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Generation of functional oocytes from male mice in vitro

Nature volume 615, pages 900–906 (2023)Cite this article

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Abstract

Sex chromosome disorders severely compromise gametogenesis in both males and females. In oogenesis, the presence of an additional Y chromosome or the loss of an X chromosome disturbs the robust production of oocytes1,2,3,4,5. Here we efficiently converted the XY chromosome set to XX without an additional Y chromosome in mouse pluripotent stem (PS) cells. In addition, this chromosomal alteration successfully eradicated trisomy 16, a model of Down’s syndrome, in PS cells. Artificially produced euploid XX PS cells differentiated into mature oocytes in culture with similar efficiency to native XX PS cells. Using this method, we differentiated induced pluripotent stem cells from the tail of a sexually mature male mouse into fully potent oocytes, which gave rise to offspring after fertilization. This study provides insights that could ameliorate infertility caused by sex chromosome or autosomal disorders, and opens the possibility of bipaternal reproduction.

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Fig. 1: Sex conversion in ES cells and oocyte production in vitro.
Fig. 2: Oocyte production from XY ES cells with autosomal aneuploidy.
Fig. 3: Pups from oocytes differentiated in vitro from sex-converted iPS cells.
Fig. 4: CD38 expression in XO and XX PS cells.

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Data availability

The DNA-seq data have been deposited at the Sequence Read Archive (SRA) database under accession number PRJNA766461 and the RNA-seq data have been deposited at the Gene Expression Omnibus (GEO) database under accession number GSE184771.  Source data are provided with this paper.

Code availability

Custom code used in this article can be accessed at https://github.com/kentamurakami1986/oocytes-from-male-mice.

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Acknowledgements

We thank K. Kitajima and C. Meno for providing microscopes; N. Konishi for technical support; H. Toh for advice about DNA-seq; and Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences for technical assistance. This study was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas from JSPS (grants 18H05544 and 18H05545 to K.H. and 18H05549 to T.S.K.); by a Grant-in-Aid for Specially Promoted Research from JSPS (grants 17H06098 and 22H04920 to M.S.); by the Takeda Science Foundation (K.H.); by the Luca Bella Foundation (K.H.); and by a Grant-in-Aid from The Open Philanthropy Project (K.H.).

Author information

Authors and Affiliations

  1. Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Kenta Murakami, Nobuhiko Hamazaki, Norio Hamada, Go Nagamatsu, Yuya Kunisaki, Fumio Arai & Katsuhiko Hayashi

  2. Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Kenta Murakami, Norio Hamada & Kiyoko Kato

  3. Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan

    Ikuhiro Okamoto, Hiroshi Ohta, Yoshiaki Nosaka, Yukiko Ishikura & Mitinori Saitou

  4. Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Ikuhiro Okamoto, Hiroshi Ohta, Yoshiaki Nosaka, Yukiko Ishikura & Mitinori Saitou

  5. Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Tomoya S. Kitajima

  6. Department of Medicine and Biosystemic Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Yuichiro Semba, Yuya Kunisaki & Koichi Akashi

  7. Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

    Mitinori Saitou

  8. Department of Genome Biology, Graduate School of Medicine, Osaka University, Suita, Japan

    Katsuhiko Hayashi

  9. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan

    Katsuhiko Hayashi

  10. Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Japan

    Katsuhiko Hayashi

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  1. Kenta Murakami
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Contributions

K.H. conceived the project. K.M., N. Hamazaki and K.H. performed the investigation. N. Hamazaki, T.S.K. and I.O. contributed the methodology. K.M. performed the DNA-seq and RNA-seq analyses. N. Hamada, G.N., H.O., Y.N., Y.I., Y.S., Y.K., F.A., K.A. and M.S. provided the resources. K.K. and K.H. supervised the project. K.M. and K.H. wrote the manuscript, incorporating feedback from all the authors.

