The full length BEND2 protein is dispensable for spermatogenesis but required for setting the ovarian reserve in mice

  1. Yan Huang
  2. Nina Bucevic
  3. Carmen Coves
  4. Natalia Felipe-Medina
  5. Marina Marcet-Ortega
  6. Nikoleta Nikou
  7. Cristina Madrid-Sandín
  8. Maria López-Panadés
  9. Carolina Buza
  10. Neus Ferrer Miralles
  11. Antoni Iborra
  12. Anna Pujol
  13. Alberto M Pendás
  14. Ignasi Roig

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    eLife Assessment

    This study provides valuable information on a novel gene that regulates meiotic progression in both male and female meiosis. The evidence supporting the conclusions of the authors is solid. This study will be of interest to developmental and reproductive biologists.

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Abstract

Abstract

Infertility affects up to 12% of couples globally, with genetic factors contributing to nearly half of the cases. Advances in genomic technologies have led to the discovery of genes like Bend2, which plays a crucial role in gametogenesis. In the testis, Bend2 expresses two protein isoforms: full-length and a smaller one. Ablation of both proteins results in an arrested spermatogenesis. Because the Bend2 locus is on the X chromosome, and the Bend2−/y mutants are sterile, BEND2’s role in oogenesis remained elusive.

In this study, we employed a novel Bend2 mutation that blocks the expression of the full-length BEND2 protein but allows the expression of the smaller BEND2 isoform. Interestingly, this mutation does not confer male sterility and mildly affects spermatogenesis. Thus, it allowed us to study the role of BEND2 in oogenesis. Our findings demonstrate that full-length BEND2 is dispensable for male fertility, and its ablation leads to a reduced establishment of the ovarian reserve. These results reveal a critical role for full-length BEND2 in oogenesis and provide insights into the mechanisms underlying the establishment of the ovarian reserve. Furthermore, these findings hold relevance for the diagnostic landscape of human infertility.

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  1. Version published to 10.7554/elife.96052.3 on eLife
  2. Version published to 10.7554/elife.96052 on eLife
  3. eLife

    eLife Assessment

    This study provides valuable information on a novel gene that regulates meiotic progression in both male and female meiosis. The evidence supporting the conclusions of the authors is solid. This study will be of interest to developmental and reproductive biologists.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, the authors investigate the role of BEND2, a novel regulator of meiosis, in both male and female fertility. Huang et al have created a mouse model where the full-length BEND2 transcript is depleted but the truncated BEND2 version remains. This mouse model is fertile, and the authors used it to study the role of BEND2 on both male and female meiosis. Overall, the full-length BEND2 appears dispensable for male meiosis. The more interesting phenotype was observed in females. Females exhibit a lower ovarian reserve suggesting that full-length BEND2 is involved in the establishment of the primordial follicle pool.

    Strengths:

    The authors generated a mouse model that enabled them to study the role of BEND2 in meiosis. The role of BEND2 in female fertility is novel and enhances our knowledge of genes involved in the establishment of the primordial follicle pool.

    Weaknesses highlighted previously:

    The manuscript extensively explores the role of BEND2 in male meiosis; however, a more interesting result was obtained from the study of female mice.

  5. eLife

    Reviewer #2 (Public review):

    In their manuscript entitled "BEND2 is a crucial player in oogenesis and reproductive aging", the authors present their findings that full-length BEND2 is important for repair of meiotic double strand break repair in spermatocytes, regulation of LINE-1 elements in spermatocytes, and proper oocyte meiosis and folliculogenesis in females. The manuscript utilizes an elegant system to specifically ablate the full-length form of BEND2 which has been historically difficult to study due to its location on the X chromosome and male sterility of global knockout animals.

    The authors have been extremely responsive to reviewer critiques and have presented strong data and appropriate conclusions, making it an excellent addition to the field.

  6. eLife

    Reviewer #3 (Public review):

    Huang et al. investigated the phenotype of Bend2 mutant mice which expressed truncated isoform. Bend2 deletion in male showed fertility and this enabled them to analyze the BEND2 function in females. They showed that Bend2 deletion in females showed decreasing follicle number which may lead to loss of ovarian reserve.

