Schematic representation of global experimental design of homologous and heterologous IVF from diverse mammalian species.

(A) Represents design of experiment shown in Tables S1-3, Figure 2A-B and Figure S1-3. (B) Represents design of experiment shown in Table S4, Figure 2C. (C) Represents design of experiment shown in Table S5-6, Figure 2D-E and Figure 3. (D) Represents design of experiment shown in Table S8-10, Figure 5 and Figure 7A. (E) Represents design of experiment shown in Table S11, and Figure 7B. (F) Represents design of experiment shown in Table S12-14, Figure 8-9.

Heterologous IVF of bovine oocytes, mouse oocytes, or empty mouse ZPs, using human, mouse, or cat sperm, before and after contact with oviductal fluid.

(A) Embryo cleavage rates resulting from the IVF of bovine oocytes with human, murine, or feline sperm including bovine sperm as homologous IVF control, and parthenogenesis as a negative control of the IVF. (B) Sperm penetration rate after the IVF of IVM mouse oocytes with murine or bovine sperm, for homologous and heterologous IVF, respectively. (C) Embryo cleavage rates resulting from the IVF of bovine IVM oocytes, preincubated 30 minutes with bovine oviductal fluid, with human sperm (IVF medium a = G-IVF™ PLUS medium (HeA); IVF medium b = Fert (HeB). (D) Penetration rates and average numbers of sperm bound to ZP after the empty zona penetration test (EZPT) using mouse ovarian IVM oocytes and murine or bovine sperm. (E) Penetration rates and average numbers of sperm bound to ZP after the EZPT using mouse oviductal oocytes and murine or bovine sperm. (F) Picture of an empty zona pellucida obtained from a mouse ovarian IVM oocyte after the EZPT using bovine sperm. Note the sperm has penetrated the zona. (G) Picture of an empty zona pellucida from a mouse oviductal oocyte after the EZPT using bovine sperm. Scale bar for ZP pictures = 50 µm. A non-fertilized parthenogenesis group is used as cleavage control in Fig. 2A and 2C. Different letters above error bars (mean ±SD) indicate significant differences (P < 0.05) among groups (ANOVA and Tukey’s post hoc test). Numbers of oocytes or ZPs used are indicated in Tables S1-S5).

Incubation of empty ZPs obtained from bovine ovarian oocytes with oviductal fluid determines the specificity of spermatozoa capable of penetrating the zona.

The empty zona penetration test (EZPT) in ZPs obtained from IVM bovine oocytes was performed after homologous (bovine sperm) or heterologous (human or murine sperm) fertilization. Similar outcomes were observed when ZPs were not treated with oviductal fluid (A), but after incubation with oviductal fluid for 30 minutes, human and murine sperm were unable to penetrate the bovine ZPs (B) (6 replicates per medium per semen sample). Different letters above error bars (mean ±SD) indicate significant differences (P < 0.05) among groups (ANOVA and Tukey’s post hoc test). Numbers of ZPs used are indicated in Table S6). (C, D, I) Representative pictures of empty bovine ZPs penetrated by bovine sperm illustrating that the penetrating sperm (J) lack an acrosome, while those unable to penetrate the zona maintain the acrosome (K). (E, F, L) Empty bovine ZP penetrated by human sperm revealing that the penetrating sperm have lost the acrosome (M), whereas those not penetrating the zona maintain the acrosome (N). (G, H, O) Representative pictures of empty bovine ZPs penetrated by murine sperm illustrating that the penetrating sperm (P) lack an acrosome, while those unable to penetrate the zona maintain the acrosome (Q). In the absence of oviductal fluid, the empty bovine ZP can be penetrated by bovine (C, I), human (E, L), or murine (G, O) sperm; however, when the ZP has been in contact with oviductal fluid, it can only be penetrated by bovine sperm (D), and not by human (F) or mouse (H) sperm. Scale bar for ZP pictures = 50 µm. Scale bar for sperm pictures = 5 µm.

Structure of oviductin, Western blots of OVGP1 recombinants and localization of these recombinants at the bovine or murine ZPs.

(A) Diagram showing the five regions (A, B, C, D and E) present in some of the oviductin proteins of human (hOVGP1), murine (mOVGP1), and bovine (bOVGP1) mammalian species (Figure taken from (29)). (B) Western blots of the three OVGP1 recombinants proteins used in this study (human, murine, and bovine). Proteins were expressed in mammalian cells, separated by SDS-PAGE and analyzed by immunoblotting using rabbit polyclonal antibody to the human OVGP1. Oviductal fluid of mice in oestrus and anoestrus indicating the presence in estrous of the OVGP1 band. Oviductal fluid from ovulated cows or anoestrus cows. ZPs from IVM murine (C) and bovine (D) oocytes were incubated for 30 minutes at RT with recombinants bOVGP1, mOVGP1, and hOVGP1. ZPs were fixed and imaged by confocal fluorescence and DIC microscopy using rabbit polyclonal antibody to the human OVGP1 for bOVGP1, and a monoclonal antibody against Flag-tag for hOVGP1 and mOVGP1. Scale bars = 20 μm.

