Oviduct-specific glycoprotein and heparin modulate sperm-zona pellucida interaction during fertilization and contribute to the control of polyspermy

Abstract

Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm-oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con- and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of approximately 1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm-ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OF-exposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoprotein-heparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Effect of different types of porcine (sow or gilt, follicular or luteal phase) and bovine (heifer at follicular phase) OF on the ZP resistance to protease digestion. The fluid from ovulating sows and from heifers at the follicular phase made the ZP highly resistant to digestion. Each bar represents the digestion time (mean ± SEM) of 30–45 IVM oocytes incubated in the fluid for 30 min and later transferred to a pronase solution (0.5% wt/vol in PBS). Experiments were carried out in triplicate. a, b, and c in each graphic indicate significant differences among groups (P < 0.001).

Fig. 2.

Effect of incubation of IVM oocytes in oviductal fluid on IVF results. In porcine IVF the parameters penetration and monospermy rate (A), number of spermatozoa per oocyte (B), and number of bound spermatozoa per ZP (C) were evaluated. In bovine IVF, the same parameters were recorded (D–F). Each bar represents mean ± SEM for each parameter. Experiments were carried out in triplicate. Each replicate consisted of 10 oocytes for pOF and 40 oocytes for bOF. Different letters (a, b) in each graphic and parameter indicate significant differences (P < 0.001).

Fig. 3.

Effect of bOF on resistance to pronase of ZP from oocytes incubated in TALP medium for different periods of time. Porcine (A) and bovine (B) IVM oocytes were incubated in bOF for 30 min and later incubated in TALPp medium for different times. The x axis indicates the time interval that the oocytes remained in TALPp after incubation in bOF and before assessing ZP resistance to proteases. The y axis indicates the time span between placement of the samples in pronase solution and complete dissolution of the ZP. Experiments were carried out in triplicate. Each replicate consisted of 20 oocytes for each time assayed. (P < 0.01).

Fig. 4.

Effect of heparin on the reversibility of the ZP resistance to proteases induced by bOF in porcine oocytes matured in vitro. Oocytes were preincubated in bOF for 30 min, transferred to TALPb medium without (A) or with (B) heparin and evaluated for resistance to proteases after 15, 60, 120, and 240 min of incubation. Each bar represents the percentage of nondigested ZP after 4 and 20 h in pronase solution (0.5% wt/vol in PBS), respectively, for each group. Experiments were carried out in triplicate. Each replicate consisted of 20 oocytes for each time assayed. (P < 0.001).

Fig. 5.

Identification of OGP as the molecule unbound from ZP after incubation in oviductal fluid (30 min) and further incubation in TALPp medium (1 h). One hundred ZPs from IVM porcine oocytes were preincubated in bOF (1 ZP/0.5 μl OF) and later in TALPp medium. Lane1 shows the SDS-PAGE electrophoresis under reducing conditions of the TALPp medium. Lane 2 shows the lysate of 100 ZPs after being removed from the medium. The two bands observed in lane 1, of ≈95 and ≈75 kDa, were analyzed on an Agilent 1100 Series HPLC. Five peptides in the 95-kDa band and five peptides in the 75-kDa band corresponding to OGP were identified.

Fig. 6.

Hypothesized mechanism by which oviductal proteins surround the oocyte in a “shell” that is responsible for the prefertilization ZP changes. (A) Oocyte in the preovulatory follicle. (B) In the oviduct, the residues (sugars?) in ZP-glycoproteins are recognized and bound by OGP. (C) Heparin-like GAGs in the oviduct fluid stabilize and reinforce the binding of OGP with residues in ZP-glycoprotein. (D) In the transit toward the uterus the system is destabilized and the OGP is partially unbound or internalized.