Corresponding author

Correspondence to Katsuhiko Hayashi.

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Competing interests

K.H. is an inventor on patent applications relating to in vitro oocyte production in mice filed by Kyushu University.

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Nature thanks Diana Laird and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Generation of XX ES cells from XO ES cells with reversine.

(a) Knock-in of DsRed into the X chromosome. The schematic diagram shows the gRNA position, primers for genotyping (arrows), and expected sizes of the amplicons. The right image shows the result of PCR using the primers. The black dots indicate the ES cell lines used in this study. M, size marker. W, wild type. (b) Removal of the Y chromosome. Shown are the results of PCR analysis of sex chromosomes. Each sex chromosome is indicated at the left side of the image. Black dots indicate XO ES cell clones. The lane with an asterisk was excluded from the count because the band was faint. (c) Effect of reversine on the proliferation of ES cells at each passage. The results of three independent experiments are shown as mean±s.e.m. (d) The effect of reversine treatment on the proliferation of ES cells at day 2. The results of three independent experiments are shown as mean±s.e.m. P value was determined by One-way ANOVA with Bonferroni adjustment. (e) Effect of reversine on DsRed intensity of XRO ES cells in the total cell population and G1 population. The top row shows the DsRed intensity of the total cell population cultured without (Control) or with reversine at each concentration. The numbers show the percentage of the cell population at each reversine concentration. The second row shows the overlay of the DsRed intensity of the total cell population cultured without (grey) and with reversine (red) at each concentration. The third row shows the overlay of the cell size measured by forward scatter (FSC) of the total cell population cultured without (grey) and with reversine (red) at each concentration. The fourth row shows the DNA content in the total cell population cultured without (Control) or with reversine at each concentration. The G1 population in each plot was analyzed in the fifth and bottom rows. The fifth row shows the overlay of the DsRed intensity of the G1 population cultured without (grey) and with reversine (red) at each concentration. The bottom row shows the overlay of the cell size measured by forward scatter (FSC) of the G1 population cultured without (grey) and with reversine (red) at each concentration. Note that DsRed-highly positive cells increased in proportion to the concentration of reversine in both the total cell and G1 populations, whereas the cell size became diverse in both the small and large fractions. (f) DNA-seq analysis of sex-converted ES cell clones. Shown are the results of sex-converted ES cell clones apart from the ES cell clone shown in Fig. 1e. Two copies are plotted at 0 in log2 ratio. The numbers below the plot are the chromosome numbers.

Source data

Extended Data Fig. 2 PGCL cell induction and transcriptome analysis of oocytes from the sex-converted XX ES cells.

(a) PGCL cell induction from XRY, XRO and XRXR ES cells. The numbers in the FACS analysis images are the percentages of PGCL cells. Scale bars, 200 µm. (b) H3K27me3 states indicative of X chromosome states during PGCL cell induction. Images show immunofluorescence analysis of ES cells and PGCL cells at day 3 of induction (d3 PGCL cells). Note that single H3K27me3 bright spots corresponding to Xi, were observed in cells at day 3 of PGCL cell induction. Scale bars, 10 μm. The right graph shows the percentage of cells with the indicated H3K27me3 state in BV positive and negative cells at day 3 of PGCL cell induction. These results are based on biologically duplicated samples. (c) Gene expression analysis of GV (left) and MII (right) oocytes differentiated from native XX ES cells (BVSCH18) and XRXR ES cells (BVSCN2-4-63R2). Genes expressed more than twofold higher (red) or twofold lower (blue) in XRXR oocytes are shown. The FDR cutoff is 0.01. Biological duplicate samples.

Source data

Extended Data Fig. 3 Reversine effect on trisomy 16 ES cells and oocyte production from trisomy 16 XY and XO ES cells.