    Strengths:

    They found the truncated isoform of Bend2 and the depletion of this isoform showed decreasing follicle number at birth.

    Weaknesses highlighted previously:

    The authors showed novel factors that impact ovarian reserve. Although the number of follicles and conception rate are reduced in mutant mice, the in vitro fertilization rate is normal and follicles remain at 40 weeks of age. It is difficult to know how critical this is when applied to the human case.

    [Editors' note: We thank the authors for considering the previous recommendations and suggested corrections.]

  7. eLife

    Author response:

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

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, the authors investigate the role of BEND2, a novel regulator of meiosis, in both male and female fertility. Huang et al have created a mouse model where the full-length BEND2 transcript is depleted but the truncated BEND2 version remains. This mouse model is fertile, and the authors used it to study the role of BEND2 on both male and female meiosis. Overall, the full-length BEND2 appears dispensable for male meiosis. The more interesting phenotype was observed in females. Females exhibit a lower ovarian reserve suggesting that full-length BEND2 is involved in the establishment of the primordial follicle pool.

    Strengths:

    The authors generated a mouse model that enabled them to study the role of BEND2 in meiosis. The role of BEND2 in female fertility is novel and enhances our knowledge of genes involved in the establishment of the primordial follicle pool.

    Weaknesses:

    The manuscript extensively explores the role of BEND2 in male meiosis; however, a more interesting result was obtained from the study of female mice.

    We sincerely appreciate the reviewer’s thoughtful evaluation of our work and recognition of the strengths of our study. We are especially grateful for the acknowledgment of the novelty of our findings regarding the role of BEND2 in female fertility. While we extensively characterized the e ects of BEND2 depletion in male meiosis, we agree that the phenotype observed in females provides particularly interesting insights into the establishment of the primordial follicle pool.

    Reviewer #2 (Public review):

    In their manuscript entitled "BEND2 is a crucial player in oogenesis and reproductive aging", the authors present their findings that full-length BEND2 is important for repair of meiotic double strand break repair in spermatocytes, regulation of LINE-1 elements in spermatocytes, and proper oocyte meiosis and folliculogenesis in females. The manuscript utilizes an elegant system to specifically ablate the full-length form of BEND2 which has been historically di icult to study due to its location on the X chromosome and male sterility of global knockout animals.

    The authors have been extremely responsive to reviewer critiques and have presented strong data and appropriate conclusions, making it an excellent addition to the field.

    We are truly grateful for the reviewer’s thoughtful review and recognition of the key contributions of our study. We appreciate the acknowledgment of how our model overcomes the challenges in studying BEND2 and the importance of our findings in both male and female meiosis. We also value the reviewer’s encouraging comments on our responsiveness to their feedback and the quality of our data and conclusions.

    Reviewer #3 (Public review):

    Huang et al. investigated the phenotype of Bend2 mutant mice which expressed truncated isoform. Bend2 deletion in male showed fertility and this enabled them to analyze the BEND2 function in females. They showed that Bend2 deletion in females showed decreasing follicle number which may lead to loss of ovarian reserve.

    Strengths:

    They found the truncated isoform of Bend2 and the depletion of this isoform showed decreasing follicle number at birth.

    Weaknesses:

    The authors showed novel factors that impact ovarian reserve. Although the number of follicles and conception rate are reduced in mutant mice, the in vitro fertilization rate is normal and follicles remain at 40 weeks of age. It is difficult to know how critical this is when applied to the human case.

    We greatly appreciate the reviewer’s comments and recognition of the strengths of our work. We are grateful for their acknowledgment of our findings related to the truncated isoform of Bend2 and its e ect on ovarian reserve. We also agree that, although our study provides important insights, we are still far from directly applying these results to human clinical scenarios. There is much further research needed before these findings can be translated.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors)::

    The authors have addressed all concerns both editorially and experimentally. This is a very nice manuscript, and I congratulate the authors on their work.

    We sincerely appreciate your kind words and thoughtful review. Your feedback has been invaluable in improving our manuscript, and we are grateful for your time and effort. Thank you for your support and encouragement!

    Reviewer #2 (Recommendations for the authors)::

    In Figure 3, graphs in panels C & D have typos in the early zygotene column where it reads "zyotene".

