Oviductin sets the species-specificity of the mammalian zona pellucida

Reviewer #1 (Public review):

Summary:

This interesting manuscript first shows that human, murine, and feline sperm penetrate the zona pellucida (ZP) of bovine oocytes recovered directly from the ovary, although first cleavage rates are reduced. Similarly, bovine sperm can penetrate superovulated murine oocytes recovered directly from the ovary. However, bovine oocytes incubated with oviduct fluid (30 min) are generally impenetrable by human sperm.

Thereafter, the cytoplasm was aspirated from murine oocytes - obtained from the ovary or oviduct. Binding and penetration by bovine and human sperm was reduced in both groups relative to homologous (murine) sperm. However, heterologous (bovine and human) sperm penetration was further reduced in oviduct vs. ovary derived empty ZP. These data show that outer (ZP) not inner (cytoplasmic) oocyte alterations reduce heterologous sperm penetration as well as homologous sperm binding.

This was repeated using empty bovine ZP incubated, or not, with bovine oviduct fluid. Prior oviduct fluid exposure reduced non-homologous (human and murine) empty ZP penetration, polyspermy, and sperm binding. This demonstrates that species-specific oviduct fluid factors regulate ZP penetrability.

To test the hypothesis that OVGP1 is responsible, the authors obtained histidiine-tagged bovine and murine OVGP1 and DDK-tagged human OVGP1 proteins. Tagging was to enable purification following over-expression in BHK-21 or HEK293T cells. The authors confirm these recombinant OVGP1 proteins bound to both murine and bovine oocytes. Moreover, previous data using oviduct fluid was mirrored using bovine oocytes supplemented with homologous (bovine) recombinant OVGP1, or not. This confirms the hypothesis, at least in cattle.

Next, the authors exposed bovine and murine empty ZP to bovine, murine, and human recombinant OVGP1, in addition to bovine, murine, or human sperm. Interestingly, both species-specific ZP and OVGP1 seem to be required for optimal sperm binding and penetration.

Lastly, empty bovine and murine ZP were treated with neuraminidase, or not, with or without pre-treatment with homologous OVGP1. In each case, neuraminidase reduced sperm binding and penetration. This further demonstrates that both ZP and OVGP1 are required for optimal sperm binding and penetration.

In summary, the authors demonstrate that two mechanisms seem to underpin mammalian sperm recognition and penetration, the first being specific (ZP-mediated) and the second non-specific (OVGP1 mediated).

Reviewer #2 (Public review):

Summary:

In the manuscript de la Fuente et al analyze the species specificity of sperm-egg recognition by looking at sperm binding and penetration of zonae pellucidae from different mammalian species and find a role for the oviductal protein OVGP1 in determining species specificity.

Strengths:

By combining sperm, oocytes, zona pellucida (ZP), and oviductal fluid from different mammalian species, they elucidate the essential role of OVGP1 in conferring species-specific fertilization.

Weaknesses:

Mice with OVGP1 deletion are viable and fertile. It would be quite interesting to investigate the species-specificity of sperm-ZP binding in this model. That would indicate whether OVGP1 is the only glycoprotein involved in determining species-specificity. Alternatively, the authors could immunodeplete OVGP1 from oviductal fluid and then ascertain whether this depleted fluid retains the ability to impede cross-species fertilization.

Reviewer #3 (Public review):

Summary:

The authors submitted a revised manuscript that reports findings from a series of experiments suggesting that bovine oviductal fluid and species-specific oviductal glycoprotein (OVGP1 or oviductin) from bovine, murine, or human sources modulate the species specificity of bovine and murine oocytes.

Strengths:

The study reported in the manuscript deals with an important topic of interest in reproductive biology.

Weaknesses:

The authors have submitted a revised manuscript with much improvement and have answered many of this reviewer's questions. However, some of the previous questions have been dealt with inadequately. There are still several issues that need to be dealt with. In particular, there are questions regarding the specificity and/or purity of the recombinant human and mouse OVGP1 which could be detrimental to the reliability of the recombinant human and mouse OVGP1s used in the study and the validity of the results presented. This Discussion should cover more broadly what has already been published in literature.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Weaknesses:

In my estimation, the following would improve this manuscript:

(1) The physiological relevance of these data could be better highlighted. For instance, future work could revolve around incubating oocytes with oviduct fluid (or OVGP1) to reduce polyspermy in porcine IVF, and naturally improve sperm selection in human IVF.

Thank you for the suggestions. We have added these physiological relevance points at the end of the discussion.

