Oviductin sets the species-specificity of the mammalian zona pellucida
- Department of Animal Reproduction, INIA-CSIC, Spain
- FivCenter Madrid Clinics, Aravaca,, Spain
- Dept. Agrarian Production, Technical University of Madrid, Spain
- Laboratorio de Biotecnología de la Reproducción Animal, Facultad de Ciencias Agropecuarias, Universidad de Cuenca (UC), Ecuador
- Programa de Medicina Veterinaria y Zootecnia, Grupo Kyron, Corporación Universitaria del Huila (CORHUILA), Colombia
- Department of Animal Medicine and Surgery, Veterinary Faculty, UCM, Spain
- Departamento de Biotecnología, INIA-CSIC, Spain
- Departamento de Biología Celular e Histología, Universidad de Murcia-Instituto Murciano de Investigación Biosanitaria Pascual Parrilla, Spain
Figures
Figure 1
Schematic representation of global experimental design of homologous and heterologous IVF from diverse mammalian species.
(A) Represents design of experiment shown in Appendix 1—table 1, Appendix 1—table 2, Appendix 1—table 3, Figure 2A and B, Figure 2—figure supplement 1, Figure 2—figure supplement 2 and Figure 2—figure supplement 3. (B) Represents design of experiment shown in Appendix 1—table 4, Figure 2C. (C) Represents design of experiment shown in Appendix 1—table 5, Appendix 1—table 6, Figure 2D and E and Figure 3. (D) Represents design of experiment shown in Appendix 1—table 8, Appendix 1—table 9, Appendix 1—table 10, Figure 5 and Figure 7A. (E) Represents design of experiment shown in Appendix 1—table 11, and Figure 7B. (F) Represents design of experiment shown in Appendix 1—table 12, Appendix 1—table 13, Appendix 1—table 14 and Figures 8 and 9.
Figure 2 with 4 supplements
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 min 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, bovine, or human sperm. (E) Penetration rates and average numbers of sperm bound to ZP after the EZPT using mouse oviductal oocytes and murine, bovine, or human 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 (A) and (C). 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 Appendix 1—table 1, Appendix 1—table 2, Appendix 1—table 3, Appendix 1—table 4 and Appendix 1—table 5.
Figure 2—figure supplement 1
Heterologous IVF between bovine oocytes and human sperm.
Sperm-oocyte binding, pronuclear formation, and cleavage after homologous (Ho) (bovine sperm) and heterologous (He) IVF: human sperm capacitated with G-IVF PLUS medium (HeA) or Tyrode’s medium (HeB). Gametes were stained with Hoechst 33342. Ho: Bound bovine sperm after 2.5 hr of co-incubation with zona-intact bovine oocytes (A); pronuclear formation at 6 hpi (B); and embryo cleavage at 48 hpi (C). HeA and HeB: Bound human sperm after 2.5 hr of co-incubation of the gametes (D, G); pronuclear formation at 18 hpi (E, H); and hybrid-embryo cleavage at 48 hpi (F, I), respectively. Arrow points to sperm head chromatin. Images were captured with a 63 X objective. Scale bar 20 µm.
Figure 2—figure supplement 2
Heterologous IVF between bovine oocytes and murine sperm.
Sperm-oocyte binding, pronuclear formation, and cleavage after homologous (Ho, bovine sperm) and heterologous (He, mouse/rodent sperm) IVF. Gametes were stained with Hoechst 33342. Ho: Bound bovine sperm after 2.5 hr of co-incubation with zona-intact bovine oocytes (A); pronuclear formation at 6 hpi (B); and embryo cleavage at 24 hpi (C). He: Bound mouse sperm after 2.5 hr of co-incubation of the gametes (D); pronuclear formation at 18 hpi (E); and hybrid-embryo cleavage at 24 hpi (F). Arrow points to sperm head chromatin. Images were captured with a 63 X objective. Scale bar 20 µm.
Figure 2—figure supplement 3
Heterologous IVF between bovine oocytes and cat sperm.
Sperm-oocyte binding, pronuclear formation, and cleavage after homologous (Ho) (bovine sperm) and heterologous (He) (cat sperm capacitated in Tyrode’s medium (HeG)) IVF. Gametes were stained with Hoechst 33342. Ho: Bound bovine sperm after 2.5 hr of co-incubation with zona-intact bovine oocytes (A); pronuclear formation at 6 hpi (B); and embryo cleavage at 48 hpi (C). HeG: Bound cat sperm after 2.5 hr of co-incubation of the gametes (D); pronuclear formation at 18 hpi (E); and hybrid-embryo cleavage at 48 hpi (F). Arrow points to sperm head chromatin. Images were captured with a 63 X objective. Scale bar 20 µm.
Figure 2—figure supplement 4
Steps (A–E) of the method used to empty the bovine zone pellucida by removing the cytoplasmic contents of the oocyte containing all the organelles, nucleus and membranes, and removing also the polar body.
Details of this procedure can be found in the Materials and methods.
Figure 3
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 min, human and murine sperm were unable to penetrate the bovine ZPs (B) (six 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 Appendix 1—table 6. (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.
Figure 4 with 3 supplements
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 (adapted from Figure 1 from Avilés et al., 2010). (B) Western blots of the three OVGP1 recombinants proteins used in this study (human, murine, and bovine). Recombinant bovine protein was expressed in BHK-21 cells and purified, whereas recombinant murine and human proteins were purchased by Origene and had been produced in HEK293T. Then, proteins were separated by SDS-PAGE and analyzed by immunoblotting using rabbit polyclonal antibody to the human OVGP1. The following lanes of the gel contain the protein extracts from oviductal fluid of female mice in oestrus and anoestrus and from oviductal fluid of ovulated cows or anoestrus cows, indicating with an arrow the presence in estrous of the OVGP1 band for both species. ZPs from IVM murine (C) and bovine (D) oocytes were incubated for 30 min 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.
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Figure 4—source data 1
Labeled data from Figure 4B.
- https://cdn.elifesciences.org/articles/101338/elife-101338-fig4-data1-v1.zip
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Figure 4—source data 2
Raw data from Figure 4B.
- https://cdn.elifesciences.org/articles/101338/elife-101338-fig4-data2-v1.zip
Figure 4—figure supplement 1
Sequence alignments of OVGP1 from Homo sapiens (NCBI AAI36407.1), Bos taurus (NP_001073685.1), and Mus musculus (AAI37996.1).
Sequences were aligned with the tool MUSCLE (Multiple Sequence Comparison by Log-Expectation) using Snapgene software. Consensus conserved amino acids are represented in the colored blocks: red = score over the 50% threshold and blue = less conserved. Amino acids corresponding to the consensus are indicated in light pink. N-glycosylation sites were predicted using the NetNGlyc 1.0 Server of DTU Health Tech Bioinformatic Services. The dark green boxes indicate possible asparagine (N) glycosylation. Residues adjacent to N in the sequons Asn-Xaa-Ser/Thr are circled in dark pink. Chitin-binding sites predicted by the UniProt server appear in turquoise boxes. Orange boxes locate the essential glutamic acid of CH18 family members.
Figure 4—figure supplement 2
Recombinant OVGP1 from human, murine and bovine recognized with anti-OVGP1 (A) and anti-His/anti-Flag (B).
Panel A is taken directly from Figure 4 in order to compare antibody recognition. In B, is represented the expression of recombinant proteins detected with an anti-His and anti-Flag co-incubated antibodies and developed simultaneously. Flag is detecting hOVGP1 and mOVGP1, while His is detecting bOVGP1.
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Figure 4—figure supplement 2—source data 1
Labeled data from Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/101338/elife-101338-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
Raw data from Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/101338/elife-101338-fig4-figsupp2-data2-v1.zip
Figure 4—figure supplement 3
Representation of immunofluorescence of control ZPs of IVM murine and bovine oocytes, ZPs incubated with murine or bovine oviductal fluid, and ZPs from murine superovulated (SOV).
