133 Cleavage rates after bovine IVF are affected by relative abundance of sperm phospholipase C zeta 1

When the membranes of the sperm and oocyte fuse, sperm phospholipase C zeta 1 (PLCZ1) causes intracellular calcium oscillations that initiate oocyte activation. In humans and horses, PLCZ1 abundance is positively associated with cleavage rates after ICSI. In bulls, genetic mutations cause genotype polymorphisms and phenotype diversity of PLCZ1 that potentially compromise sperm quality traits and fertility in vivo or in vitro. Frozen-thawed bull sperm are routinely used for AI and IVF. However, sperm undergo cryoinjuries and protein modifications during cryopreservation, which could affect PLCZ1 abundance. Limited information is available on bovine sperm PLCZ1 abundance and its association with fertility in vivo and in vitro. We hypothesized that the PLCZ1 abundance in frozen-thawed bull sperm is associated with cleavage after IVF. Frozen sperm from six bulls with variable fertility in vitro were evaluated in duplicate to assess acrosome integrity (PNA-Alexa647, Invitrogen) and relative PLCZ1 abundance (antibovine PLCZ1, MyBioSource; anti-IgG-Alexa488, Invitrogen) using flow cytometry in one session. Unstained and single-stained sperm with PNA-Alexa647 or the secondary antibody were used to set voltages, compensations, and quadrants. Sperm displaying fluorescence from the primary–secondary antibody complex to the right of the marker from single-stained sperm with the secondary antibody were considered positive for PLCZ1 (PLCZ1+). Immunofluorescence was used to localize PLCZ1 in frozen-thawed bull sperm. Bull sperm samples were grouped based on cleavage rates after IVF (>5 IVF sessions per bull and >60 oocytes per session) as high cleavge (HC, ≥70%, n = 3) and low cleavage (LC, <70%, n = 3). Data were analyzed by a generalized linear model and Tukey pairwise comparisons and expressed as mean ± s.e.m. PLCZ1 was found in the acrosomal region and equatorial segment of the bull sperm, exhibiting four patterns: acrosomal + equatorial, only acrosomal, only equatorial, and devoid. For flow cytometric analysis, the percentage of PLCZ1+ sperm from HC and LC (81.8 ± 2.2% vs. 80.8 ± 2.2%) samples did not differ. However, PLCZ1 mean florescence intensity (MFI, arbitrary units) from HC samples was greater in the whole sperm population than samples from LC (1621 ± 179 vs. 847 ± 179; P = 0.004). The PLCZ1+ sperm subpopulation from HC samples also had greater MFI than did that from LC (1960 ± 179 vs. 1015 ± 179; P = 0.0008). Similarly, PLCZ1+ sperm with intact acrosomes had greater MFI in samples from HC than LC (1970 ± 137 vs. 1006 ± 137; P < 0.0001). We concluded that frozen-thawed bull sperm samples vary in PLCZ1 abundance, which is closely related to acrosome integrity, and that PLCZ1 abundance is positively associated with cleavage rates after IVF.