(2) Biological and technical replicate values for each experiment are unclear - for semen, oocytes, and oviduct fluid pools. I suggest providing in the Materials and Methods and/or Figure legends.

Biological and technical replicates are now indicated in M&M. Number of oocytes or ZPs used were already indicated in every Supplementary Table.

(3) Although differences presented in the bar charts seem obvious, providing statistical analyses would strengthen the manuscript.

Statistical analyses are now indicated in each bar chart.

(4) Results are presented as {plus minus} SEM (line 677); however, I believe standard deviation is more appropriate.

This was a mistake; all the results are indicated as standard deviation.

(5) Given the many independent experimental variables and combinations, a schematic depiction of the experimental design may benefit readers.

A schematic depiction of the experimental design is now included as Figure 1. This new Figure modifies the number assigned to the rest of Figures.

(6) Attention to detail can be improved in parts, as delineated in the "author recommendation" review section.

Done

Reviewer #2 (Public review):

Weaknesses:

The authors postulate a role for oviductal fluid in species-specific fertilization, but in my opinion, they cannot rule out hormonal effects or differences in the method of oocyte maturation employed.

As we indicate below, the effect of hormones has been analyzed, and we have demonstrated that it is not the cause of zona pellucida specificity.

They also cannot unequivocally prove that OVGP1 is the oviductal protein involved in the effect. Additional experiments are necessary to rule out these alternative explanations.

Our work does not demonstrate that other proteins could be involved, but it does show that OVGP1 is involved in the process.

When performing the EZPT assay on mouse oocytes obtained either from the ovary or from the oviduct, the oocytes obtained from the ovary came from mice primed with eCG, whereas the ones collected from the oviduct were obtained from superovulated mice (eCG plus hCG). This difference in the hormonal environment may make a difference in the properties of the ZP. Additionally, the ones obtained from the ovary were in vitro matured, which is also different from the freshly ovulated eggs and, again, may change the properties of the ZP. I suggest doing this experiment superovulating both groups of mice but collecting the fully matured MII eggs from the ovary before they get ovulated. In that way the hormonal environment will be the same in both groups and in both groups, oocytes will be matured in vivo. Hence, the only difference will be the exposure to oviductal fluids.

In Figure 2, we compare ZPs from murine oocytes obtained from the ovary using only PMSG with ZPs from oviductal oocytes treated with both HCG and PMSG. But in Figure 7, however, we compared ZPs from murine oocytes exposed only to PMSG, with the only difference being whether or not they had been in contact with OVGP1. This shows that it is not the effect of the hormone but rather the contact with OVGP1 that determines their specificity.

Mice with OVGP1 deletion are viable and fertile. It would be quite interesting to investigate the species-specificity of sperm-ZP binding in this model. That would indicate whether OVGP1 is the only glycoprotein involved in determining species-specificity. Alternatively, the authors could immunodeplete OVGP1 from oviductal fluid and then ascertain whether this depleted fluid retains the ability to impede cross-species fertilization.

We agree with the reviewer that it would be interesting to investigate sperm-ZP binding in this model. Unfortunately, we do not have the OVGP1 knockout mouse strain. We also believe that immunodepletion of OVGP1 would not completely remove the protein, so its effect would likely not be entirely eliminated.

What is the concentration of OVGP1 in the oviduct? How did the authors decide what concentration of protein to use in the experiments where they exposed ZPs to purified OVGP1? Why did they use this experimental design to check the structure of the ZP by SEM? Why not do it on oocytes exposed to oviductal fluid, which would be more physiological?

We have included in the manuscript that the concentration of OVGP1 in the oviductal fluid was quantified using ImageJ software by comparing the mean gray value of the band in the oviductal fluid to the band in the recombinant protein lane. By establishing this relationship, along with the known concentration of protein amount in the recombinant one and in the total protein amount of oviductal fluid, the concentration of OVGP1 in the oviductal fluid was determined as the average of three western blots. The concentration of OVGP1 in oviductal fluids was in the range of 100-150 ng/µl in mice and 150-200 ng/µL in cow. We have included also in the manuscript the concentration that we have use for the EZPTs, 30 ng/µL of recombinants OVGP1 (bovine, murine and human) for 30 minutes in 20µL drops. With this concentration, we observed a clear effect on zona specificity with no negative impact on the gametes.

As you can see in supplementary Fig S8B, we already realized SEM of oocytes exposed to oviductal fluid.

None of the figures show any statistical analysis. Please perform analysis for all the data presented, include p values, and indicate which statistical tests were performed. The Statistical analysis section in the Methods indicating that repeated measures ANOVA was used must refer to the tables. Was normality tested? I doubt all the data are normally distributed, in which case using ANOVA is not appropriate.

