Immunization Against a Conserved Short 13-Amino Acid Receptor-Binding Epitope of FSHβ Reduces Spermatogenesis and Sperm Motility in Male Mice
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript “Immunization against a conserved short 13-amino acid receptor-binding epitope of FSHb reduces spermatogenesis and sperm motility in male mice” provides information on the role of FSHb receptor in male fertility in mice. Unfortunately, in mice, the immunization of FSHb in male fertilization exhibits a functional decline than we expected, but it needs to be further investigated and discussed. Also, it needs to add the schemes of conclusion to make it easier for readers to understand.
- It would be easier to understand if a diagram showed which part of the FSHb receptor was targeted.
- The authors need to discuss why the results were not impacted in mice. Are there differences depending on the strain? Is there a relationship between multiple and single pregnancies?
- It is necessary to briefly describe the potential clinical application of these results in the future. If we were to apply this to clinical practice, it would be easier to use methods such as oral or topical administration rather than immunization. The feasibility of this should be described.
- Summarizing the results in a diagram will make it easier for readers to understand (including the difference between human and mice).
Author Response
Responds to the comments of Reviewer #1:
General comments
The manuscript “Immunization against a conserved short 13-amino acid receptor-binding epitope of FSHb reduces spermatogenesis and sperm motility in male mice” provides information on the role of FSHb receptor in male fertility in mice. Unfortunately, in mice, the immunization of FSHb in male fertilization exhibits a functional decline than we expected, but it needs to be further investigated and discussed. Also, it needs to add the schemes of conclusion to make it easier for readers to understand.
Response: Thank you for your valuable comments! These suggestions have been highly insightful for improving the quality of the manuscript. We have addressed these issues you raised point by point below.
Specific comments
Comments 1. It would be easier to understand if a diagram showed which part of the FSHb receptor was targeted.
Response: According to your good suggestion, we have drawn a diagram to shown the FSHb receptor targeted domain (Figure 1A).
Comments 2. The authors need to discuss why the results were not impacted in mice. Are there differences depending on the strain? Is there a relationship between multiple and single pregnancies?
Response: Thank you for these constructive comments. We have added a detailed discussion in the revised manuscript to clarify why no significant effects on fertility were observed in mice (see Line 423-432; marked in red). The observed species differences are largely attributed to distinct FSH dependence for spermatogenesis rather than mouse strain or reproductive strategy (single vs. multiple pregnancies). Specifically, rodents can complete spermatogenesis in the absence of FSH stimulation (Reference 4), whereas men harboring loss-of-function mutations in FSHβ exhibit azoospermia and infertility (Reference 3). These points have been clearly addressed in the revised text (see Line 425-432; marked in red).
Comments 3. It is necessary to briefly describe the potential clinical application of these results in the future. If we were to apply this to clinical practice, it would be easier to use methods such as oral or topical administration rather than immunization. The feasibility of this should be described.
Response: According to your good suggestion, we have added a brief description of the potential clinical applications and future translational directions, including a discussion on the feasibility of more convenient delivery routes (e.g., oral or topical administration) compared with immunization, to improve clinical practicality. The relevant content has been updated in the manuscript (see Line 434-439; marked in red).
Comments 4. Summarizing the results in a diagram will make it easier for readers to understand (including the difference between human and mice).
Response: Thank you for this constructive suggestion. We have added a schematic diagram (i.e.,Table 2) to summarize the key findings of this study, including the distinct spermatogenic responses to FSH inhibition between mice and humans, to improve clarity for readers (see line 327-341; marked in red).
Comments 5: The English could be improved to more clearly express the research.
Quality of Figures.
Response: We have invited a professional English language expert to revise and edit our manuscript. Should any typos or grammatical errors remain, we will utilize the expert editing services recommended by the journal for further refinement.
Special thanks to your good comments and suggestions!
Reviewer 2 Report
Comments and Suggestions for AuthorsReview Report
The manuscript entitled «Immunization against a conserved short 13-amino acid receptor-binding epitope of FSHβ reduces spermatogenesis and sperm motility in male mice» describes the development of a novel male contraceptive vaccine based on a tandem repeat of a 13-amino acid epitope of the FSHβ subunit (FSHβ13AA-T). The study demonstrates that this targeted immunization effectively reduces sperm production and motility in male mice without disrupting the HPG axis (LH and Testosterone levels) or sexual behavior (libido). This research is highly significant as it addresses the "specificity" gap in male contraceptive development.
General Comments
The manuscript is well-written, and the experimental design in the Materials and Methods section is robust. The study provides important mechanistic insights into how FSH-neutralization affects the testicular microenvironment. However, some minor revisions are required to strengthen the narrative and clarify specific experimental choices.
Specific Revisions
Introduction
Mechanistic Bridging: While the introduction is logically sound and follows a clear progression, it could be further strengthened. I suggest the authors better bridge the gap between the functional outcome (infertility) and the molecular disruption Specifically, foreshadowing the downregulation of the Cyp19a1 (aromatase) pathway would help set the stage for the novel mechanistic findings presented in the Results section.
