Mammalian Fertilization: Identifying the second sperm factor that induces residual calcium oscillations and its contributions to egg activation - There is a persistent gap in our knowledge of the sperm molecules and mechanisms that induce Ca2+ oscillations in mammalian eggs. Ca2+ release triggers egg activation and initiation of embryo development in all mammals, including human beings. A sperm molecule, the sperm factor, SF, later identified as Phospholipase zeta, PLCz, was thought to be the only SF responsible for inducing Ca2+ release, and PLCz inactivating mutations cause infertility in human males. However, Plcz knockout mice are subfertile and evoke residual Ca2+ oscillations, suggesting the function of a backup mechanism. The component(s) and the physiological role of the backup system are gaps in our knowledge. These gaps represent serious impediments, as until we overcome them, we do not understand the Ca2+ regulation of egg activation events or can objectively diagnose, prevent or treat infertility associated with egg activation failure. The long-term goal is to understand the molecular basis of the Ca2+ oscillations that unfold the developmental program at fertilization. The objective is to identify the sperm molecules that trigger these Ca2+ oscillations in the egg. The mouse and its gametes are perfect models because they are amenable to manipulation and genetic modifications. The central hypothesis underpinning this proposal is that one or more sperm PLCs besides PLCz contribute to delivering the Ca2+ signal and could serve as the backup mechanism in case of Plcz disfunction. This hypothesis was conceived based on extensive preliminary data. The rationale for the proposed research is that once all sperm molecules that induce oscillations are known, their regulation assessed, and their diagnostic value and application to the reproductive management of species fully explored. We plan to test our central hypothesis by pursuing the following specific aims: 1) Identify the molecule(s) in PLC1z-KO sperm that induce Ca2+ oscillations at fertilization; 2) Determine the contribution of the backup system to the Ca2+ responses and egg activation events of typical fertilization. Under Aim 1, we will use Trim-21 mRNA, specific antibodies, CRISPR-generated KO mouse lines, immunofluorescence, Ca2+ monitoring, and embryo culture to determine if PLCd4 is the active factor of the backup system. Under Aim 2, we will employ similar methods but target different molecules and mechanisms to ascertain the contribution of the Ca2+ backup system to the Ca2+ signal of typical fertilization. The research in this application is innovative because it represents a fresh examination of the role of other sperm PLCs, including one whose deletion causes male infertility, in the oscillations of fertilization. The contributions of the proposed project are significant because we aim to perfect the treatment of egg activation failure, improve embryo development, discover indicators of male fertility, and identify novel targets of contraception. Finally, we anticipate progress in understanding the molecules and regulatory mechanisms that induce embryo development in mammals.