Statistical results are now included in each Figure and Table. All the statistical analysis are included, all the data pass normality, homogeneity of variance and independence; for this reason the data analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Significance level was set at p <0.05.

Why was OVGP1 selected as the probable culprit of the species specificity? In the Results section entitled "Homology of bovine, human and murine OVGP1 proteins..." the authors delve into the possible role of this protein without any rationale for investigating it. What about other oviductal proteins?

A sentence indicating this rationale for investigating OVGP1 has been introduced in this paragraph.

Reviewer #3 (Public review):

Weaknesses:

The manuscript began with a well-written introduction, but problems started to surface in the Results section, in the Discussion, as well as in the Materials and Methods. Major concerns include inconsistencies, misinterpretation of results, lacking up-to-date literature search, numerous errors found in the figure legends, misleading and incorrect information given in the Materials and Methods, missing information regarding statistical analysis, and inadequate discussion. These concerns raise questions regarding the authenticity of the study, reliability of the findings, and interpretation of the results. The manuscript does not provide solid and convincing findings to support the conclusion.

We have modified and clarified all the issues, some of which are misunderstandings, we have also performed the suggested experiment of putting sperm in contact with OVGP1.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Ensure consistency in (past) tense, for example, "decondensed" (line 102), "induced" (line 103), and elsewhere.

Done

(2) Replace "table" with "Table" throughout.

Done

(3) The authors often refer to "co-incubation". I believe this should read "incubation". My understanding is that oocytes were incubated with oviduct fluid or sperm but never both simultaneously as "co-incubation" implies.

Done

(4) Synonymous terms "OVGP1" and "oviductin" are used interchangeably. Consider using one or the other for consistency.

We believe that by using both terms, reading is more fluid.

(5) Delete "around" on line 256 and "approximately" on line 263 and provide actual percentages.

Done

(6) The point of the sentence on lines 311-313 is unclear to me.

Rewritten

(7) Suggest specifying "wildtype" on line 419.

All the mice used in this work are wildtype

(8) Do the authors have details regarding cattle oocyte donor breeds?

Done

(9) What do the authors mean by "strengthen" on line 500?

The word strengthen has been changed to carefully isolated

(10) Ponceau and vinculin (Figure 3) details are not provided in the manuscript.

Ponceau and vinculin details are now included in the manuscript

(11) Address formatting issues (e.g. citation 26 among others).

Done

(12) Primary and secondary antibody controls for immunofluorescent imaging (to fully exclude autofluorescence) are lacking.

Controls for immunofluorescent imaging are indicated in Supplementary Figure S7.

(13) The corresponding author on the manuscript and in the eLife submission system are different

It was a problem during submission, now it is corrected.

Reviewer #2 (Recommendations for the authors):

(1) For the experiment depicted in Figures 3C and D, the authors need to perform a negative control to demonstrate that this fluorescent signal is specific. What happens if they express a different FLAG-tagged protein instead of bOVGP1 and mOVGP1? FLAG antibodies give quite strong non-specific binding. Or if they expressed untagged bovine and mouse OVGP1?

The negative controls are in the supplementary Figure S7. A rabbit polyclonal antibody to the human OVGP1 was used for murine and bovine IVM ZPs from ovaries and murine superovulated ZPs recovered from mouse oviducts. There is a remarkable difference in the ones that are not incubated with any OVGP1 and the endogenous one, given the specificity of the antibody.

Also, IVM mouse and bovine oocytes incubated or not with OF were immunoblotted with anti-Flag-tag antibody. Since any of them present OVGP1 tagged to Flag, there is not signal in the immunofluorescence.

(2) For the Western blots of recombinant proteins, why are the authors not showing the blots using His and FLAG tag antibodies? Is the 50-kDa band observed for the mouse OVGP1 detected with His-Tag antibody?

We have included a supplementary figure S6 with the western blot with anti-His and anti-Flag. The protein around 50 kDa is not a specific band (there is not signal with anti-Flag). This new figure modifies the number assigned to the rest of supplementary figures (S6-S8).

(3) How was the estrous cycle stage determined in mice? It is not described in the Methods.

Estrous cycle stage was determined in mice by visual examination of the vaginal opening and cytological examination of the vagina smear. This is now included in the M&M

(4) For sperm binding, what does the percentage mean?

It was a mistake, percentages were related to pronuclear formation and cleavage not to sperm binding, this is now corrected.