    We appreciate your careful review and for pointing out the typos in Figure 4, which has been corrected in the new version of the manuscript.

    Reviewer #3 (Recommendations for the authors):

    ・Since there are two isoforms of Bend2, and the authors depleted one isoform, this is not suitable to use "full length" in the titles and in the manuscripts.

    We respectfully disagree with the reviewer’s comment. In our mouse model, we specifically remove the full-length isoform of Bend2. Therefore, we consider it appropriate to refer to it as such in the manuscript. Our results indicate that the full-length isoform is not required to complete meiotic prophase in males but is indispensable for setting up the ovarian reserve in females. We appreciate the reviewer’s input and are happy to clarify this point further if needed.

    ・Is there any reason why authors used 7 month old females for in vitro fertilization? It may not be recognized as aged mice but it seems a bit old to perform IVF especially when the ovarian reserve in mutant mice is decreased. If there is any reason, please clarify it. In addition, since the authors added IVF data, which showed similar fertilization ratio between control and mutant, the authors need to discuss why the litter size was decreased in mutant mice. It may be to strong to conclude "subfertility".

    We used 7-month-old females for IVF because this falls within the age range of the samples analyzed for ovarian reserve, with the oldest females being 8 months old. Regarding the apparent discrepancy between IVF results and litter size, we addressed this in the discussion section of the manuscript: 'Interestingly, our mutant oocyte quality analysis suggests that mature oocytes from mutant females are equally competent to develop into a blastocyst as control ones. These data suggest that the subfertility observed in Bend2 mutants may be due to errors in later developmental stages, such as implantation or organogenesis.' We appreciate the reviewer’s feedback and hope this clarification helps.

  8. eLife

    eLife Assessment

    This study provides valuable information on a novel gene that regulates meiotic progression in both male and female meiosis. The evidence supporting the conclusions of the authors is solid. This study will be of interest to developmental and reproductive biologists.

  9. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, the authors investigate the role of BEND2, a novel regulator of meiosis, in both male and female fertility. Huang et al have created a mouse model where the full-length BEND2 transcript is depleted but the truncated BEND2 version remains. This mouse model is fertile, and the authors used it to study the role of BEND2 on both male and female meiosis. Overall, the full-length BEND2 appears dispensable for male meiosis. The more interesting phenotype was observed in females. Females exhibit a lower ovarian reserve suggesting that full-length BEND2 is involved in the establishment of the primordial follicle pool.

    Strengths:

    The authors generated a mouse model that enabled them to study the role of BEND2 in meiosis. The role of BEND2 in female fertility is novel and enhances our knowledge of genes involved in the establishment of the primordial follicle pool.

    Weaknesses:

    The manuscript extensively explores the role of BEND2 in male meiosis; however, a more interesting result was obtained from the study of female mice.

  10. eLife

    Reviewer #2 (Public review):

    In their manuscript entitled "BEND2 is a crucial player in oogenesis and reproductive aging", the authors present their findings that full-length BEND2 is important for repair of meiotic double strand break repair in spermatocytes, regulation of LINE-1 elements in spermatocytes, and proper oocyte meiosis and folliculogenesis in females. The manuscript utilizes an elegant system to specifically ablate the full-length form of BEND2 which has been historically difficult to study due to its location on the X chromosome and male sterility of global knockout animals.

    The authors have been extremely responsive to reviewer critiques and have presented strong data and appropriate conclusions, making it an excellent addition to the field.

  11. eLife

    Reviewer #3 (Public review):

    Huang et al. investigated the phenotype of Bend2 mutant mice which expressed truncated isoform. Bend2 deletion in male showed fertility and this enabled them to analyze the BEND2 function in females. They showed that Bend2 deletion in females showed decreasing follicle number which may lead to loss of ovarian reserve.

    Strengths:

    They found the truncated isoform of Bend2 and the depletion of this isoform showed decreasing follicle number at birth.

    Weaknesses:

    The authors showed novel factors that impact ovarian reserve. Although the number of follicles and conception rate are reduced in mutant mice, the in vitro fertilization rate is normal and follicles remain at 40 weeks of age. It is difficult to know how critical this is when applied to the human case.

