PLCζ disruption with complete fertilization failure in normozoospermia

Abstract

Purpose Intracytoplasmic sperm injection (ICSI) is widely used to achieve fertilization in the presence of severe male factor, resulting in high fertilization rates. Nevertheless, 1-3 % of couples experience complete fertilization failure after ICSI. When a male factor is identified, assisted oocyte activation (AOA) can help overcome fertilization failures. The objective of this study is to describe a case of repeated complete fertilization failures after ICSI with donor oocytes, and to investigate the molecular and functional aspects of phospholipase C zeta (PLCζ) protein in the patient semen. Methods The patient was a normozoospermic male who had previously fathered, through natural conception, four children by a different partner. Molecular and functional analysis of sperm-specific PLCζ in the patient and control samples by means of gene sequencing, immunocytochemistry, Western blot, mouse oocyte activation test (MOAT), and mouse oocyte calcium analysis (MOCA) were used.Results PLCζ expression levels and distribution were significantly disrupted, although MOAT and MOCA did not indicate a decrease in activation ability. Conclusions Normozoospermic males can have disrupted expression and distribution of PLCζ, and reduced activation ability after ICSI in human oocytes, despite their normal activation potential in functional testing using mouse oocytes. Discrepancy among molecular and functional data might exist, as mutations in the gene sequence may not be the only cause of alteration in PLCζ protein related to activation failures.

Ca2+ dynamics in oocytes from naturally-aged mice

“…The role of paternal age in ICSI failure is often hard to analyse in clinical studies 4 5 6 . Investigations into PLCζ protein levels with paternal age would therefore be extremely valuable, as multiple studies show that human sperm with little to no PLCζ expression fail to initiate egg activation 56 57 . Alternatively, zinc has recently emerged as an intriguing potential determinant of egg fertilisation success, sperm-egg fusion inducing a series of ‘zinc sparks’ that occur rapidly in response to Ca 2+ oscillations 58 .…”

PLCζ sequence, protein levels, and distribution in human sperm do not correlate with semen characteristics and fertilization rates after ICSI

“…Genomic DNA (gDNA) was isolated with QIAamp DNA Blood Mini Kit (QIAGEN, Germany) following the manufacturer's instructions. Amplicons of PLCζ were amplified by polymerase chain reaction (PCR) with Phusion HighFidelity DNA polymerase (NEB, USA) using previously described primer pairs [20,21,30].…”

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

“…Previous studies have shown that failed fertilization after ICSI in some cases of infertile men (i.e., globozoospermic, oligoasthenoteratozoospermic, normozoospermic patients) is related to reduced expression and abnormal localization of PLCζ protein (Azad et al, ; Chithiwala et al, ; Durban et al, ; Lee et al, ; Tavalaee, Nomikos, Lai, & Nasr‐Esfahani, ). Despite the key role of PLCζ in the fertilization process and its potential as biomarker for the ability of sperm to induce oocyte activation, the status of this protein in unexplained infertile (UI) and asthenoteratozoospermic (AT) men remains unclear.…”