Artificial oocyte activation (AOA) and phospholipase C zeta (PLCζ) in assisted reproductive technology (ART)

Oocyte activation deficiency (OAD) is the main cause of total fertilisation failure/low fertilisation rate in assisted reproductive technology (ART). Phospholipase C zeta (PLCζ) is the dominant sperm-specific factor responsible for triggering oocyte activation in mammals. Abnormalities in PLCζ have been linked with OAD. Artificial oocyte activation (AOA) is currently the only option for OAD in clinic but lacks reliable diagnostic approach and may cause abnormal Ca2+ release in oocytes. Previous studies have attempted to synthesise recombinant human PLCζ (rhPLCζ) protein but struggled to purify the protein or maintain functionality and stability. This thesis aimed to address these shortfalls by investigating the mechanism(s) of OAD and improving the applicability of PLCζ as a diagnostic and therapeutic agent for male infertility. This thesis first compared the efficiency of two immunofluorescence methods to assess PLCζ expression in male sperm (n=26 males) and demonstrated that our in-house assay showed the best labelling results when compared with a recently-published antigen-unmasking (AUM) method. I further developed our in-house PLCζ assay into a clinical diagnostic assay, which targets on infertile candidates for the AOA treatment (i.e., Ca2+ ionophores). Four out of five couples received AOA and achieved healthy live-births (fertilisation rate from 18.6% to 56.8%, p<0.001). Cut-offs for the key PLCζ characteristics were also determined for the first time (mean PLCζ levels: 15.57 arbitrary units [a.u.], and the proportion of sperm exhibiting PLCζ: 71%). Globally, eleven mutations have been identified in the PLCζ gene; however, the XY-linker that connects the main X and Y domains has received for less research attention. In addition, CAPZA3 shares a bidirectional promoter with PLCζ and is responsible for sperm morphology and function. In this thesis, Sanger sequencing and next-generation sequencing (NGS) were used to screen PLCζ exons (n=61 males), and PLCζ promoter, XY-linker and CAPZA3 regions (n=33 males). Ten single polymorphisms (SNPs) in PLCζ introns and three mutations in CAPZA3 exon were identified, some of which may partially explain the causes for male infertility. In addition, this study attempted to synthesise a novel rhPLCζ with uses of a pHLsec vector feathered with secretion signal sequence and further examined the functionality of the protein. Surprisingly, simple incubation of the protein with mouse oocytes triggered activation (22/52 developed to the 2-cell stage, p<0.001), which not only suggests that the protein may be functional, but also indicates the possible existence of a novel plasma membrane-bound mechanism that may act alongside the sperm factor.