Regulation of the functional expression of the TRPM7 chanzyme essential for Mg2+ homeostasis in mouse oocytes and cleaving embryos to improve assisted reproductive technologies and modulate fertility - There is a persistent gap in our knowledge about the channels that modulate the divalent cations essential for oocyte maturation, fertilization, and embryo development in mammals, including humans. The transient receptor potential melastatin 7, TRPM7, a plasma membrane (PM) chanzyme with a channel and a kinase domain, is expressed in gametes and embryos. TRPM7 permeates three vital divalent ions, Ca2+, Mg2+, and Zn2+, but Trpm7-null embryos fail to develop to the blastocyst (BL) stage because of Mg2+ deficiency. The long-term goal is to unravel the molecules and mechanisms controlling the homeostasis of divalent cations that underpin fertilization and embryo development and are crucial for human and animal fertility. The objective here is to elucidate the regulation and function of the main domains of the TRPM7 chanzyme in oocytes and embryos, focusing on its ion channel activity essential for development. Our central hypothesis is that the function of TRPM7 is modulated in a stage-specific manner by post-translational modifications, downregulation, and sub-cellular localization. We also hypothesize that the channel and kinase domains crosstalk. The effect of these changes and interactions, including one with TRPM6 at the BL stage, is to optimize embryo development. The rationale for our study is to uncover the regulation and role of TRPM7 in gametes and embryos. This knowledge will allow us to optimize the Mg2+ requirements, potentially improving fertility treatments and unveiling regulatory insights that may apply to diverse cellular systems. We will test our central hypothesis by pursuing the following specific aims: 1) Determine the function and significance of TRPM7 downregulation during oocyte maturation. We plan to use monoclonal antibodies, imaging, biochemical and genetic approaches, expression of mutant channel versions, inhibitors, and electrophysiology to elucidate the mechanisms underlying TRPM7 functional expression inactivation and the consequences of bypassing it. 2) Identify the mechanisms that regulate TRPM7’s channel function in preimplantation embryos. We will use electrophysiology of zygotes and blastomeres, inhibitors, IF, and expression of WT and mutant versions of TRPM7 to determine if its clustered organization enhances the channel function and is regulated by the actomyosin cytoskeleton. We will also investigate if the role of TRPM7’s and Mg2+ homeostasis on BL hatching and growth is due to the progressive association of TRPM7 with TRPM6. The research in this application is innovative because it will uncover unknown TRPM7 regulatory mechanisms that only became evident after our studies employing Trpm7-gamete-specific null lines and a specific monoclonal antibody. The proposed project is significant because it is necessary to modulate divalent cation influx, especially Mg2+, which is essential for egg activation, initiation of development, blastomere proliferation, and differentiation required for implantation and pregnancy.