(5) In Figure 3A, the labels for regions C, D, and E are mixed up. It is regions A and C that are conserved (or orange and blue, if the letters are incorrect). The purple region is only present in the mouse (E?), and the red region (D?) is only in the human form. Also, the legend for this panel is repeated verbatim in the Results section. Please remove one of them.

Errors in Figure 3a have been corrected. Legend repetition is removed.

(6) In the title of Figure 1B and in different places in the text, it should be mouse (not mice) oocytes.

Done

(7) In line 140, I would change the part indicating "We extracted the cytoplasmic contents from the oocytes". It is not only the cytoplasm, but all the oocyte, including the nucleus and membranes, that are being removed.

Done

(8) Please rephrase the sentence in lines 245-247, as it is quite confusing.

Done

(9) In line 236, the authors indicate that "During in vitro maturation (IVM), oocytes displayed a porous ZP structure...". Do they mean after IVM? When were those oocytes collected for SEM?

The sentence has been modified by “after IVF”. Bovine oocytes were collected from slaughterhouse ovaries and were similar to those used in the rest of the experiments in the manuscript.

(10) In the legend of Figure 1, please indicate what the parthenogenic group is.

Done

(11) In the legend to Figure 1G, the text indicates "Note sperm only appear outside the zona". However, I cannot see any sperm in that image.

The phrase has been removed, as when enlarging the image to better see the sperm that are inside the area, the vision of those that are outside has been lost.

(12) In the legend to Figure 2 describing the different zona pictures, the letters of the panels are not correct.

Done

(13) In line 999, please provide the right concentration for NMase (it indicates 10 μ/mL).

Done

(14) Where does the model depicted at the end of the manuscript go? Is it a Figure? A graphical abstract? In that model, please correct some typos: it should be "ZP obtained from ovarian oocytes"; and change specie for species in all three panels.

Done. It is a model (Fig. 10)

(15) The FITC-PNA staining to visualize acrosomes is not described in the Methods section.

Done

Reviewer #3 (Recommendations for the authors):

The present study reports findings from a series of experiments suggesting that bovine oviductal fluid and species-specific oviductal glycoprotein (OVGP1 or oviductin) from bovine, murine, or human sources modulate the species specificity of bovine and murine oocytes. The manuscript began with a well-written introduction, but problems started to surface in the Results section, Discussion as well as in the Materials and Methods. Major concerns include inconsistencies, misinterpretation of results, lacking up-to-date literature search, numerous errors found in the figure legends, misleading and incorrect information given in the Materials and Methods, missing information regarding statistical analysis, and inadequate discussion.

We have modified and clarified all the issues, some of which are misunderstandings, we have also performed the suggested experiment of putting sperm in contact with OVGP1.

Specific comments:

(1) Lines 142 to 143 on page 5: It is stated that "Because this experiment was done on empty ZPs, we called this test "empty zona penetration test" (EZPT)". In fact, the experiment was not actually done on empty ZPs, but on oocytes with the ooplasm extracted. Therefore, the zona pellucidae used in the experiment were not empty but contained an intact zona matrix of glycoproteins. The term "EZPT" used by the authors in the manuscript is a misnomer. A better term should be used to reflect the ZPs which were intact and not empty.

We extracted the cytoplasmic containing all the organelles, nucleus and membranes, and the polar body. This has been clarified in the text.

(2) The authors need to distinguish between sperm penetration and sperm binding in the manuscript. In lines 169 to 177 on page 6, the authors mixed up the terms "penetration" and "binding" in the text. In writing about events leading to fertilization in reproductive biology, the term "sperm binding" refers to the interaction between the sperm plasma membrane and the oocyte zona pellucida (ZP), whereas the term "sperm penetration" refers to the passage of the sperm through the ZP. Therefore, the statements in lines 169 to 177 describing the binding of bovine, murine, and human sperm to bovine oocytes with and without prior treatment with oviductal fluid are misleading and not correct. In fact, Figure 2 and Table 6 show sperm penetration and not sperm binding.

Figure 2A and B (now 3A and 3B), and Tables S6 show both sperm penetration (% penetration rate and average sperm in penetrated ZPs) and sperm binding (average sperm bound to ZPs). Throughout the manuscript, a clear distinction is made between sperm attached to the ZP and sperm that have penetrated it.

