The acrosome proteins arylsulfatase F and zona pellucida-binding protein 2 are candidate markers associated with Impaired Acrosomal Exocytosis in sperm from subfertile Thoroughbred stallions

Abstract

Thoroughbred (TB) stallions that carry the linked susceptibility genotype A/A-A/A in exon 5 of the FKBP6 gene (ECA13; EquCab 3.0) are profoundly subfertile due to Impaired Acrosomal Exocytosis (IAE). A clear causation of the FKBP6 genotype and IAE is still unknown. In the current study, the sperm proteome in frozen/thawed semen from three fertile TB stallions (n = 3) and three subfertile TB stallions (FKBP6 genotype A/A-A/A; n = 3) was studied using mass spectrometry. Sperm from both stallion groups were incubated in Modified Whitten's-lactate media (MW-Lac) to induce acrosomal exocytosis in viable sperm (AE/Viable). At 0h, 2h, 4h, and 6h, sperm aliquots were analyzed for AE/Viable using flow cytometry (Fixable Live/Dead Red + FITC-PSA), while the sperm proteome was analyzed via Data-Independent Acquisition mass spectrometry (DIA-MS). Student's t-tests and two-way ANOVA with Benjamini-Hochberg multiple testing correction (FDR q-value 0.05) were used to determine differences in AE/Viable and protein relative abundance between experimental groups and incubation periods. At 4h and 6h of incubation, the mean AE/Viable was higher in the fertile than in the subfertile stallions (41 and 44% vs. 14 and 16%, respectively; P < 0.05). A total of 2220 proteins were identified by DIA-MS. Usingstrict selection criteria (FDR 1.0%, q-value < 0.05, and fold-change < 1.5 or > 1.5), 140 proteins were found to be differentially abundant in sperm from the subfertile stallions when compared to that of the fertile stallions (83 less and 57 more abundant). Using bioinformatic analyses, most of the proteins of lower abundance in sperm from subfertile stallions were found to be overrepresented in the “metabolism” (32 proteins) and “metabolism of lipids” (18 proteins) pathways (Homo sapiens orthologs; Reactome database). Two of these proteins, arylsulfatase F (ARSF; fold-change < 4.02; P < 0.05) and zona pellucida-binding protein 2 (ZPBP2; fold-change < 3.36; P < 0.05), are acrosomal proteins known to play a fundamental role in sperm-oocyte binding. By using immunofluorescence, at 0h of incubation in MW-Lac, ARSF was identified at the acrosome, mid-, and principal piece locations in sperm from fertile TB stallions, while only at the mid- and principal piece regions in sperm from subfertile TB stallions. No evidence of ZPBP2 was observed in sperm from both stallion groups at 0h incubation in Lac-MW. Current studies are focused on determining whether these proteins undergo changes in localization within the sperm during acrosomal exocytosis.