Sex reversal caused by Mus musculus domesticus Y chromosomes linked to variant expression of the testis-determining gene Sry

When the Y chromosomes from certain populations of Mus musculus domesticus are introduced into the mouse strain C57BL/6 (B6), testis determination can fail, resulting in gonads developing either as ovotestes (with both ovarian and testicular components) or as ovaries. Not all Y(DOM) chromosomes cause sex reversal. Y(DOM) chromosomes are divided into three classes based upon their ability to induce testes in B6. The molecular basis underlying the three Y(DOM) classes is an enigma. The simplest explanation is that they harbor different alleles of the testis-determining gene, Sry. Sequencing of Sry(DOM) genes has indeed identified polymorphisms. However, none were unequivocally linked to the sex-reversal trait. It was concluded that all SRY(DOM) proteins are functionally equivalent. Using a semiquantitative RT-PCR assay, we now show that representatives of the three Y(DOM) classes have variant Sry expression patterns, that severity of sex reversal correlates with Sry mRNA titers, and that genetic correction of the sex reversal results in the upregulation of Sry expression. We propose that the variant Sry expression patterns result from polymorphisms at the site of a putative Sry enhancer.     

Sex reversal in C57BL/6J XY mice caused by increased expression of ovarian genes and insufficient activation of the testis determining pathway

Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XY(B6) mice impaired testis differentiation, but no ovarian tissue developed. If, however, a Y(AKR) chromosome replaced the Y(B6) chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/- B6 XY(AKR), Wt1+/- B6 XY(AKR), B6 XY(POS), and B6 XY(AKR) fetuses. We propose that Wt1(B6) and Sf1(B6) are hypomorphic alleles of testis-determining pathway genes and that Wnt4(B6) is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1(B6) and/or Sf1(B6) alleles that compromise testis differentiation and a Wnt4(B6) allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a "weak" Sry allele, such as the one on the Y(POS) chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal.     

Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y(DOM) sex reversal

The phenomenon of B6-Y(DOM) sex reversal arises when certain variants of the Mus domesticus Y chromosome are crossed onto the genetic background of the C57BL/6J (B6) inbred mouse strain, which normally carries a Mus musculus-derived Y chromosome. While the sex reversal has been assumed to involve strain-specific variations in structure or expression of Sry, the actual cause has not been identified. Here we used in situ hybridization to study expression of Sry, and the critical downstream gene Sox9, in strains containing different chromosome combinations to investigate the cause of B6-Y(DOM) sex reversal. Our findings establish that a delay of expression of Sry(DOM) relative to Sry(B6) underlies B6-Y(DOM) sex reversal and provide the first molecular confirmation that Sry must act during a critical time window to appropriately activate Sox9 and effect male testis determination before the onset of the ovarian-determining pathway.     

Regulation of Sex Determination in Mice by a Non-coding Genomic Region

To identify novel genomic regions that regulate sex determination, we utilized the powerful C57BL/6J-Y^POS (B6-Y^POS) model of XY sex reversal where mice with autosomes from the B6 strain and a Y chromosome from a wild-derived strain, Mus domesticus poschiavinus (Y^POS), show complete sex reversal. In B6-Y^POS, the presence of a 55-Mb congenic region on chromosome 11 protects from sex reversal in a dose-dependent manner. Using mouse genetic backcross designs and high-density SNP arrays, we narrowed the congenic region to a 1.62-Mb genomic region on chromosome 11 that confers 80% protection from B6-Y^POS sex reversal when one copy is present and complete protection when two copies are present. It was previously believed that the protective congenic region originated from the 129S1/SviMJ (129) strain. However, genomic analysis revealed that this region is not derived from 129 and most likely is derived from the semi-inbred strain POSA. We show that the small 1.62-Mb congenic region that protects against B6-Y^POS sex reversal is located within the Sox9 promoter and promotes the expression of Sox9, thereby driving testis development within the B6-Y^POS background. Through 30 years of backcrossing, this congenic region was maintained, as it promoted male sex determination and fertility despite the female-promoting B6-Y^POS genetic background. Our findings demonstrate that long-range enhancer regions are critical to developmental processes and can be used to identify the complex interplay between genome variants, epigenetics, and developmental gene regulation.     