(3) Lines 182 to 187 on page 6: What is being described in the text here does not match what is being shown in Figure 3A. As a result, the information provided in lines 182 to 187 is not correct and misleading. For example, it is stated in lines 182 to 183 that "As depicted in Fig. 3A, the sequences of these three OVGP1 have five distinct regions (A, B, C, D and E)." However, Figure 3A shows that hOVGP1 and mOVGP1 both have only 4 regions and bOVGP1 has only 3 regions. None of the three has 5 regions. In lines 183 to 184, the authors continued to state that "Regions A and D are conserved in the different mammals." This statement is also not true because Figure 3A shows that only region A is conserved in all three species but not region D which is found only in the human. What is stated in lines 186 to 187 is also not correct based on the information provided in Figure 3A. It is stated here that "Region C is an insertion present only in the mouse (Mus) and region E is typical of human oviductin." However, based on the color codes provided in Figure 3A, region C is present in all three species while region E is present only in the mouse.

Errors with naming regions in Figure 3A (now 4A) have been corrected.

(4) In lines 195 to 197 on page 6, the authors stated that "Western blots of the three OVGP1 recombinants indicated expected sizes based on those of the proteins: 75 kDa for human and murine OVGP1 and around 60 kDa for bovine OVGP1 (Fig. 3B)." However, the expected size of the recombinant human OVGP1 is not in agreement with what has been published in literature regarding the molecular weight of recombinant human OVGP1. It has been previously reported that a single protein band of approximately 110-150 kDa was detected for recombinant human OVGP1 using an antibody against human OVGP1. The authors provided Western blots of murine oviductal fluid and bovine oviductal fluid in Figure 3B but not a Western blot of native human oviductal fluid. The latter should have been included for a comparison with the recombinant human OVGP1.

We do not have human oviductal fluid, but we have included now a supplementary figure 6S of a western blot with antibody again His and Flag (present in the recombinant OVGP1) which shows that the size of the recombinant protein is as indicated in the Figure 3B (now 4B).

(5) Lines 220 to 229 on page 7: In this experiment, the authors conducted the EZPT using ZPs from bovine oocytes that were either treated with or without bOVGP1 followed by incubation, respectively, with homologous sperm (bovine) and heterologous sperm (human and murine). This is a logical experiment to determine if OVGP1 plays a species-specific role in setting the specificity of the zona pellucida. However, in the in vivo situation, sperm that reach the lumen of the ampulla region of the oviduct where fertilization takes place are also exposed to oviductal fluid of which OVGP1 is a major constituent. Therefore, an additional experiment in which sperm are treated with OVGP1 prior to incubation with ZP should be carried out for a comparison.

The additional experiment in which sperm are treated with OVGP1 prior to incubation with ZP has been done (Table S9). No effects were observed. This is now included in the manuscript.

(6) Regarding the results obtained with the use of neuraminidase (lines 278 to 293 on pages 8 to 9), if neuraminidase treatment of bovine ZP prevented bovine sperm penetration regardless of whether ZPs had been or had not been in contact with OVGP1, that means OVGP1 is not responsible for penetration despite the description of earlier findings in the manuscript. Sialic acid is likely associated with the sugar side chains of ZP glycoproteins and not sugar side chains of OVGP1. To attribute the species-specific property of sialic acid to OVGP1 for sperm binding, an experiment in which OVGP1 will be treated with neuraminidase prior to performing the EZPT is needed.

We conducted the experiment by treating only OVGP1 with neuraminidase and then isolating OVGP1 from the enzyme previously to incubate treated OVGP1 with ZPs. The results agree with our previous findings, indicating the importance of sialic acid on OVGP1 for sperm binding and penetration, and confirming that OVGP1 is responsible for species-specific penetration. Results are shown in Fig. 9 and Table S14.

(7) The Discussion appears superficial and a more in-depth discussion regarding the results obtained in the present study in relation to other reports about OVGP1 published in literature is needed (e.g. a recent paper published by Kenji Yamatoya et al. (2023) Biology of Reproduction https://doi.org/10.1093/biolre/ioad159). Lines 317 to 342 of the Discussion on pages 10 to 11 should belong to the Introduction.

Results of Yamatoya are now included in discussion. Part of the discussion from 317 to 342 are now in the introduction

(8) In is not clear what the authors exactly want to say in lines 343 to 344 of the Discussion on page 11. It is stated here that "The empty zona penetration test (EZPT) enables heterologous sperm to overcome the oocyte's second barrier, the plasma membrane or oolemma." Do the authors mean that the sperm can now enter the empty space encircled by the ZP without having to go through the plasma membrane or oolemma? In Figure S4 which depicts the method used to empty the ooplasm in the bovine oocyte, does the method extract only the ooplasm (or cytoplasmic contents) leaving behind the intact plasma membrane or oolemma? This needs to be clearly shown and clearly explained. High magnifications of the zona pellucida are also needed to show whether the plasma membrane (or oolemma) is still present and intact after extraction of the ooplasm.

