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Gadd45g Is Essential for Primary Sex Determination, Male Fertility and Testis Development

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Gadd45g Is Essential for Primary Sex Determination, Male Fertility and Testis Development Gadd45g Is Essential for Primary Sex Determination, Male Fertility and Testis Development Heiko Johnen1, Laura Gonza´lez-Silva1, Laura Carramolino2, Juana Maria Flores3, Miguel Torres2, Jesu´ s M. Salvador1* 1 Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a/Consejo Superior de Investigaciones Cientı´ficas, Campus Cantoblanco, Madrid, Spain, 2 Department of Cardiovascular Development and Repair, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain, 3 Animal Surgery and Medicine Department, Veterinary School, Universidad Complutense de Madrid, Madrid, Spain Abstract In humans and most mammals, differentiation of the embryonic gonad into ovaries or testes is controlled by the Y-linked gene SRY. Here we show a role for the Gadd45g protein in this primary sex differentiation. We characterized mice deficient in Gadd45a, Gadd45b and Gadd45g, as well as double-knockout mice for Gadd45ab, Gadd45ag and Gadd45bg, and found a specific role for Gadd45g in male fertility and testis development. Gadd45g-deficient XY mice on a mixed 129/C57BL/6 background showed varying degrees of disorders of sexual development (DSD), ranging from male infertility to an intersex phenotype or complete gonadal dysgenesis (CGD). On a pure C57BL/6 (B6) background, all Gadd45g2/2 XY mice were born as completely sex-reversed XY-females, whereas lack of Gadd45a and/or Gadd45b did not affect primary sex determination or testis development. Gadd45g expression was similar in female and male embryonic gonads, and peaked around the time of sex differentiation at 11.5 days post-coitum (dpc). The molecular cause of the sex reversal was the failure of Gadd45g2/2 XY gonads to achieve the SRY expression threshold necessary for testes differentiation, resulting in ovary and Mu¨llerian duct development. These results identify Gadd45g as a candidate gene for male infertility and 46,XY sex reversal in humans. Citation: Johnen H, Gonza´lez-Silva L, Carramolino L, Flores JM, Torres M, et al. (2013) Gadd45g Is Essential for Primary Sex Determination, Male Fertility and Testis Development. PLoS ONE 8(3): e58751. doi:10.1371/journal.pone.0058751 Editor: Yann Herault, IGBMC/ICS, France Received December 11, 2012; Accepted February 5, 2013; Published March 13, 2013 Copyright: ß 2013 Johnen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the European Research Council (ERC; FP7-208250-1), the Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias, PI081718, PI1101411), and the Ministerio de Ciencia e Innovacio´n (200820I109) to JMS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction developmental pathway is activated [12]. These pathways are mutually antagonistic, and disturbances in their molecular The Gadd45 (growth arrest and DNA-damage-inducible network can lead to sex reversal and other disorders of sexual protein 45) family members Gadd45a, -b and -g are small nuclear development. We found that Gadd45g, but not Gadd45a or and cytoplasmic proteins that bind to and modify the activity of Gadd45b, is necessary for activation of the male sex-determining other intracellular proteins, including p21 [1,2], PCNA [1,2], pathway in mice and its absence leads to development of female CRIF [3], CDK1 [4] and the MAP kinases p38 [5] and MAP3K4 gonads. Lack of Gadd45g decreased SRY expression, resulting in [6]. They are implicated in the regulation of apoptosis, survival, ovary and Mu¨llerian duct development, whereas lack of Gadd45a senescence, cell cycle control, DNA repair and the response to and/or Gadd45b had no effect on testis development. physiological or environmental stress in mammalian cells. All family members appear to have overlapping but non-identical functions and binding partners, and are induced by different Materials and Methods stimuli [7]. Little is known of their role in embryonic development. Mouse Strains and Genotyping It was suggested that loss of an enhancer region that drives brain- Gadd45a2/2 [13] and Gadd45b2/2 mice [14] were generated specific Gadd45g expression leads to increased growth of brain on a mixed 129/C57BL/6 genetic background. Gadd45g2/2 regions in humans compared to chimpanzees [8]. Another group mice were generated by Drs. J.M. Salvador and C. Hollander on a studied the expression pattern during mouse embryonic develop- mixed 129/C57BL/6 genetic background (Fig. S1) [15]. Mice ment up to 10.5 dpc and proposed a conserved role for Gadd45g 2 2 were maintained in the CNB animal facility. Gadd45a / , in vertebrate neurogenesis [9] as well as involvement in embryonic 2 2 2 2 Gadd45b / , and Gadd45g / mice on a pure C57BL/6 neural cell development and exit from pluripotency in Xenopus background were generated by backcrossing for seven generations. [10]. We mated Gadd45a2/2 mice with