Production of Offspring from Azoospermic Mice with Meiotic Failure: Precise Biparental Meiosis within Halved Oocytes

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While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% ( P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest.

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    Referee #3

    Evidence, reproducibility and clarity

    In mice, failures in conducting meiosis during spermatogenesis can be rescued by injecting prophase I male chromosomes into oocytes, to allow them to undergo the two meiotic divisions within the oocyte, together with the chromosomes of the oocyte. However, segregations are highly error prone and rarely lead to a live birth when the resulting embryos are reimplanted into foster mothers. In this study, the authors show that segregation errors in meiosis I oocytes harboring both male and female chromosomes are mainly affecting the male chromosome set. Most errors are due to precocious segregation of sister chromatids in unpaired male chromosomes (univalents). A delay in alignemnt of male chromosomes compared to female chromosomes was also observed. Reducing the volume of the oocyte cytoplams to half leads to a signifncant reduction in the errors occuring, and hence, a significant increase in successful birth after re-implantation. Excitingly, with this technique, live births were obtained from male mice with a spermatogenic arrest phenotype.

    Main points:

    1)The authors conclude that halving the oocyte cell size is helping in proper segregation of male meiosis I chromosomes in the cytoplasm of meiosis I oocytes. It is also possible that the experimental procedure involved in removing half of the cytoplasm is promoting proper segregation for some unknown reason. The authors should include a condition where half of the cytoplasm is aspirated but then put back again, so oocytes have the same volume as before but the cytoplasm underwent the same treatment as in the halved oocytes. Also, increasing the cytoplasm volume of the oocyte should not lead to a better segregation of male chromosomes but make things worse, have the authors checked for that?

    2)The authors mention that male chromosomes align with a delay, compared to the female chromosomes. Does this delay depend on activation of error correction, or the spindle asembly checkpoint? Is it possible that dilution of factors required for checkpoint control and hence, assuring proper chromosome segregation, are the reason for error prone segregation in oocytes harboring twice the amount of chromosomes? If yes, have the authors stained for SAC proteins at the kinetochores? Maybe slight overepxression of the SAC protein were sufficient to rescue male meiotic divisions in the oocyte- have the authors tested this hypothesis?

    1. The authors state that male chromosomes have a hard time segregating in the hugh cytoplasm of the oocytes. Maybe it is not the fact that the chromosomes came from a male pronucleus, but this is just a manner of double the chromosomes that have to be segregated in the oocyte cytoplams. How do male chromosomes behave in enucleated oocytes undergoing meiosis I? Conversely, if female chromosomes coming from another oocyte are injected into the recipient oocyte instead of ale chromosomes, are those segregating correctly, or the delay in chromosome alignment and error rate comparable to the situation when the additional chromosome set comes from the male?

    2. In the rescue of mice with spermatogenic arrest the authors find aneuploidies of sex-chromosomes in the off-spring, not of autosomes. To my best of knowledge, autosome aneuploidies are not viable in the mouse, hence this result does not indicate that sex-chromosomes are the main source of aneuploidies. Nevertheless, it is attractive to speculate that aneuploidies are mainly due to sex chromosomes, because the oocyte is not prepared to segregate a male sex-chromosome bivalent. The authors should determine whether the segregation errors in meiosis I in oocytes harboring the additional male chromosome set concern mainly the male sex-chromosomes, by doing Fish analysis after meiosis I.


    This study is very interesting and of high significance, and very well executed. I think the study can go much further as far as mechanistic insights are concerned, only requiring techniques and tools that the authors have at their disposition.

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    Referee #2

    Evidence, reproducibility and clarity


    Previously, the team has shown that primary spermatocyte nucleus can undergo meiosis when transplanted into immature oocytes, and later obtained normal mice from the fertilized oocytes (Zygotes 1997, PMID: 9276513; PNAS 1998, PMID: 9576931). However, the efficiency was quite low (~ 1%) due to chromosome aberration, thus not feasible for basic/clinical research applications. In this study, Ogonuki et al., extrapolated from the recent study showing the reduction of the ooplasm ameliorate the error of chromosome segregation during meiosis (Dev Cell 2017, PMID: 28486131), injected the spermatocyte nucleus into the half-sized GV oocytes, and succeeded to obtain live murine pups with a high incidence (the birth rate improved from 1% with full-sized oocytes to 19% with half-sized oocytes). Further, through detailed observation with high-resolution 3D live imaging, the authors clarified that the misalignment of paternal chromosomes could be ameliorated by reducing the volume of ooplasm. Finally, the authors applied this technology and obtained live pups from azoospermic mice, suggesting the potential application in human infertility treatment.

    Major comments:

    This is a great study combining the expertise on both sperm and oocytes. The experiments are well designed and performed. The key conclusions are convincing.

