Biallelic variants in MAD2L1BP (p31comet) cause female infertility characterized by oocyte maturation arrest
Curation statements for this article:-
Curated by eLife
eLife assessment
This valuable study identifies three independent patient mutations in MAD2L1BP (p31 comet) that cause infertility. Consistent with the known functions of p31 comet, solid experiments in mouse oocytes imply that infertility could be caused by a failure to silence the spindle assembly checkpoint, though the mechanism was not determined. Although the sample size is small, a rescue experiment in human oocytes promises the potential for therapy.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Developmental Biology (eLife)
Abstract
Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human disease remain largely unknown. The spindle assembly checkpoint (SAC) is an intricate surveillance mechanism that ensures accurate segregation of chromosomes throughout cell cycles. Once the kinetochores of chromosomes are correctly attached to bipolar spindles and the SAC is satisfied, the MAD2L1BP, best known as p31 comet , binds mitosis arrest deficient 2 (MAD2) and recruits the AAA+-ATPase TRIP13 to disassemble the mitotic checkpoint complex (MCC), leading to the cell-cycle progression. In this study, by whole-exome sequencing (WES), we identified homozygous and compound heterozygous MAD2L1BP variants in three families with female patients diagnosed with primary infertility owing to oocyte metaphase I (MI) arrest. Functional studies revealed that the protein variants resulting from the C-terminal truncation of MAD2L1BP lost their binding ability to MAD2. cRNA microinjection of full-length or truncated MAD2L1BP uncovered their discordant roles in driving the extrusion of polar body 1 (PB1) in mouse oocytes. Furthermore, the patient’s oocytes carrying the mutated MAD2L1BP resumed polar body extrusion (PBE) when rescued by microinjection of full-length MAD2L1BP cRNAs. Together, our studies identified and characterized novel biallelic variants in MAD2L1BP responsible for human oocyte maturation arrest at MI, and thus prompted new therapeutic avenues for curing female primary infertility.
Article activity feed
-
-
Author Response
Reviewer #1 (Public Review):
The manuscript by Huang, Li, et al. describes the identification of variants in the gene coding for p31 comet, a protein required for silencing the spindle assembly checkpoint or SAC, in women with recurrent pregnancy loss upon IVF. In three families mutations affecting splicing or expression of full-length protein were identified. The authors show that oocytes of the patients arrest in meiosis I, are most likely to fail to inactivate the SAC without a fully functional p31 comet. Indeed, the metaphase I arrest occurring in mouse oocytes upon overexpression of Mad2 can be rescued by overexpression of wild-type p31 comet, but not a truncated version. Injection of wt p31 comet into 6 human oocytes from one patient rescued the meiosis I arrest.
Main points:
The fact that inactivation of …
Author Response
Reviewer #1 (Public Review):
The manuscript by Huang, Li, et al. describes the identification of variants in the gene coding for p31 comet, a protein required for silencing the spindle assembly checkpoint or SAC, in women with recurrent pregnancy loss upon IVF. In three families mutations affecting splicing or expression of full-length protein were identified. The authors show that oocytes of the patients arrest in meiosis I, are most likely to fail to inactivate the SAC without a fully functional p31 comet. Indeed, the metaphase I arrest occurring in mouse oocytes upon overexpression of Mad2 can be rescued by overexpression of wild-type p31 comet, but not a truncated version. Injection of wt p31 comet into 6 human oocytes from one patient rescued the meiosis I arrest.
Main points:
The fact that inactivation of the SAC is required for anaphase I onset in human oocytes is not novel. Biallelic mutations of TRIP13 were shown to lead to the same phenotype (Zhang et al. Am J. Hum Gen., 2020).
As pointed out by the editors and both other reviewers, the strength of this study is highlighted by the identification of genetic variants responsible for oocyte meiosis I arrest in human patients. As a fact, very few genetic variants that cause female oocyte meiotic failure are identified (Ref: Qing Sang, et al. Understanding the genetics of human infertility. Science. 2023). In this study, we for the first time reported the novel deleterious p31comet variants causing human oocyte MI arrest. Without exploring the etiological landscape of human genetic variants, it is impossible to comprehensively invent diagnostic and therapeutic approaches for female patients.