IVM murine (A) and bovine (B) ZPs were fixed and imaged by conventional fluorescence and DIC microscopy using rabbit polyclonal antibody to the human OVGP1 (I, II) or a mouse monoclonal antibody against Flag-tag (III, IV). IVM ZPs from murine and bovine oocytes were incubated for 30 min with murine or bovine oviductal fluid (respectively) and imaged using anti-Flag tag (V, 1452 54 VI). ZPs obtained from oocytes of the oviducts of superovulated mice females (SOV) incubated with anti-OVGP1 (VII). Scale bars = 20 μm.
Figure 5
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 min, 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 Appendix 1—table 8.
Figure 6 with 1 supplement
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.
Figure 6—figure supplement 1
Scanning electron micrographs (SEM) of the outer surface of the bovine ZP.
SEM of the outer surface of the bovine ZP treated (B) or not (A) with bovine OF, mOVGP1 (C), or hOVGP1 (D). (E) Image showing bovine sperm on the surface of the bovine ZP after having been in contact with OVGP1, showing the comparison between the size of the sperm and the size of the pores of the ZP.
Figure 7
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 Appendix 1—table 10, Appendix 1—table 11.
Figure 8
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 min 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 min of incubation with mOVGP1. ZPs of both species were co-incubated with acetate buffer (PH 4.5) at 38 °C for 18 hr 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 Appendix 1—table 12, Appendix 1—table 13.
Figure 9
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 Appendix 1—table 14.
Figure 10
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) This specificity is lost when OVGP1 is heterologous to the ZP (right diagram).
Tables
Appendix 1—table 1
Rates of sperm-ZP binding, pronuclear formation, and cleavage after homologous (bovine sperm) and heterologous (human sperm, media A or B) co-incubation with bovine ovarian oocytes recorded at different times post-insemination.
| Semen group (N) | Sperm binding | Pronuclear formation | Cleavage rate | |||
|---|---|---|---|---|---|---|
| 2.5 hpi, n | 6 hpi, n (%) | 12 hpi, n (%) | 18 hpi, n (%) | 22 hpi, n (%) | 48 hpi, n (%) | |
| Ho (133) | 32 (0.5±0.17) | 36 (72.1±7.3)a | 65 (86.0±7.8)a | |||
| HeA (200) | 31 (0.3±0.17) | 32 (34.4±4,7) | 35 (25.5±6.1) | 31 (54.8±7.1)b | 30 (56.6±8.1) | 41 (51.2±8.3)b |
| HeB (236) | 33 (0.2±0.17) | 36 (30.5±5.4) | 37 (32.1±6.8) | 36 (33.5±7.3)c | 38 (34.3±6.6) | 56 (34.4±7.1)b |
| Parth (28) | 28 (3.3±3.6)c | |||||
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Ho = homologous IVF with bovine sperm; HeA = heterologous IVF with human sperm using G-IVF PLUS medium; HeB = heterologous IVF with human sperm using Fert medium; Parth = parthenogenic non-fertilized oocytes. Sperm binding was expressed as the average number of spermatozoa that remained bound to the ZP. ‘N’ refers to the total number of oocytes fertilized per treatment or non-fertilized oocytes (Parth); ‘n’ refers to the total number of fertilized oocytes/presumptive zygotes or non-fertilized oocytes (Parth) recorded at each time point to determine rates of sperm binding, pronuclear formation, and embryo cleavage. Pronuclear formation was expressed as the percentage of oocytes with two pronuclei at each time point over the number (n) of oocytes examined. Cleavage rate was expressed as the percentage of two-cell embryos over the number (n) of fertilized or non-fertilized (Parth) oocytes. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Significance level was set at p<0.05. Different superscripts (a, b, c) in the same column indicate significant differences (p<0.05; n=3); Values are expressed as the mean ± SD.
Appendix 1—table 2
Rates of sperm-ZP binding, pronuclear formation, and cleavage after homologous (bovine sperm) and heterologous (murine sperm) co-incubation with bovine ovarian oocytes at different times post-insemination.
| Semen group (N) | Sperm binding | Pronuclear formation | Cleavage rate | ||
|---|---|---|---|---|---|
| 2.5 hpi, n | 18 hpi, n (%) | 20 hpi, n (%) | 26 hpi, n (%) | 48 hpi, n (%) | |
| Ho (231) | 46 (0.6±1.0) | 86 (75.8±10.21)a | 99 (81.8±1.9)a | ||
| He (436) | 47 (0.6±1.0) | 86 (44.3±6.7)b | 87 (44.9±8.8) | 85 (44.7±3.6) | 131 (52.6±3.1)b |
| Parth (43) | 43 (4.4±8.7)c | ||||
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Ho = homologous IVF with bovine sperm; He = heterologous IVF with murine sperm. Parth = parthenogenic non-fertilized oocytes. Sperm binding was expressed as the average number of spermatozoa that remained bound to the ZP. ‘N’ refers to the total number of oocytes fertilized per treatment or non-fertilized oocytes (Parth); ‘n’ refers to the total number of fertilized oocytes/presumptive zygotes or non-fertilized oocytes (Parth) recorded at each time point to determine rates of sperm binding, pronuclear formation, and embryo cleavage. Pronuclear formation was expressed as the percentage of oocytes with two pronuclei at each time point over the number (n) of oocytes examined. Cleavage rate was expressed as the percentage of two- cell embryos over the number (n) of fertilized or non-fertilized (Parth) oocytes. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Significance level was set at p<0.05. Different superscripts (a, b, c) in the same column indicate significant differences (p<0.05; n=3); Values are expressed as the mean ± SD.
Appendix 1—table 3
Rates of sperm-ZP binding, pronuclear formation, and embryo cleavage after homologous (bovine sperm) and heterologous (cat sperm) co-incubation with bovine ovarian oocytes at different times post-insemination.
| Semen group (N) | Sperm binding | Pronuclear formation | Cleavage | |
|---|---|---|---|---|
| 2.5 hpi, n | 6 hpi, n (%) | 18 hpi, n (%) | 48 hpi, n (%) | |
| Ho (184) | 43 (0.5±0.9) | 62 (71.0±2.3)a | 79 (83.4±1.7)a | |
| He (259) | 41 (0.2±0.7) | 48 (31.3±17.14) | 58 (34.1±8.8)b | 112 (41.3±2.1)b |
| Parth (44) | 44 (2.2±6.6)c | |||
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Ho = homologous IVF with bovine sperm; He = heterologous IVF with cat sperm using Tyrode’s medium; Parth = parthenogenic non-fertilized oocytes. Sperm binding was expressed as the average number of spermatozoa that remained bound to the ZP. ‘N’ refers to the total number of oocytes fertilized per treatment or non-fertilized oocytes (Parth); ‘n’ refers to the total number of fertilized oocytes/presumptive zygotes or non-fertilized oocytes (Parth) recorded at each time point to determine rates of sperm-ZP binding, pronuclear formation, and cleavage. Pronuclear formation was expressed as the percentage of oocytes with two pronuclei at each time point over the number (n) of oocytes examined. Cleavage rate was expressed as the percentage of two-cell embryos over the number (n) of fertilized or non-fertilized (Parth) oocytes. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Significance level was set at p<0.05. Different superscripts (a, b, c) in the same column indicate significant differences (p<0.05; n=3); Values are expressed as the mean ± SD.
