Proteomic profile of stallion seminal plasma

Abstract

Proteins mediate all physiological changes. Seminal plasma (SP), the product of testes, epididymis, and accessory sex glands, is a fluid released during ejaculation and represents up to 98% of the voluminous stallion ejaculate. The SP plays an essential role in reproduction as a transit medium and a source of energy, antioxidants, enzymes, and minerals. This study aimed to investigate proteomic profile of the seminal plasma of stallions. Twenty-four Criollo stallions with a known reproductive history and per-cycle concept rate data calculated from at least 30 inseminations were used. One ejaculate was collected from each stallion during the breeding season. Pregnancy rates ranged at day 16 after artificial insemination from 20.2% to 95.6%. The animals were divided into two groups: High Pregnancy (HP), with a pregnancy rate per cycle ≥51%, and Low Pregnancy (LP), with a pregnancy rate of <50%. Sperm concentration, kinetics, morphology, and plasma membrane integrity and functionality were evaluated. The sample was centrifuged at 400xg for 10min. SP was then transferred to a 2 mL microcentrifuge tube and centrifuged again (10.000xg, 60min, 4°C). Proteins were separated using 2D SDS PAGE gel electrophoresis in duplicate. Spots in at least 80% of the gels in one of the groups, with significant abundance (P<0.05) and at least a 1.5-fold magnitude difference between groups, were selected. Thespectra were acquired in a nano LC-MS/MS spectrometer, and proteins were identified by the MASCOT application and validated by SCAFFOLD. From a total of 716 analyzed spots, four were selected. For non-parametric data the Kruskal Wallis and for parametric data Anova and t-test were used. The cysteine-rich secretory protein 3 (CRISP3) and SP protein HSP-1-like (HSP1) showed higher abundance in the SP in group LP than HP (P< 0.05). However, kallikrein-1E2 (KLK) and phospholipase A2 (PLA2) showed higher abundance in HP than LP (P<0.05). In the horse, CRISP-3 is synthesized in great amounts in the accessory sexual glands, ampulla and seminal vesicle. HSP1 has an essential role as a molecular chaperone for sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume, and possibly in the interaction between gametes. KLK is one of the major proteins found in seminal equine plasma. The kallikreins are serine proteases, and the glandular kallikrein is regulated by androgens and secreted by the prostate. The PLA2 plays an essential role in the late maturational events of spermatozoa, the acrosomal reaction, and sperm egg fusion. In conclusion, the proteins are essential to the biological processes related to stallion fertility, providing insight into SP proteins that could serve as biomarkers for fertility in stallions.