No new mechanistic insights are obtained.
To gain the molecular mechanism, we have optimized and performed a modified Smart-seq2 protocol using frozen single-cell human oocytes (Page 11 and Figure 4-figure supplement 1). These data were in well agreement with the phenotypes as reported.
The authors propose a role for female fertility, however, also a male patient with a p31 comet variant is sterile.
This manuscript focuses on screening the genetics variants responsible for the oocyte failure in female patients, rather than male patients. In addition, we had difficulties with collection of more detailed information from this male patient because he rejected to provide the consent to us. We currently only have limited information after we tried every effort to get in touch with the male patient. We have added more discussion in the MS. Certainly, further exploration of the roles of MAD2L1BP variants in the male meiosis, for example, by collection of a cohort of male patients’ samples with meiotic defects, would be an interesting direction in the future, but this is beyond the scope of this study.
The fact that the C-terminus of p31 comet is required for interaction with Mad2 and hence, turning off the SAC, is already known.
The interaction between p31comet and Mad2 is known in somatic cells, but not in oocytes. As it is widely known that the oocytes are distinct from somatic cells in that the SAC in oocytes is not effective because oocytes can proceed to anaphase I in the presence of even one unattached kinetochore, as compared with somatic cells. We provided evidence that the overexpression of Mad2 can only be rescued by overexpression of wild-type p31comet, but not the truncated p31comet variant in both mouse and human oocytes (Fig.3 and 4), which sufficiently characterized the causative roles of p31comet variants underlying female infertility.
Reviewer #2 (Public Review):
In this manuscript by Huang et al. the authors explore the genetic underpinnings that may cause human oocyte meiotic arrest. The meiotic arrest of oocytes can cause female infertility leading patients to seek treatment at IVF clinics to assist in having genetically related babies. However, because oocytes fail to develop to MII, oocytes from these patients cannot be fertilized, leaving no current options for genetically related babies for patients with this pathology. Huang et al identified 50 IVF patients with this phenotype, and after the whole exome sequence, 3 patients had mutations in a spindle assembly checkpoint regulator, Mad1bp1. This study describes these mutations in detail, shows how these mutations affect Mad1bp1 expression, evaluates gross function in mouse oocytes, and explores therapeutic treatment in human oocytes. Overall, this is an important translational study that adds to the growing body of literature that genetic mutations impact oocyte quality and fertility.
Thank you for your favorable comments.
In its current form, I find that the strengths exist in the analysis of the patients' genomes and pedigree information. This is unique data and is important for the field. The expression in oocytes, structure modeling, and conservation in evolution, while not essential for this study, add interesting information for the reader to consider. I sometimes find these distracting in manuscripts, but appreciate them here in this context. The conclusion using human oocytes to propose possible treatment takes the study to completion and is not an easy approach to carry out.
Thank you for your positive comments on this manuscript.
I do find some weaknesses that weaken the conclusions. The conclusion described is that the SAC is not satisfied in oocytes from these patients. The authors attempt to show this by analysis of mouse oocytes using polar body extrusion and its timing as an assay. There could be many reasons contributing to arrest, therefore a singular assay is not ideal to justify the conclusions. While I do suspect the authors are correct, an intact SAC should be shown at the molecular level to fully justify this conclusion. There are many assays routinely performed in mouse oocytes that the authors can consider (check papers by authors from Wassmann, FitzHarris, and Schindler labs for example).
Thanks for your good comments. Following your advice, we have performed the immunofluorescence assay to evaluate the SAC integrity using mouse oocytes by microinjection of WT and Mut Mad2l1bp cRNA, which clearly validated the intact SAC activation with Mut Mad2l1bp cRNA injection. Please see the reply as detailed below.