This is clearly explained in the text. To obtain empty ZP, everything except ZP (nucleus, organelles, membranes and cytoplasmic contents of the oocytes) was removed using a micromanipulator, following the procedure outlined in Figure S4.

(9) The authors stated in the Discussion in lines 383 to 383 on page 12 that "After ovulation, the changes reported in the carbohydrate composition of the ZP (3, 25) are likely induced by the addition of glycoproteins of oviductal origin, as we have seen here with OVGP1." There is no evidence in the present study to suggest that OVGP1 or glycoproteins of oviductal origin have changed or can change the carbohydrate composition of the ZP. At present, it is not known if OVGP1 or glycoproteins of oviductal origin directly interact with ZP glycoproteins (including ZP1, ZP2, ZP3 and/or ZP4) that make up the zona matrix.

There is scientific evidence suggesting that oviductal glycoproteins, including OVGP1, interact with the zona pellucida (ZP) glycoproteins of the oocyte. Studies have shown that OVGP1 binds to the ZP of the oocyte. Specifically, OVGP1 is thought to interact with ZP glycoproteins, such as ZP2 and ZP3, in a way that may help stabilize the oocyte or modify the ZP structure during its passage through the oviduct. This interaction is believed to influence processes like sperm binding, oocyte maturation, and potentially the prevention of polyspermy during fertilization. For example, in several studies, the absence of OVGP1 in knockout animals (such as in Ovgp1-KO hamsters) has been associated with impaired fertilization and embryonic development, which indicates the importance of this interaction. However, the detailed molecular mechanisms and functional significance of these interactions require further exploration. We have use the work “likely” to soften this statement.

Velásquez, J. G., Canovas, S., Barajas, P., Marcos, J., Jiménez‐Movilla, M., Gallego, R. G., ... & Coy, P. (2007). Role of sialic acid in bovine sperm–zona pellucida binding. Molecular reproduction and development, 74(5), 617-628.

Kunz, P., et al. (2013). "The role of oviductal glycoprotein 1 in sperm–egg interaction and early embryonic development." Reproduction, 145(3), 225-233. DOI: 10.1530/REP-12-0300

Yamatoya, K., Kurosawa, M., Hirose, M., Miura, Y., Taka, H., Nakano, T., ... & Araki, Y. (2024). The fluid factor OVGP1 provides a significant oviductal microenvironment for the reproductive process in golden hamster. Biology of reproduction, 110(3), 465-475.

(10) Lines 390 to 391 page 12: The statement "This determines that OVGP1 modifications are critical to define the barrier among the different species of mammals." needs to be rephrased because there is no evidence in the present study showing that OVGP1 has been modified. There are many concerns with errors, important information that is missing, and inconsistencies as well as wrong and misleading information in the Materials and Methods which are troublesome. These concerns raise questions regarding the authenticity and reliability of the study. Some of the major concerns are listed below:

All concerns have been fixed

(11) It says in line 399 on page 13 that "Human semen samples were obtained from a normozoospermic donor...". Do the authors really mean that the semen samples were obtained from only one donor?

Samples were obtained from 3 normozoospermic donor, this is now indicated in M&M

(12) In lines 409 to 411 on page 13, what do the authors mean by "...the samples were frozen into pellets..."? Was centrifugation of the samples carried out prior to freezing the samples? Secondly, what do the authors mean by "....and stored in liquid nitrogen at -196{degree sign}C or lower.", particularly what do the authors mean by "or lower"? The temperature of liquid nitrogen is -196{degree sign}C. What is the "lower" temperature?

Centrifugation of the samples were no carried out at this time. A more detailed protocol is now included The word lower has been removed.

(13) Line 424 on page 13: Provide the full name of "M2" when it is first used in the text then followed by the abbreviation.

Done

(14) Is there a reason why different counting chambers were used to determine sperm concentrations? In line 432 on page 13, a Thomas cell counting chamber was used to determine the sperm count of epididymal mouse sperm whereas it is mentioned in line 441 on page 14 that a Neubauer cell counting chamber was used to determine epididymal cat sperm. Furthermore, where did the cat's sperm come from?

The cat sperm was obtained and processed at the Faculty of Veterinary Medicine and the rest of the samples were processed in the INIA-CSIC lab, and different chambers were used in both places.

(15) The mention of the use of cat spermatozoa in line 439 on page 14 is a worrisome problem of the manuscript. The present study used bovine, mouse, and human sperm and not cat. Therefore, the sudden mentioning of the use of cat spermatozoa in the Materials and Methods is troublesome and worrisome. It appears that the paragraph from lines 439 to 450 was directly copied and pasted from previously published work. Furthermore, lines 441 to 445 do not flow and do not make sense. In fact, what is described in this paragraph (lines 439 to 450) does not appear to correspond to the method(s) used to obtain the results presented in the Results section of the manuscript.

