Molecular Cloning and Characterization of Phospholipase C Zeta in Equine Sperm and Testis Reveals Species-Specific Differences in Expression of Catalytically Active Protein1

Abstract

Oocyte activation at fertilization is brought about by the testis-specific phospholipase C zeta (PLCZ), owing to its ability to induce oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Whereas this is a highly conserved mechanism among mammals, important species-specific differences in PLCZ sequence, activity, and expression have been reported. Thus, the objectives of this research were to clone and characterize the intracellular Ca(2+)-releasing activity and expression of equine PLCZ in sperm and testis. Molecular cloning of equine PLCZ yielded a 1914-bp sequence that translated into a protein of the appropriate size (~73 kDa), as detected with an anti-PLCZ-specific antibody. Microinjection of 1 μg/μl of equine PLCZ cRNA supported [Ca(2+)](i) oscillations in murine oocytes that were of a higher relative frequency than those generated by an equivalent concentration of murine Plcz cRNA. Immunofluorescence revealed expression of PLCZ over the acrosome, equatorial segment, and head-midpiece junction; unexpectedly, PLCZ also localized to the principal piece of the flagellum in all epididymal, uncapacitated, and capacitated sperm. Immunostaining over the acrosome was abrogated after induction of acrosomal exocytosis. Moreover, injection of either sperm heads or tails into mouse oocytes showed that PLCZ in both fractions is catalytically active. Immunohistochemistry on equine testis revealed expression as early as the round spermatid stage, and injection of these cells supported [Ca(2+)](i) oscillations in oocytes. In summary, we report that equine PLCZ displays higher intrinsic intracellular Ca(2+)-releasing activity than murine PLCZ and that catalytically active protein is expressed in round spermatids as well as the sperm flagellum, emphasizing important species-specific differences. Moreover, some of these results may suggest potential novel roles for PLCZ in sperm physiology.

Human PLC exhibits superior fertilization potency over mouse PLC in triggering the Ca2+ oscillations required for mammalian oocyte activation

“…There are significant species-specific differences in the relative potency of PLCz from different species (Swann et al, 2006;Saunders et al, 2007;Cooney et al, 2010;Bedford-Guaus et al, 2011). In the present study, we have compared quantitatively and qualitatively the relative potencies of human and mouse PLCz to induce Ca 2+ oscillations in unfertilized mouse oocytes (Fig.…”

“…This makes PLCz the smallest known mammalian PLC with a molecular mass of 70 kDa in humans and 74 kDa in mice Saunders et al, 2002). Notably, there appear to be substantial differences in the relative potency of PLCz from different species (Swann et al, 2006;Saunders et al, 2007;Cooney et al, 2010;Bedford-Guaus et al, 2011). Previous reports have suggested that it takes nearly .10 times less human PLCz cRNA than mouse PLCz cRNA to trigger Ca 2+ oscillations in mouse oocytes .…”

Pre-treatment of sperm reduces success of ICSI in the pig

“…In mice, it is known that the components of the tail are not required for induction of oocyte activation (Kuretake et al 1996). However, subsequent to the submission of this manuscript, the existence of PLCz in equine sperm tail was suggested (Bedford-Guaus et al 2011). In this study, we also suggest that PLCz was detectable in both the post-acrosomal region and the tail region of pig sperm (Figs 2 and 5), suggesting that pig sperm tails may be capable of triggering oocyte activation and embryo development.…”

Characterization of Equine Phospholipase C Zeta: A Review and Preliminary Results on Expression Defects in Subfertile Stallions

“…After decades of work, the sperm-delivered protein responsible for these events was cloned from mouse testis and identified as testis-specific phospholipase C zeta (PLCZ) [15]. Since then, PLCZ has been sequenced in the human, monkey [16], chicken [17], pig [18], medaka fish, rat [19], hamster [20], quail [21], and horse [22] owing to a highly conserved mechanism for triggering egg activation in mammals and potentially other vertebrate species.…”

“…For instance, injection of sperm extracts, sperm (intracytoplasmic sperm injection [ICSI]), or murine PLCZ cRNA into mare oocytes triggered [Ca 2þ ] i oscillations and activation of the developmental program [4,8,9]. Moreover, stallion sperm and testis express PLCZ that was shown to be enzymatically active when injected into mouse oocytes [22].…”