Reviewer #3 (Public Review):
The spindle checkpoint ensures the accuracy of chromosome segregation by sensing unattached kinetochores during mitosis and meiosis and delays the onset of anaphase. Unattached kinetochores catalyze the conformational activation of the latent open MAD2 (O-MAD2) to the active closed MAD2 (C-MAD2). C-MAD2 is then incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C) to delay anaphase. When all kinetochores are properly unattached, the MAD2-binding protein p31comet and the ATPase TRIP13 extract C-MAD2 from the MCC, leading to MCC disassembly and the conversion of C-MAD2 back to O-MAD2. This action turns off the spindle checkpoint, resulting in APC/C activation and anaphase onset. Cells deficient in p31comet exhibit mitotic delays.
In the current study, Huang et al. have linked p31comet mutations to female infertility. Biallelic loss-of-function alleles of p31comet cause delays in the exiting metaphase of meiosis I and polar body extrusion. The p31comet mutant proteins contain C-terminal truncations and fail to bind to MAD2. Reintroducing full-length p31comet into patient oocytes can bypass the metaphase arrest. Together with a previous study that showed biallelic mutations of TRIP13 caused female infertility, this work established a critical role of the p31comet-TRIP13 module in regulating meiotic progression during oogenesis. As such, this is a significant study.
Thank you for the very positive comments on this manuscript.
-
eLife assessment
This valuable study identifies three independent patient mutations in MAD2L1BP (p31 comet) that cause infertility. Consistent with the known functions of p31 comet, solid experiments in mouse oocytes imply that infertility could be caused by a failure to silence the spindle assembly checkpoint, though the mechanism was not determined. Although the sample size is small, a rescue experiment in human oocytes promises the potential for therapy.
-
Reviewer #1 (Public Review):
The manuscript by Huang, Li, et al. describes the identification of variants in the gene coding for p31 comet, a protein required for silencing the spindle assembly checkpoint or SAC, in women with recurrent pregnancy loss upon IVF. In three families mutations affecting splicing or expression of full-length protein were identified. The authors show that oocytes of the patients arrest in meiosis I, are most likely to fail to inactivate the SAC without a fully functional p31 comet. Indeed, the metaphase I arrest occurring in mouse oocytes upon overexpression of Mad2 can be rescued by overexpression of wild-type p31 comet, but not a truncated version. Injection of wt p31 comet into 6 human oocytes from one patient rescued the meiosis I arrest.
Main points:
The fact that inactivation of the SAC is required for …
Reviewer #1 (Public Review):
The manuscript by Huang, Li, et al. describes the identification of variants in the gene coding for p31 comet, a protein required for silencing the spindle assembly checkpoint or SAC, in women with recurrent pregnancy loss upon IVF. In three families mutations affecting splicing or expression of full-length protein were identified. The authors show that oocytes of the patients arrest in meiosis I, are most likely to fail to inactivate the SAC without a fully functional p31 comet. Indeed, the metaphase I arrest occurring in mouse oocytes upon overexpression of Mad2 can be rescued by overexpression of wild-type p31 comet, but not a truncated version. Injection of wt p31 comet into 6 human oocytes from one patient rescued the meiosis I arrest.
Main points:
The fact that inactivation of the SAC is required for anaphase I onset in human oocytes is not novel. Biallelic mutations of TRIP13 were shown to lead to the same phenotype (Zhang et al. Am J. Hum Gen., 2020).
No new mechanistic insights are obtained.
The authors propose a role for female fertility, however, also a male patient with a p31 comet variant is sterile.
The fact that the C-terminus of p31 comet is required for interaction with Mad2 and hence, turning off the SAC, is already known.