Appendix 1—table 4
Rates of sperm-ZP binding, pronuclear formation, and cleavage after homologous (bovine sperm) and heterologous (human sperm, media A or B) co-incubation with bovine ovarian oocytes (previously incubated with oviductal fluid for 30 min) at different times post-insemination.
| Semen group (N) | Sperm binding | Pronuclear formation | Cleavage | |||
|---|---|---|---|---|---|---|
| 2.5 hpi n | 6 hpi n (%) | 12 hpi n (%) | 18 hpi n (%) | 22 hpi n (%) | 48 hpi n (%) | |
| Ho (135) | 32 (0.5±0.9)a | 33 (66.7±3.6)a | 70 (85.7±3.4)a | |||
| HeA (267) | 34 (0.01±0.2)b | 38 (0.0±0.0) | 43 (0.0±0.0) | 49 (0.0±0.0)b | 44 (0.0±0.0) | 59 (3.3±2.6)b |
| HeB (235) | 31 (0.03±0.2)b | 36 (0.0±0.0) | 34 (0.0±0.0) | 36 (0.0±0.0)b | 36 (0.0±0.0) | 62 (3.1±4.5)b |
| Parth (46) | 28 (1.8±4.1)b | |||||
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Ho = homologous IVF with bovine sperm; HeA = heterologous IVF with human sperm in IVF-G medium; HeB = heterologous IVF with human sperm in Fert medium; Parth = parthenogenic non-fertilized oocytes. ‘N’ refers to the total number of oocytes fertilized per treatment or non-fertilized oocytes (Parth); ‘n’ refers to the total number of fertilized oocytes/presumptive zygotes or non-fertilized oocytes (Parth) recorded at each time point to determine rates of sperm-ZP binding, pronuclear formation and cleavage. Sperm binding was expressed as the average number of spermatozoa that remained bound to the ZP. Pronuclear formation was expressed as the percentage of oocytes with two pronuclei at each time point over the number (n) of oocytes examined. Cleavage rate was expressed as the percentage of two-cell embryos over the number (n) of fertilized or non fertilized (Parth) oocytes. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 5
Rates of sperm penetration and binding to the murine ZP after the homologous (murine sperm) and heterologous (bovine sperm) EZPT using ZPs prepared from oocytes from murine ovaries (HoA, HeA) and from the oviducts of superovulated female mice (HoB, HeB).
| Group N | Sperm | ZPs from murine oocytes | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|
| HoA 56 | Murine | Ovary | 8.76±1.07 | 56 (100±0)a | 848 (15.08±1.19)a | 29 ± 5.50a | 56 (100±0)a | 1042 (19.02±2.21)a | 32 ± 2.06a |
| HeAb 97 | Bovine | Ovary | 3.67±0.19 | 56 (57.85±5.45)b | 167 (2.98±0.02)b | 10±1.4b | 91 (93.87±2.65)b | 303 (3.33±0.52)b | 9±1.42b |
| HeAh 85 | Human | Ovary | 6.12±0.17 | 60 (70.83±5.89)b | 177 (2.95±0,07)b | 9±0.71b | 79 (92.92±0.59)b | 263 (3.34±0.35)b | 11±1.41b |
| HoB 37 | Murine | Oviduct | 8.76±1.07 | 37 (100±0)a | 423 (23.86±0.79)a | 18 ± 4.24a | 37 (100±0)a | 645 (10.02±1.81)a | 28 ± 2.83a |
| HeBb 131 | Bovine | Oviduct | 3.67±0.19 | 3 (6.08±1.36)c | 3 (1±0)c | 1 ± 0c | 21 (21.46±2.06)c | 62 (2.96±0.33)c | 4 ± 0.71c |
| HeBh 65 | Human | Oviduct | 6.12±0.17 | 3 (4.72±0,39)c | 3 (0.05±0.01)c | 1 ± 0c | 14 (22.50±3.54)c | 39 (2.70±0.42)c | 5 ± 2.83c |
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HoA = homologous EZPT with murine sperm (oocytes obtained from ovary). HeAb = heterologous EZPT with bovine sperm (oocytes obtained from ovary). HeAh = heterologous EZPT with human sperm (oocytes obtained from ovary). HoB = homologous EZPT with murine sperm (oocytes obtained from the oviduct of SOV females). HeBb = heterologous EZPT with bovine sperm (oocytes obtained from the oviduct of SOV females). HeBh = heterologous EZPT with human sperm (oocytes obtained from the oviduct of SOV females). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZPs refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZP with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. ‘N’ refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b, c) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 6
Rates of sperm penetration and binding to the bovine ZP after homologous (bovine sperm) and heterologous (human or murine sperm) EZPT using bovine ZPs co-incubated without bovine oviductal fluid (HoA, HeA) or with bovine oviductal fluid (HoB, HeB).
| Group N | Sperm | Oviductal fluid | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|
| HoA 90 | Bovine | - | 4.16±0.27 | 90 (100±0)a | 853 (9.13±1.23)a | 30 ± 1.41a | 90 (100±0)a | 830 (9.27±0.58)a | 23 ± 1.12a |
| HeAh 406 | Human | - | 9.14±3.98 | 283 (69.7±12.1)b | 1588 (5.61±2.83)b | 23±4.2b | 96 (23.65±8.59)b | 188 (1.49±0.24)b | 6±1.27b |
| HeAm 83 | Murine | - | 8.76±1.07 | 80 (96.25±5.3)a | 300 (3.73±0.31)C | 21±2.12b | 83 (100±0)a | 490 (5.92±0.81)c | 20 ± 3.54a |
| HoB 56 | Bovine | + | 4.16±0.27 | 56 (100±0)a | 585 (10.52±0.56)a | 24±2.82b | 56 (100±0)a | 574 (10.02±1.81)a | 23 ± 2.12a |
| HeBh 131 | Human | + | 9.14±3.98 | 0 (0±0)c | 0 (0±0)c | 0 ± 0c | 10 (7.42±2.53)c | 10 (1±0)d | 1 ± 0c |
| HeBm 48 | Murine | + | 9.71±0.61 | 4 (0.08±0.005)c | 4 (1±0)c | 1 ± 0c | 20 (41.74±2.46)b | 45 (0.94±0.08)d | 5±0.71b |
-
HoA = homologous EZPT with bovine sperm. HeAh = heterologous EZPT with human sperm. HeAm = heterologous EZPT with murine sperm. HoB = homologous EZPT with bovine sperm (ZPs were co-incubated with bovine oviductal fluid for 30 min). HeBh = heterologous EZPT with human sperm (ZPs were co-incubated with bovine oviductal fluid for 30 min). HeBm = heterologous EZPT with murine sperm (ZPs were co-incubated with bovine oviductal fluid for 30 min). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZPs refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZPs with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. ‘N’ refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b, c, d) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 7
O-glycosylation prediction sites in human, mouse and bull OVGP1.
Identification and location of the GalNAc―Type O-glycosylation sites with the online server NetOGlyc 4.0 (Steentoft et al., 2013).