  12. eLife

    Author response:

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

    Reviewer #1 (Public Review):

    Summary:

    In this manuscript, the authors investigate the role of BEND2, a novel regulator of meiosis, in both male and female fertility. Huang et al have created a mouse model where the fulllength BEND2 transcript is depleted but the truncated BEND2 version remains. This mouse model is fertile, and the authors used it to study the role of BEND2 on both male and female meiosis. Overall, the full-length BEND2 appears dispensable for male meiosis. The more interesting phenotype was observed in females. Females exhibit a lower ovarian reserve suggesting that full-length BEND2 is involved in the establishment of the primordial follicle pool.

    Strengths:

    The authors generated a mouse model that enabled them to study the role of BEND2 in meiosis. The role of BEND2 in female fertility is novel and enhances our knowledge of genes involved in the establishment of the primordial follicle pool.

    Weaknesses:

    The manuscript extensively explores the role of BEND2 in male meiosis; however, a more interesting result was obtained from the study of female mice. Only a few experiments were performed using female mice, therefore, more experiments should be performed to complete the story of the role of BEND2 on female fertility. In addition, the title and abstract of the manuscript do not align with the story, as female fertility is only a small portion of the data compared to the male fertility section.

    We appreciate the reviewer’s thoughtful summary, recognition of the strengths of our study, and constructive feedback. In the revised manuscript, we have performed additional experiments to enhance our understanding of the role of BEND2 in female gametogenesis. These new experiments provide further insights into the establishment of the ovarian reserve and the role of BEND2 in female fertility.

    Additionally, we have rewritten the title, abstract, and introduction to better align with the content of the manuscript and to reflect the balance between the male and female fertility results. We believe these changes address the reviewer’s concerns and improve the overall clarity and focus of the manuscript.

    Reviewer #1 (Recommendations For The Authors):

    • I recommend that the authors re-organize their abstract and introduction to accurately reflect the manuscript's primary focus on male fertility. Right now, the title of the manuscript is misleading. The manuscript does not investigate reproductive aging; rather, it primarily describes the depletion of primordial follicle number. The mechanism behind this depletion and whether this phenotype accelerates reproductive aging, are not explored. Clarifying these points will help align the title and content of the manuscript more accurately.

    We thank the reviewer for this suggestion. We agree that the original title and abstract did not fully capture the focus of the study. In response, we have rewritten the title, abstract, and introduction to better align with the results presented, focusing more clearly on the implications of the effects of the full-length BEND2 depletion for spermatogenesis and oogenesis. These revisions ensure that the title, the abstract, and the manuscript's introduction are now more accurately reflective of the work performed.

    • Figure 1: I couldn't find the validation of the polyclonal antibody against BEND2 that the authors generated.

    Regarding this query about the validation of the polyclonal antibody against BEND2, we apologize for any confusion. We would like to clarify that this validation is indeed presented in Figure 2 of our manuscript. To ensure this information is easily accessible, we have revised the text to explicitly mention the validation in Figure 2.

    • Figure 2A: Could you provide the actual numbers for the weight of the mice testis?

    In response to this question regarding Figure 2A and the weights of the mice testis, we have now included this data in a graph in Fig 2A and Table S1 and added this information in the results section.

    • Figure 2C and D: I am confused by the fact that in the WB we can appreciate a high expression of the p75 protein, but the signal is very low in the IF (Figure 2D).

    We thank the reviewer for raising this point. We acknowledge the apparent discrepancy between the strong p75 signal observed in the Western blot (Fig. 2C) and the weaker signal seen in the immunofluorescence (Fig. 2D). We think several factors could contribute to this difference, such as differences in sensitivity and detection methods, epitope accessibility, protein localization or differences in sample preparation, antibody affinity, and experimental conditions between Western blot and IF.

    • In the same figure, the authors also mention that the p75 protein is functional. On what basis do they rely on reaching this conclusion?

    We acknowledge that we cannot definitively confirm the functionality of the p75 protein. Our assumption was based on the observed fertility of the male mice and existing literature indicating that BEND2 is essential for completing meiosis (Ma et al., 2022). However, we understand the importance of clarity in our claims. To avoid any potential confusion, we have revised the sentence to read: "The p75 BEND2 protein—likely corresponding to an exon 11-skipped transcript—is present and might be functional in our mutant testis, based on the observed phenotype (see below)."

