Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation

Abstract Early embryonic arrest and fragmentation (EEAF) is a common phenomenon leading to female infertility, but the genetic determinants remain largely unknown. The Moloney sarcoma oncogene (MOS) encodes a serine/threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Here, we identified four rare variants of MOS in three infertile female individuals with EEAF that followed a recessive inheritance pattern. These MOS variants encoded proteins that resulted in decreased phosphorylated ERK1/2 level in cells and oocytes, and displayed attenuated rescuing effects on cortical F‐actin assembly. Using oocyte‐specific Erk1/2 knockout mice, we verified that MOS‐ERK signal pathway inactivation in oocytes caused EEAF as human. The RNA sequencing data revealed that maternal mRNA clearance was disrupted in human mature oocytes either with MOS homozygous variant or with U0126 treatment, especially genes relative to mitochondrial function. Mitochondrial dysfunction was observed in oocytes with ERK1/2 deficiency or inactivation. In conclusion, this study not only uncovers biallelic MOS variants causes EEAF but also demonstrates that MOS‐ERK signaling pathway drives human oocyte cytoplasmic maturation to prevent EEAF.

In this work Yin-Li Zhang and colleagues study the mechanism of early embryonic arrest and fragmentation (EEAF) through genetic, cellular and molecular studies. They studied three patients with EEAF by WES and found pathogenic variants in of the same gene MOS a serine-threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates.
Using cellular system i.e. HEK cells, human oocytes with overexpression of the MOS control or mutated mRNAs and a rodent model (ERK1/2 knock out mice generated by this group), they could demonstrate that the variants alter MOS expression or ERK1/2 activation, and the MOS-ERK pathway is required in oocytes for cytoskeleton assembly and prevents embryo fragmentation. RNA sequencing in single oocytes of one patient has shown that MOS regulates the degradation of maternal mRNAs during oocyte maturation and that the variants detected in patients mainly prevent the decrease in transcripts from human GV to MII oocytes, similarly to the inhibition of ERK1/2. Finally they studied mitochondrial functions in human and murine MII oocytes after U0126 treatment and in mature oocytes in vivo from WT and ERK1/ -/-mice that this group has generated. They conclude that the human MOS/ ERK cascade is required to maintain mitochondrial function through accelerating mitochondrial mRNA clearance during oocyte maturation. This work is original, well done, with a large spectrum of combined approaches, in vitro or in vivo, in humans or in rodents, to be able to understand the mechanism of EEAF and associated female infertility, which was not explained by the previously reported genetic causes. It provides important information on the signaling pathway that drives human oocyte maturation to prevent embryo arrest and fragmentation. These results might have a therapeutic impact in the future. Specific comments: Patients: are they siblings in the families with potential fertility problems? This is not shown in the pedigrees presented. Are the patients from the same country? No information on their origin is given. It is striking to observe in the various experiments a minor inhibition of ERK1/2 or MEK activation with the combined p.Met139Thr/p. Arg 246His variants (Fig 2C, EV1D). This is not discussed in the work presented. Are there differences in the EEAF phenotype between the different patients that might be correlated with these differences in ERK1/2 inhibition? It appears that there is only about 25% alteration of resumption of meiosis with the combined variants, while this alteration is much greater for the other Asn95Lys and Cys320Ter variants. Precise quantification must be performed. The question is raised as to whether they might be a threshold for ERK activation to allow correct oocyte maturation. In this respect a range of transfected RNAs (control or mutated) could provide the answer. A single dose 500 ng is used in all experiments.
RNA sequencing could also be performed in the oocytes of patient 2 harboring the combined mutations. Fewer RNAs are expected to be deregulated. This could lead to the identification of the functionally most important RNAs deregulated with this milder variant. Since there is transient expression of MOS, overexpression of WT MOS could be used to compensate for the molecular defects. Do the authors try such experimental approaches in vitro as proof of concept or in mice in vivo and observe the consequences on blastocyst formation and embryonic development?