| Start | End | Score | Comment | Start | End | Score | Comment | Start | End | Score | Comment | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HOMO | 29 | 29 | 0.148743 | MUS | 17 | 17 | 0.0664484 | BOS | 2 | 2 | 0.477639 | |||
| HOMO | 34 | 34 | 0.513945 | + | MUS | 20 | 20 | 0.0136154 | BOS | 33 | 33 | 0.0141907 | ||
| HOMO | 40 | 40 | 0.133798 | MUS | 29 | 29 | 0.13254 | BOS | 36 | 36 | 0.0521786 | |||
| HOMO | 52 | 52 | 0.0144943 | MUS | 34 | 34 | 0.5 | + | BOS | 44 | 44 | 0.211234 | ||
| HOMO | 60 | 60 | 0.0428766 | MUS | 40 | 40 | 0.153781 | BOS | 48 | 48 | 0.201186 | |||
| HOMO | 93 | 93 | 0.0158923 | MUS | 52 | 52 | 0.0118199 | BOS | 49 | 49 | 0.283351 | |||
| HOMO | 96 | 96 | 0.0261905 | MUS | 60 | 60 | 0.0436814 | BOS | 52 | 52 | 0.124074 | |||
| HOMO | 104 | 104 | 0.216291 | MUS | 62 | 62 | 0.0894996 | BOS | 53 | 53 | 0.100269 | |||
| HOMO | 105 | 105 | 0.252093 | MUS | 93 | 93 | 0.0153551 | BOS | 55 | 55 | 0.207505 | |||
| HOMO | 108 | 108 | 0.14834 | MUS | 96 | 96 | 0.0240849 | BOS | 62 | 62 | 0.226985 | |||
| HOMO | 109 | 109 | 0.315057 | MUS | 104 | 104 | 0.143094 | BOS | 63 | 63 | 0.0478178 | |||
| HOMO | 112 | 112 | 0.180764 | MUS | 105 | 105 | 0.199945 | BOS | 70 | 70 | 0.0242045 | |||
| HOMO | 113 | 113 | 0.114855 | MUS | 108 | 108 | 0.111836 | BOS | 88 | 88 | 0.081231 | |||
| HOMO | 123 | 123 | 0.030659 | MUS | 112 | 112 | 0.0957522 | BOS | 95 | 95 | 0.0428404 | |||
| HOMO | 126 | 126 | 0.0314989 | MUS | 113 | 113 | 0.100753 | BOS | 114 | 114 | 0.078206 | |||
| HOMO | 130 | 130 | 0.0220408 | MUS | 123 | 123 | 0.00798697 | BOS | 122 | 122 | 0.240522 | |||
| HOMO | 148 | 148 | 0.0445812 | MUS | 126 | 126 | 0.0128272 | BOS | 126 | 126 | 0.474913 | |||
| HOMO | 155 | 155 | 0.0372563 | MUS | 148 | 148 | 0.0325529 | BOS | 149 | 149 | 0.0380635 | |||
| HOMO | 174 | 174 | 0.0488918 | MUS | 174 | 174 | 0.0484669 | BOS | 152 | 152 | 0.00857951 | |||
| HOMO | 182 | 182 | 0.160501 | MUS | 182 | 182 | 0.166503 | BOS | 158 | 158 | 0.0631895 | |||
| HOMO | 186 | 186 | 0.462315 | MUS | 186 | 186 | 0.388758 | BOS | 163 | 163 | 0.275655 | |||
| HOMO | 194 | 194 | 0.11187 | MUS | 190 | 190 | 0.0693216 | BOS | 167 | 167 | 0.487753 | |||
| HOMO | 195 | 195 | 0.0571954 | MUS | 194 | 194 | 0.0973003 | BOS | 171 | 171 | 0.482996 | |||
| HOMO | 212 | 212 | 0.00914751 | MUS | 195 | 195 | 0.057626 | BOS | 178 | 178 | 0.0814828 | |||
| HOMO | 218 | 218 | 0.0565867 | MUS | 212 | 212 | 0.00829588 | BOS | 179 | 179 | 0.348292 | |||
| HOMO | 223 | 223 | 0.195033 | MUS | 218 | 218 | 0.047986 | BOS | 202 | 202 | 0.139565 | |||
| HOMO | 227 | 227 | 0.375705 | MUS | 223 | 223 | 0.18869 | BOS | 206 | 206 | 0.199712 | |||
| HOMO | 231 | 231 | 0.310035 | MUS | 227 | 227 | 0.246007 | BOS | 213 | 213 | 0.5 | + | ||
| HOMO | 238 | 238 | 0.115751 | MUS | 231 | 231 | 0.246397 | BOS | 226 | 226 | 0.696097 | + | ||
| HOMO | 239 | 239 | 0.335736 | MUS | 236 | 236 | 0.17176 | BOS | 231 | 231 | 0.428867 | |||
| HOMO | 253 | 253 | 0.0093353 | MUS | 238 | 238 | 0.102913 | BOS | 276 | 276 | 0.0105995 | |||
| HOMO | 262 | 262 | 0.09816 | MUS | 239 | 239 | 0.289418 | BOS | 296 | 296 | 0.0180258 | |||
| HOMO | 266 | 266 | 0.142253 | MUS | 251 | 251 | 0.029901 | BOS | 309 | 309 | 0.0717533 | |||
| HOMO | 273 | 273 | 0.4854 | MUS | 262 | 262 | 0.133467 | BOS | 317 | 317 | 0.183042 | |||
| HOMO | 286 | 286 | 0.829831 | + | MUS | 273 | 273 | 0.193716 | BOS | 328 | 328 | 0.222882 | ||
| HOMO | 291 | 291 | 0.174597 | MUS | 279 | 279 | 0.662371 | + | BOS | 329 | 329 | 0.779465 | + | |
| HOMO | 304 | 304 | 0.0267057 | MUS | 281 | 281 | 0.959249 | + | BOS | 330 | 330 | 0.813643 | + | |
| HOMO | 336 | 336 | 0.0314593 | MUS | 286 | 286 | 0.791757 | + | BOS | 332 | 332 | 0.728598 | + | |
| HOMO | 338 | 338 | 0.0119623 | MUS | 291 | 291 | 0.32144 | BOS | 338 | 338 | 0.930032 | + | ||
| HOMO | 356 | 356 | 0.0239896 | MUS | 294 | 294 | 0.286168 | BOS | 339 | 339 | 0.896458 | + | ||
| HOMO | 365 | 365 | 0.131311 | MUS | 304 | 304 | 0.048395 | BOS | 343 | 343 | 0.821513 | + | ||
| HOMO | 369 | 369 | 0.120416 | MUS | 334 | 334 | 0.00966718 | BOS | 344 | 344 | 0.892312 | + | ||
| HOMO | 388 | 388 | 0.491023 | MUS | 336 | 336 | 0.0104758 | BOS | 352 | 352 | 0.862842 | + | ||
| HOMO | 389 | 389 | 0.824032 | + | MUS | 338 | 338 | 0.0196048 | BOS | 354 | 354 | 0.816318 | + | |
| HOMO | 390 | 390 | 0.6421 | + | MUS | 356 | 356 | 0.0175912 | BOS | 358 | 358 | 0.767588 | + | |
| HOMO | 391 | 391 | 0.739353 | + | MUS | 365 | 365 | 0.035171 | BOS | 359 | 359 | 0.60832 | + | |
| HOMO | 398 | 398 | 0.757269 | + | MUS | 385 | 385 | 0.265295 | BOS | 373 | 373 | 0.426953 | ||
| HOMO | 399 | 399 | 0.872818 | + | MUS | 387 | 387 | 0.549473 | + | BOS | 375 | 375 | 0.726137 | + |
| HOMO | 403 | 403 | 0.797847 | + | MUS | 389 | 389 | 0.61239 | + | BOS | 379 | 379 | 0.817664 | + |
| HOMO | 404 | 404 | 0.793866 | + | MUS | 390 | 390 | 0.360483 | BOS | 381 | 381 | 0.78998 | + | |
| HOMO | 405 | 405 | 0.955482 | + | MUS | 398 | 398 | 0.76942 | + | BOS | 383 | 383 | 0.829896 | + |
| HOMO | 406 | 406 | 0.740014 | + | MUS | 399 | 399 | 0.793433 | + | BOS | 385 | 385 | 0.746896 | + |
| HOMO | 414 | 414 | 0.978317 | + | MUS | 400 | 400 | 0.887109 | + | BOS | 392 | 392 | 0.65328 | + |
| HOMO | 415 | 415 | 0.912469 | + | MUS | 404 | 404 | 0.813503 | + | BOS | 394 | 394 | 0.763928 | + |
| HOMO | 418 | 418 | 0.926281 | + | MUS | 408 | 408 | 0.793101 | + | BOS | 396 | 396 | 0.718114 | + |
| HOMO | 419 | 419 | 0.913712 | + | MUS | 411 | 411 | 0.761464 | + | BOS | 399 | 399 | 0.571659 | + |
| HOMO | 421 | 421 | 0.780885 | + | MUS | 414 | 414 | 0.725498 | + | BOS | 404 | 404 | 0.875147 | + |
| HOMO | 433 | 433 | 0.403671 | MUS | 418 | 418 | 0.690982 | + | BOS | 411 | 411 | 0.852652 | + | |
| HOMO | 443 | 443 | 0.803539 | + | MUS | 419 | 419 | 0.677789 | + | BOS | 413 | 413 | 0.827119 | + |
| HOMO | 445 | 445 | 0.760132 | + | MUS | 421 | 421 | 0.746303 | + | BOS | 420 | 420 | 0.640815 | + |
| HOMO | 449 | 449 | 0.950001 | + | MUS | 433 | 433 | 0.757383 | + | BOS | 421 | 421 | 0.566064 | + |
| HOMO | 450 | 450 | 0.883589 | + | MUS | 434 | 434 | 0.316116 | BOS | 428 | 428 | 0.77777 | + | |
| HOMO | 452 | 452 | 0.897374 | + | MUS | 444 | 444 | 0.751447 | + | BOS | 441 | 441 | 0.403374 | |
| HOMO | 454 | 454 | 0.952252 | + | MUS | 445 | 445 | 0.394782 | BOS | 446 | 446 | 0.770962 | + | |
| HOMO | 457 | 457 | 0.844377 | + | MUS | 448 | 448 | 0.787216 | + | BOS | 459 | 459 | 0.760108 | + |
