Functional disparity between human PAWP and PLCζ in the generation of Ca²⁺oscillations for oocyte activation

Abstract

Mammalian oocyte activation is mediated by cytosolic calcium (Ca(2+)) oscillations initiated upon delivery of a putative 'sperm factor' by the fertilizing sperm. Previous studies suggest the identity of this sperm factor as the testis-specific phospholipase C-zeta (PLCζ). Recently, a post-acrosomal sheath WW domain-binding protein (PAWP) has been proposed as an alternative sperm factor candidate, following a report that human PAWP protein and cRNA elicited Ca(2+) oscillations in mouse and human oocytes. Those Ca(2+) oscillations were inhibited by a PAWP-derived peptide corresponding to a functional PPGY binding motif. Herein, using a series of human PAWP expression constructs, we demonstrate that both human PAWP protein and cRNA are, in our experiments, unable to elicit Ca(2+) release following microinjection into mouse oocytes. Parallel experiments performed with human PLCζ elicited the characteristic Ca(2+) oscillations present at mammalian fertilization, which produced oocyte activation and embryo development. Furthermore, sperm-induced Ca(2+) oscillations were not inhibited by the PAWP-derived PPGY peptide following in vitro fertilization or intracytoplasmic sperm injection. Thus, the functional disparity with PLCζ leads us to conclude that human PAWP is neither sufficient nor necessary for the Ca(2+) oscillations that initiate mammalian oocyte activation at fertilization.

Expression profile ofPLCζ,PAWP,andTR-KITin association with fertilization potential, embryo development, and pregnancy outcomes in globozoospermic candidates for intra-cytoplasmic sperm injection and artificial oocyte activation

“…Among these factors, PLCf has emerged as the strongest candidate and several studies have shown the correlation between absence or deficiency of PLCf with failed fertilization in globozoospermic men (Heytens et al, 2009;and Aghajanpour et al, 2011). However, a growing body of evidence has challenged the importance of PLCf as the key SOAF and proposed other candidate proteins, particularly PAWP and TR-KIT, as the major SOAFs (Muciaccia et al, 2010;Aarabi et al, 2014b;Amdani et al, 2015;Nomikos et al, 2015).…”

Molecular triggers of egg activation at fertilization in mammals

“…The expression of PAWP protein from injected RNA was validated in these experiment with tagged or untagged RNA, and the levels that were similar or greater than those levels reported to be found in sperm (Wu et al 2007). In addition, injecting the same PY-containing peptide (used by Aarabi et al) into eggs did not result in any inhibition of Ca 2+ i oscillations induced by IVF or ICSI (Nomikos et al 2015a). Thirdly, it has now been shown that PAWP null male mice are fertile and produce sperm that can trigger a normal pattern of Ca 2+ i oscillations and embryonic development after ICSI (Satouh et al 2015).…”

“…SOAF activity is present in the perinuclear extracts of the mouse sperm and such extracts were shown to contain several different proteins as well as PLCζ, but these extracts did not contain PAWP (Fujimoto et al 2004). Secondly, in marked contrast to PLCζ, the data showing PAWP's ability to cause Ca 2+ i increases have not been reproducible (Nomikos et al 2014(Nomikos et al , 2015a. Independent studies injecting mouse eggs with human or mouse PAWP protein or cRNA failed to detect any Ca 2+ i transients (Nomikos et al 2014(Nomikos et al , 2015a).…”

“…Secondly, in marked contrast to PLCζ, the data showing PAWP's ability to cause Ca 2+ i increases have not been reproducible (Nomikos et al 2014(Nomikos et al , 2015a. Independent studies injecting mouse eggs with human or mouse PAWP protein or cRNA failed to detect any Ca 2+ i transients (Nomikos et al 2014(Nomikos et al , 2015a). The expression of PAWP protein from injected RNA was validated in these experiment with tagged or untagged RNA, and the levels that were similar or greater than those levels reported to be found in sperm (Wu et al 2007).…”

Ultrastructure of Spermatozoa from Infertility Patients

“…The oocyte-activating factors are transmitted from the sperm PT into the oocyte cytoplasm after the incorporation and rapid dissolution of the PT. In the normal fertilization cycle, the PT dissolves in the oocyte cytoplasm simultaneously with decondensation of the sperm nucleus and initiates division of the maternal pronucleus by hydrolyzing a membrane-bound phospholipid substrate, triggering cytoplasmic Ca 2+ oscillations [42]. In the case of ICSI, activation occurs only in the oocytes that contain a partly or completely dissolved PT.…”