I don't understand why the reviewer says we don't use cat sperm. This study uses cat sperm. Results of cat sperm are indicated in the Figure 1A (now 2A). We have modified the M&M to clarify frozen description.

(16) Similarly, several problems are also found in the paragraphs (lines 453-478 on page 14) describing the methods and procedures to obtain homologous and heterologous IVF of bovine oocytes. Firstly, it is mentioned here (in line 460) that COCs were co-incubated with selected sperm without removing the cumulus cells. However, the results of the sperm penetration experiment indicated otherwise. Figures 2 and 3 show that the oocytes were denuded of cumulus cells. Secondly, it is very worrisome and troublesome to read what is written in line 468 on page 14 that "...from other species (cat, human, mouse, and rabbit)." One wonders where the cat and rabbit came from. Again, it appears that this paragraph was directly copied and pasted from previously published work.

Cat sperm was used in this manuscript and it is correctly indicated in every section and figures. About IVF and EZPT protocols, in the protocol of IVF for bovine oocytes, COCs were used without removing the cumulus cells. For the EZPT cumulus cells were removed, this is described in the following sections of the material and methods. The word rabbit was a mistake and it has been removed.

(17) In lines 468 to 469 on page 14, it is mentioned that "Sperm-egg interactions were assessed through a sperm-ZP binding assay...". The authors only examined sperm penetration in their study. Therefore, this needs to be specified in the Materials and Methods. Secondly, the authors did not use the conventional sperm-ZP binding assay in their study. Instead, they used the EZPT in their study. There appear to be many inconsistencies throughout the manuscript.

When the IVF experiments using bovine COCs were done (Fig 2A and C, Fig 1S to 3S, and Tables 1S to 4S) conventional sperm-egg interaction was assessed at 2.5 hours after IVF. EZPT was used in the rest of experiments. IVF with COCs and EZPT with ZPs are different experiments.

(18) Lines 480 to 489 on page 15 under the sub-heading of "In vitro culture of presumptive zygotes to first cleavage embryos on Day 2" do not provide the correct methodology used for obtaining the results presented in the manuscript. In line 482, it is not clear where the "synthetic oviductal fluid" came from. In fact, in the Results section, none of the results came from the use of synthetic oviductal fluid. In line 487, humans and rabbits are mentioned here. However, human and rabbit oocytes were not used in the present study. It is very strange indeed to read human and rabbit in the sentence.

SOF reference is now included. Human results are in Fig 1A; the sentence is referred about the cultures of bovine oocytes inseminated with sperm of bull, human, mouse or cat). Rabbit word is a mistake and is now eliminated of the manuscript.

(19) In line 500 on page 15, what do the authors mean by "Each oviduct was strengthen by removing the adjacent tissue..."?

The sentence has been modified.

(20) On page 15 in the Materials and Methods, the authors described the collection of bovine and mouse oviductal fluid. However, there is no mention of human oviductal fluid and how it was collected. This important information is missing.

We have not use human oviductal fluid in this manuscript.

(21) Line 510 on page 15: The sub-heading of "Preparation of empty zonae pellucidae from bovine ovarian oocytes" should be rephrased. As pointed out earlier in my review, the ZPs prepared by the authors were intact and not "empty". It was the oocyte which was empty after extraction of the ooplasm.

Everything except the ZP were removed from the oocyte, this is now clarified in the manuscript.

(22) Line 518 on page 16 and line 553 on page 17: "Figure S5" should be "Figure 4S".

Done

(23) Line 538 and line 547 on page 16: "mice oocytes" should be "mouse oocytes".

Done

(24) On page 17, the procedures for in vitro fertilization, sperm penetration, and binding assessment in mice were described here in lines 560 to 574. Several problems are noted in this paragraph as listed below:
a. As mentioned earlier the authors in the present manuscript mixed up sperm penetration and sperm binding which are two separate events. Based on the results presented in the manuscript, they represent sperm penetration and not sperm binding. Therefore, the authors need to precisely explain in the manuscript whether the results presented refer to sperm penetration or sperm binding.

Both sperm penetration and binding have been analyzed in this work.

b. In line 570 on page 17, the term "insemination" is wrongly used here. Insemination is the introduction of semen into the female reproductive tract either through sexual intercourse or through an instrument. The procedure used in the present study was carried out in vitro in a co-incubation manner and not by transferring sperm into the female reproductive tract.