Materials and Methods
Line 102: "at 7 weeks of a" — This appears to be a typographical error. Please remove or correct this phrase.
Line 128 (Offspring Data): The authors recorded the body weight of offspring from birth to weaning.
Query: Could the authors clarify the rationale for this measurement? If the primary goal is to assess male fertility/contraception, please explain if this was intended to check for any "leaky" fertility or to ensure that the health of the progeny (if any are born) remains unaffected by the paternal vaccination.
Results
The results are presented clearly and the statistical analysis appears appropriate. The selective impact on FSH without affecting Testosterone is a key strength of the data.
Discussion
Line 294 (Animal Management): The authors mention the potential use of this vaccine for population management of domestic animals and wildlife.
- Request: This is a very interesting application; please provide a relevant reference to support the use of immunocontraception in wildlife management (e.g., studies on GnRH or FSH vaccines in wild populations).
The study represents a significant contribution to the field of reproductive biology. The claim that this is the first study to link FSH vaccination to the suppression of local testicular estrogen production via the Cyp19a1 pathway is a major novelty.
Author Response
Responds to the comments of Reviewer #2:
General Comments:
The manuscript is well-written, and the experimental design in the Materials and Methods section is robust. The study provides important mechanistic insights into how FSH-neutralization affects the testicular microenvironment. However, some minor revisions are required to strengthen the narrative and clarify specific experimental choices.
Response: Thanks for your positive comments. All minor revisions suggested by the reviewer have been addressed point-by-point below.
Specific comments:
Comments 1. Introduction- Mechanistic Bridging: While the introduction is logically sound and follows a clear progression, it could be further strengthened. I suggest the authors better bridge the gap between the functional outcome (infertility) and the molecular disruption. Specifically, foreshadowing the downregulation of the Cyp19a1 (aromatase) pathway would help set the stage for the novel mechanistic findings presented in the Results section.
Response: Thank you for this valuable suggestion. In light of this, we have added a discussion of the role of Cyp19a1 (aromatase) in the regulation of male fertility to the Introduction section, thereby establishing a clear mechanistic link between FSH blockage-associated infertility/subfertility and the underlying molecular perturbations (see line 70-81; marked in red).
Comments 2. Line 102: "at 7 weeks of a" — This appears to be a typographical error. Please remove or correct this phrase.
Response: This error has been corrected (see line 117; marked in red).
Comments 3. Line 128 (Offspring Data): The authors recorded the body weight of offspring from birth to weaning.
Query: Could the authors clarify the rationale for this measurement? If the primary goal is to assess male fertility/contraception, please explain if this was intended to check for any "leaky" fertility or to ensure that the health of the progeny (if any are born) remains unaffected by the paternal vaccination.
Response: We apologize for the lack of clarification regarding the purpose. Indeed, this approach was intended to guarantee that the health of the offspring (if any were produced) would remain unaffected by paternal vaccination. In the revised manuscript, we have clarified the purpose of this measurement (see line 148-149; marked in red).
Comments 4. Results---The results are presented clearly and the statistical analysis appears appropriate. The selective impact on FSH without affecting Testosterone is a key strength of the data.
Response: Thanks for the positive comments.
Comments 5. Discussion---Line 294 (Animal Management): The authors mention the potential use of this vaccine for population management of domestic animals and wildlife. Request: This is a very interesting application; please provide a relevant reference to support the use of immunocontraception in wildlife management (e.g., studies on GnRH or FSH vaccines in wild populations).
The study represents a significant contribution to the field of reproductive biology. The claim that this is the first study to link FSH vaccination to the suppression of local testicular estrogen production via the Cyp19a1 pathway is a major novelty.
Response: According to the reviewer’s good suggestion, we have added relevant references to support the use of immunocontraception in wildlife management (i.e., reference 19,20) (see line 345; marked in red). Thanks for the positive comments on the novelty of our key finding.
Special thanks to your good comments and suggestions!
Reviewer 3 Report
Comments and Suggestions for AuthorsThe principal novelty lies in (i) the use of a conserved 13-AA FSHβ receptor-binding epitope, and (ii) the mechanistic link between FSH neutralization, reduced testicular aromatase (Cyp19a1) expression, and local estrogen depletion (lines ~260–335). The latter is explicitly claimed as a first report (lines ~334–336) and represents a genuine conceptual advance beyond descriptive antifertility outcomes.
The experimental design is generally sound, with two independent experiments addressing physiology and fertility/libido (lines ~98–129). The vaccination protocol, controls, and endpoints are clearly described. Use of DSP counts, CASA for motility, histology, and RT-qPCR across the HPT axis provides a comprehensive assessment.
