Osteopontin enhances sperm capacitation and in vitro fertilization efficiency in boars

Abstract

In this study, we used more reliable experimental materials and methods to detect the effects of osteopontin (OPN) on boar sperm in vitro capacitation, acrosome reaction, and fertilization efficiency. We reorganized and obtained the OPN protein of the porcine source. Immunofluorescence and Western blot show the localization and expression of the OPN protein before and after sperm capacitation. To determine whether OPN can affect sperm during sperm capacitation, we examined cyclic adenosine monophosphate (cAMP) concentrations after sperm capacitation, and the results showed that OPN significantly increased the cAMP concentration in sperm (p < 0.05). Flow cytometry showed that 0.1 μg/mL OPN-treated sperm had better acrosome reaction ability. In vitro fertilization (IVF) showed that 0.1 μg/mL OPN significantly increased the rate of embryo division. In conclusion, this study found that 0.1 μg/mL porcine OPN protein can significantly improve porcine capacitated sperm motility, cAMP concentration after capacitation sperm, acrosome reaction ability, and embryo division during IVF and provides new clues to explore the mechanism of OPN's function on sperm.

Keywords: Acrosome reaction; Capacitation; In vitro fertilization; Osteopontin; Sperm.

Fig. 1.. Changes in the localization of OPN during sperm capacitation.

(A and D) Images of the nucleus before and after capacitation (Hoechst, blue). (B and E) Images of OPN protein expression before and after capacitation (green). a: head. b: neck. c: tail. (C and F) Merged images of the nucleus (Hoechst, blue) and OPN protein (green) images. The merged images were obtained using a confocal Nikon TS-1000 microscope and NIS Elements image software (Nikon, Japan). Bar=10 μm. (G and H) OPN protein and phosphotyrosine were probed with anti-OPN and anti-phosphotyrosine antibodies, respectively. OPN, osteopontin; BC, before capacitation; AC, after capacitation.

Fig. 2.. Effect of OPN on the acrosome reaction of sperm during IVF.

(A) FITC-PSA flow diagram of the acrosome reaction in untreated sperm (4 h). (B) FITC-PSA flow diagram of the IgG-treated acrosome reaction (negative control). (C) FITC-PSA flow diagram (anti-OPN group) of the acrosome reaction after sperm incubation with the anti-OPN antibody for 4 h in culture medium. (D) FITC-PSA flow diagram of the acrosome reaction incubation with the OPN protein in culture medium for 4 h (OPN protein group). Q1, dead sperm; Q2, near-dead sperm; Q3, acrosome reaction; Q4, intact acrosome. (E) Percentage of acrosome reaction. Different letters represent significant differences (p < 0.05). OPN, osteopontin; IVF, in vitro fertilization; FITC-PSA, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin; IgG, Immunoglobulin G.