Fujitani K et al. (2016), Sexually dimorphic expression of Dmrt1 and γH2A...

XB-ART-52177

FEBS Open Bio 2016 Feb 26;64:276-84. doi: 10.1002/2211-5463.12035.

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Sexually dimorphic expression of Dmrt1 and γH2AX in germ stem cells during gonadal development in Xenopus laevis.

Fujitani K , Otomo A , Wada M , Takamatsu N , Ito M .

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In many animals, primordial germ cells (PGCs) migrate into developing gonads. There, they proliferate and differentiate into female and male germ stem cells (GSCs), oogonia and spermatogonia, respectively. Few studies have focused on the molecular mechanisms underlying the development of GSC sex determination. Here, we investigated the expression of the transcription factor Dmrt1 and a phosphorylated form of the histone variant H2AX (γH2AX) during gonadal development in Xenopus laevis. During early sexual differentiation, Dmrt1 was expressed in the GSCs of the ZW (female) and ZZ (male) gonads as well as somatic cells of the ZZ gonads. Notably, the PGCs and primary GSCs contained large, unstructured nuclei, whereas condensed, rounder nuclei appeared only in primary oogonia during tadpole development. After metamorphosis, Dmrt1 showed its expression in secondary spermatogonia, but not in secondary oogonia. Like Dmrt1, γH2AX was expressed in the nuclei of primary GSCs in early developing gonads. However, after metamorphosis, γH2AX expression continued in primary and secondary spermatogonia, but was barely detected in the condensed nuclei of primary oogonia. Taken together, these observations indicate that spermatogonia tend to retain PGC characteristics, compared to oogonia, which undergo substantial changes during gonadal differentiation in X. laevis. Our findings suggest that Dmrt1 and γH2AX may contribute to the maintenance of stem cell identity by controlling gene expression and epigenetic changes, respectively.