(a) DNA-seq analysis of a mosaic trisomy 16 clone. (b) DNA-seq analysis of trisomy 16 XRO ES cells. Another trisomy 16 XRO cell line is shown in Fig. 2b. (c) Q-PCR analysis to determine the X chromosome number. Blue and orange dots show the X chromosome content determined using chromosome 7 and chromosome 11, respectively, as references. Details are described in Fig. 1c. The genotypes of sex chromosomes and individual clone IDs are shown below the graph. (d) DNA-seq analysis of FACS sorted trisomy 16 XRO ES cells without (control) or with reversine treatment. The left column shows the whole population, and the right column shows the top 5% fraction of the DsRed-high population. (e) Log2 ratio of autosomes and the X chromosome under each condition in the DNA-seq analysis. Note that the ratio of XRXR ES cells was significantly increased in the top 5% fraction compared to the total cell fraction with 0.02 µM reversine, while autosomes were relatively unaffected. There was no significant difference (P > 0.11) in the ratio of XRXR ES cells in the top 5% fraction without and with 0.02 µM reversine, consistent with the results shown in Fig. 1b. P value was determined by One-way ANOVA with Bonferroni adjustment. The values were compiled from biologically triplicated experiments. (f) PGCL cell induction from trisomy 16 XRY ES cells and trisomy 16 XRO ES cells. Shown are representative images and FACS analyses of the induction of PGCL cells from the ES cells indicated. The numbers in the FACS analysis are the percentages of PGCL cells. Scale bars, 200 µm. (g) IVDi culture using PGCL cells from trisomy 16 XRY ES cells and trisomy 16 XRO ES cells. Representative images of the rOvaries at the days indicated are shown. Scale bars, 200 µm. Similar results were obtained in biologically duplicated experiments.

Source data

Extended Data Fig. 4 Generation of XX iPS cells from XY iPS cells followed by oocyte production.

(a) Genotype of XY iPS cells used in this study. Shown are the results of PCR analysis of sex chromosomes (top) and knock-in of DsRed into the X chromosome (bottom). Details for the knock-in are shown in Extended Data Fig. 1a. All six iPS cell lines tested had X and Y chromosomes (top). The black dot in each image indicates the iPS cell clone used in this study. The knock-in alleles were detected in four of the six iPS cell lines. The same results were obtained in PCR experiment repeated twice. (b) Removal of the Y chromosome. Shown are the results of PCR analysis of sex chromosomes after 9 passages. The black dot indicates XRO iPS cells used in this study. The same results were obtained in PCR experiment repeated twice. (c) Q-PCR analysis to determine the X chromosome number. Blue and orange dots show the X chromosome content determined using chromosome 7 and chromosome 11, respectively, as references. Details are described in Fig. 1c. The genotypes of sex chromosomes and individual clone IDs are shown below the graph. (d) DNA-seq analysis of sex-converted iPS cell clones. Two copies are plotted at 0 in log2 ratio. The numbers below the plot are the chromosome numbers. The genotypes and the IDs of the individual clones are displayed on the left side of the plot. (e) PGCL cell induction from XRY, XRO and XRXR iPS cells. Shown are representative images and FACS analyses of the induction of PGCL cells from the iPS cells indicated. The numbers in the FACS analysis are the percentages of PGCL cells. Scale bars, 200 µm. Similar results were obtained in biologically duplicated experiments. (f) IVDi culture using PGCL cells from XRY, XRO and XRXR iPS cells. Shown are representative images of the rOvaries at the days indicated. As a negative control, a representative rOvary containing only embryonic gonadal somatic cells is shown. The images at day 21 of culture are the same as in Fig. 3b. Scale bars, 200 µm. Similar results were obtained in biologically duplicated experiments.

Source data

Extended Data Fig. 5 Derivation of offspring from a single iPS cell line.

