Abstract
Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.
Highlights
The mammalian oviduct is a strategic site in the female reproductive tract where it provides a luminal microenvironment for gamete transport and maturation, sperm capacitation, and fertilization as well as early embryo development by secreting an oviductal fluid consisted of a combination of plasma exudates and secretory components from oviductal epithelial cells [1,2,3]
human embryonic kidney 293 (HEK293) cells derived from clones stably expressing high levels of rHamOVGP1 were selected and maintained in the medium and green fluorescent protein (GFP) expression was monitored on a daily basis
lentivirus-derived vectors (LVs)-mediated gene transfer was applied to the establishment of stable cell lines co-expressing rHamOVGP1and GFP
Summary
The mammalian oviduct is a strategic site in the female reproductive tract where it provides a luminal microenvironment for gamete transport and maturation, sperm capacitation, and fertilization as well as early embryo development by secreting an oviductal fluid consisted of a combination of plasma exudates and secretory components from oviductal epithelial cells [1,2,3]. Bovine OVGP1 has been shown to enhance sperm capacitation and increase in vitro fertilization rates [27]. Recent studies in our laboratory have demonstrated that estrus stage-specific HamOVGP1 enhances tyrosine phosphorylation of a sub-set of sperm proteins during in vitro capacitation [32]. These accumulative findings all point to several important roles played by OVGP1 during the early events of mammalian reproduction. To circumvent the problem of obtaining adequate amounts of native OVGP1 for future studies, the production of recombinant OVGP1 can be envisaged as an alternative
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