    Line 228. The authors claimed that all the pups born following the injection of wild-type or mutant spermatocytes grew into fertile adults.

    Because the authors tested 3 males from wt spermatocytes (line 197), the above sentence should be rephrased.

    The authors found one XXY male among the three male mice from wt spermatocytes. Was the XYY male mouse fully fertile without XY/XYY mosaicism?

    How many females and males were obtained from wt spermatocytes?

    Minor comments:

    The authors clearly showed the technique can be applied to rescue the spermatogenic arrest. The readers would appreciate if the authors include any unsuccessful cases.

    To prevent sex-chromosome aberration, are there any potential markers for selecting most developed spermatocytes?


    One in six couples suffers from infertility, and 70-90% of male infertility cases are related to defects in spermatogenesis. Clinically, intracytoplasmic injection of sperm is common, but it is not applicable to men who lack haploid germ cells. Injection of primary spermatocyte nucleus can give pups but the efficiency was poor (~1%, PNAS 1998, PMID: 9576931). In the present study, by using halved oocytes as recipient, the authors improved the efficiency from 1% to 19%. With the great improvement, they further obtained healthy fertile offspring from the male mice genetically lacking haploid cells. This approach opens up the window for the infertile patients suffering from spermatogenic arrest.

    The reviewer's field of expertise: knockout mice, male infertility, spermatogenesis, sperm function, fertilization.

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    Referee #1

    Evidence, reproducibility and clarity

    Ogonuki et al developed a new technique using primary spermatocyte-injected oocytes for offspring production. They examined chromosome segregation error in biparental meiosis using spermatocyte-injected oocytes. They showed that artificially reducing ooplasmic volume rescued highly error-prone chromosome segregation by preventing sister separation in biparental meiosis. Their live-imaging analysis demonstrated that erroneous chromosome segregation derived from univalent-like chromosomes followed by predivision of sister chromatids during prometaphase I in biparental meiosis. They showed that the birth rate was improved using halved oocytes. Furthermore, they showed that production of offspring was successful using spermatocyte from azoospermic mice.

    Overall data are convincing and the manuscript addresses important questions. The data was produced in a technically high level. Presented data are sufficient to support conclusions of the authors, and further provide a significant insight into application to production of offspring for azoospermia animals. Thus, the manuscript could be open for the fields and are supposed to deserve publication, if they could address following minor concerns.

    Fig1A, Line 117 This is an amazing experiment to set up biparental meiosis using spermatocyte nuclei. Since spermatocytes are in different stages during progression through meiotic prophase, some of them (late pachytene) should yield crossover but others (before mid-pachytene) are yet to complete recombination. Thus, whether donor paternal chromosomes have bivalents or univalents depends on which stage spermatocytes derived from. The authors should describe how spermatocytes were picked up for injection and whether they used a particular stage of spermatocytes.

    Line 159-160 The authors stated that paternal chromosomes are susceptible to errors in ooplasm-hosted biparental meiosis. This is nice demonstration to trace the origin of separated chromatids. In Fig2C right graph, 1 to 2 paternal chromosomes showed misalignment. It is unclear whether premature separation is biased to any particular paternal chromosome, eg XY ? The authors should discuss more about it.

    Line 176-177 The authors stated that most of errors were preceded by premature separation of bivalent chromosomes into univalent-like structures. This implies that premature separation of bivalent chromosomes happens prior to anaphase onset. Does this depend on spindle force? Or is cohesion intrinsically fragile in donor spermatocyte chromosomes? The authors should discuss more about it.

    Fig3E, The authors depicted that in normal sized oocytes, univalent-like chromosomes undergo predivision at anaphase. This is somewhat too simplified, because Fig3B shows that a certain population exhibits nondisjunction. This model and description should be corrected to fit the data they demonstrated. If sister segregation at anaphase is predominant, I wonder what happens to sister kinetochore mono-orientation and sister centromeric protection in such univalent-like chromosomes. It would be nice to show centromeric proteins MEIKIN, SGO2 in donor spermatocyte chromosomes versus those of oocyte to examine centromeric cohesion. The authors should clarify this issue.

    Line296-294 What do the authors mean by the sentence " It is known that sex chromosomes are prepared to undergo meiosis later than autosomes."?


    The manuscript will provide biological significance for the reproduction fields. There are two major biological significances : They addressed the mechanism of erroneous chromosome segregation in biparental meiosis. They showed that biparental meiosis using spermatocyte-injected oocytes can be applied to production of offspring of azoospermic mice, which would have great impact on reproductive biology field. The data was produced with their high level of technique.

    Referee Cross-commenting

    I agree to the point described in Reviewer #3's Main points2. It would be better to see SAC proteins.