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Proteins associated with Impaired Acrosomal Exocytosis (IAE) can be identified in sperm from subfertile Thoroughbred stallions by using data-independent acquisition mass spectrometry (DIA-MS)

ABSTRACT: Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). However, clear causation of this condition has not been determined. In the current study, the sperm proteome in frozen/thawed semen from three fertile TB stallions and three subfertile TB stallions (FKBP6 genotype A/A-A/A) was studied using mass spectrometry-based global proteomics. Sperm from both stallion groups were incubated in Lactate-Modified Whitten's (Lac-MW) medium to induce spontaneous acrosomal exocytosis in viable sperm (AE/Viable). At 0h, 2h, 4h, and 6h, sperm aliquots were removed for analysis for AE/Viable using flow cytometry (Fixable Live/Dead Red + FITC-PSA) global proteomics 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 was identified by DIA-MS. Using strict 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) at 0h incubation. 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; log2 fold change = -2.0; p < 0.05) and zona pellucida-binding protein 2 (ZPBP2; log2 fold change = -1.7; 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 in sperm from fertile TB stallions, while only at the mid-and principal piece in sperm from subfertile TB stallions. No evidence of ZPBP2 was observed in sperm from both stallion groups at 0h incubation in Lac-MW. Sperm from subfertile Thoroughbred stallions were bound to porcine zonae pellucidae at a lower frequency than sperm from fertile Thoroughbred stallions. In conclusion, DIA-MS is a powerful tool to identify candidate proteins that contribute to the etiology of IAE in Thoroughbred stallions, including proteins of acrosome origin that have a role in the sperm-oocyte binding process.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Equus Caballus (ncbitaxon:9796)

SUBMITTER: Charles C. Love, D.V.M., Ph.D.

PROVIDER: MSV000093730 | MassIVE | Wed Dec 27 11:54:00 GMT 2023

SECONDARY ACCESSION(S): PXD048150

REPOSITORIES: MassIVE

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Project description:Globally invasive Aedes aegypti mosquitoes disseminate numerous arboviruses that impact human health. One promising method to control Ae. aegypti populations is transinfection with the intracellular bacterium Wolbachia pipientis, a symbiont that naturally infects ~40-52% of insects but is normally absent from Ae. aegypti. Transinfection of Ae. aegypti with the wMel Wolbachia strain induces cytoplasmic incompatibility (CI), allowing infected individuals to rapidly invade native populations. Further, wMel Wolbachia-infected females display refractoriness to medically relevant arboviruses. Thus, wMel Wolbachia-infected Ae. aegypti are being released in several areas to replace native populations, thereby suppressing disease transmission by this species. Wolbachia is reported to have minimal effects on Ae. aegypti fertility, but its influence on other reproductive processes is unknown. Female insects undergo several post-mating physiological and behavioral changes required for optimal fertility. Post-mating responses (PMRs) in female insects are typically elicited by receipt of male seminal fluid proteins (SFPs) transferred with sperm during mating, but can be modified by other factors, such as adult age, nutritional status, and microbiome composition. To assess how Wolbachia infection influences Ae. aegypti female PMRs, we collected wMel Wolbachia-infected Ae. aegypti from the field in Medellín, Colombia and introduced the bacterium into our laboratory strain. We found that Wolbachia influences female fecundity, fertility, and re-mating incidence. Further, we observed that Wolbachia significantly extends longevity of virgin females. Changes in female PMRs are not due to defects in sperm transfer by infected males, or sperm storage by infected females. Using proteomic methods to examine the seminal proteome of infected males, we found that Wolbachia infection has a moderate effect on SFP composition. However, we identified 125 Wolbachia proteins that are paternally transferred to females by infected males. Surprisingly, the CI factor proteins (Cifs), were not detected in the ejaculates of Wolbachia-infected males. Our findings indicate that Wolbachia infection of Ae. aegypti alters female post-mating responses, potentially influencing control programs that utilize Wolbachia-infected individuals.

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Proteins associated with Impaired Acrosomal Exocytosis (IAE) can be identified in sperm from subfertile Thoroughbred stallions by using data-independent acquisition mass spectrometry (DIA-MS)

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