Studies on the Acrosomal Function of Sperm from Fertile and Subfertile Stallions

Abstract

The current studies determined from a clinical and molecular standpoint some potential causes associated with the occurrence of impaired acrosomal exocytosis (IAE) in stallions that carry the IAE-susceptibility genotype, i.e., A/A-A/A in the FKBP6 gene exon 5. An extensive review of the literature regarding acrosomal function, with emphasis on the stallion, was conducted to identify potential gaps in the knowledge regarding current methodologies for evaluation of acrosomal function in stallions, the role of acrosomal function in stallion fertility, and the use of new technologies to identify potential markers of acrosomal function (Chapter 1). In a retrospective clinical study at our laboratory (Chapter 2), a total of twenty-one (21) stallions (1.86% total population analyzed; 1,128 stallions between 2003 – 2020) were identified to have reduced fertility despite having acceptable sperm quality. Of these, 8 (38.1%) were diagnosed with IAE, as determined by calcium ionophore A23187-induced AE, and 7 of these 8 were found to carry the susceptibility genotype for IAE in the gene FKBP6. All the positive stallions for the susceptibility genotype were of the Thoroughbred (TB) registry. Despite the method to study acrosomal function (e.g., calcium ionophore A23187) being able to detect differences in the acrosomal function of some subfertile stallions, its use also resulted in loss of sperm motility and viability during incubation. Thus, an in vitro model that relies on the use of specific sperm culture conditions was validated as a test for acrosomal function in stallions of different fertility levels, including subfertile TB stallions that carry the susceptibility genotype for IAE (Chapter 3). This method resulted in higher sperm viability (as determined by flow cytometry) during in vitro incubation when compared to the calcium ionophore-based method (P < 0.05). Additionally, common methods for storage of stallion sperm (e.g., cooled storage or freezing/thawing) did not affect negatively the acrosomal response of stallion sperm incubated in this medium (P > 0.05). This model, but not the calcium ionophore-based test, detected differences of acrosomal exocytosis between a group of fertile stallions with variable in vivo fertility (P < 0.05), and was linearly associated with both seasonal and per-cycle pregnancy rates (r = 0.6556, and r = 0.9275, respectively). The acrosomal response in sperm from fertile and subfertile TB stallions was evaluated using this model, and differences on the rate of AE in viable sperm were detected at 4 and 6 hours of incubation between stallion groups (P > 0.05). Using this new model, the sperm proteome in frozen/thawed semen from fertile and subfertile TB stallions was analyzed using data-independent acquisition mass spectrometry (DIA-MS; Chapter 4). A total of 2220 proteins were identified by DIA-MS in sperm from both stallion groups. Using strict selection criteria (FDR 1.0%, q-value < 0.05, and log2fold > 0.584 or < -0.584 [e.g., fold change in protein abundance between fertile and subfertile stallions > 1.5]), 140 proteins were found to be differentially abundant in sperm from the subfertile stallions when compared to that of fertile stallions (83 less and 57 more abundant). Principal component analysis revealed that these proteins explained 66% of the difference in the sperm proteome between both stallion groups. Analysis using the Reactome database and Homo sapiens orthologs revealed that the proteins of lower abundance in sperm from the subfertile stallions were mostly overrepresented in the “metabolism” (32 proteins) and the “metabolism of lipids” (18 proteins) pathways. Some of the identified proteins included those of acrosomal origin, such as arylsulfatase F and zona pellucida-binding protein. None of the proteins identified by DIA-MS was related to the protein codified by the gene FKBP6.

In summary, the prevalence of IAE in a population of breeding stallions with “idiopathic” subfertility can reach up to 40%, and is mostly associated with the susceptibility genotype for IAE, A/A-A/A. A more physiological method to induce spontaneous AE in stallion sperm can be used to determine differences on the fertility potential of stallions that otherwise would not be detected by the use of other methodologies to stimulate AE (i.e., high dose of calcium ionophore A23817). The analysis of the sperm proteome in subfertile TB stallions that carry the susceptibility genotype for IAE suggested that proteins associated with metabolism and lipid metabolism might be related to the occurrence of this condition.