| HOMO | 459 | 459 | 0.950975 | + | MUS | 451 | 451 | 0.5 | + | BOS | 460 | 460 | 0.831493 | + |
| HOMO | 464 | 464 | 0.852675 | + | MUS | 453 | 453 | 0.541804 | + | BOS | 462 | 462 | 0.757798 | + |
| HOMO | 471 | 471 | 0.696304 | + | MUS | 457 | 457 | 0.835698 | + | BOS | 466 | 466 | 0.653766 | + |
| HOMO | 474 | 474 | 0.846582 | + | MUS | 459 | 459 | 0.749447 | + | BOS | 468 | 468 | 0.823171 | + |
| HOMO | 478 | 478 | 0.776336 | + | MUS | 466 | 466 | 0.72813 | + | BOS | 475 | 475 | 0.116021 | |
| HOMO | 480 | 480 | 0.745116 | + | MUS | 471 | 471 | 0.756103 | + | |||||
| HOMO | 481 | 481 | 0.794363 | + | MUS | 478 | 478 | 0.539962 | + | |||||
| HOMO | 486 | 486 | 0.819105 | + | MUS | 480 | 480 | 0.748887 | + | |||||
| HOMO | 488 | 488 | 0.884396 | + | MUS | 481 | 481 | 0.616459 | + | |||||
| HOMO | 495 | 495 | 0.723935 | + | MUS | 486 | 486 | 0.774382 | + | |||||
| HOMO | 501 | 501 | 0.820028 | + | MUS | 488 | 488 | 0.664138 | + | |||||
| HOMO | 503 | 503 | 0.97727 | + | MUS | 489 | 489 | 0.858047 | + | |||||
| HOMO | 504 | 504 | 0.886534 | + | MUS | 490 | 490 | 0.843136 | + | |||||
| HOMO | 508 | 508 | 0.780642 | + | MUS | 493 | 493 | 0.871285 | + | |||||
| HOMO | 510 | 510 | 0.739161 | + | MUS | 495 | 495 | 0.77954 | + | |||||
| HOMO | 511 | 511 | 0.895653 | + | MUS | 496 | 496 | 0.965151 | + | |||||
| HOMO | 516 | 516 | 0.863073 | + | MUS | 497 | 497 | 0.917272 | + | |||||
| HOMO | 518 | 518 | 0.913466 | + | MUS | 500 | 500 | 0.91103 | + | |||||
| HOMO | 523 | 523 | 0.767302 | + | MUS | 502 | 502 | 0.736343 | + | |||||
| HOMO | 525 | 525 | 0.753387 | + | MUS | 503 | 503 | 0.961134 | + | |||||
| HOMO | 531 | 531 | 0.808482 | + | MUS | 504 | 504 | 0.901609 | + | |||||
| HOMO | 533 | 533 | 0.898742 | + | MUS | 507 | 507 | 0.921979 | + | |||||
| HOMO | 536 | 536 | 0.828278 | + | MUS | 509 | 509 | 0.729352 | + | |||||
| HOMO | 539 | 539 | 0.891049 | + | MUS | 510 | 510 | 0.950811 | + | |||||
| HOMO | 541 | 541 | 0.95368 | + | MUS | 511 | 511 | 0.896131 | + | |||||
| HOMO | 545 | 545 | 0.897366 | + | MUS | 514 | 514 | 0.889054 | + | |||||
| HOMO | 546 | 546 | 0.894875 | + | MUS | 516 | 516 | 0.727671 | + | |||||
| HOMO | 548 | 548 | 0.726574 | + | MUS | 517 | 517 | 0.975857 | + | |||||
| HOMO | 554 | 554 | 0.7768 | + | MUS | 518 | 518 | 0.936021 | + | |||||
| HOMO | 556 | 556 | 0.938298 | + | MUS | 521 | 521 | 0.906595 | + | |||||
| HOMO | 561 | 561 | 0.917442 | + | MUS | 523 | 523 | 0.623161 | + | |||||
| HOMO | 575 | 575 | 0.93519 | + | MUS | 524 | 524 | 0.94394 | + | |||||
| HOMO | 578 | 578 | 0.910072 | + | MUS | 525 | 525 | 0.898366 | + | |||||
| HOMO | 582 | 582 | 0.93418 | + | MUS | 528 | 528 | 0.923861 | + | |||||
| HOMO | 584 | 584 | 0.85508 | + | MUS | 531 | 531 | 0.871652 | + | |||||
| HOMO | 589 | 589 | 0.855402 | + | MUS | 535 | 535 | 0.672261 | + | |||||
| HOMO | 598 | 598 | 0.640589 | + | MUS | 537 | 537 | 0.382735 | ||||||
| HOMO | 599 | 599 | 0.929108 | + | MUS | 542 | 542 | 0.777675 | + | |||||
| HOMO | 603 | 603 | 0.806488 | + | MUS | 545 | 545 | 0.908425 | + | |||||
| HOMO | 623 | 623 | 0.774533 | + | MUS | 549 | 549 | 0.882743 | + | |||||
| HOMO | 624 | 624 | 0.894222 | + | MUS | 551 | 551 | 0.683389 | + | |||||
| HOMO | 625 | 625 | 0.890486 | + | MUS | 553 | 553 | 0.925689 | + | |||||
| HOMO | 634 | 634 | 0.826692 | + | MUS | 556 | 556 | 0.931487 | + | |||||
| HOMO | 650 | 650 | 0.733632 | + | MUS | 558 | 558 | 0.691547 | + | |||||
| HOMO | 651 | 651 | 0.915455 | + | MUS | 559 | 559 | 0.971129 | + | |||||
| HOMO | 654 | 654 | 0.858169 | + | MUS | 560 | 560 | 0.938113 | + | |||||
| HOMO | 656 | 656 | 0.926624 | + | MUS | 563 | 563 | 0.954235 | + | |||||
| HOMO | 659 | 659 | 0.87521 | + | MUS | 566 | 566 | 0.967467 | + | |||||
| HOMO | 660 | 660 | 0.942906 | + | MUS | 567 | 567 | 0.932682 | + | |||||
| HOMO | 663 | 663 | 0.864385 | + | MUS | 570 | 570 | 0.951799 | + | |||||
| HOMO | 672 | 672 | 0.582959 | + | MUS | 572 | 572 | 0.839915 | + | |||||
| MUS | 573 | 573 | 0.963663 | + | ||||||||||
| MUS | 574 | 574 | 0.928422 | + | ||||||||||
| MUS | 577 | 577 | 0.899768 | + | ||||||||||
| MUS | 579 | 579 | 0.767341 | + | ||||||||||
| MUS | 580 | 580 | 0.946279 | + | ||||||||||
| MUS | 581 | 581 | 0.89457 | + | ||||||||||
| MUS | 584 | 584 | 0.886062 | + | ||||||||||
| MUS | 586 | 586 | 0.83614 | + | ||||||||||
| MUS | 587 | 587 | 0.939191 | + | ||||||||||
| MUS | 588 | 588 | 0.905539 | + | ||||||||||
| MUS | 591 | 591 | 0.880483 | + | ||||||||||
| MUS | 593 | 593 | 0.856288 | + | ||||||||||
| MUS | 594 | 594 | 0.959942 | + | ||||||||||
| MUS | 595 | 595 | 0.898068 | + | ||||||||||
| MUS | 598 | 598 | 0.773555 | + | ||||||||||
| MUS | 600 | 600 | 0.838638 | + | ||||||||||
| MUS | 601 | 601 | 0.945883 | + | ||||||||||
| MUS | 602 | 602 | 0.902728 | + | ||||||||||
| MUS | 605 | 605 | 0.900001 | + | ||||||||||
| MUS | 607 | 607 | 0.877611 | + | ||||||||||
| MUS | 608 | 608 | 0.962159 | + | ||||||||||
| MUS | 612 | 612 | 0.909255 | + | ||||||||||
| MUS | 614 | 614 | 0.699823 | + | ||||||||||
| MUS | 615 | 615 | 0.909116 | + | ||||||||||
| MUS | 619 | 619 | 0.763155 | + | ||||||||||
| MUS | 624 | 624 | 0.836727 | + | ||||||||||
| MUS | 631 | 631 | 0.957254 | + | ||||||||||
| MUS | 632 | 632 | 0.5 | + | ||||||||||
| MUS | 634 | 634 | 0.560477 | + | ||||||||||
| MUS | 639 | 639 | 0.867656 | + | ||||||||||
| MUS | 641 | 641 | 0.831486 | + | ||||||||||
| MUS | 643 | 643 | 0.864738 | + | ||||||||||
| MUS | 647 | 647 | 0.510238 | + | ||||||||||
| MUS | 648 | 648 | 0.532835 | + | ||||||||||
| MUS | 656 | 656 | 0.70976 | + | ||||||||||
| MUS | 658 | 658 | 0.725052 | + | ||||||||||
| MUS | 662 | 662 | 0.732086 | + | ||||||||||
| MUS | 663 | 663 | 0.682057 | + | ||||||||||
| MUS | 667 | 667 | 0.400653 | |||||||||||
| MUS | 674 | 674 | 0.731579 | + | ||||||||||
| MUS | 676 | 676 | 0.683573 | + | ||||||||||
| MUS | 681 | 681 | 0.684517 | + | ||||||||||
| MUS | 683 | 683 | 0.602425 | + | ||||||||||
| MUS | 688 | 688 | 0.565729 | + | ||||||||||
| MUS | 690 | 690 | 0.315646 | |||||||||||
| MUS | 695 | 695 | 0.476853 | |||||||||||
| MUS | 702 | 702 | 0.0864334 | |||||||||||
| MUS | 704 | 704 | 0.115475 |
Appendix 1—table 8
Rates of sperm penetration and binding to bovine ZP after homologous (bovine sperm) or heterologous (human and murine sperm) EZPT using bovine ZPs co-incubated without bovine OVGP1 (HoA, HeAh, HeAm) or with bovine OVGP1 (HoB, HeBh, HeBm).