    • The phenotype in females is very interesting. The authors conclude that BEND2 influences primordial follicle formation, oocyte quality, fertility, and reproductive aging by (1) performing follicle counts, (2) analyzing the litter size, and (3) analyzing meiotic progression. Given that the authors build their story around these experiments, I strongly encourage them to expand the section on female fertility, or reorganize the manuscript, or be more cautious with some of their conclusions. They might consider performing additional experiments such as:

    - Oocyte quality: To determine whether BEND2 impacts oocyte quality, mice should be stimulated with hormones and oocyte quality should be analyzed (GV, MI, MII progression, spindle morphology and/or fertilization, and embryo development). Does the decrease in primordial follicles correlate with the number of ovulated oocytes, or is the impact only on oocyte quality?

    We appreciate the reviewer's suggestion to assess the impact of BEND2 on oocyte quality. Following the reviewer’s recommendation, we stimulated three control and three mutant mice. We analyzed the number of ovulated oocytes, their fertilization rate, and the percentage of embryos that developed to the blastocyst stage. These new results are included in the revised manuscript (see Results section and new Table 1). Our analyses indicate that for all parameters assessed, control and mutant oocytes behaved similarly. Specifically, there were no significant differences in the number of ovulated oocytes, fertilization rates, or the ability of embryos to progress to the blastocyst stage between the control and mutant groups. These findings suggest that mutant oocyte quality is comparable to control mice of a similar age. We have incorporated these new results into the manuscript.

    - Reproductive aging: A fertility trial would provide more information on whether BEND2 depletion triggers an acceleration of reproductive aging. In addition, the oldest mice used by the authors are 9 months old, and at this point, fertility has not declined yet.

    We appreciate the reviewer's suggestion regarding the assessment of reproductive aging. However, we respectfully disagree with the assertion that fertility has not declined by 9 months of age. In our colony, we have observed a significant decline in fertility around 10 months of age. Specifically, out of 18 10-month-old female mice placed in breeding cages, we observed only three pregnancies within the first 30 days (N.N. and I.R., data not published). Based on these observations, we determined that fertility begins to decline around this age in our colony, which informed our decision to use 9-month-old mice as the oldest age group for our analysis. Thus, this age is appropriate for evaluating the potential effects of BEND2 depletion on reproductive aging in our specific mouse population.

    - The observation that the primordial follicle pool is already diminished in mice that are 1 week old is very interesting. Some experiments that the authors could perform to figure out the mechanism are: (1) Analyzing apoptosis. Are the primordial follicles dying during the pool's establishment, or is this an ongoing apoptotic process throughout the mice's lifespan? (2) If the authors still have ovaries from mice younger than 1 week of age (when the primordial pool is forming), they could perform DDX4 staining and quantify the number of oocytes in follicles and the total number of oocytes. These experiments would provide mechanistic insights into whether BEND2 impacts the formation of the primordial follicle pool or if the pool forms but is then depleted.

    We appreciate the reviewer's suggestion to further explore the mechanism behind the reduced primordial follicle pool. In response, we have analyzed the number of DDX4positive cells (DDX4 labels oocytes) in newborn mutant and wild-type animals. Our results show that mutant ovaries contain significantly fewer oocytes compared to controls (see new Fig. 5). This finding supports the hypothesis that BEND2 is critical for the establishment of a normal ovarian reserve. We are grateful for this suggestion, as these additional data reinforce our conclusion that BEND2 is required to determine a normal ovarian reserve in mice.

    • What is the red signal in Supplementary Figure 1C?

    This image depicts the BEND2 staining pattern in 16 days post-coitum (dpc) wild-type mouse ovaries. To clarify this and prevent any confusion, we have updated the figure legend to explicitly state that the sample shown is from a wild-type mouse.

    • Please spell out the full term of all the acronyms.

    We apologize for the oversight in not fully spelling out some acronyms in the original manuscript. We have carefully reviewed the entire manuscript and have ensured that all acronyms are now spelled out in full upon their first use in the revised version. We want to thank the reviewer for bringing this to our attention.

