Tamara Leahy

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction‐specific proteins that show demonstrable links to sperm fertility. Many of these membrane‐bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.

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.

Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode’s capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and dl-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu2+ to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1). Reproduction (2016) 151 1–10

Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7±2.7% to 2.8±1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

Cholesterol is an essential component of the mammalian plasma membrane because it promotes membrane stability without comprising membrane fluidity. Given this important cellular role, cholesterol levels are tightly controlled at multiple levels. It has been clearly shown that cholesterol redistribution and depletion from the sperm membrane is a key part of the spermatozoon's preparation for fertilization. Some factors that regulate these events are described (e.g., bicarbonate, calcium) but the mechanisms underlying cholesterol export are poorly understood. How does a hydrophobic cholesterol molecule inserted in the sperm plasma membrane enter the energetically unfavorable aqueous surroundings? This review will provide an overview of knowledge in this area and highlight our gaps in understanding. The overall aim is to better understand cholesterol redistribution in the sperm plasma membrane, its relation to the possible activation of a cholesterol transporter and the role of cholesterol acceptors. Armed with such knowledge, sperm handling techniques can be adapted to better prepare spermatozoa for in vitro and in vivo fertilization.

While the mechanisms that underpin maturation, capacitation, and sperm–egg interactions remain elusive it is known that these essential fertilisation events are driven by the protein complement of the sperm surface. Understanding these processes is critical to the regulation of animal reproduction, but few studies have attempted to define the full repertoire of sperm surface proteins in animals of agricultural importance. Recent developments in proteomics technologies, subcellular fractionation, and optimised solubilisation strategies have enhanced the potential for the comprehensive characterisation of the sperm surface proteome. Here we report the identification of 419 proteins from a mature bull sperm plasma membrane fraction. Protein domain enrichment analyses indicate that 67% of all the proteins identified may be membrane associated. A large number of the proteins identified are conserved between mammalian species and are reported to play key roles in sperm–egg communication, capacitation and fertility. The major functional pathways identified were related to protein catabolism (26S proteasome complex), chaperonin‐containing TCP‐1 (CCT) complex and fundamental metabolic processes such as glycolysis and energy production. We have also identified 118 predicted transmembrane proteins, some of which are implicated in cell adhesion, acrosomal exocytosis, vesicle transport and immunity and fertilisation events, while others have not been reported in mammalian LC‐MS‐derived sperm proteomes to date. Comparative proteomics and functional network analyses of these proteins expand our system's level of understanding of the bull sperm proteome and provide important clues toward finding the essential conserved function of these proteins.