Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization

Abstract

Osteopontin (OPN) is a secreted extracellular matrix phosphoprotein identified in various tissues and fluids including those of the male and female reproductive tracts. OPN was previously identified as a 55 kDa high fertility marker in Holstein bull seminal plasma, produced by the ampulla and the vesicular gland. The objectives of this study were to characterize OPN on ejaculated and cauda epididymal sperm using immunofluorescence and western blot analysis, and to assess the role of sperm OPN in fertilization. Solubilized sperm membrane proteins from ejaculated and cauda epididymal sperm were separated by 1D SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to bovine milk OPN. A 35 kDa protein was detected by this antibody in both ejaculated and cauda epididymal sperm membranes. Analyses also recognized OPN at 55 and 25 kDa in cauda epididymal fluid and testicular parenchyma homogenates respectively. Immunofluorescent analysis of ejaculated and cauda epididymal sperm showed OPN localization in a well-defined band in the postacrosomal region of the sperm head and also on the midpiece. Results of in vitro fertilization experiments showed that sperm treated with an antibody to OPN fertilized fewer oocytes than sperm treated with control medium while increasing incidence of polyspermy, suggesting a role of sperm-associated OPN in fertilization and a block to polyspermy. These studies demonstrate that OPN exists at multiple molecular weight forms in the bull reproductive tract and its presence on ejaculated sperm may signal its importance in fertilization by interacting with integrins or other proteins on the oocyte plasma membrane. Reproduction (2007) 133 909-917

Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls

“…AS is particularly prevalent in the testes and it plays an important role in several developmental pathways. Several studies have shown that deviations in aberrant transcripts are one of the causal factors in the reduced reproductive performance of bulls, including sperm maturation and fertilization (Erikson et al 2007, Brandenburger et al 2011, Noda et al 2013. Single-nucleotide polymorphisms (SNPs) in exonic splice enhancers (ESEs) or exonic splice silencers are associated with spliced pre-mRNA by causing aberrant splicing in or near cis-acting elements, including exon skipping and/or intron retention; the mutation increases the frequency of the skipping of exons in the transcription process and gives rise to aberrant transcripts (Liegel et al 2011).…”

Seminal Plasma Proteins as Potential Markers of Relative Fertility in Boars

“…In stallions, Brandon et al (1999) reported that a 72-kDa seminal plasma protein, also identified as horse osteopontin, was positively correlated with fertility. Osteopontin has recently been localized on ejaculated bull sperm and may play a role in fertilization and also as a block to polyspermy (Erikson et al, 2007). In the pig, addition of osteopontin during in vitro fertilization also reduced polyspermy rates (Hao et al, 2006) and improved embryo development after fertilization (Hao et al, 2008).…”

Protein biomarkers for male artificial insemination subfertility in bovine spermatozoa

“…The osteopontin (Ca 2+ ‐binding protein) of the seminal plasma originates from the epithelial cells of the seminal vesicle and ampulla 106. This protein also was detectable in the cauda epididymal fluid and the testicular parenchyma homogenates as the 55 kDa form and the 25 kDa form, respectively, and was localized as the 35 kDa form in the postacrosomal region and middle piece of the ejaculated spermatozoa 108. In contrast, the lipocalin‐type prostaglandin D synthase was detectable in the elongating spermatids and Sertoli cells of the seminiferous tubules, cuboidal epithelial cells of the rete testis and efferent ducts, and the epithelial principal cells of the epididymides and was localized in the apical ridge of the acrosome on the ejaculated spermatozoa 109.…”