    • Is Line-1 also dysregulated in the ovary? This was one of the main findings from the male part. It would be interesting to perform the same analysis in the ovary since Line1 has a role in establishing the ovarian reserve (PDMI: 31949138).

    We thank the reviewer for this insightful suggestion. We have analyzed the number of LINE1 and SYCP3-positive cells in wild-type and mutant newborn ovaries (new Fig. S4). Our results show no significant difference between the two genotypes, suggesting that LINE-1 is not dysregulated in newborn Bend2 mutant oocytes. These findings indicate that, at least in the context of the newborn ovary, LINE-1 does not appear to be affected by BEND2 depletion.

    Reviewer #2 (Public Review):

    In their manuscript entitled "BEND2 is a crucial player in oogenesis and reproductive aging", the authors present their findings that full-length BEND2 is important for repair of meiotic double strand break repair in spermatocytes, regulation of LINE-1 elements in spermatocytes, and proper oocyte meiosis and folliculogenesis in females. The manuscript utilizes an elegant system to specifically ablate the full-length form of BEND2 which has been historically difficult to study due to its location on the X chromosome and male sterility of global knockout animals.

    While the manuscript is an overall excellent addition to the field, it would significantly benefit from a few additional experiments, as well as some additional clarification/elaboration.

    The claim that BEND2 is required for ovarian reserve establishment is not supported, as the authors only look at folliculogenesis and oocyte abundance starting at one week of age, after the reserve is formed. Analysis of earlier time points would be much more convincing and would parse the role of BEND2 in the establishment vs. maintenance of this cell population. In spermatocytes, the authors demonstrate a loss of nuclear BEND2 in their mutant but do not comment on the change in localization (which is now cytoplasmic) of the remaining protein in these animals. This may have true biological significance and a discussion of this should be more thoroughly explored.

    We thank the reviewer for their thoughtful feedback and constructive suggestions to improve our manuscript.

    In response to the comment regarding the establishment of the ovarian reserve, we have now analyzed Bend2 mutant and control newborn ovaries. Our results show a significant reduction in the number of DDX4-positive cells in mutant ovaries compared to controls. These findings demonstrate that BEND2 is required for the establishment of the ovarian reserve, as the reduction is evident at birth.

    Regarding the cytoplasmic staining of BEND2 in mutant spermatocytes, we did perform secondary-antibody-only controls using goat anti-rabbit Cy3 to address the specificity of the signal. The staining observed in the Bend2 mutants closely resembles background staining, suggesting that the cytoplasmic signal is nonspecific. Therefore, we do not believe this represents a meaningful change in the localization of BEND2 protein in the mutants. We have clarified this in the revised manuscript to address this point.

    We hope these additional experiments and clarifications strengthen the manuscript and address the reviewer’s concerns.

    Reviewer #2 (Recommendations For The Authors):

    Major points:

    (1) The title of the manuscript does not accurately capture the content of the work. The vast majority of the data presented here is from the male, which is not reflected at all in the title - perhaps considering revising it?

    Thank you for your valuable suggestion. We agree that the original title did not fully reflect the focus of the manuscript. In response, we have revised the title, along with the abstract and introduction, to more accurately capture the content of the study and the emphasis on the male data. These changes ensure that the manuscript more clearly aligns with the results presented.

    (2) In Figure 2D, the authors demonstrate that WT BEND2 expression and localization are lost in the mutant, but staining is still apparent, just in the cytoplasm. Did the authors perform secondary-antibody-only controls to determine if this was background staining or real staining? If real, can they comment on the change in localization of the protein?

    We thank the reviewer for this insightful question. We have indeed performed secondary antibody-only controls using goat anti-rabbit Cy3. The staining observed in the Bend2 mutants closely resembles background staining, suggesting that the signal in the cytoplasm is not specific. Therefore, we do not believe this staining represents any real or meaningful expression of the BEND2 protein in the mutants.

    (3) In Figure S2A, the authors show Ku70 staining and describe that it is similar between the genotypes, but - to my eye - it looks quite distinctly different. It appears to stain in patches in WT SYCP3+ spermatocytes, versus staining in patches in the more mature, SYCP3- germ cells closer to the lumen in the mutant. Can the authors please clarify, or provide arrows to point which foci they are referring to?

