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Sperm proteomes have emerged for several species; however, the extent of species similarity is unknown. Sheep are an important agricultural species for which a comprehensive sperm proteome has not been produced. In addition, potential proteomic factors from seminal plasma that may contribute to improved fertility after cervical insemination are yet to be explored. Here we use liquid chromatography-tandem mass spectrometry to investigate the proteome of ejaculated ram spermatozoa, with quantitative comparison to epididymal spermatozoa. We also present a comparison to published proteomes of five other species. We identified 685 proteins in ejaculated ram spermatozoa, with the most abundant proteins involved in metabolic pathways. Only 5% of ram sperm proteins were not detected in other species, which suggest highly conserved structures and pathways. Of the proteins present in both epididymal and ejaculated ram spermatozoa, 7% were more abundant in ejaculated spermatozoa. Only two membrane-bound proteins were detected solely in ejaculated sperm lysates: liver enriched gene 1 (LEG1/C6orf58) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3). This is the first evidence that despite its relatively complex proteomic composition, seminal plasma exposure leads to few novel proteins binding tightly to the ram sperm plasma membrane.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Purpose In men, obesity may lead to poor semen parameters and reduced fertility. However, the causative links between obesity and male infertility are not totally clear, particularly on a molecular level. As such, we investigated how obesity modifies the human sperm proteome, to elucidate any important implications for fertility. Methods Sperm protein lysates from 5 men per treatment, classified as a healthy weight (body mass index (BMI) ≤ 25 kg/m 2) or obese (BMI ≥ 30 kg/m 2), were FASP digested, submitted to liquid chromatography tandem mass spectrometry, and compared by label-free quantification. Findings were confirmed for several proteins by qualitative immunofluorescence and a quantitative protein immunoassay. Results A total of 2034 proteins were confidently identified, with 24 proteins being significantly (p < 0.05) less abundant (fold change < 0.05) in the spermatozoa of obese men and 3 being more abundant (fold change > 1.5) compared with healthy weight controls. Proteins with altered abundance were involved in a variety of biological processes, including oxidative stress (GSS, NDUFS2, JAGN1, USP14, ADH5), inflammation (SUGT1, LTA4H), translation (EIF3F, EIF4A2, CSNK1G1), DNA damage repair (UBEA4), and sperm function (NAPA, RNPEP, BANF2). Conclusion These results suggest that oxidative stress and inflammation are closely tied to reproductive dysfunction in obese men. These processes likely impact protein translation and folding during spermatogenesis, leading to poor sperm function and subfertility. The observation of these changes in obese men with no overt andrological diagnosis further suggests that traditional clinical semen assessments fail to detect important biochemical changes in spermatozoa which may compromise fertility.

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