-
Reviewer #2 (Public Review):
In this manuscript by Huang et al. the authors explore the genetic underpinnings that may cause human oocyte meiotic arrest. The meiotic arrest of oocytes can cause female infertility leading patients to seek treatment at IVF clinics to assist in having genetically related babies. However, because oocytes fail to develop to MII, oocytes from these patients cannot be fertilized, leaving no current options for genetically related babies for patients with this pathology. Huang et al identified 50 IVF patients with this phenotype, and after the whole exome sequence, 3 patients had mutations in a spindle assembly checkpoint regulator, Mad1bp1. This study describes these mutations in detail, shows how these mutations affect Mad1bp1 expression, evaluates gross function in mouse oocytes, and explores therapeutic …
Reviewer #2 (Public Review):
In this manuscript by Huang et al. the authors explore the genetic underpinnings that may cause human oocyte meiotic arrest. The meiotic arrest of oocytes can cause female infertility leading patients to seek treatment at IVF clinics to assist in having genetically related babies. However, because oocytes fail to develop to MII, oocytes from these patients cannot be fertilized, leaving no current options for genetically related babies for patients with this pathology. Huang et al identified 50 IVF patients with this phenotype, and after the whole exome sequence, 3 patients had mutations in a spindle assembly checkpoint regulator, Mad1bp1. This study describes these mutations in detail, shows how these mutations affect Mad1bp1 expression, evaluates gross function in mouse oocytes, and explores therapeutic treatment in human oocytes. Overall, this is an important translational study that adds to the growing body of literature that genetic mutations impact oocyte quality and fertility.
In its current form, I find that the strengths exist in the analysis of the patients' genomes and pedigree information. This is unique data and is important for the field. The expression in oocytes, structure modeling, and conservation in evolution, while not essential for this study, add interesting information for the reader to consider. I sometimes find these distracting in manuscripts, but appreciate them here in this context. The conclusion using human oocytes to propose possible treatment takes the study to completion and is not an easy approach to carry out.
I do find some weaknesses that weaken the conclusions. The conclusion described is that the SAC is not satisfied in oocytes from these patients. The authors attempt to show this by analysis of mouse oocytes using polar body extrusion and its timing as an assay. There could be many reasons contributing to arrest, therefore a singular assay is not ideal to justify the conclusions. While I do suspect the authors are correct, an intact SAC should be shown at the molecular level to fully justify this conclusion. There are many assays routinely performed in mouse oocytes that the authors can consider (check papers by authors from Wassmann, FitzHarris, and Schindler labs for example).
-
Reviewer #3 (Public Review):
The spindle checkpoint ensures the accuracy of chromosome segregation by sensing unattached kinetochores during mitosis and meiosis and delays the onset of anaphase. Unattached kinetochores catalyze the conformational activation of the latent open MAD2 (O-MAD2) to the active closed MAD2 (C-MAD2). C-MAD2 is then incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C) to delay anaphase. When all kinetochores are properly unattached, the MAD2-binding protein p31comet and the ATPase TRIP13 extract C-MAD2 from the MCC, leading to MCC disassembly and the conversion of C-MAD2 back to O-MAD2. This action turns off the spindle checkpoint, resulting in APC/C activation and anaphase onset. Cells deficient in p31comet exhibit mitotic delays.
In the …
Reviewer #3 (Public Review):
The spindle checkpoint ensures the accuracy of chromosome segregation by sensing unattached kinetochores during mitosis and meiosis and delays the onset of anaphase. Unattached kinetochores catalyze the conformational activation of the latent open MAD2 (O-MAD2) to the active closed MAD2 (C-MAD2). C-MAD2 is then incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C) to delay anaphase. When all kinetochores are properly unattached, the MAD2-binding protein p31comet and the ATPase TRIP13 extract C-MAD2 from the MCC, leading to MCC disassembly and the conversion of C-MAD2 back to O-MAD2. This action turns off the spindle checkpoint, resulting in APC/C activation and anaphase onset. Cells deficient in p31comet exhibit mitotic delays.
In the current study, Huang et al. have linked p31comet mutations to female infertility. Biallelic loss-of-function alleles of p31comet cause delays in the exiting metaphase of meiosis I and polar body extrusion. The p31comet mutant proteins contain C-terminal truncations and fail to bind to MAD2. Reintroducing full-length p31comet into patient oocytes can bypass the metaphase arrest. Together with a previous study that showed biallelic mutations of TRIP13 caused female infertility, this work established a critical role of the p31comet-TRIP13 module in regulating meiotic progression during oogenesis. As such, this is a significant study.
-
