Dataset Information

Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX<-->XY chimeric testes.

ABSTRACT: We produced XX<-->XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X*), making the fluorescent green female (XX*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX* germ cells developed as "eggs" within the seminiferous tubules of XX*<-->XY chimeric testes. These cells were indistinguishable from XX* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the "testicular eggs" was examined by recovering the eggs from chimeric testes. The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.

SUBMITTER: Isotani A

PROVIDER: S-EPMC554793 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX<-->XY chimeric testes.

Isotani Ayako A Nakanishi Tomoko T Kobayashi Shin S Lee Jiyoung J Chuma Shinichiro S Nakatsuji Norio N Ishino Fumitoshi F Okabe Masaru M

Proceedings of the National Academy of Sciences of the United States of America 20050303 11

We produced XX<-->XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X*), making the fluorescent green female (XX*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indic ...[more]

PMID: 15746241

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Project description:Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.

Project description:The sex chromosome complement, XX or XY, determines sexual differentiation of the gonadal primordium into a testis or an ovary, which in turn directs differentiation of the germ cells into sperm and oocytes, respectively, in eutherian mammals. When the X monosomy or XY sex reversal occurs, XO and XY females exhibit subfertility and infertility in the mouse on the C57BL/6J genetic background, suggesting that functional germ cell differentiation requires the proper sex chromosome complement. Using these mouse models, we asked how the sex chromosome complement affects gene transcription in the oocytes during follicular growth. An oocyte accumulates cytoplasmic components such as mRNAs and proteins during follicular growth to support subsequent meiotic progression, fertilization, and early embryonic development without de novo transcription. However, how gene transcription is regulated during oocyte growth is not well understood. Our results revealed that XY oocytes became abnormal in chromatin configuration, mitochondria distribution, and de novo transcription compared to XX or XO oocytes near the end of growth phase. Therefore, we compared transcriptomes by RNA-sequencing among the XX, XO, and XY oocytes of 50-60 µm in diameter, which were still morphologically comparable. The results showed that the X chromosome dosage limited the X-linked and autosomal gene transcript levels in XO oocytes whereas many genes were transcribed from the Y chromosome and made the transcriptome in XY oocytes closer to that in XX oocytes. We then compared the transcript levels of 3 X-linked, 3 Y-linked and 2 autosomal genes in the XX, XO, and XY oocytes during the entire growth phase as well as at the end of growth phase using quantitative RT-PCR. The results indicated that the transcript levels of most genes increased with oocyte growth while largely maintaining the X chromosome dosage dependence. Near the end of growth phase, however, transcript levels of some X-linked genes did not increase in XY oocytes as much as XX or XO oocytes, rendering their levels much lower than those in XX oocytes. Thus, XY oocytes established a distinct transcriptome at the end of growth phase, which may be associated with abnormal chromatin configuration and mitochondria distribution.

Dataset Information

Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX<-->XY chimeric testes.

Publications

Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX<-->XY chimeric testes.

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