148 EXPRESSION OF OVIDUCT-SPECIFIC GLYCOPROTEIN IN THE CANINE OVIDUCT DURING THE PERIOVULATORY PERIOD

Abstract

In the canine species, the oocyte is ovulated at the immature germinal vesicle (GV) stage and will reach metaphase II stage after 3 to 4 days spent in the oviduct. Fertilization and embryonic development to the blastocyst stage also take place in the oviduct. In a previous study (Tahir et al. 2012 Reprod. Domest. Anim. 47, 487), we reported the expression of oviductin (oviduct-specific glycoprotein) mRNA in the oviduct. The present study aimed to describe the oviductin protein expression (immunolocalization and Western blot quantification) and the effect of the oviducal region and the ovarian cycle. Beagle bitches were ovariectomized at 6 stages (6 bitches/stage): anestrus, after the onset of proestrus and before the LH peak (Pre-LH), after the LH peak and before ovulation (Pre-ov), 1 day (Day 1), 4 days (Day 4), and 7 days (Day 7) after ovulation. Three oviducal regions were collected [i.e. ampulla, isthmus, and ampulla-isthmus junction (AIJ)]. Ampulla and isthmus were fixed in paraformaldehyde, embedded in paraffin, and 7-μm sections were used for immuno-histochemistry using a goat polyclonal anti-human oviductin (N20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ImmPress kit (Vector Laboratories, Burlingame, CA, USA). Total protein from the AIJ was extracted and used for Western Blot using a mouse monoclonal anti-mouse antibody (H8; Santa Cruz Biotechnology). The expression of oviductin in AIJ was quantified in duplicate on blots using ImageJ software and normalized with actin levels. Relative amounts of oviductin were compared between stages by ANOVA followed by a Tukey test. Immuno-histochemistry revealed that oviductin was specifically expressed in the nonciliated cells of the oviducal epithelium from Pre-LH to Day 7, with a stronger staining in the isthmus than in the ampulla at all stages. Furthermore, the expression of oviduct-specific glycoprotein, detected by Western Blot, varied significantly with the stage (P < 0.0001). The oviductin protein expression was at its lowest level at anestrus, then increased significantly at Pre-LH and Pre-ov (35- and 41-fold higher levels than anestrus, respectively), reached a maximal level at Day 1 (66-fold higher than anestrus), then decreased at Days 4 and 7 (47- and 20-fold higher than anestrus, respectively). In conclusion, this is the first report of oviductin protein expression in the canine oviduct. The region-specific higher expression of oviductin at Day 1 post-ovulation suggests a potential role of this glycoprotein in gamete maturation and fertilization in the bitch.