Incubation of empty ZPs obtained from bovine ovarian oocytes with OVGP1 determines the specificity of spermatozoa capable of penetrating the zona.

The empty zona penetration test (EZPT) was performed after homologous (bovine sperm) or heterologous (human or murine sperm) fertilization. Similar penetration rates were observed when ZPs were not treated with bOVGP1 protein (A), but after incubation with bOVGP1 for 30 minutes, human or murine sperm were unable to penetrate the bovine ZPs (B). A drastic reduction was also observed for fertilization by heterologous sperm, both in the average number of sperm penetrating the ZPs, and in the average of number of sperm binding to the ZPs, when fertilization without pretreatment of the ZP with bOVGP1 (A) was compared to fertilization after the zona had been in contact with bOVGP1 (B). (6 replicates per medium per semen sample). Different letters above error bars (mean ±SD) indicate significant differences (P < 0.05) among groups (ANOVA and Tukey’s post hoc test). Numbers of ZPs used are indicated in Table S8).

Scanning electron micrographs (SEM) of the outer surface of the bovine ZP treated or not with OVGP1.

The ZP of an in vitro matured (IVM) bovine oocyte: Magnification x2000 (A) and x4000 (B); and bovine oocytes IVM in the presence of bovine OVGP1 (bOVGP1): Magnification x2000 (C) and x4000 (D). High magnification reveals the ultrastructural characteristics of the ZP’s pores on the IVM oocytes without bOVGP1 (B) and with bOVGP1 (D). Scale bars = 10 μm.

Species-specific OVGP1 confers complete species-specificity to the zona pellucida.

Experiment using the EZPT to analyze several combinations whereby ZPs from bovine and murine ovarian IVM oocytes were co-incubated with bovine, murine, or human oviductin, and exposed to sperm of all three species. The variables ZP penetration rate, average number of sperm penetrating the ZPs, and average number of sperm bound to ZPs were examined using bovine ZPs (A) or murine ZPs (B) and combinations of one of the three oviductins with sperm of one of the three species. Only by combining ZP, OVGP1 and sperm of the same species, was species-specific fertilization possible. However, when the same species was matched only with the oviductin and sperm, or only with the ZP and sperm, penetration and binding to the ZP of the spermatozoa also occurred, although in much smaller measure. No penetration was observed when only the ZP and OVGP1 species matched. Different letters above error bars (mean ±SD) indicate significant differences (P < 0.05) among groups (ANOVA and Tukey’s post hoc test). Numbers of ZPs used are indicated in Table S10-11).

Effect of neuraminidase (NMase) treatment of bovine or murine ZPs before and after contact with OVGP1 on sperm penetration.

Penetration and sperm binding rates were measured after homologous EZPT with bovine sperm using ZPs not subjected (A) or subjected (B) to 30 minutes of incubation with bOVGP1. Penetration and sperm binding rates were measured after homologous EZPT with murine sperm using ZPs not subjected (C) or subjected (D) to 30 minutes of incubation with mOVGP1. ZPs of both species were co-incubated with acetate buffer (PH 4.5) at 38°C for 18 hours in the presence or absence of neuraminidase diluted at 5 UI/mL. Different letters above error bars (mean ±SD) indicate significant differences (P < 0.05) among groups (ANOVA and Tukey’s post hoc test). Numbers of ZPs used are indicated in Table S12-13).

Effect of neuraminidase (NMase) treatment of OVGP1 on EZPT using bovine ZPs with homologous or heterologous sperm.

(A) Penetration rate, average number of sperm within penetrated ZPs, and average of number of sperm bound to ZPs were measured after homologous EZPT with bovine sperm, using ZPs untreated with NMase and bOVGP1 either treated or untreated with NMase. (B) Penetration rate, average number of sperm within penetrated ZPs, and average of number of sperm bound to ZPs were measured after homologous (bovine sperm) or heterologous (mouse sperm) EZPT, using ZPs untreated with NMase, either without bOVGP1 or with bOVGP1 treated or untreated with NMase. Numbers of ZPs used are indicated in Table S14

Model. Schematic representation of the heterologous fertilization model via EZPT under multiple conditions.

The figure illustrates how sperm penetration into the zona pellucida (ZP) occurs non-specifically among various mammalian species when the ZP has not been exposed to homologous oviductal fluid or OVGP1 (left diagram). However, this interaction becomes species-specific when the ZP is incubated with oviductal fluid or OVGP1 from the same species (central diagram); being this specificity lost when OVGP1 is heterologous to the ZP (right diagram).