| GroupN | Sperm | Bovine OVGP1 | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|
| HoA 67 | Bovine | - | 4.15±0.61 | 67 (100±0)a | 730 (10.89±0.40)a | 33±5.59 | 67 (100±0) | 918 (13.70±0.21)a | 25±2.45 |
| HeAh 30 | Human | - | 8,81±0.74 | 21 (70.0±10.0)b | 182 (8.66±0.68)b | 13±2.08 | 10 (33.33±5.77) | 18 (0.60±0.66)b | 3±0.58 |
| HeAm 83 | Murine | - | 8.76±1.07 | 80 (96.25±5.3)c | 300 (3.75±0.31)c | 11±2.12 | 83 (100±0) | 490 (5.90±0.81)c | 20±3.54 |
| HoB 70 | Bovine | + | 4.15±0.61 | 69 (98.57±1.36)a | 708 (10.26±1.51)a | 29±4.30 | 70 (100±0) | 768 (10.97±1.31)a | 22±2.38 |
| HeBh 90 | Human | + | 8,81±0.74 | 0 (0±0)d | 0 (0±0)d | 0±0 | 12 (13.33±3.33) | 12 (0.13±0.03)d | 1±0 |
| HeBm 81 | Murine | + | 8.76±1.07 | 3 (3.79±2.08)d | 3 (1±0)d | 1±0 | 36 (44.31±3.16) | 94 (1.16±0.28)d | 6±0.71 |
-
HoA = homologous EZPT with bovine sperm. HeAh = heterologous EZPT with human sperm. HeAm = heterologous EZPT with murine sperm. HoB = homologous EZPT with bovine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min). HeBh = heterologous EZPT with human sperm (ZPs were co-incubated with bovine OVGP1 for 30 min). HeBm = heterologous EZPT with murine sperm (ZPs were co incubated with bovine OVGP1 for 30 min). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZPs refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZP with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. ‘N’ refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b, c, d) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 9
Rates of sperm penetration and binding to bovine ZP after homologous (bovine sperm) and heterologous (murine sperm) EZPT using bovine ZPs co-incubated without OVGP1 (HoA, HeA) or with bovine OVGP1 (HoB, HeB).
Sperm was also previously co-incubated without OVGP1 or with bovine OVGP1 (HoAb*, HeAb*, HoBb*, HeBb*) or with murine OVGP1 (HeBm*).
| Group N | Sperm | ZPs OVGP1 | Sperm OVGP1 | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|---|
| HoA 15 | Bovine | - | - | 5.74±1.64 | 15 (100±0)a | 272 (18.13±1.32)a | 32±2.08 | 15 (100±0) | 338 (22.53±2.08)a | 25±2.51 |
| HoAb* 61 | Bovine | - | Bovine | 5.74±1.64 | 61 (100.0±10.00)a | 963 (15.80±1.18)a | 31±1.53 | 61 (100±0) | 1093 (17.97±3.01)a | 31±1.15 |
| HeA 60 | Murine | - | - | 4.31±0.94 | 55 (91.67±8.15)b | 236 (4.24±0.52)b | 16±1.15 | 60 (100±0) | 338 (5.63±0.48)b | 21±1.00 |
| HeAb* 55 | Murine | - | Bovine | 4.31±0.94 | 49 (88.24±11.76)b | 198 (4.11±0.51)b | 14±1.00 | 55 (100±0) | 276 (5.00±0.31)b | 18±2.65 |
| HeAm* 58 | Murine | - | Murine | 4.31±0.94 | 52 (89.65±9.75)b | 233 (4.02±0.66)b | 15±1.73 | 58 (100±0) | 294 (5.08±0.32)b | 22±0.58 |
| HoB 14 | Bovine | Bovine | - | 5.74±1.64 | 14 (100±0)b | 232 (16.65±0.99)a | 33±1.52 | 14 (100±0) | 288 (20.88±3.88)a | 33±2.08 |
| HoBb* 59 | Bovine | Bovine | Bovine | 5.74±1.64 | 59 (100±0)a | 906 (15.34±0.66)a | 29±1.00 | 59 (100±0) | 1001 (16.87±2.34)a | 32±1.52 |
| HeB 16 | Murine | Bovine | - | 4.31±0.94 | 0 (0±0)c | 0 (0±0)c | 0±0 | 15 (94.44±9.62) | 78 (4.90±0.61)b | 8±0.57 |
| HeBb* 48 | Murine | Bovine | Bovine | 4.31±0.94 | 0 (0±0)c | 0 (0±0)c | 0±0 | 41 (84.81±10.67) | 115 (2,39±0.16)c | 11±1.52 |
| HeBm* 51 | Murine | Bovine | Murine | 4.31±0.94 | 2 (3.98±3.51)c | 2 (1±0)c | 1±0 | 51 (100±0) | 229 (4.48±0.57)b | 9±1 |
-
HoA = homologous EZPT with bovine sperm. HoAb*=homologous EZPT with bovine sperm (sperm was previously co-incubated with bovine OVGP1 for 30 min). HeA = heterologous EZPT with murine sperm. HeAb*=heterologous EZPT with murine sperm (sperm was previously co-incubated with bovine OVGP1 for 30 min). HeAm*=heterologous EZPT with murine sperm (sperm was previously co-incubated with murine OVGP1 for 30 min). HoB = homologous EZPT with bovine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min). HoBb*=homologous EZPT with bovine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min) (sperm was previously co-incubated with bovine OVGP1 for 30 min). HeB = heterologous EZPT with murine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min). HeBb*=heterologous EZPT with murine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min) (sperm was previously co-incubated with bovine OVGP1 for 30 min). HeBm*=heterologous EZPT with murine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min) (sperm was previously co-incubated with murine OVGP1 for 30 min). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZP refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZP with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. ‘N’ refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b, c) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 10
Rates of sperm penetration and binding to bovine ZP after homologous (bovine sperm) and heterologous (human and murine sperm) EZPT using bovine ZPs co-incubated without OVGP1 (HoA, HeAh, HeAm) or with bovine OVGP1 (HoBb, HeBh, HeBm), human OVGP1 (HoC, HeCh, HeCm) or murine OVGP1 (HoD, HeDh, HeDm).