Referee #2 (Remarks for Author):
This is a solid and comprehensive paper reporting that deleterious alleles of the MOS gene can cause infertility in human females. The authors find 3 pedigrees in which IVF attempts failed due to early arrest of embryos. Exome sequence revealed biallelic mutations of Mos in probands that were inherited from parents. What sets this paper apart from other papers that stop at this gene:phenotype correlative stage is that the authors go on to conduct a number of studies proving that these alleles encode functionally defective proteins that compromise oocyte development and embryo progression. They even make some interesting fundamental observations using genomic transcriptome studies that resulted in the discovery of mitochondrial defects when the MOS s defective. I found that the paper was well-prepared, especially the figures, although some light English editing would help refine it. I only have a few minor suggestions for this otherwise welcome report: Line 116: "....confirming the possible genetic contribution of MOS to human EFAF." The word "confirming" is not appropriate. "Suggesting" would be a more accurate word at this point in the paper.
Line 131/ Table 2: It would be useful to provide the actual SIFT and PolyPhen scores in the Table. Also, many computational  people like to see CADD scores, for what they're worth. In the Introduction, it would be helpful to further explain how ERK1/2 phosphorylation maintains MII arrest.
Line 156: It is not obvious or rigorously demonstrated that ".... MOS and MOS variants led to decreased MOS protein levels ( Figure 2C and D)." On line 161, explain the purpose of milrinone.
Referee #3 (Comments on Novelty/Model System for Author): Early embryonic arrest and fragmentation (EEAF) is a common phenomenon in human embryos. However, the role of MOS in human oocyte maturation and early embryo development is not fully uncovered. In this study, the authors validated the pathogenic roles of four rare MOS variants identified in three individuals exhibiting EEAF. The pathogenic effects of MOS variants were investigated in HEK293 cells and mouse oocytes, including the Erk1/2 conditional knockout mice. Combined with in vivo and in vitro RNA-seq results from control, patient oocytes and U0126-treated oocytes, they concluded that MOS variants caused ERK1/2 signal cascade inactivation, resulted in cortical F-actin assembly defect and impaired maternal mRNA decay, especially on genes relative to mitochondrial function. The study reveals the important role of maternal MOS in human embryo development and stability. The conclusion is interesting in identifying the association of MOS pathogenic variants and female infertility and establishing MOS-ERK1/2 signal pathway as the critical factors for human oocyte cytoplasmic maturation by maternal mRNA clearance. The conclusion is appropriate and evidenced by the results. Besides, the manuscript is well written. I support for the publication of this manuscript, with an expectation that some aspects of current manuscript could be improved as mentioned in remarks to the author.
Dear editor, Thanks for editor and reviewers' constructive comments to improve the manuscript. As suggested, we have re-submitted a .docx formatted version of revised manuscript with "tracked-changes mode". We have checked each figure according to the guidance of "Figure Guide PDF" of EMBO Molecular Medicine journal. We included the information of ORCID IDs of corresponding authors, Data Availability and Author Contributions in the manuscript, and checked data quality as required. The checklist and source data of each figures have been submitted to the editorial system. In the light of reviewers' comments, we have made point-by-point response and performed some experiments and added these new results in Appendix Figure S1, S3 and S4. These Supplementary information is uploaded as pdf (without highlighted changes). Please find the point-by-point response to reviewers' comments below: ***** Reviewer's comments ***** Referee #1 (Remarks for Author): In this work Yin-Li Zhang and colleagues study the mechanism of early embryonic arrest and fragmentation (EEAF) through genetic, cellular and molecular studies. They studied three patients with EEAF by WES and found pathogenic variants in of the same gene MOS a serine-threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Using cellular system i.e. HEK cells, human oocytes with overexpression of the MOS control or mutated mRNAs and a rodent model (ERK1/2 knock out mice generated by this group), they could demonstrate that the variants alter MOS expression or ERK1/2 activation, and the MOS-ERK pathway is required in oocytes for cytoskeleton assembly and prevents embryo fragmentation. RNA sequencing in single oocytes of one patient has shown that MOS regulates the degradation of maternal mRNAs during oocyte maturation and that the variants detected in patients mainly prevent the decrease in transcripts from human GV to MII oocytes, similarly to the inhibition of ERK1/2. Finally they studied mitochondrial functions in human and murine MII oocytes after U0126 treatment and in mature oocytes in vivo from WT and ERK1/ -/-mice that this group has generated. They conclude that the human MOS/ ERK cascade is required to maintain mitochondrial function through accelerating mitochondrial mRNA clearance during oocyte maturation. This work is original, well done, with a large spectrum of combined approaches, in vitro or in vivo, in humans or in rodents, to be able to understand the mechanism of EEAF and associated female infertility, which was not explained by the previously reported genetic causes. It provides important information on the signaling pathway that drives human oocyte maturation to prevent embryo arrest and fragmentation. These results might have a therapeutic impact in the future. Response: Thank you for your positive assessment. Specific comments: Patients: are they siblings in the families with potential fertility problems? This is not shown in the pedigrees presented. Are the patients from the same country? No information on their origin is given. Response: The three patients are not from the families with potential fertility problems. They all 15th Sep 2021 1st Authors' Response to Reviewers are from China and belong to Chinese Han population. We have included this information into Line 104-105 in the revised manuscript.
