Proteomics of seminal plasma and porcine spermatozoa

Resumen

Proteins are essential for sperm function, including their fertilizing capacity. Consequently, the aim of the PhD Thesis was to describe and update the proteomes of porcine seminal plasma (SP) and spermatozoa, respectively; identifying proteins related to sperm freezability and fertility after artificial insemination (AI). Five experimental studies were performed, three focused on the SP proteome (studies 1, 2 and 3) and the other two on the sperm proteome (studies 4 and 5). Sperm and SP samples were derived from 313 ejaculates (89 boars), collected either in fractions (the 10 first mL of the sperm rich fraction [SRF], the remainder of the SRF and the post-SRF) or as an entire ejaculate. In addition, epididymal (study 4) and cryopreserved spermatozoa (study 5) were also used. For proteomic analysis, the SP and sperm proteins were digested by trypsin, the resulting peptide mixtures subjected to different fractionation approaches before mass spectrometry (MS) analysis. Liquid chromatography-tandem MS was used for proteome analysis. Quantitative differences in protein composition were analyzed either by SWATH (Sequential window acquisition of all theoretical mass spectra; studies 1, 2, 3 and 5) or by iTRAQ (Isobaric tags for relative and absolute quantitation, study 4). The fertility of the boars (study 3), as farrowing rates (FR, number of farrowing sows respect to the number of inseminated sows) and litter size (LS, total number of piglets born per litter) was recorded from 25,069 sows after AI. Sperm function (study 5) was assessed in terms of total and progressive motility, viability, nuclear DNA fragmentation, membrane lipid peroxidation and early signs of apoptosis. A total of 872 proteins in SP (studies 1 and 2) and of 1,723 in spermatozoa (study 4) were identified; of which 679 respectively 1,602 were quantified. Several proteins, many of them relevant for sperm function, were identified as quantitatively differently expressed among ejaculate fractions in both SP (study 1) and sperm (study 4) proteomes. Some of these were common for SP and spermatozoa, revealing that the proteome of pig spermatozoa is remodeled during ejaculation through interaction with the surrounding SP. In study 3, a total of 11 and 4 differentially expressed proteins were identified in SP from boars with differences in farrowing rate and litter size, respectively. These proteins are revealed as potential fertility biomarkers of boar used in AI programs. Finally, study 5 identified 26 sperm proteins differentially expressed between samples showing differences in freezability. This PhD Thesis provides the most extensive and updated description of SP and pig sperm proteomes. It also evidences that the pig sperm proteome is remodeled during ejaculation and identifies proteins directly involved in sperm freezability and boar fertility in the proteome of spermatozoa and SP, respectively.