piRNA expression patterns in high vs. low fertility bovine sperm.

PIWI-interacting RNAs (piRNAs) are 24-32 nucleotide RNA sequences primarily expressed in germ cells and developing embryos that suppress transposable element expression to protect genomic integrity during epigenetic reprogramming events. We characterized the expression of piRNA sequences and their encoding clusters in sperm samples from an idiopathic fertility model of Holstein bulls with high and low Sire Conception Rates. The piRNA populations were determined to be mostly similar between fertility conditions when investigated by principal component and differential expression analysis, suggesting that a high degree of conservation in the piRNA system is likely necessary for the production of viable sperm. Both fertility conditions demonstrated evidence of 'ping-pong' activity - a secondary biogenesis pathway associated with active transposable element targeting and suppression. Most sperm-borne piRNAs were between 29-30 nucleotides in length and originated from 226 clusters across the genome, with the exception of chromosome 20. Mapping analysis revealed abundant targeting of several transposable element families, suggesting a suppressive function of sperm piRNAs consistent with their established roles. Expression of genes targeted by sperm-borne piRNAs is significantly reduced throughout early embryogenesis compared to the mRNA population. Limited transposable element expression is known to be essential for spermatogenesis, thus epigenetic regulation of this pathway is likely to influence sperm quality and fertilizing capacity.

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.

Characteristics of miRNAs Present in Bovine Sperm and Associations With Differences in Fertility.

Small non-coding RNAs have been linked to different phenotypes in bovine sperm, however attempts to identify sperm-borne molecular biomarkers of male fertility have thus far failed to identify a robust profile of expressed miRNAs related to fertility. We hypothesized that some differences in bull fertility may be reflected in the levels of different miRNAs in sperm. To explore such differences in fertility that are not due to differences in visible metrics of sperm quality, we employed Next Generation Sequencing to compare the miRNA populations in Bos taurus sperm from bulls with comparable motility and morphology but varying Sire Conception Rates. We identified the most abundant miRNAs in both populations (miRs -34b-3p; -100-5p; -191-5p; -30d-4p; -21-5p) and evaluated differences in the overall levels and specific patterns of isomiR expression. We also explored correlations between specific pairs of miRNAs in each population and identified 10 distinct pairs of miRNAs that were positively correlated in bulls with higher fertility and negatively correlated in comparatively less fertile individuals. Furthermore, 8 additional miRNA pairs demonstrated the opposite trend; negatively correlated in high fertility animals and positively correlated in less fertile bulls. Finally, we performed pathway analysis to identify potential roles of miRNAs present in bull sperm in the regulation of specific genes that impact spermatogenesis and embryo development. Together, these results present a comprehensive picture of the bovine sperm miRNAome that suggests multiple potential roles in fertility.

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.