Sample size justification and power calculations are not provided (e.g., n=10 in Experiment 1; n=15 in Experiment 2, lines ~99–124). While effects are statistically significant, a brief power rationale would strengthen robustness.
The use of Duncan’s multiple range test (lines ~202–203) is dated and relatively liberal; justification or confirmation with a more conservative post-hoc method would improve statistical rigor.
Terminological inconsistency appears in statistical analysis (“pig within treatment,” line ~196), likely a residual template error and should be corrected.
Effect sizes (e.g., percentage reduction in DSP, motility) are not consistently reported alongside P-values (Figures 3D–E; lines ~241–244). Including effect sizes would aid biological interpretation.
The conclusion that the mouse is “not a good animal model” for contraceptive efficacy (lines ~387–389) is correct but should be framed more precisely: mice are suboptimal for efficacy assessment, but remain informative for mechanistic dissection.
Limitations are acknowledged, particularly species differences in FSH dependence (lines ~374–381). However, translational extrapolation to humans (lines ~379–381) would benefit from a more explicit discussion of immunogenicity, reversibility, and safety—key requirements for human contraceptive vaccines.
Author Response
Responds to the comments of Reviewer #3:
General comments
The principal novelty lies in (i) the use of a conserved 13-AA FSHβ receptor-binding epitope, and (ii) the mechanistic link between FSH neutralization, reduced testicular aromatase (Cyp19a1) expression, and local estrogen depletion (lines ~260–335). The latter is explicitly claimed as a first report (lines ~334–336) and represents a genuine conceptual advance beyond descriptive antifertility outcomes. The experimental design is generally sound, with two independent experiments addressing physiology and fertility/libido (lines ~98–129). The vaccination protocol, controls, and endpoints are clearly described. Use of DSP counts, CASA for motility, histology, and RT-qPCR across the HPT axis provides a comprehensive assessment.
Response: Thank you for these positive comments.
Specific comments
Comments 1. Sample size justification and power calculations are not provided (e.g., n=10 in Experiment 1; n=15 in Experiment 2, lines ~99–124). While effects are statistically significant, a brief power rationale would strengthen robustness.
Response: Thank you for the suggestion. We apologize for not including sample size justification and power calculations, and have now added these details to the Materials and Methods section. Sample sizes (n=10 for Exp 1; n=15 for Exp 2) were determined via a priori power analysis (G*Power 3.1) for two-tailed t-tests (d=0.8, α=0.05, 1-β=0.90), ensuring adequate power to detect large effects consistent with prior FSH/reproductive biology studies. A concise power rationale is now included to strengthen methodological robustness (see line 219-223; marked in red).
Comments 2. The use of Duncan’s multiple range test (lines ~202–203) is dated and relatively liberal; justification or confirmation with a more conservative post-hoc method would improve statistical rigor.
Response: Thank you for this important comment and valuable suggestion. Following your advice, we have replaced Duncan’s multiple range test with the unpaired two-tailed Student’s t-test throughout the statistical analysis, as our study includes only two experimental groups. This more conservative and widely accepted post-hoc approach enhances the statistical rigor and reliability of our results (see line 235-236; marked in red).
Comments 3. Terminological inconsistency appears in statistical analysis (“pig within treatment,” line ~196), likely a residual template error and should be corrected.
Effect sizes (e.g., percentage reduction in DSP, motility) are not consistently reported alongside P-values (Figures 3D–E; lines ~241–244). Including effect sizes would aid biological interpretation.
Response: We sincerely apologize for the terminological inconsistency (“pig within treatment”), which was a residual template error and has now been fully removed and corrected in the revised manuscript. Following your valuable suggestion, we have also consistently added effect sizes alongside the corresponding P-values to improve biological interpretability, as recommended (see line 277-278; marked in red).
Comments 4. The conclusion that the mouse is “not a good animal model” for contraceptive efficacy (lines ~387–389) is correct but should be framed more precisely: mice are suboptimal for efficacy assessment, but remain informative for mechanistic dissection. Limitations are acknowledged, particularly species differences in FSH dependence (lines ~374–381). However, translational extrapolation to humans (lines ~379–381) would benefit from a more explicit discussion of immunogenicity, reversibility, and safety—key requirements for human contraceptive vaccines.
Response: Thank you for this constructive and insightful comments. We have revised our statement to be more precise: mice are suboptimal for contraceptive efficacy assessment, but remain highly informative for mechanistic dissection, completely according to your good suggestion (see line 444-447; marked in red).
In addition, according to your good suggestion, we have expanded the discussion to provide a more explicit evaluation of translational potential to humans, with specific emphasis on immunogenicity, reversibility, and safety—critical criteria for the future development of contraceptive vaccines in humans (see line 432-439; marked in red).
Special thanks to your good comments and suggestions!
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI think that the revised manuscript has been improved.
Comments on the Quality of English LanguageI think that the revised manuscript has been improved. However, we need to think carefully again about whether it is at a level that can be published in this journal (Vaccines)
