Quality of fresh and cryopreserved bovine sperm is reduced by BPA and BPF exposure.

Bisphenol A (BPA) is an endocrine disrupting compound, used as the key monomer of polycarbonate plastics and epoxy resins. BPA has been detected in both humans and farm animals and has been correlated with decreased sperm counts and motility. BPS and BPF are structural analogs of BPA and are increasingly being used in manufacturing as BPA substitutes. In this study we aim to assess the direct outcomes of BPA, BPS and BPF exposure on bovine sperm parameters in vitro to elucidate how they affect sperm quality and fertilization potential, and to assess whether BPS and/or BPF are less harmful than BPA. Sperm from three or more bulls was obtained from either fresh samples or cryopreserved straws and exposed to 0.05 mg/mL of BPA, BPS and BPF in vitro. After 4h incubation, motility, capacitation, apoptosis/necrosis, and mitochondrial membrane potential levels were measured by CASA or computational flow cytometry. Results showed that BPA exposure significantly reduced both fresh and cryopreserved sperm motility, capacitation, viability and mitochondrial membrane potential levels. Furthermore, BPF significantly decreased motility, capacitation and mitochondrial membrane potential in cryopreserved sperm only. BPS did not have any significant effects on any of the parameters measured. Our results suggest that BPA is the most harmful to sperm, while BPF is toxic under certain conditions, and BPS seems to be the least detrimental. Overall, this study provides an understanding of how the ubiquitous environmental chemicals, bisphenols, may impact male fertility even after ejaculation.

Characterization of Na+K+-ATPase in bovine sperm.

Existing as a ubiquitous transmembrane protein, Na(+)K(+)-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na(+)K(+)-ATPase is a dimer of α and ß subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na(+)K(+)-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all ß isoforms. Relative quantity of Na(+)K(+)-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of ß1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of ß1 revealed three distinct spots. Based on the unique quantity, location and structure Na(+)K(+)-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm.

Lipid bilayer composition affects transmembrane protein orientation and function.

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na(+) K(+)-ATPase or α-amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the ß subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na(+) K(+)-ATPase within the membranes.