Homozygous inactivation of Sox9 causes complete XY sex reversal in mice

In the presence of the Y-chromosomal gene Sry, the bipotential mouse gonads develop as testes rather than as ovaries. The autosomal gene Sox9, a likely and possibly direct Sry target, can induce testis development in the absence of Sry. Sox9 is thus sufficient but not necessarily essential for testis induction. Mutational inactivation of one allele of SOX9/Sox9 causes sex reversal in humans but not in mice. Because Sox9(-/-) embryos die around Embryonic Day 11.5 (E11.5) at the onset of testicular morphogenesis, differentiation of the mutant XY gonad can be analyzed only ex vivo in organ culture. We have therefore conditionally inactivated both Sox9 alleles in the gonadal anlagen using the CRE/loxP recombination system, whereby CRE recombinase is under control of the cytokeratin 19 promoter. Analysis of resulting Sox9(-/-) XY gonads up to E15.5 reveals immediate, complete sex reversal, as shown by expression of the early ovary-specific markers Wnt4 and Foxl2 and by lack of testis cord and Leydig cell formation. Sry expression in mutant XY gonads indicates that downregulation of Wnt4 and Foxl2 is dependent on Sox9 rather than on Sry. Our results provide in vivo proof that, in contrast to the situation in humans, complete XY sex reversal in mice requires inactivation of both Sox9 alleles and that Sox9 is essential for testogenesis in mice.     

A Single Genetic Determinant that Prevents Sex Reversal in C57BL-YPOS Congenic Mice

Sex determination in the mammalian embryo begins with the activation of a gene on the Y chromosome which triggers a cascade of events that lead to male development. The mechanism by which this gene, designated SRY in humans and Sry in mice (sex determining region of the Y chromosome), is activated remains unknown. Likewise, the downstream target genes for Sry remain unidentified at present. C57BL mice carrying a Y chromosome from Mus musculus musculus or molossinus develop normally as males. In contrast, C57BL/6 mice with the Y chromosome from M. m. domesticus often show sex reversal, i.e., develop as XY females. It has been documented that C57BL mice with the Y chromosome from Poschiavinus (Y^POS), a domesticus subtype, always develop as females or hermaphrodites. This suggests that a C57BL gene either up- or downstream of Sry is ineffective in interacting with Sry, which then compromises the processes that lead to normal male sex development. Nonetheless, by selective breeding, we have been able to generate a sex reversal-resistant C57BL/6-congenic strain of mice in which the XY^POS individuals consistently develop as normal males with bilateral testes. Because the resistance to sex reversal was transferred from strain 129S1/Sv (nonalbino) by simple selection over 13 backcross generations, it is inferred that a single autosomal gene or chromosomal region confers resistance to the sex reversal that would otherwise result. XY^POS normal males generated in these crosses were compared to XY^POS abnormal individuals and to C57BL/6 controls for sexual phenotype, gonadal weight, serum testosterone, and major urinary protein (MUP) level. A clear correlation was found among phenotypic sex, MUP level, and testis weight in the males and in the incompletely masculinized XY^POS mice. The fully masculinized males of the congenic strain resemble C57BL/6 males in the tested parameters. DNA analysis confirmed that these males, in fact, carry the Y^POS Sry gene.     

Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary

Mammalian sex determination is controlled by antagonistic pathways that are initially co-expressed in the bipotential gonad and subsequently become male- or female-specific. In XY gonads, testis development is initiated by upregulation of Sox9 by SRY in pre-Sertoli cells. Disruption of either gene leads to complete male-to-female sex reversal. Ovarian development is dependent on canonical Wnt signaling through Wnt4, Rspo1 and β-catenin. However, only a partial female-to-male sex reversal results from disruption of these ovary-promoting genes. In Wnt4 and Rspo1 mutants, there is evidence of pregranulosa cell-to-Sertoli cell transdifferentiation near birth, following a severe decline in germ cells. It is currently unclear why primary sex reversal does not occur at the sex-determining stage, but instead occurs near birth in these mutants. Here we show that Wnt4-null and Rspo1-null pregranulosa cells transition through a differentiated granulosa cell state prior to transdifferentiating towards a Sertoli cell fate. This transition is preceded by a wave of germ cell death that is closely associated with the disruption of pregranulosa cell quiescence. Our results suggest that maintenance of mitotic arrest in pregranulosa cells may preclude upregulation of Sox9 in cases where female sex-determining genes are disrupted. This may explain the lack of complete sex reversal in such mutants at the sex-determining stage.     

Sexual differentiation in the developing mouse brain: contributions of sex chromosome genes

Neural sexual differentiation begins during embryogenesis and continues after birth for a variable amount of time depending on the species and brain region. Because gonadal hormones were the first factors identified in neural sexual differentiation, their role in this process has eclipsed investigation of other factors. Here, we use a mouse with a spontaneous translocation that produces four different unique sets of sex chromosomes. Each genotype has one normal X‐chromosome and a unique second sex chromosome creating the following genotypes: XY^*x, XX, XY^*, XX^Y*. This Y^* mouse line is used by several laboratories to study two human aneuploid conditions: Turner and Klinefelter syndromes. As sex chromosome number affects behavior and brain morphology, we surveyed brain gene expression at embryonic days 11.5 and 18.5 to isolate X‐chromosome dose effects in the developing brain as possible mechanistic changes underlying the phenotypes. We compared gene expression differences between gonadal males and females as well as individuals with one vs. two X‐chromosomes. We present data showing, in addition to genes reported to escape X‐inactivation, a number of autosomal genes are differentially expressed between the sexes and in mice with different numbers of X‐chromosomes. Based on our results, we can now identify the genes present in the region around the chromosomal break point that produces the Y^* model. Our results also indicate an interaction between gonadal development and sex chromosome number that could further elucidate the role of sex chromosome genes and hormones in the sexual differentiation of behavior.     

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co‐expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth‐promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial–mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co‐transfectants exhibited invasive properties in three‐dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf‐1, a specific antagonist of the Wnt/β‐catenin pathway, or short hairpin RNA targeting β‐catenin expression demonstrated that the invasive activity was not mediated by β‐catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context‐dependent. While Rspo2 and Wnt1 act synergistically in the β‐catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures. J. Cell. Physiol. 227: 1960–1971, 2012. © 2011 Wiley Periodicals, Inc.     

Sry基因的研究进展

性别决定基因（Sex region of Y chromosome, 人类以SRY,小鼠以Sry表示）的研究进展是近几年来人类在性别决定,性别分化研究中获得的最大的突破性成果,该文从SRY（Sry)发现前关于性别决定因子的研究,SRY（Sry)的确定,小鼠Sry的结构研究,小鼠Sry的表达研究及Sry下游基因的确定等5个方面对小鼠Sry的研究进展进行综述,对进一步深入研究Sry下游基因存在的瓶颈问题人了一定的分析,并提出核移植技术可能对研究Sry的调节及其下游基因所需的特殊实验材料展现了新的希望。     

Rspo1对雌性脊椎动物性别分化的功能

长期以来雌性脊椎动物的性别分化被认为是一个“默认”的程序.但是近些年研究发现,Rspo1基因的突变或缺失可导致哺乳动物XX型个体性反转为雄性.Rspo1在鱼类、两栖爬行类、鸟类和哺乳类动物性腺发育的不同阶段表达,其表达在雌雄个体性别分化时期有差异,是潜在的性别调控基因.Rspo1在性别发育早期可通过Wnt/β-catenin信号通路调控性腺分化相关因子的表达,影响原始生殖细胞分裂增殖、细胞周期和生长发育,参与调控性腺中体细胞的分化.本文总结了近年来Rspo1在脊椎动物中的表达调控及其在雌性性别决定方面功能的研究进展.