It is striking to observe in the various experiments a minor inhibition of ERK1/2 or MEK activation with the combined p.Met139Thr/p. Arg 246His variants (Fig 2C, EV1D). This is not discussed in the work presented. Are there differences in the EEAF phenotype between the different patients that might be correlated with these differences in ERK1/2 inhibition? Response: Thanks for pointing it out. Based on our new added results in Appendix Figure S1, the MOS p.Met139Thr variant caused ERK1/2 inactivation, similar to the other two homozygous variants p.Asn95Lys and p.Cys320Ter, yet the MOS p.Arg246His variant decreased the ability of binding with MEK1 and thereby just decreased ERK1/2 activation to some extent. We microinjected the combined variants with same dose (250 ng/ul mRNAs for each variant, reaching a total dose of 500 ng/ul) in mouse oocyte to mimic the compound heterozygous variants of patient 2. However, we neglect there exists allelic gene expression imbalance in human. If possible, we might validate this by comparing the expression level of the two alleles in future through performing the RNA-seq using oocyte from patient 2. We have added this into Discussion part (Line 381-390). We approve that the differences in the EEAF phenotype between the different patients are correlated with these differences in ERK1/2 inhibition. The patient 3 carries with a homozygous nonsense variant in MOS gene, causing premature termination at 320 th amino acid. The p.C320Ter variant displayed the weakest interaction with MEK1 (Appendix Figure S1B-C) and resulted in lowest ERK1/2 activation ( Figure 2C and Figure EV1D). Consistently, the patient 3 exhibited the earliest embryonic arrest (2-3 cell stage) than those of other two patients (2-7 cell stage). We have added this into Discussion part (Line 372-378).
It appears that there is only about 25% alteration of resumption of meiosis with the combined variants, while this alteration is much greater for the other Asn95Lys and Cys320Ter variants. Precise quantification must be performed. Response: We are apologized for your confusion due to inaccurate description. We have re-checked the mRNA concentration used for microinjection and we make sure that a total 500 ng/ul mRNAs (each variant with 250 ng/ul in the combined p.Met139Thr/p.Arg246His variants group) were used for microinjection in three independent experiments. We have added this into Materials and Method (Line 566-568). We also found the ability of resumption of meiosis is associate with the level of MOS-mediated ERK1/2 signal cascade activation. The source data of this result is provided.
The question is raised as to whether they might be a threshold for ERK activation to allow correct oocyte maturation. In this respect a range of transfected RNAs (control or mutated) could provide the answer. A single dose 500 ng is used in all experiments. Response: Thank you for this important and helpful idea. Because the 500 ng/ul is commonly used concentration in other published papers and this microinjection dose is sufficient, we selected this single dose 500 ng/ul to confirm the pathogenic roles of four MOS variants. As you suggested, we microinjected different concentrations of MOS mRNAs (0, 50, 125, 250 and 500 ng/ul) into mouse GV oocytes to determine the threshold of MOS mRNAs on ERK1/2 activation to allow correct oocyte maturation. We found 50 ng/ul and 125ng/ul MOS mRNAs are sufficient to promote 35% and 60% oocytes to resume meiotic maturation, respectively. With the increase of MOS mRNAs from concentrations of 125ng/ul to 500 ng/ul, oocyte maturation percentage maintained around 50%-60%. Thus, we can conclude that there exists a threshold of MOS mRNAs for ERK1/2 activation. We have added this into Discussion part (Line 390-396). Based on this threshold theory, we could answer your last question "It appears that there is only about 25% alteration of resumption of meiosis with the combined variants…". Except to the imbalanced allelic gene expression, we thought that the 500 ng/ul of mRNAs of the combined MOS p.Met139Thr/p.Arg246His variants are probably overdosed, resulting in relative high maturation rate. We microinjected a total dose of 125 ng/ul wild-type MOS mRNAs or the combined p.Met139Thr/p.Arg246His variants mRNAs (each with 62.5 ng/ul) into immature mouse oocytes. We found that the wild-type MOS promotes 53% oocyte maturation, whereas only 12% oocytes resumed meiotic maturation in the combined p.Met139Thr/p.Arg246His variants group, which verified our hypothesis. These results were included in newly Appendix Figure S4 and described in Discussion section (Line 396-402) in the revised manuscript.
RNA sequencing could also be performed in the oocytes of patient 2 harboring the combined mutations. Fewer RNAs are expected to be deregulated. This could lead to the identification of the functionally most important RNAs deregulated with this milder variant. Response: Thanks for your suggestion. As patient 2 did not agree to donate oocyte to perform experiment, we didn't perform RNA-seq. Unfortunately, we also didn't obtain the oocytes in patient 3. Indeed, we can identify the functionally most important RNAs regulated by MOS through comparing the RNA-seq data from oocytes with different variants. Thanks again for your helpful suggestion.
Since there is transient expression of MOS, overexpression of WT MOS could be used to compensate for the molecular defects. Do the authors try such experimental approaches in vitro as proof of concept or in mice in vivo and observe the consequences on blastocyst formation and embryonic development? Response: Thank you for your professional and helpful suggestion. In restriction of Mos deficiency mice is in the process of generation and is available at least half a year, we didn't perform this experiment. In the our next study, we will focus on the rescuing effect of MOS mRNAs on Mos -/mice and patients with MOS deleterious variants. At that time, your question can be answered. I believe an appropriate dose of MOS mRNA or MOS protein could compensate for the molecular defects of oocyte and embryos from Mos -/mice and patients. Furthermore, the rescuing effect and the potential pathogenic effect of overexpression of MOS should carefully evaluated. This question indeed requires many experiments to investigate.