(a) Chimeric mice harbouring XRY or sex-converted XRXR iPS cells. The graph below the images shows the ratio of iPS cell chimeric mice. The number in the top graph is the percentage of male chimeras harbouring XRY iPS cells and the numbers in the middle and bottom graphs are the percentages of female chimeras harbouring XRXR iPS cells. (b) Offspring from chimeric mice harbouring XRY or sex-converted XRXR iPS cells. The top images show offspring from male chimeric mice harbouring XRY iPS cells (left) and offspring female chimeric mice harbouring XRXR iPS cells (right). The middle graph shows the coat colour of offspring from a mating between chimeric mice and wild-type albino mice. The number in the graph is the percentage of non-albino mice from a mating between chimeric mice and wild type albino mice. The bottom gel image show genotyping of the offspring from iPS cell chimeric mice harbouring XRY iPS cells (left) or sex-converted XRXR iPS cells (right). PCR products from BV and DsRed transgenes, exogenous retroviral Sox2 and Pou5f1, and the Prdm 14 locus as a positive control are shown. The coat colour of number 10 was albino. P, positive control. N, negative control. Details of the positive control and negative control are described in Fig. 3d. (c) Pups from a mating between an XRY iPS cell-chimeric male and XRXR iPS cell-chimeric female mouse. (d) FACS analysis of H-2K haplotypes of offspring obtained from crosses between the chimeric mice. Shown are the FACS plot of H-2K haplotypes in lymphocytes of C57BL/6J (H-2Kb), 129X1/SvJ (H-2Kb), Balb/c (H-2Kd), and F1 mice from a mating between chimeric mice. Note that one of the F1 mice had only H-2Kb, whereas the other had a hybrid haplotype. (e) SNP analysis of offspring. Shown are SNPs at rs3022980 and rs3022981 in C57BL/6J, 129X1/SvJ, BALB/c and F1 mice from a mating between chimeric mice. (f) Ratio of homozygous F1 from a mating between an XRY iPS cell-chimeric mouse and XRXR iPS cell-chimeric mouse. (g) An adult mouse from (c) at 10 months after birth.

Source data

Extended Data Fig. 6 Generation of XX ES cells from XO ES cells using anti-CD38 antibodies.

(a) FACS analysis of CD38 expression in C57BL/6J (originally XX) ES cells. Note that there are two types of CD38 expression: low and high. (b) FACS analysis of DsRed and CD38 in XRO and XRXR ES cells. Note that the CD38 intensity was increased in XRXR ES cells in proportion to the intensity of DsRed. (c) The gates used to select XX ES cells from XO ES cells using anti-CD38 antibodies. The gate at left (solid line) was used for the CD38-high cell population and the gate at right (dashed line) was used for the CD38-extremely high cell population. (d) Q-PCR analysis to determine the X chromosome number. The left graph shows the results using BVSC XO ES cells with high CD38 (the solid gate in (c)) and the right graph shows the results using BVSC XO ES cells with extremely high CD38 (the dashed gate in (c)). (e) The graph shows the results using non-reporter XO ES cells with high CD38. The genotypes of sex chromosomes and individual clone IDs are shown below the graph. Blue and orange dots show the X chromosome content determined using chromosome 7 and chromosome 11, respectively, as references. Details are described in Fig. 1c. (f) FACS analysis of PGCL cells derived from non-reporter and X-duplicated ESCs. PGCL cells were identified by antibodies against SSEA1 and CD6126. Similar results were obtained in biologically duplicated experiments.

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Extended Data Table 1 Frequency of Y-chromosome loss in XY ES cells and iPS cells
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Extended Data Table 2 Percentage of MII oocytes after IVM
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Extended Data Table 3 Percentage of 2-cell embryos after IVF using iPS93C-49R6
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Extended Data Table 4 Birth rate of the 2-cell embryos transferred using iPS93C-49R6
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Murakami, K., Hamazaki, N., Hamada, N. et al. Generation of functional oocytes from male mice in vitro. Nature 615, 900–906 (2023). https://doi.org/10.1038/s41586-023-05834-x

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