Gadd45b2/2 and Gadd45g2/ Here we identify a specific role for Gadd45g in mammalian sex 2 mice and intercrossed F1 double heterozygotes to obtain double- determination. In male gonads, SRY expression triggers differen- null mice. Mouse embryos were obtained by timed mating of tiation of a somatic support cell lineage into Sertoli cells, which Gadd45g2/2 or Gadd45g+/2 females with Gadd45g+/2 males. direct the male developmental pathway [11]; in the absence of For embryo staging, midday on the day of vaginal plug SRY (in XX gonads), granulosa cells differentiate and the female PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e58751 Gadd45 Family in Development appearance was considered 0.5 dpc. Embryos were staged more (Fig. 1B,H), and histological analysis of the gonads revealed no accurately by counting tail somites posterior to the hind limb bud. obvious abnormalities. Hematoxylin/eosin-stained ovaries from all Genotyping was done by PCR using the following primers: B6 XY-F mice analyzed had oocytes and follicles at different Gadd45g 59-GCTGTGCTTTCCGGAACTGTA-39,59- developmental stages (Fig. 1O,U). Corpora lutea (Fig. 1U) were CGGCAGATTTGAGGCT GTGT-39 and 59-AGTTGCCAGC- found in all ovaries, indicating that ovulation took place in B6 XY- CATCTGTTGT-39; SRY 59-TCTTAAACTCTGAAGAAG F mice. AGAC-39 and 59-GTCTTGCCTGTATGTGATGG-39. Experi- In contrast, the internal and external reproductive organs of ments were approved by the CNB Ethical Committee, and mice 129/B6 Gadd45g2/2 XY mice showed a range of DSD that could were handled according to national and European Union animal be broadly categorized in three groups. In the first group, mice care standards. had a male phenotype and reproductive system (XY-M), although with testis hypoplasia (Fig. 1D,J). All fertility-tested Gadd452/2 Isolation of Fetal Gonads from Embryos of Timed- XY-M mice were infertile. Histological analysis showed hypoplas- pregnant Mice tic seminiferous tubules with reduced spermatogenesis and interstitial cell hyperplasia (frequent in infertility; Fig. 1P). No Dissection was carried out around noon each day. Pregnant spermatozoa were found in the cauda epididymis and vas deferens females were sacrificed and embryos extracted. Gonads (including (Fig. 1V). Mice in the second group had a female phenotype and mesonephros) from embryos at different stages were isolated, fixed reproductive system (XY-F, Fig. 1E,K). These mice had female in 4% paraformaldehyde (1 h) and stored in methanol at 280uC reproductive organs (ovary, oviduct, uterus), although the ovaries or flash frozen for RNA extraction [16]. usually contained mainly interstitial cells and multiple primordial follicles, but no other stages of oocyte and follicle development Histological Analysis and Immunohistochemistry (Fig. 1Q,W). All fertility-tested XY-F mice were infertile. The third Ovaries and testes from adult mice were fixed in 10% formalin, group consisted of intersex mice (XY-intersex, Fig. 1F,L) with dehydrated in ascending ethanol concentrations, cleared in xylene male, female, or ambiguous external genitals and internal and embedded in paraffin wax. Embedded samples were sectioned reproductive organs with both female and male characteristics. and hematoxylin/eosin-stained. Gonads were whole-mount For example, one side developed a hypoplastic testes/epididymis/ stained with anti-SOX9 (sc 20095), -AMH (sc-6886, both from vas deferens (Fig. 1L) and the contralateral side, an ovary/ Santa Cruz,) and -PECAM-1-FITC antibodies (Pharmingen). oviduct/uterus or an ovotestis with mixed ovarian and testicular Gonads were rehydrated with TBS, incubated in blocking buffer tissue in the same gonad (Fig. 1L,R,X). (1 h), stained with antibodies (overnight, 4uC), mounted with The cause for the variability of this phenotype is likely to be the Vectashield Hardset (Source) and analyzed using a Leica TCS SP5 variable contribution of the B6 background in individual mixed- confocal microscope and LAS AF imaging software. background mice. Compared to the 129 strain, B6 mice are more responsive to disturbances in early testis development [17]. As a Quantitative PCR result, all B6 Gadd45g2/2 XY, and a small number of B6 RNA was extracted from both embryonic gonads (including Gadd45g+/2 XY mice showed complete sex reversal (Fig. 2A), mesonephros) using the RNeasy Micro Kit (Qiagen). cDNA was whereas some 129/B6 Gadd45g2/2 XY mice still developed as synthesized from 100 ng RNA using a High Capacity cDNA (infertile) males (Fig. 2B). Chromosome painting of metaphase X Reverse Transcription Kit with RNase Inhibitor (Applied Biosys- and Y chromosomes confirmed that mice identified as XY-F by tems). SRY and Gadd45g transcript amounts were quantified PCR (Fig. 2C) carried the Y chromosome (Fig. 2D). using the Taqman gene expression assays Mm00441712_s1 (SRY) We evaluated the role of Gadd45a and/or Gadd45b in testis and Mm00442225_m1 (Gadd45g)(Applied Biosystems) on an development and sex determination.
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