    We apologize for the confusion caused by the image provided in the original submission. Upon review, we realized that the mutant image was not fully representative of the staining pattern observed in the majority of mutant samples. We have replaced this image with a new one in the revised manuscript, which more accurately reflects the similarity in Ku70 staining between wild-type and mutant testis. In this updated Figure S2, we have also included arrowheads to indicate the relevant foci, making it clearer to the reader. We have updated the figure legend to correspond with these changes as well.

    (4) The authors state that BEND2 is "required to establish the ovarian reserve during oogenesis" but this has not been demonstrated. The authors do show a reduced density of primordial follicles at one week of age. While this is compelling data, the ovarian reserve is established earlier in the mouse, around postnatal days 0-1, so it is not clear from this manuscript whether BEND2 is required for the maintenance of this population after PND1, leading to reduced numbers by 1 week of age, OR if it is required for the establishment of this population, which would result in reduced numbers of oocytes around the time of birth. This is a critical experiment that should be performed in order to determine which of these possibilities is likely the case. Ideally, looking at embryonic through early postnatal time points during ovarian development would be very helpful.

    We thank the reviewer for raising this important point. As mentioned earlier in response to Reviewer 1, we have performed the experiment suggested by Reviewer 2 and analyzed the number of DDX4-positive cells in newborn ovaries. Our results show that Bend2 mutant ovaries have fewer oocytes at birth than wild-type controls (Fig. 5H). This finding reinforces our conclusion that BEND2 is indeed required to establish the ovarian reserve, as the reduction in oocyte number is evident at the time of birth. We agree that this additional data strengthens our original claim, so we have included these results in the revised manuscript.

    Reviewer #3 (Public Review):

    Summary:

    Huang et al. investigated the phenotype of Bend2 mutant mice which expressed a truncated isoform. This mutant male showed increasing apoptosis due to unrepaired double-strand breaks. However, this mutant male has fertility, and this enabled them to analyze Bend2 function in females. They revealed that Bend2 mutation in females showed decreasing follicle numbers which leads to loss of ovarian reserve.

    Strengths:

    Since their Bend2 mutant males were fertile, they were able to analyze the function of Bend2 in females and they revealed that loss of Bend2 causes less follicle formation.

    Weaknesses:

    Why the phenotype of their mutant male is different from previous work (Ma et al.) is not clear enough although they discuss it.

    We appreciate the reviewer’s comment regarding the differences between our Bend2 mutant male phenotype and the previously reported phenotype by Ma et al., 2022. We believe this discrepancy is due to the fact that the Bend2 locus encodes two BEND2 isoforms: p140 and p80. In contrast to the previous study, where both proteins were ablated by mutation employed (the deletion of exons 12 and 13), our exon 11 deletion specifically ablates p140 expression while allowing the expression of p80 in the testis.

    Based on the distinct phenotypes observed in the two Bend2 mutant mouse models, we hypothesize that p80 is sufficient to fulfill BEND2’s roles in meiosis, which could explain why our Bend2 mutant males remain fertile. We have rewritten the relevant sections in the results and discussion to better articulate this hypothesis and clarify the potential mechanisms behind the observed phenotypic differences.

    We hope these clarifications and additional details adequately address the reviewer’s concerns.

    Reviewer #3 (Recommendations For The Authors):

    (1) The authors showed that Bend2 mutant females had decreased fertility. This may be due to decreased ovarian reserve. Did the authors check if the mutant mice decreased or lost fertility faster than WT? If the authors have the data, please refer to it in the manuscript.

    We followed the breeding performance of a small number of control and Bend2 mutant females, and preliminary observations suggested no clear differences between the two groups. However, due to the limited sample size, we felt that these data were not conclusive enough to be included in the manuscript. We agree that a more thorough analysis of fertility decline over time would be valuable, and we plan to address this question in a future study.

    (2) In Figure 1 A, there is no exon1 in the upper figure.

    We thank the reviewer for pointing this out. We have revised Figure 1A to include exon 1 and ensure the schematic is accurate. The updated figure is included in the revised version of the manuscript.

    (3) Figure 3A, it would be nice to show several tubules of the testis section as well as an enlarged one.