The word insemination has been changed to incubation

c. Information regarding procedures for treatment with various oviductal fluid and OVGP1s are all missing in the Materials and Methods.

This information is now in M&M

d. The concentrations of various oviductal fluids and OVGP1s used and the number of ZPs used in each incubation are also missing.

Concentrations are now indicated in the manuscript. All the numbers and ZPs used are indicated in supplementary figures.

(25) Lines 577 to 603 on pages 17 to 18: Were recombinant bovine and murine glycoproteins prepared using the same methodology? In line 595 on page 18, it is stated that "Supernatant was saved in subsequent experiments." It is not clear exactly what experiments the supernatant was subsequently used in.

Details about how the bovine and murine glycoproteins were prepared are now included. Sentence about subsequent experiment is delete; supernatant was used for the next steps of protein purification.

(26) What is being described in lines 604 to 609 on page 18 is problematic. The paragraph starts by saying that "Human recombinant oviductin was obtained from Origene Technologies....". Strangely, the paragraph continues by saying that the recombinant proteins were produced by transfection in HEK293T...". If recombinant human OVGP1 had already been obtained from Origene Technologies, why did the authors want to produce it again? It does not make sense.

We briefly described the method that Origene used for the production of the human recombinant OVGP1

(27) In lines 626 to 627 on page 18, it is stated that "Zonae pellucidae previously incubated with OVGP1 proteins from several species and murine oviductal fluid...". Were the zonae pellucidae previously incubated with only murine oviductal fluid or also with others?

It was only incubated with OVGP1 or with oviductal fluid, this is now clarified in the text.

(28) In lines 638 and 639 on page 19, can the authors please explain the difference between "endogenous OVGP1 and bOVGP1" and "exogenous recombinant hOVGP1 and mOVGP1"?

This is now clarified

(29) As stated in lines 676 to 679 on page 20, statistical analysis was performed in the study. Strangely, no "n" numbers and p values were provided in any of the figures that require statistical analysis. This is problematic.

Statistical analysis and significant differences are now included in the figures, all the numbers used are included in the supplementary tables that are related with the figures.

There are also many errors noted in the Figure Legends. These concerns raise questions regarding the reliability of the findings and interpretation of the results. Some major ones that require attention are listed below:

(30) Figure legend 1 on page 27: In line 912, where did the "cat sperm" come from? In line 913, where did the "feline sperm" come from? In line 918, as pointed out earlier, the term "empty zona penetration test (EZPT)" is a misnomer and should be replaced with a better term. In line 924, it is stated that "Note sperm only appear outside the zona." However, no sperm can be seen outside the zona pellucida shown in Figure 1.

Cat sperm is used in this manuscript. Term EZPT is now clarified The sentence about sperm outside of ZP is removed

(31) Figure legend 2 on page 27 (lines 928 to 940) needs to be rewritten. Some of the sentences are not clearly written. Authors, please check all the capital labeling letters some of which appear to be wrong.

Done

(32) As is written, Figure legend 3 on pages 28 and 29 (lines 943 to 959) presents many problems:

a. Contrary to what is stated in the figure legend, not all five regions are present in the hOVGP1, mOVGP1, and bOVGP1.

Done

b. Contrary to what is stated in line 946, region D is not conserved in the mouse and bull as shown in Figure 3A, and region C is not present only in the mouse.

Done

c. Based on what is shown in Figure 3A, region E is present only in the mouse and not in the human.

Done

d. What is stated in line 951 that "Proteins were expressed in mammalian cells..." is not correct. Based on the information provided in the manuscript, recombinant human OVGP1 was obtained from Origene Technologies and was not expressed in mammalian cells as claimed.

All the recombinant proteins were produced in mammalian cells.

(33) Figure legend 6 on page 28: In lines 985 to 986, what do the authors mean by "...and combinations of the three oviductins with sperm of the three species."? As is written, it appears that the bovine ZPs were pretreated with a combination of all three oviductins and then co-incubated with sperm from the bull, mouse and human together.

We have clarified this sentence

(34) What is described in the figure legend for the supplemental figure (Figure S7) does not make sense.

Legend of Fig S7 (now S8) is related to pictures A to E, the legend is now clarified.

(35) In addition to the figures and supplemental figures provided in the manuscript, there is also an additional figure labeled with "Model" showing three diagrams. Strangely, there is no mention of this additional figure in the manuscript. There is no figure legend for or description of this figure. It is not clear what is being shown in this figure, and it is not clear about the purpose of the use of this figure.

We have included a legend to the model that is now Figure 10.