oocyte Health

Shams M. Saad, Janice P. Evans

Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA

Abstract Text:

The progression of female gametes through meiosis is crucial for producing competent eggs capable of supporting fertilization and normal embryonic development. The actin cytoskeleton and actin-binding proteins (ABPs) within oocytes play a vital role in facilitating the highly asymmetric cell division necessary for this process. Nexilin is a novel F-actin-binding protein, and our lab is the first to demonstrate that nexilin has a role in oocyte meiotic maturation. Nexilin is best known for its role in cardiac muscle, with evidence for a role in stabilizing Z-disks within sarcomeres, and in initiation and formation of transverse (T)-tubules in junctional membrane complexes (JMCs) in cardiomyocytes. Mutations in the NEXN gene are associated with various cardiovascular disorders (CVDs), particularly dilated cardiomyopathy (DCM), a condition in which the heart becomes enlarged and weakened, impairing its ability to pump blood effectively. Interestingly, a transcriptome database also revealed that Nexn transcripts are highly enriched in mouse oocytes and fertilized eggs. We assessed nexilin protein expression in oocytes at different meiotic stages, finding consistent expression throughout meiosis. We also examined the localization of nexilin at different meiotic stages, showing that nexilin is localized in the cytoplasm and cortex from prophase I to metaphase II. Additionally, nexilin is found in the actin-rich cap overlying the meiotic spindle of metaphase II eggs, with some of these eggs also displaying meiotic spindle localization. Using siRNA-mediated knockdown, we demonstrated that nexilin-deficient oocytes become arrested in meiosis I with disrupted actin organization and spindle localization. To further investigate the effects of nexilin depletion in oocytes over a shorter time frame, we will utilize a post-translational reverse genetics protein depletion method called Trim-Away. Currently, we are validating the specificity of two anti-nexilin antibodies for Trim-Away, through immunoblotting of proteins separated by SDS and native gel electrophoresis, as well as immunofluorescence. Nexilin is known to have two main splice variants, B-nexilin and S-nexilin, which differ by 64 amino acids and the presence of one actin-binding domain. We examined the expression of these in oocytes using reverse transcription (RT)-PCR and immunoblotting. These analyses revealed that S-nexilin is the predominant form in oocytes, while B-nexilin is barely detectable in oocytes, but is the predominant form in heart. To test the hypothesis that expression of B-nexilin in oocytes could have effects on meiotic maturation, we evaluated in vitro maturation of prophase I oocytes exogenously expressing B-nexilin or S-nexilin. The percentage of oocytes that matured to metaphase II did not differ significantly between the two groups or compared to the controls. Additionally, polar body sizes were assessed, and no significant differences were observed among the groups. We are extending our studies of nexilin by generating an oocyte-specific conditional nexilin knockout to assess nexilin’s role in oocytes and female fertility. This study is the first to investigate the role of nexilin, a novel actin-binding protein, in the progression of oocytes through meiosis, which could have implications for infertility. With growing evidence suggesting a link between fertility status and other health conditions such as CVDs, nexilin may act as a molecular mediator of female fertility and cardiovascular health. 

Supported by NIH grant R01HD091117 and the EMBRIO Institute under contract #2120200, funded by the National Science Foundation (NSF) Biology Integration Institute.