Potential effects of assisted reproductive technology upon the abundance and localisation of two vital sperm proteins

Assisted reproductive technology (ART) uses advanced techniques such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) to combat human infertility. However, the success rate of ART is poor and can, at least in part, be attributed to detrimental (iatrogenic) damage incurred by gametes and embryos during laboratory treatment or manipulation, thus compromising their functional role and reducing the chances of fertilisation. The sperm plays two fundamental roles upon gamete fusion: (1) to deliver paternal genomic DNA of optimal integrity into the oocyte, and (2) to activate the oocyte to initiate embryogenesis. Protamine and phospholipase C zeta (PLCζ) are two critical sperm proteins fundamentally responsible for facilitating these two key roles, respectively. The essential role of these sperm proteins with regards to male fertility, and fertilisation outcome following ART treatment, has been widely reported. This thesis was predominantly designed to investigate the potential effects of cryopreservation and sperm immobilisation via polyvinylpyrrolidone (PVP) upon the abundance and localisation of protamine and PLCζ in mouse and human sperm, respectively. Deficiency of these proteins could lead to reduced sperm DNA integrity and oocyte activation ability, respectively. An immunofluorescent quantitative assay was first designed and optimised for the determination of protamine 1 (P1) and 2 (P2) levels in sperm. This assay demonstrated that the total levels of P1 and P2, but not the P1:P2 ratio, were significantly reduced (by approximately 50%) in mouse sperm following cryopreservation. This novel assay may represent a useful clinical tool to predict DNA integrity and help select sperm with the best quality DNA. Clinical screening of PLCζ was also carried out in the largest dataset reported to date and confirmed that total levels of PLCζ in human sperm varied significantly between samples (P ≤ 0.05). Cluster analysis led to the development of a PLCζ scoring system with significant potential as a clinical prognostic and diagnostic assay. Regression models also correlated fertilisation rate and PLCζ content in a total of 30 clinical samples. Collectively, these novel tools show significant promise as predictors of oocyte activation ability. Specific case studies involving vasectomy, oocyte activation deficiency (OAD), and globozoospermia, were identified and shown to be associated with significantly reduced levels of PLCζ (P ≤ 0.05). In two of these case studies, a single nucleotide polymorphism (SNP) was identified in the PLCζ promoter region, potentially indicating a novel mechanism for PLCζ expression in human sperm. Another case of OAD suggested the apparent deficiency of a crucial interacting factor in the oocyte, emphasising that OAD is not exclusively linked to sperm abnormalities. For the first time, efforts were made to assess whether PLCζ expression was linked to male age; total levels and the proportion of sperm exhibiting PLCζ were found not to differ significantly amongst a total of 46 males. Furthermore, in a pilot experiment, levels of PLCζ were significantly reduced (by 23% to 89%) in PVP-treated sperm from 9 controls and 3 infertile patients, with patient sperm showing higher susceptibility to the effects of PVP compared to controls. However, a more robust experiment featuring sperm from 16 fertile donors, failed to show any significant effect of PVP upon PLCζ. Collectively, data arising from this thesis generated a series of potential clinical tools to quantify protamine and PLCζ in sperm, provides strong evidence that levels of protamine are significantly reduced by cryopreservation, and has provided at least some evidence that PVP may cause detrimental effect upon the level of PLCζ in human sperm. Further work on the effects of vasectomy and the relative functional importance of the SNP detected in the PLCζ promoter are highly warranted. Further investigation and clinical translation of these findings may help to improve the success rate of ART.

Keywords: sperm biology
Assisted reproductive technology
reproductive science
Advisor: Coward, K