| Group N | Sperm | OVGP1 | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|
| HoA 67 | Bovine | - | 4.15±0.61 | 67 (100±0)a | 730 (10.89±0.40)a | 33±5.59 | 67 (100±0) | 918 (13.70±0.21)a | 25±2.51 |
| HeAh 30 | Human | - | 8,81±0.74 | 21 (70.0±10.0)c | 182 (8.66±0.68)c | 13±2.08 | 10 (33.33±5.77) | 18 (0.60±0.66)b | 3±0.58 |
| HeAm 83 | Murine | - | 8.76±1.07 | 80 (96.25±5.3)a | 300 (3.75±0.31)c | 21±2.12 | 83 (100±0) | 490 (5.90±0.81)c | 20±3.54 |
| HoB 70 | Bovine | Bovine | 4.15±0.61 | 69 (98.57±1.36)a | 708 (10.26±1.51)a | 29±4.30 | 70 (100±0) | 768 (10.97±1.31)a | 22±2.38 |
| HeBh 90 | Human | Bovine | 8,81±0.74 | 0 (0±0)b | 0 (0±0)b | 0±0 | 12 (13.33±3.33) | 12 (0.13±0.03)b | 1±0 |
| HeBm 81 | Murine | Bovine | 8.76±1.07 | 3 (3.79±2.08)b | 3 (1±0)b | 1±0 | 36 (44.31±3.16) | 94 (1.17±0.28)b | 6±0.71 |
| HoC 84 | Bovine | Human | 5.03±0.66 | 49 (58.3±1.12)c | 235 (4.80±0.02)c | 11±1.41 | 84 (100±0) | 820 (4.80±0.02)c | 27±0.71 |
| HeCh 87 | Human | Human | 4.79±0.15 | 53 (61.11±7.85)c | 249 (4.73±0.73)c | 8±0.71 | 87 (100±0) | 832 (9.56±0.02)c | 27±1.41 |
| HeCm 30 | Murine | Human | 6.90±0.67 | 0 (0±0)b | 0 (0±0)b | 0±0 | 2 (6.67±0) | 2 (0.08±0.03)b | 1±0 |
| HoD 68 | Bovine | Murine | 4.35±0.46 | 43 (63.16±3.61)c | 199 (4.66±0.55)c | 7±0.71 | 68 (100±0) | 602 (8.86±0.20)c | 16±0.71 |
| HeDh 23 | Human | Murine | 4.79±0.15 | 0 (0±0)b | 0 (0±0)b | 0±0 | 1 (12.5±8.84) | 1 (0.06±0.09)b | 1±0.71 |
| HeDm 73 | Murine | Murine | 5.23±0.43 | 46 (63.23±7.55)c | 211 (4.59±0.13)c | 7±1.41 | 73 (100±0) | 980 (13.42±0.27)a | 25±3.53 |
-
HoA = homologous EZPT with bovine sperm. HeAh = heterologous EZPT with human sperm. HeAm = heterologous EZPT with murine sperm. HoB = homologous EZPT with bovine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min). HeBh = heterologous EZPT with human sperm (ZPs were co-incubated with bovine OVGP1 for 30 min). HeBm = heterologous EZPT with murine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min). HoC = homologous EZPT with bovine sperm (ZPs were co-incubated with human OVGP1 for 30 min). HeCh = heterologous EZPT with human sperm (ZPs were co-incubated with human OVGP1). HeCm = heterologous EZPT with murine sperm (ZPs were co-incubated with human OVGP1 for 30 min). HoD = homologous EZPT with bovine sperm (ZPs were co-incubated with murine OVGP1 for 30 min). HeDh = heterologous EZPT with human sperm (ZPs were co-incubated with murine OVGP1). HeDm = heterologous EZPT with murine sperm (ZPs were co-incubated with murine OVGP1 for 30 min). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZP refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZP with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. ‘N’ refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b, c) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 11
Rates of sperm penetration and binding to murine ZP after homologous (murine sperm) and heterologous (human and bovine sperm) EZPT using bovine ZPs co-incubated without OVGP1 (HoA, HeAh, HeAb) or with in vivo-matured murine OVGP1 (oocytes from superovulated females) (HoBb, HeBh, HeBb), murine OVGP1 (HoC, HeCh, HeCb), human OVGP1 (HoD, HeDh, HeDb) or bovine OVGP1 (HoE, HeEh, HeEb).
| Group N | Sperm | OVGP1 | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|
| HoA 56 | Murine | - | 8.76±1.08 | 56 (100±0)b | 848 (15.14±1.19)b | 29±5.50 | 56 (100±0) | 1042 (18.60±2.21) | 32±2.06 |
| HeAh 85 | Human | - | 6.12±0.17 | 60 (70.83±5.89)c | 177 (2.95±0.07)c | 9±0.71 | 79 (92.92±0.59) | 263 (3.32±0.31)d | 11±1.41 |
| HeAb 97 | Bovine | - | 3.67±0.19 | 56 (57.85±5.45)c | 167 (2.95±0.02)c | 6±0.00 | 91 (93.87±2.65) | 303 (3.32±0.40)d | 10±1.41 |
| HoB 19 | Murine | Murine | 5.35±0.07 | 19 (100±0)b | 228 (12.00±0.15)b | 31±7.78 | 19 (100±0) | 313 (16.47±0.64)b | 34±2.83 |
| HeBh 30 | Human | Murine | 6.12±0.17 | 1 (6.67±4.71)a | 1 (1±0)a | 1±0 | 3 (10.00±4.71) | 3 (1.00±0.00)a | 1±0 |
| HeBb 27 | Bovine | Murine | 4.26±0.09 | 1 (6.25±4.42)a | 1 (1±0)a | 1±0 | 3 (10.80±2.41) | 3 (1.00±0.00)a | 1±0 |
| HoC 99 | Murine | Human | 6.33±1.46 | 67 (67.49±2.39)c | 228 (3.40±0.24)c | 11±1.41 | 97 (98.00±2.82) | 449 (4.63±0.09)d | 19±0.71 |
| HeCh 104 | Human | Human | 5.60±0.91 | 67 (64.39±2.32)c | 185 (2.76±0.02)c | 8±0.71 | 101 (10.38±0.54) | 1079 (10.68±0.54)c | 29±2.12 |
| HeCb 30 | Bovine | Human | 6.09±1.31 | 0 (0±0)a | 0 (0±0)a | 0±0 | 2 (6.67±0) | 2 (1.00±0)a | 1±0 |
| HoD 88 | Murine | Bovine | 5.29±0.43 | 56 (63.80±1.49)c | 219 (3.91±0.21)c | 8±0.71 | 87 (99.01±1.39) | 459 (5.28±0.62)d | 19±1.41 |
| HeDh 25 | Human | Bovine | 5.45±0.75 | 0 (0±0)a | 0 (0±0)a | 0±0 | 1 (3.33±4.71) | 1 (1.00±0.04)a | 1±0.71 |
| HeDb 90 | Bovine | Bovine | 4.35±0.46 | 54 (59.88±1.90)c | 182 (3.37±1.38)c | 7±0.71 | 90 (100±0) | 956 (10.62±0.08)c | 28±1.41 |
-
HoA = homologous EZPT with murine sperm. HeAh = heterologous EZPT with human sperm. HeAb = heterologous EZPT with bovine sperm. HoB = homologous EZPT with murine sperm (ZPs were co-incubated with murine OVGP1 for 30 min). HeBh = heterologous EZPT with human sperm (ZPs were co-incubated with murine OVGP1 for 30 min). HeBb = heterologous EZPT with bovine sperm (ZPs were co-incubated with murine OVGP1 for 30 min). HoC = homologous EZPT with murine sperm (ZPs were co-incubated with human OVGP1 for 30 min). HeCh = heterologous EZPT with human sperm (ZPs were co-incubated with human OVGP1). HeCb = heterologous EZPT with bovine sperm (ZPs were co-incubated with human OVGP1 for 30 min). HoD = homologous EZPT with murine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min). HeDh = heterologous EZPT with human sperm (ZPs were co-incubated with bovine OVGP1). HeDb = heterologous EZPT with bovine sperm (ZPs were co incubated with bovine OVGP1 for 30 min). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZP refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZP with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. “N” refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b, c. d) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 12
Rates of sperm penetration and binding to bovine ZP after homologous (bovine sperm) EZPT using bovine ZPs co-incubated with neuraminidase (NMase) and without NMase and with bovine OVGP1 or without bovine OVGP1.