Referee #2 (Remarks for Author):
This is a solid and comprehensive paper reporting that deleterious alleles of the MOS gene can cause infertility in human females. The authors find 3 pedigrees in which IVF attempts failed due to early arrest of embryos. Exome sequence revealed biallelic mutations of Mos in probands that were inherited from parents. What sets this paper apart from other papers that stop at this gene:phenotype correlative stage is that the authors go on to conduct a number of studies proving that these alleles encode functionally defective proteins that compromise oocyte development and embryo progression. They even make some interesting fundamental observations using genomic transcriptome studies that resulted in the discovery of mitochondrial defects when the MOS s defective. Response: Thank you for your positive evaluation of this study. I found that the paper was well-prepared, especially the figures, although some light English editing would help refine it. I only have a few minor suggestions for this otherwise welcome report: Line 116: "....confirming the possible genetic contribution of MOS to human EFAF." The word "confirming" is not appropriate. "Suggesting" would be a more accurate word at this point in the paper. Response: Thanks for your advice for improving our manuscript. We have corrected it in Line 132 in the new version of manuscript.
Line 131/ Table 2: It would be useful to provide the actual SIFT and PolyPhen scores in the Table. Also, many computational people like to see CADD scores, for what they're worth. Response: This is a very professional comment. We have added these scores in Table 2 and described it in Line 147-149.
In the Introduction, it would be helpful to further explain how ERK1/2 phosphorylation maintains MII arrest. Response: Thanks for your helpful suggestion to improve the readability of the manuscript. We have added the description of mechanism about ERK1/2 phosphorylation maintaining MII arrest in Line 79-80. Also, we explained the mechanism of MOS-ERK signal cascade in promotion of meiotic maturation in Line 84-87 in the revised manuscript.
Line 156: It is not obvious or rigorously demonstrated that ".... MOS<Asn95Lys> and MOS<Cys320Ter> variants led to decreased MOS protein levels ( Figure 2C and D)." Response: Yes, this description is not rigorous. We have modified this to "MOS Asn95Lys and MOS Cys320Ter variants encoded MOS protein with slight decrease" in Line175-176 in the revised manuscript.
On line 161, explain the purpose of milrinone. Response: The milrinone is a PDE3 inhibitor, which is used to prevent oocyte from meiotic resumption by maintaining elevated cAMP levels in oocytes. We have added this information into Line 190-191.

