48 Semiquantitative and quantitative assessments of phospholipase C zeta 1 in stallion sperm

Phospholipase C zeta 1 (PLCZ1) is considered the primary sperm-borne factor for oocyte activation. However, the PLCZ1 content can vary among sperm from individual stallions and after cryopreservation, resulting in plasma membrane damage and protein modifications. The PLCZ1 content of frozen sperm is associated with cleavage and embryo development rates after equine intracytoplasmic sperm injection (ICSI); therefore, methods to assess PLCZ1 content of sperm samples could have clinical relevance and provide markers for improving cryopreservation methods. We hypothesise that PLCZ1 is a soluble protein that can be lost from the sperm plasma membrane, and such loss is associated with reduced membrane integrity and is quantifiable by immunoassay. To assess the loss of PLCZ1 associated with cryopreservation, sperm (n = 12 stallions) were frozen in lactose-EDTA cryodiluent, thawed at 37°C for 60 s, diluted 10-fold (EZ Freezin^® CryoMax MFR5 [Animal Reproduction Systems Inc.]) and refrozen. Fresh (FR), frozen (FZ), and refrozen (RFZ) sperm were assessed for membrane integrity and PLCZ1 expression by flow cytometry using LIVE/DEAD™ Fixable Far-Red Kit (molecular probes [ThermoFisher]), antihuman-PLCZ1 antibody (Santa Cruz Biotechnology) previously validated for stallion sperm, and anti-rabbit IgG-H⁺L-Alexa Fluor^®488 (Invitrogen). Assessments of PLCZ1 were performed in cryopreserved sperm from a single stallion by Western blot, immunofluorescence, and ELISA (MyBioSource). Sperm were sequentially incubated in nonionic (1% Nonidet-P40 [NP]) and anionic (2% sodium dodecyl sulfate [SDS]) detergents to obtain extractable and nonextractable fractions and detergent-extracted sperm. Data were analysed by mixed model for repeated-measures, ANOVA, and Tukey pairwise comparison. Percentages of intact plasma membrane sperm decreased (P < 0.05) after each freezing cycle (FR: 67.9 ± 2.8, FZ: 34.0 ± 2.8, RFZ: 17.7 ± 2.7, mean ± s.e.M). Percentages of positive-labelled sperm (FR: 84.3 ± 1.9, FZ: 58.8 ± 1.9, RFZ: 56.9 ± 1.9) and mean fluorescence intensity (FR: 325.3 ± 29.6, FZ: 228.5 ± 29.6, RFZ: 230.1 ± 28.3 arbitrary units [au]) for PLCZ1 were also reduced (P < 0.05) after cryopreservation. Semiquantitative and quantitative reduction (P < 0.05) of PLCZ1 after freezing were found by immunofluorescence (FR: 935.8 ± 55.7, FZ: 584.9 ± 41.7, RFZ: 474.3 ± 52.8 au of corrected fluorescence intensity) and ELISA (FR: 0.82 ± 0.02, FZ: 0.67 ± 0.03, RFZ: 0.70 ± 0.02 ng/mL). Detergent-extracted sperm by NP (456.9 ± 45.7 au) and SDS (386.7 ± 33.9 au) had lower (P < 0.05) PLCZ1 corrected fluorescence intensity than FR and FZ sperm. Western blot confirmed a relative PLCZ1 reduction in sperm after freezing and PLCZ1 presence in cryodiluents and detergent extractable fractions. A detergent-nonextractable fraction of PLCZ1 persisted in the postacrosomal region, confirmed by Western blot and immunofluorescence. We concluded that most, but not all, of PLCZ1 can be extracted from the stallion sperm membrane; freezing and refreezing is detrimental to sperm plasma membrane integrity and results in loss of PLCZ1. However, a nonextractable PLCZ1 fraction remains in the sperm after cryopreservation.