Oviductin sets the species-specificity of the mammalian zona pellucida

Reviewer #1 (Public review):

Summary:

This very 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 (Figure 1A). Similarly, bovine sperm can penetrate superovulated murine oocytes recovered directly from the ovary (Figure 1B). However, bovine oocytes incubated with oviduct fluid (30 min) are generally impenetrable by human sperm (Figure 1C).

Thereafter, the cytoplasm was aspirated from murine oocytes - obtained from the ovary (Figure 1D) or oviduct (Figure 1D). Binding and penetration by bovine and human sperm were 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 compelling 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 (Figure 2B), or not (Figure 2A) 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 his-tagged bovine and murine OVGP1 and DDK-tagged human OVGP1 proteins. Tagging was to enable purification following overexpression in BHK-21 or HEK293T cells. The authors confirm these recombinant OVGP1 proteins bound to both murine (Figure 3C) and bovine (Figure 3D) oocytes. Moreover, previous data using oviduct fluid (Figure 1D-E and 2A-B) was mirrored using bovine oocytes supplemented with homologous (bovine) recombinant OVGP1 (Figure 4B) or not (Figure 4A). This confirms the hypothesis, at least in cattle.

Next, the authors exposed bovine (Figure 6A) and murine (Figure 6B) 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 (Figures 7A-B) and murine (Figures 7C-D) 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.

Strengths:

The authors convincingly demonstrate that two mechanisms underpin mammalian sperm recognition and penetration, the first being specific (ZP-mediated) and the second non-specific (OVGP1-mediated). This may prove useful for improving porcine in vitro fertilization (IVF), which is notoriously prone to polyspermy, in addition to human IVF, for better intrinsic individual sperm selection.

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.

(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.

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

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

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

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

Reviewer #2 (Public review):

In the manuscript entitled "Oviductin sets the species-specificity of the mammalian zona pellucida." The study analyzes 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:

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.

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.

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.

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.

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?

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.

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?

Reviewer #3 (Public review):

Summary:

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.

Strengths:

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

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.

Author response

We appreciate the positive comments and constructive suggestions from the editors and reviewers, which will help us improve our manuscript. We will implement the changes as requested by the reviewers, focusing primarily on revising and clarifying the following aspects:

First, we will clarify the use of biological and technical replicates in each experiment and provide more details about the statistical analyses conducted. Additionally, we plan to include a schematic representation of the experimental design.

Second, we will explain the experiment conducted to rule out hormonal effects or differences in the oocyte maturation method used. We will also indicate the concentration of OVGP1 in the oviduct and explain why we selected OVGP1 as the probable cause of species specificity.

Third, by addressing all of the reviewers' suggestions, we aim to resolve any concerns, inconsistencies, or minor errors identified by the reviewers.

We are committed to addressing all the issues raised by the reviewers and believe that the manuscript will greatly benefit from the insightful suggestions and invaluable contributions of the editors and reviewers.