    Following the reviewer's advice, we have revised Figure 3A to include new images showing several tubules and an enlarged view of one section of a tubule. These updates are included in the revised manuscript to better represent the testis sections.

    (4) Please be consistent with the format of the graph, especially Supplemental figures 2C and 4D.

    We have revised the figures, including Supplemental Figures 2C and 4D, to ensure consistency in the format throughout the manuscript. We have made modifications to the figures to align them more closely and improve the overall presentation.

  13. Version published to 10.7554/elife.96052.2 on eLife
  14. Version published to 10.1101/2024.02.06.579114v3 on bioRxiv
  15. Version published to 10.1101/2024.02.06.579114v2 on bioRxiv
  16. Version published to 10.7554/elife.96052.1 on eLife
  17. eLife

    eLife assessment

    This study provides valuable information on a novel gene that regulates meiotic progression in both male and female meiosis, but the evidence supporting the conclusions of the authors on the role of BEND2 in oogenesis and reproductive aging is incomplete. This study will be of interest to developmental and reproductive biologists.

  18. eLife

    Reviewer #1 (Public Review):

    Summary:

    In this manuscript, the authors investigate the role of BEND2, a novel regulator of meiosis, in both male and female fertility. Huang et al have created a mouse model where the full-length BEND2 transcript is depleted but the truncated BEND2 version remains. This mouse model is fertile, and the authors used it to study the role of BEND2 on both male and female meiosis. Overall, the full-length BEND2 appears dispensable for male meiosis. The more interesting phenotype was observed in females. Females exhibit a lower ovarian reserve suggesting that full-length BEND2 is involved in the establishment of the primordial follicle pool.

    Strengths:

    The authors generated a mouse model that enabled them to study the role of BEND2 in meiosis. The role of BEND2 in female fertility is novel and enhances our knowledge of genes involved in the establishment of the primordial follicle pool.

    Weaknesses:

    The manuscript extensively explores the role of BEND2 in male meiosis; however, a more interesting result was obtained from the study of female mice. Only a few experiments were performed using female mice, therefore, more experiments should be performed to complete the story of the role of BEND2 on female fertility. In addition, the title and abstract of the manuscript do not align with the story, as female fertility is only a small portion of the data compared to the male fertility section.

  19. eLife

    Reviewer #2 (Public Review):

    In their manuscript entitled "BEND2 is a crucial player in oogenesis and reproductive aging", the authors present their findings that full-length BEND2 is important for repair of meiotic double strand break repair in spermatocytes, regulation of LINE-1 elements in spermatocytes, and proper oocyte meiosis and folliculogenesis in females. The manuscript utilizes an elegant system to specifically ablate the full-length form of BEND2 which has been historically difficult to study due to its location on the X chromosome and male sterility of global knockout animals.

    While the manuscript is an overall excellent addition to the field, it would significantly benefit from a few additional experiments, as well as some additional clarification/elaboration.

    The claim that BEND2 is required for ovarian reserve establishment is not supported, as the authors only look at folliculogenesis and oocyte abundance starting at one week of age, after the reserve is formed. Analysis of earlier time points would be much more convincing and would parse the role of BEND2 in the establishment vs. maintenance of this cell population. In spermatocytes, the authors demonstrate a loss of nuclear BEND2 in their mutant but do not comment on the change in localization (which is now cytoplasmic) of the remaining protein in these animals. This may have true biological significance and a discussion of this should be more thoroughly explored.

  20. eLife

    Reviewer #3 (Public Review):

    Summary:

    Huang et al. investigated the phenotype of Bend2 mutant mice which expressed a truncated isoform. This mutant male showed increasing apoptosis due to unrepaired double-strand breaks. However, this mutant male has fertility, and this enabled them to analyze Bend2 function in females. They revealed that Bend2 mutation in females showed decreasing follicle numbers which leads to loss of ovarian reserve.

    Strengths:

    Since their Bend2 mutant males were fertile, they were able to analyze the function of Bend2 in females and they revealed that loss of Bend2 causes less follicle formation.

    Weaknesses:

    Why the phenotype of their mutant male is different from previous work (Ma et al.) is not clear enough although they discuss it.

  21. Version published to 10.1101/2024.02.06.579114v1 on bioRxiv