| Group N | NMase | Bovine OVGP1 | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|
| HoA 55 | + | - | 5.89±0.22 | 2 (3.67±0.47)b | 2 (1±0.0)b | 1±0 | 53 (96.67±4.71) | 159 (2.89±0.35)b | 4±0.00 |
| HoAb 53 | + | + | 5.89±0.22 | 3 (5.89±3.45)b | 3 (1±0)b | 1±0 | 52 (98.27±2.44) | 202 (3.81±0.73)b | 8±0.71 |
| HoB 46 | - | - | 5.89±0.22 | 46 (100±0)a | 492 (10.62±0.82)a | 33±2.12 | 46 (100±0) | 759 (16.50±2.56)a | 33±2.82 |
| HoBb 45 | - | + | 5.89±0.22 | 45 (100±0)a | 499 (11.00±0.82)a | 32±1.41 | 45 (100±0) | 757 (16.82±2.34)a | 31±0.71 |
-
HoA = homologous EZPT with bovine sperm (ZPs were co-incubated with NMase at 10 U/mL in acetate buffer, PH 4.5 at 38 °C for 18 hr). HoAb = homologous EZPT with bovine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min and then co-incubated with NMase diluted to 10µ/mL in acetate buffer, PH 4.5 at 38 °C for 18 hr). HoB = homologous EZPT with bovine sperm (ZPs were co-incubated with acetate buffer, PH 4.5 at 38 °C for 18 hr). HoBb = homologous EZPT with bovine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min and then co-incubated with acetate buffer, PH 4.5 at 38 °C for 18 hr). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZPs refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZP with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. ‘N’ refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 13
Rates of sperm penetration and binding to murine ZP after homologous (murine sperm) EZPT using murine ZPs co-incubated with neuraminidase (NMase) and without NMase and with murine OVGP1 or without murine OVGP1.
| Group N | NMase | Murine OVGP1 | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|
| HoA 55 | + | - | 6.90±0.94 | 3 (5.45±2.16)b | 3 (1±0)b | 1±0 | 53 (96.15±5.43) | 257 (4.84±0.59)b | 9±0.71 |
| HoAm 53 | + | + | 6.90±0.94 | 3 (5.66±2.31)b | 3 (1±0)b | 1±0 | 50 (93.98±5.36) | 186 (3.50±1.40)b | 10±0 |
| HoB 28 | - | - | 6.90±0.94 | 28 (100±0)a | 345 (12.29±0.74)a | 33±1.99 | 28 (100±0) | 512 (18.28±0.67)a | 31±7.99 |
| HoBm 40 | - | + | 6.90±0.94 | 40 (100±0)a | 398 (9,97±0.8)a | 31±0,99 | 40 (100±0) | 363 (9.08±0.18)a | 30±0.98 |
-
HoA = homologous EZPT with murine sperm (ZPs were co-incubated with NMase diluted to 10µ/mL in acetate buffer, PH 4.5 at 38 °C for 18 hr). HoAm = homologous EZPT with murine sperm (ZPs were co-incubated with murine OVGP1 for 30 min and then co-incubated with NMase at 10 U/mL in acetate buffer, PH 4.5 at 38 °C for 18 hr). HoB = homologous EZPT with murine sperm (ZPs were co-incubated with acetate buffer, PH 4.5 at 38 °C for 18 hr). HoBm = homologous EZPT with murine sperm (ZPs were co-incubated with murine OVGP1 for 30 min and then co-incubated with acetate buffer, PH 4.5 at 38 °C for 18 hr). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZPs refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZP with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. ‘N’ refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b) in the same column indicate significant differences (p<0.05; n=3). Values are expressed as the mean ± SD.
Appendix 1—table 14
Rates of sperm penetration and binding to bovine ZP after homologous (bovine sperm) and heterologous (murine sperm) EZPT using bovine ZPs co-incubated with bovine OVGP1 previously treated with Neuraminidase (NMase) (HoA, HeA) and with non-treated bOVGP1 (HoB, HeB).
Control groups without ZPs treatment was added for both, homologous (HoC) and heterologous (HeC).
| Group N | Bovine OVGP1 | NMase treatment to bOVGP1 | TMC | Penetrated ZPs (%) | Sperm penetration inside ZP (mean) | Max. no. of sperm inside a single ZP | ZP with bound sperm (%) | Sperm bound to ZPs (mean) | Max. no. of sperm bound to a single ZP |
|---|---|---|---|---|---|---|---|---|---|
| HoA 15 | + | + | 5.25±0.60 | 76 (84.49±9.91)a | 473 (5.11±1.12)a | 22±1.53 | 87 (100±0) | 720 (8.02±1.06)a | 29±3.05 |
| HoB 87 | + | - | 5.25±0.60 | 15 (100±0)b | 254 (21.07±7.61)b | 29±3.05 | 15 (100±0) | 355 (23.67±7.11)b | 33±5.51 |
| HeA 82 | + | + | 5.34±0.05 | 45 (52.78±12.73)c | 123 (1.46±0.27)c | 6.00±1.00 | 65 (79.58±7.88) | 286 (3.51±0.31)c | 11±1.52 |
| HeB 15 | + | - | 5.34±0.05 | 0 (0±0)d | 0 (0±0)d | 0±0 | 4 (26.67±11.54) | 5 (0.33±0.23)d | 2±0.57 |
| HeC 20 | - | - | 5.34±0.05 | 18 (88.10±6.73)a | 113 (5.70±0.19)a | 13±2.12 | 20 (100±0) | 185 (9.98±2.61)a | 19±3.54 |
-
HoA = homologous EZPT with bovine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min. Bovine OVGP1 was previously treated by incubation with NMase at 10U/mL in acetate buffer, PH 4.5 at 38ºC for 18 hr). HoB = homologous EZPT with bovine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min. Bovine OVGP1 was previously incubated in acetate buffer, PH 4.5 at 38ºC for 18 hr). HeA = heterologous EZPT with murine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min. Bovine OVGP1 was previously treated with NMase at 10U/mL in acetate buffer, PH 4.5 at 38ºC for 18 hr). HeB = Heterologous EZPT with murine sperm (ZPs were co-incubated with bovine OVGP1 for 30 min. Bovine OVGP1 was previously treated in acetate buffer, PH 4.5 at 38ºC for 18 hr). HeC = heterologous EZPT with murine sperm (ZPs were co-incubated with acetate buffer, PH 4.5 at 38ºC for 18 hr). TMC = total motile sperm count after capacitation calculated by multiplying the volume by concentration (million sperm/mL) and by the motility (%) after capacitation. Penetrated ZPs refers to the mean number of ZPs penetrated by at least one sperm. ZPs with bound sperm was calculated as the mean number of ZP with, at least, one bound sperm. Sperm penetration was taken as the total number of sperm observed inside the zonas. Bound sperm was expressed as the mean number of sperm that remained bound to the ZP. ‘N’ refers to the total number of ZPs fertilized per treatment. Analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Different superscripts (a, b, c, d) in the same column indicate significant differences (p <0.05; n=3). Values are expressed as the mean ± SD.
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Oviductin sets the species-specificity of the mammalian zona pellucida
eLife 13:RP101338.
https://doi.org/10.7554/eLife.101338.4