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Species referenced: Xenopus laevis
Genes referenced: ddx4 dmrt1 gsc h2ax h2ax pgc stra8
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	Fig. 1. Real-time PCR analysis of dmrt1 and stra8 in ZZ and ZW gonads during sexual development. qPCR was performed for dmrt1 (A) or stra8 (B) using cDNAs derived from the total RNAs of three ZZ or ZW gonads from tadpoles at different stages of development and adult frogs. The dmrt1 or stra8 primer pairs were designed within common sequences shared between the two dmrt1 (dmrt1.L and dmrt1.S) and two stra8 cDNAs in X. laevis. W, weeks; Y, years.
	Fig. 2. Distribution of Dmrt1 in ZZ and ZW gonads during tadpole development. Immunostaining with anti-Dmrt1 and anti- VASA antibodies was performed on gonadal sections from ZW and ZZ tadpoles at stages 56 (A) and 62 (B). Nuclei were stained with TO-PRO-3. Oc, oocyte; Og, secondary oogonium; POg, primary oogonium; PSg, primary spermatogonium; pSe, pre-Sertoli cell. White arrowheads and arrows indicate Dmrt1-expressing pre- Sertoli cells and primary oogonia, respectively. Typical GSCs with somatic cells and their 2.59 magnification images are shown in the dashed squares. A dashed polygon indicates a typical cyst consisting of secondary oogonia.
	Fig. 3. Distribution of Dmrt1 in the immature ovary and mature testis after metamorphosis. Immunostaining with anti- Dmrt1 and anti-VASA antibodies was performed using the immature ovary from a ZW frog 2 weeks after metamorphosis (A) and the mature testis of an adult ZZ frog (B). Nuclei were stained with TOPRO- 3. Oc, Oocyte; POg, primary oogonium; PSg, primary spermatogonium; Se, Sertoli cell; Sg, secondary spermatogonium; Sc, spermatocyte; St, spermatid. pSe, pre-Sertoli cell. Dmrt1- expressing primary oogonia and their 59 magnification images are shown in the dashed squares. White arrows and arrowhead in (A) indicate primary oogonia and diplotene oocytes, respectively. White arrowheads in (B) indicate Dmrt1- expressing primary spermatogonium.
	Fig. 4. BrdU incorporation and Dmrt1 expression in the ZW developing ovary and ZZ adult testis. Immunostaining with anti-Dmrt1 and anti-BrdU antibodies was performed on the immature tadpole ovary at stage 62 and the mature testis of an adult frog. Nuclei were stained with TOPRO- 3. Og, secondary oogonium; POg, primary oogonium; PSg, primary spermatogonium; Sg, secondary spermatogonium; Sc, spermatocyte.
	Fig. 5. Distribution of cH2AX in ZZ and ZW gonads during development. Immunostaining with anti-Dmrt1 and anticH2AX antibodies was performed on gonadal sections from ZW and ZZ tadpoles at stages 53 and 62 (A) and on gonadal sections from a ZZ adult frog (B). Nuclei were stained with TO-PRO. Oc, oocyte; POg, primary oogonium; PSg, primary spermatogonium; Sg, secondary spermatogonium; Sc, spermatocyte. White arrowheads and arrows indicate Dmrt1- or cH2AX-positive primary GSCs and leptotene/zygotene oocytes, respectively.
	dmrt1 (doublesex and mab-3 related transcription factor 1) gene expression in Xenopus laevis ovary, assayed via immunohistochemistry, NF stage 62.
	dmrt1 (doublesex and mab-3 related transcription factor 1) gene expression in Xenopus laevis testis, assayed via immunohistochemistry, adult frog stage.
	Figure 1. Real‐time PCR analysis of dmrt1 and stra8 in ZZ and ZW gonads during sexual development. qPCR was performed for dmrt1 (A) or stra8 (B) using cDNAs derived from the total RNAs of three ZZ or ZW gonads from tadpoles at different stages of development and adult frogs. The dmrt1 or stra8 primer pairs were designed within common sequences shared between the two dmrt1 (dmrt1.L and dmrt1.S) and two stra8 cDNAs in X. laevis. W, weeks; Y, years.
	Figure 2. Distribution of Dmrt1 in ZZ and ZW gonads during tadpole development. Immunostaining with anti‐Dmrt1 and anti‐VASA antibodies was performed on gonadal sections from ZW and ZZ tadpoles at stages 56 (A) and 62 (B). Nuclei were stained with TO‐PRO‐3. Oc, oocyte; Og, secondary oogonium; POg, primary oogonium; PSg, primary spermatogonium; pSe, pre‐Sertoli cell. White arrowheads and arrows indicate Dmrt1‐expressing pre‐Sertoli cells and primary oogonia, respectively. Typical GSCs with somatic cells and their 2.5× magnification images are shown in the dashed squares. A dashed polygon indicates a typical cyst consisting of secondary oogonia.
	Figure 3. Distribution of Dmrt1 in the immature ovary and mature testis after metamorphosis. Immunostaining with anti‐Dmrt1 and anti‐VASA antibodies was performed using the immature ovary from a ZW frog 2 weeks after metamorphosis (A) and the mature testis of an adult ZZ frog (B). Nuclei were stained with TO‐PRO‐3. Oc, Oocyte; POg, primary oogonium; PSg, primary spermatogonium; Se, Sertoli cell; Sg, secondary spermatogonium; Sc, spermatocyte; St, spermatid. pSe, pre‐Sertoli cell. Dmrt1‐expressing primary oogonia and their 5× magnification images are shown in the dashed squares. White arrows and arrowhead in (A) indicate primary oogonia and diplotene oocytes, respectively. White arrowheads in (B) indicate Dmrt1‐expressing primary spermatogonium.
	Figure 4. BrdU incorporation and Dmrt1 expression in the ZW developing ovary and ZZ adult testis. Immunostaining with anti‐Dmrt1 and anti‐BrdU antibodies was performed on the immature tadpole ovary at stage 62 and the mature testis of an adult frog. Nuclei were stained with TO‐PRO‐3. Og, secondary oogonium; POg, primary oogonium; PSg, primary spermatogonium; Sg, secondary spermatogonium; Sc, spermatocyte.
	Figure 5. Distribution of γH2AX in ZZ and ZW gonads during development. Immunostaining with anti‐Dmrt1 and anti‐γH2AX antibodies was performed on gonadal sections from ZW and ZZ tadpoles at stages 53 and 62 (A) and on gonadal sections from a ZZ adult frog (B). Nuclei were stained with TO‐PRO. Oc, oocyte; POg, primary oogonium; PSg, primary spermatogonium; Sg, secondary spermatogonium; Sc, spermatocyte. White arrowheads and arrows indicate Dmrt1‐ or γH2AX‐positive primary GSCs and leptotene/zygotene oocytes, respectively.

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