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A critical function of Mad2l2 in primordial germ cell development of mice.

Pirouz M, Pilarski S, Kessel M - PLoS Genet. (2013)

Bottom Line: By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2.Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status.The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. mehdi.pirouz@mpibpc.mpg.de

ABSTRACT
The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2(-/-) embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.

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Mad2l2 expression and loss of germ cells from mutant ovaries and testes.(A) Mad2l2 mRNA expression in adult murine organs and E14.5 embryos. For an actin loading control of this northern blot see [74]. (B) Hematoxylin and Eosin (HE) staining of ovaries with low (upper panel) and high (lower panel) magnifications. Mad2l2−/− ovaries (P80) are smaller, and do not contain follicular or germ cells. (C) Testes (P70) are significantly smaller in Mad2l2−/− animals. (D) Morphologic analysis of testes (upper panel) and epididymis (lower panel) by HE staining reveals the absence of germ cells in mutant organs (P70). (E) Mad2l2 protein is expressed in pachytene spermatocytes (P10). (F–H) Mad2l2−/− seminiferous tubules (P14) lack spermatogonial cells as identified by Plzf, pre-meiotic cells as identified by Stra8, and meiotic cells as identified by γH2AX. (I) Mad2l2−/− seminiferous tubules (P70) contain highly vacuolated (red arrow) and miss-localized (arrowhead) Sertoli cells as identified by Wt1. Note hyperplasia of Leydig cells between seminiferous tubules (black arrow). Scale bars in B, E–I, 100 µm, in D, 200 µm.
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pgen-1003712-g001: Mad2l2 expression and loss of germ cells from mutant ovaries and testes.(A) Mad2l2 mRNA expression in adult murine organs and E14.5 embryos. For an actin loading control of this northern blot see [74]. (B) Hematoxylin and Eosin (HE) staining of ovaries with low (upper panel) and high (lower panel) magnifications. Mad2l2−/− ovaries (P80) are smaller, and do not contain follicular or germ cells. (C) Testes (P70) are significantly smaller in Mad2l2−/− animals. (D) Morphologic analysis of testes (upper panel) and epididymis (lower panel) by HE staining reveals the absence of germ cells in mutant organs (P70). (E) Mad2l2 protein is expressed in pachytene spermatocytes (P10). (F–H) Mad2l2−/− seminiferous tubules (P14) lack spermatogonial cells as identified by Plzf, pre-meiotic cells as identified by Stra8, and meiotic cells as identified by γH2AX. (I) Mad2l2−/− seminiferous tubules (P70) contain highly vacuolated (red arrow) and miss-localized (arrowhead) Sertoli cells as identified by Wt1. Note hyperplasia of Leydig cells between seminiferous tubules (black arrow). Scale bars in B, E–I, 100 µm, in D, 200 µm.

Mentions: Low levels of Mad2l2 mRNA are widely expressed in adult and E14.5 embryonic cells, with a particularly high level in testis (Figure 1A). High levels of Mad2l2 protein were detected in pachytene spermatocytes by immunohistochemistry (Figure 1E), while the antibody did not lead to specific signals above background in other tissues, including PGCs. Significant amounts of Mad2l2 RNA were previously detected in E9.5 PGCs by microarray analysis (NCBI database Gene Expression Omnibus GEO; Hayashi et al., 2011).


A critical function of Mad2l2 in primordial germ cell development of mice.

Pirouz M, Pilarski S, Kessel M - PLoS Genet. (2013)

Mad2l2 expression and loss of germ cells from mutant ovaries and testes.(A) Mad2l2 mRNA expression in adult murine organs and E14.5 embryos. For an actin loading control of this northern blot see [74]. (B) Hematoxylin and Eosin (HE) staining of ovaries with low (upper panel) and high (lower panel) magnifications. Mad2l2−/− ovaries (P80) are smaller, and do not contain follicular or germ cells. (C) Testes (P70) are significantly smaller in Mad2l2−/− animals. (D) Morphologic analysis of testes (upper panel) and epididymis (lower panel) by HE staining reveals the absence of germ cells in mutant organs (P70). (E) Mad2l2 protein is expressed in pachytene spermatocytes (P10). (F–H) Mad2l2−/− seminiferous tubules (P14) lack spermatogonial cells as identified by Plzf, pre-meiotic cells as identified by Stra8, and meiotic cells as identified by γH2AX. (I) Mad2l2−/− seminiferous tubules (P70) contain highly vacuolated (red arrow) and miss-localized (arrowhead) Sertoli cells as identified by Wt1. Note hyperplasia of Leydig cells between seminiferous tubules (black arrow). Scale bars in B, E–I, 100 µm, in D, 200 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757036&req=5

pgen-1003712-g001: Mad2l2 expression and loss of germ cells from mutant ovaries and testes.(A) Mad2l2 mRNA expression in adult murine organs and E14.5 embryos. For an actin loading control of this northern blot see [74]. (B) Hematoxylin and Eosin (HE) staining of ovaries with low (upper panel) and high (lower panel) magnifications. Mad2l2−/− ovaries (P80) are smaller, and do not contain follicular or germ cells. (C) Testes (P70) are significantly smaller in Mad2l2−/− animals. (D) Morphologic analysis of testes (upper panel) and epididymis (lower panel) by HE staining reveals the absence of germ cells in mutant organs (P70). (E) Mad2l2 protein is expressed in pachytene spermatocytes (P10). (F–H) Mad2l2−/− seminiferous tubules (P14) lack spermatogonial cells as identified by Plzf, pre-meiotic cells as identified by Stra8, and meiotic cells as identified by γH2AX. (I) Mad2l2−/− seminiferous tubules (P70) contain highly vacuolated (red arrow) and miss-localized (arrowhead) Sertoli cells as identified by Wt1. Note hyperplasia of Leydig cells between seminiferous tubules (black arrow). Scale bars in B, E–I, 100 µm, in D, 200 µm.
Mentions: Low levels of Mad2l2 mRNA are widely expressed in adult and E14.5 embryonic cells, with a particularly high level in testis (Figure 1A). High levels of Mad2l2 protein were detected in pachytene spermatocytes by immunohistochemistry (Figure 1E), while the antibody did not lead to specific signals above background in other tissues, including PGCs. Significant amounts of Mad2l2 RNA were previously detected in E9.5 PGCs by microarray analysis (NCBI database Gene Expression Omnibus GEO; Hayashi et al., 2011).

Bottom Line: By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2.Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status.The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. mehdi.pirouz@mpibpc.mpg.de

ABSTRACT
The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2(-/-) embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.

Show MeSH
Related in: MedlinePlus