Combined albumin and bicarbonate induces head-to-head sperm agglutination which physically prevents equine sperm–oviduct binding

Abstract

In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition.D-galactose has previously been characterized as a key molecule that facilitates sperm-oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates; glycosaminoglycans; lectins; S-S reductants; and the capacitating factors albumin, Ca 2C and HCO 3 K on sperm-oviduct binding in the horse. Carbohydratespecific lectin staining indicated that N-acetylgalactosamine, N-acetylneuraminic acid (sialic acid) and D-mannose or D-glucose were the most abundant carbohydrates on equine oviduct epithelia, whereas D-galactose moieties were not detected. However, in a competitive binding assay, sperm-oviduct binding density was not influenced by any tested carbohydrates, glycosaminoglycans, lectins or D-penicillamine, nor did the glycosaminoglycans induce sperm tail-associated protein tyrosine phosphorylation. Furthermore, N-glycosidase F (PNGase) pretreatment of oviduct explants and APM did not alter sperm-oviduct binding density. By contrast, a combination of the sperm-capacitating factors albumin and HCO 3 K severely reduced (O10-fold) equine sperm-oviduct binding density by inducing rapid head-to-head agglutination, both of which events were independent of Ca 2C and an elevated pH (7.9). Conversely, neither albumin and HCO 3 K nor any other capacitating factor could induce release of oviduct-bound sperm. In conclusion, a combination of albumin and HCO 3 K markedly induced sperm head-to-head agglutination which physically prevented stallion sperm to bind to oviduct epithelium.

Why doesn’t conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization

“…These residues were less highly expressed in the ampulla where cycle-related differences were observed, characterized by maximum galactosyl expression during oestrus (Ball et al 1997). Using an oviduct explant or apical plasma membrane model, we were unable to identify d-galactose as key molecule in equine sperm-oviduct interaction (Leemans et al 2016). Similarly, immunohistochemistry failed to reveal expression of d-galactose on the equine oviduct epithelial plasma membrane, whereas other galactosyl carbohydrates were present, which supported the findings of a previous study (Desantis et al 2004).…”

“…Although various carbohydrates, including galactosyl moieties, were highly expressed on the epithelium of oviduct explants, we were unable to competitively inhibit sperm-oviduct epithelium binding using any of a variety of candidate mono-and polysaccharides, including d-galactose, and lectins. We did, however, find that, in contrast to some other mammalian species (Suarez 2001), equine sperm-oviduct explant binding was neither dependent on calcium nor on disulphide (S-S) covalent bonds (Leemans et al 2016). In short, it appears that equine sperm-oviduct epithelium interaction in vivo is not based on a single lectin or S-S covalent bond.…”

“…Therefore, spermbinding studies are often performed using equine oviduct monolayers, which have a flat surface (Thomas et al 1995b, Dobrinski et al 1999. Alternatively, accurate quantification of sperm binding can be performed using oviduct apical plasma membranes coated onto nitrocellulose (Leemans et al 2016). However, when using oviduct explants and apical plasma membranes in parallel, we were unable to identify significant differences in sperm binding, even though the number of spermatozoa bound per mm 2 varied more using the explants with their irregular surface (Leemans et al 2016).…”

Protein kinase C activity in boar sperm

“…The most notorious cytological effect of PKC activation with PMA is head-to-head agglutination with permanent flagellar motility, which provides clusters of sperm in motion (see video in Supporting information). Previous works have shown that sperm head-to-head agglutination occurs between spermatozoa with intact plasma acrosome membranes (Yang et al, 2012;Leemans et al, 2016). Moreover, Harayama et al (2003) showed that boar sperm agglutination is promoted by bicarbonate and PKA activation.…”

“…In addition, they concluded that cytoplasmic free Ca 2+ is involved in sperm head-to-head agglutination and that agglutination is not the resultant of acrosome reaction (Harayama et al, 2003). Sperm head-tohead agglutination has been proposed to be an early stage of the capacitation process in vitro (Harayama et al, 2000), which physically prevents sperm from binding to the oviduct epithelium (Leemans et al, 2016). It is to note that flagellar activity is maintained, suggesting that the fluidity of the flagellar plasma membrane is not affected.…”