Referee #3 (Comments on Novelty/Model System for Author):
Early embryonic arrest and fragmentation (EEAF) is a common phenomenon in human embryos. However, the role of MOS in human oocyte maturation and early embryo development is not fully uncovered. In this study, the authors validated the pathogenic roles of four rare MOS variants identified in three individuals exhibiting EEAF. The pathogenic effects of MOS variants were investigated in HEK293 cells and mouse oocytes, including the Erk1/2 conditional knockout mice. Combined with in vivo and in vitro RNA-seq results from control, patient oocytes and U0126-treated oocytes, they concluded that MOS variants caused ERK1/2 signal cascade inactivation, resulted in cortical F-actin assembly defect and impaired maternal mRNA decay, especially on genes relative to mitochondrial function. The study reveals the important role of maternal MOS in human embryo development and stability. The conclusion is interesting in identifying the association of MOS pathogenic variants and female infertility and establishing MOS-ERK1/2 signal pathway as the critical factors for human oocyte cytoplasmic maturation by maternal mRNA clearance. The conclusion is appropriate and evidenced by the results. Besides, the manuscript is well written. I support for the publication of this manuscript, with an expectation that some aspects of current manuscript could be improved as mentioned in remarks to the author. Response: Thank you for your appreciation on this work.

Referee #3 (Remarks for Author):
Major points: 1. How many patients were selected for whole exon sequencing in this study? What are the inclusion criteria for the patient with EEAF? Please indicate them in the method or the results. Why do the authors focus on these three patients? Response: We apologize for the unclear description of this part. The EEAF patients were screened according to the following criteria: primary infertility; normal karyotype in both couples; male factors excluded; experienced recurrent failure of IVF/ICSI attempts suffering embryonic arrest and more than 50% of the cleaved embryo represented severer embryo fragmentation occurred in at least one attempt. We have selected 120 patients that experienced EEAF to WES, and just three patients herein carried biallelic mutations in MOS. We have added the relevant description to the Materials and Methods section (Line 479-485). 2. Please descript the characteristics of 100-control individuals you needed in this study. Response: The 100-controls were women who had conceived naturally, without medical intervention. This has been clearly described in the results of the revised manuscript (Line 132).
3. Please provide more detail clinical information for the patients, such as hormone and chromosome status. Response: These three females possessed the normal endocrine level and karyotype, we have added related information in Appendix Table S1 and described it in Line 106 of revised manuscript. 4. Was the functional role of MOS the same between human and mouse? I noticed that the corresponding amino acids of two identified variants were not conserved between human and mouse. Response: These are good and very interesting questions. I think your first question is focus on the function of MOS protein, whereas the second question is focus on the different amino acids on MOS's function. MOS gene is firstly discovered in Xenopus for its role in maintaining MII arrest. Then, many studies demonstrated that MOS is required in mouse oocyte maturation and MII arrest. 5. Except RNA-seq data, RT-qPCR was needed to further validate the affected mitochondrial mRNA clearance. Response: Thanks for your professional suggestion. We have performed RT-qPCR of human oocyte with or without U0126 treatment and included these results into Appendix Figure S4. 6. Apart from U0126 treatment, it is better to detect mitochondrial dysfunction using Erk1/2 deficient mice. Response: Thanks for your professional suggestion. We used Erk1/2 deficient oocytes to determine the mitochondrial function. Consistent results were obtained with those after U0126 treatment. We found a very interesting phenomenon that many mitochondria is extruded into polar bodies by staining JC-1. These results have been presented in Figure 6 E-I and corresponding descriptions have been added in results (Line 323-332) in the manuscript.
Minor points: 1. How to explain the decreased pMEK1/2 and pERK1/2 level with unchanged protein levels in patient 2? Response: MOS is a serine/threonine kinase that directly interacts and phosphorylates MEK1/2 and thereby activates ERK1/2 phosphorylation. We performed co-immunoprecipitation of MEK1 and MOS variants, we found MOS p.Arg246His interacted weaker than wild-type MOS with MEK1 (Appendix Figure S1B-C), which may lead to decreased pMEK1/2 and pERK1/2 level with unchanged protein levels.
2. In Fig. 1D, "Blastocst" should be "Blastocyst". Response: Thanks for your careful revision. We have corrected it in Figure 1D. 3. In the discussion, the relationship between MOS-ERK1/2 pathway and mitochondrial function should be added. Response: As suggested, we have added the relationship between MOS-ERK1/2 pathway and mitochondrial function in Discussion part (Line 453-468) in the revised manuscript.

