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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Animal Biotechnology & Biomedical Sciences

Year Degree Awarded

2016

Month Degree Awarded

February

First Advisor

Rafael A. Fissore

Second Advisor

Dominic L. Poccia

Third Advisor

Pablo E. Visconti

Subject Categories

Cell Biology | Developmental Biology

Abstract

Calcium channels at the plasma membrane have been suggested to mediate Ca2+ influx during egg activation. The transient receptor potential (TRP) Ca2+ channel, TRPV3, is differentially expressed in oocytes during maturation, being fully active at MII stage. Specific stimulation of TRPV3 channels in mouse eggs promotes Ca2+ influx sufficient to induce egg activation and parthenogenesis. In chapter 2, we explore the function and distribution dynamics of the TRPV3 channel protein during oocyte maturation. Using dsRNA, TrpV3 overexpression, and inhibitors of protein synthesis, we modified the native expression of the channel and showed that the TRPV3 protein is synthesized and translocated to the plasma membrane during maturation. We demonstrated that 2-APB at the concentration used to promote Ca2+ influx in eggs, specifically and reversibly targets TRPV3 channels, and does not block the IP3 receptor. Finally, we showed that TRPV3 channels functionally interact with the actin cytoskeleton indicating an actin-based regulation of its expression on the plasma membrane. Our results indicated that TRPV3 is a target of 2-APB in eggs, a condition that can be used to induce parthenogenesis. The association of endogenous TRPV3 channels with the actin cytoskeleton is a novel finding, and suggests that the rearrangements of actin that occur during maturation could regulate both plasma membrane presence and function of TRPV3 Ca2+ channels involved in oocyte maturation and fertilization. We show that TRPV3 is required for strontium influx because TRPV3-KO eggs failed to conduct Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation.

In chapter 3, we conducted studies to determine if expression of the testis-specific PLCZ1 correlated with low success or fertilization failure after ICSI in patients with normal parameters after standard semen analysis (SA). The presence of PLCZ1 in sperm was ascertained using western blotting and immunofluorescence (IF) analysis. The ability of sperm to initiate changes in the intracellular concentrations of [Ca2+]i was also examined. Male partners of couples with failed or low success ICSI fertilization but with normal SA parameters showed low expression levels of PLCZ1 as determined by western blotting and reduced fluorescent signal during IF studies. In addition, fewer of these males’ sperm showed PLCZ1 expression and were able to initiate robust [Ca2+]i oscillations upon injection into eggs. In the second part of this chapter, we examined two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intra cytoplasmic sperm injection (ICSI). Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, converting Ile 489 into Phe (Ile489Phe). We showed that the mutation is deleterious, leading to absence of the protein in sperm. Injection of cRNA into GV and MII oocytes showed that the protein was mislocalized and produced highly abnormal Ca2+ transients and early embryonic arrest. Altogether these alterations are consistent with our patients’ sperm inability to induce oocyte activation and initiate embryo development. In contrast, no mutations were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans that the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes, opening the door for structure function studies to define the conserved amino acids on the b1 strand of the C2 domain that might regulate the selectivity of PLCZ1 for its lipid substrate(s).

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