140 Subcellular localization of phospholipase C zeta 1 in stallion sperm

Phospholipase C zeta 1 (PLCZ1) meets the criteria of a sperm-borne oocyte activating factor. Oocyte activation is initiated when a fertilizing sperm delivers a “soluble factor” that diffuses into the ooplasm after gamete fusion. The activating factor likely locates in the perinuclear theca, which is the first sperm cytosol component to enter in the ooplasm, and its disassembly triggers calcium oscillations and oocyte activation cascade. Due to the soluble nature of PLCZ1, we hypothesise that PLCZ1 could be differentially extracted to determine the subcellular localization in stallion sperm. We aimed to assess PLCZ1 in extracts of fresh stallion sperm by sequential exposure to nonionic (Nonidet-P40, NP) and ionic detergents (sodium dodecyl sulfate; SDS) to examine PLCZ1 binding to surface or internal sperm structures, respectively. Fresh sperm from six stallions were washed in PBS, incubated in 1% NP, washed again, and then incubated in 2% SDS. The remaining sperm contained the nonextractable fraction of PLCZ1. Fresh sperm, NP-extract, SDS-extract, and nonextractable fraction were evaluated by immunoblotting using antihuman-PLCZ1 antibody (Santa Cruz Biotechnology) that was previously validated for stallion sperm and anti-rabbit IgG-HRP (Invitrogen). Amido Black Staining Solution (Millipore Sigma) was used to assess total protein for the loading control and densitometry analysis. Fresh, NP- and SDS-extracted sperm were evaluated by immunofluorescence using antihuman-PLCZ1 antibody and anti-rabbit IgG-AlexaFluor^® 488 (Invitrogen). Data were analysed by mixed model for repeated-measures and Tukey pairwise comparison and presented as mean ± s.e.m. Immunoblotting confirmed that equine PLCZ1 was ~72 kDa. Based on the relative PLCZ1 abundance of fresh sperm samples, fresh sperm (1.00 ± 0.16 relative units, ru) had greater (P < 0.05) PLCZ1 than SDS-extract (0.05 ± 0.16 ru) and nonextractable fraction (0.02 ± 0.16 ru), but fresh sperm did not differ and NP-extract (1.16 ± 0.16 ru). Immunofluorescence revealed that PLCZ1 was located in the acrosomal and postacrosomal regions and tail of fresh sperm, with a progressive reduction in the acrosomal localization after detergent extraction. Quantitative fluorescence analysis of PLCZ1 in sperm heads demonstrated lower (P < 0.05) intensity in sperm exposed to NP (720 ± 45 × 10³ arbitrary units, au) and SDS (600 ± 47 × 10³ au) than fresh sperm (1076 ± 43 × 10³ au). However, the nonextractable fraction retained a weak PLCZ1 fluorescence signal in the postacrosomal region. Tail fluorescence was notably reduced after detergent extraction. In conclusion, nonionic detergent extraction removed a significant proportion of PLCZ1, demonstrating that PLCZ1 is soluble and interacts primarily with sperm surface structures such as the acrosome and, to a lesser extent, with interior sperm structures. However, a detergent-resistant, nonextractable fraction is retained in the postacrosomal region of equine sperm, congruent to the perinuclear theca localization and strategically located to be available for sperm-oocyte interactions.