5th Oct 2021 1st Revision -Editorial Decision
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Referee #3 (Comments on Novelty/Model System for Author): The mechanism was well clarified.

Referee #3 (Remarks for Author):
The present version is suitable for publication. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
We used at least 3 Erk1/2 conditional knockout mice to collect oocytes or embryos for phenotype analysis, which at least 60 oocytes or embryos were obtained. In each independent microinjection experiment, GV oocytes were collected from 25-30 wild-type female mice and randomly divided into 5-6 groups with 150-200 oocytes in each group.

NA
All collected oocytes from wild-type mice were randomized into experimental groups for subsequent microinjection. Then, the survived oocytes in each group were used for experiments and analysis.

Manuscript Number: EMM-2021-14887-V2
Yes.Differences between two groups were compared using the unpaired two-tailed Student's t-test. In the case three or more groups, one-way ANOVA was used, followed by post hoc Tukey's test for multiple comparisons. P < 0.05 was considered statistically significant. The statistical methods are indicated in the figure legends.
Yes. Data are presented as mean ± standard deviation (SD) of minimal independent samples. Studies with sufficient N's, we used the box plot to confirm data variability and the degree of normality.

NA
In order to minimize the effects of subjective bias, cell transfection, western blot and immunofluorescence staining were performed by two independent investigators. The confocal images were captured with the same parameters in each group.
For the oocyte or embryo phenotype analysis from wild-type and Erk1-/-;Erk2f/f;Gdf9-Cre mice, age-matched animals were grouped based on their genotype, which was not blinded.

Data
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The sample size is similar to previous reports in the same field. We screened 120 infertile patients exhibiting early embryonic arrest and fragmentation and only biallelic variants of MOS (OMIM ID: 190060; NM_005372.1) were found in three affected individuals, yet absent in our 100-controls undergoing natural conception database. This suggested the possible genetic contribution of MOS to human female infertility.
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