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Epidemiologic studies have shown sex differences in ischemic stroke. The four core genotype (FCG) mouse model, in which the testes determining gene, Sry, has been moved from Y chromosome to an autosome, was used to dissociate the effects of sex hormones from sex chromosome in ischemic stroke outcome. Middle cerebral artery occlusion (MCAO) in gonad intact FCG mice revealed that gonadal males (XXM and XYM) had significantly higher infarct volumes as compared with gonadal females (XXF and XYF). Serum testosterone levels were equivalent in adult XXM and XYM, as was serum estrogen in XXF and XYF mice. To remove the effects of gonadal hormones, gonadectomized FCG mice were subjected to MCAO. Gonadectomy significantly increased infarct volumes in females, while no change was seen in gonadectomized males, indicating that estrogen loss increases ischemic sensitivity. Estradiol supplementation in gonadectomized FCG mice rescued this phenotype. Interestingly, FCG male mice were less sensitive to effects of hormones. This may be due to enhanced expression of the transgene Sry in brains of FCG male mice. Sex differences in ischemic stroke sensitivity appear to be shaped by organizational and activational effects of sex hormones, rather than sex chromosomal complement.

BackgroundTesticular dysgenesis syndrome (TDS) is a common disease that links testicular germ cell cancer, cryptorchidism and some cases of hypospadias and male infertility with impaired development of the testis. The incidence of these disorders has increased over the last few decades, and testicular cancer now affects 1% of the Danish and Norwegian male population.MethodsTo identify genetic variants that span the four TDS phenotypes, the authors performed a genome-wide association study (GWAS) using Affymetrix Human SNP Array 6.0 to screen 488 patients with symptoms of TDS and 439 selected controls with excellent reproductive health. Furthermore, they developed a novel integrative method that combines GWAS data with other TDS-relevant data types and identified additional TDS markers. The most significant findings were replicated in an independent cohort of 671 Nordic men.ResultsMarkers located in the region of TGFBR3 and BMP7 showed association with all TDS phenotypes in both the discovery and replication cohorts. An immunohistochemistry investigation confirmed the presence of transforming growth factor β receptor type III (TGFBR3) in peritubular and Leydig cells, in both fetal and adult testis. Single-nucleotide polymorphisms in the KITLG gene showed significant associations, but only with testicular cancer.ConclusionsThe association of single-nucleotide polymorphisms in the TGFBR3 and BMP7 genes, which belong to the transforming growth factor β signalling pathway, suggests a role for this pathway in the pathogenesis of TDS. Integrating data from multiple layers can highlight findings in GWAS that are biologically relevant despite having border significance at currently accepted statistical levels.

Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY DSD gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY DSD in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

Sex determination in mammals hinges on a cell fate decision in the fetal bipotential gonad between formation of male Sertoli cells or female granulosa cells. While this decision normally is permanent, loss of key cell fate regulators such as the transcription factors Dmrt1 and Foxl2 can cause postnatal transdifferentiation from Sertoli to granulosa-like (Dmrt1) or vice versa (Foxl2). Here, we examine the mechanism of male-to-female transdifferentiation in mice carrying either a null mutation of Dmrt1 or a point mutation, R111G, that alters the DNA-binding motif and causes human XY gonadal dysgenesis and sex reversal. We first define genes misexpressed during transdifferentiation and then show that female transcriptional regulators driving transdifferentiation in the mutant XY gonad (ESR2, LRH1, FOXL2) bind chromatin sites related to those normally bound in the XX ovary. We next define gene expression changes and abnormal chromatin compartments at the onset of transdifferentiation that may help destabilize cell fate and initiate the transdifferentiation process. We model the R111G mutation in mice and show that it causes dominant gonadal dysgenesis, analogous to its human phenotype but less severe. We show that R111G partially feminizes the testicular transcriptome and causes dominant disruption of DMRT1 binding specificity in vivo. These data help illuminate how transdifferentiation occurs when sexual cell fate maintenance is disrupted and identify chromatin sites and transcripts that may play key roles in the transdifferentiation process.

The testicular dysgenesis syndrome (TDS) hypothesis proposes that maldevelopment of the testis, irrespective of cause, leads to malfunction of the somatic (Leydig, Sertoli) cells and consequent downstream TDS disorders. Studies in rats exposed in utero to di(n-butyl) phthalate (DBP) have strongly supported the TDS concept, but so far no direct evidence has been produced that links dysgenesis per se to somatic cell dysfunction, in particular to androgen production/action during the 'masculinization programming window' (MPW; e15.5-e18.5). Normal reproductive tract development and anogenital distance (AGD) are programmed within the MPW, and TDS disorders arise because of deficiencies in this programming. However, DBP-induced focal testicular dysgenesis (Leydig cell aggregation, ectopic Sertoli cells, malformed seminiferous cords) is not evident until after the MPW. Therefore, we used AGD as a read-out of androgen exposure in the MPW, and investigated if this measure was related to objectively quantified dysgenesis (Leydig cell aggregation) at e21.5 in male fetuses exposed to vehicle, DBP (500 or 750 mg/kg/day) or the synthetic glucocorticoid dexamethasone (Dex; alone or plus DBP-500) from e15.5-e18.5 (MPW), e13.5-e20.5 or e19.5-e20.5 (late window). Dysgenesis was found only in animals exposed to DBP during the MPW, and was negatively correlated (R² = -0.5) with AGD at e21.5 and at postnatal day 8, irrespective of treatment period. Dysgenesis was also negatively correlated (R² = -0.5) with intratesticular testosterone (ITT) at e21.5, but only when treatments in short windows (MPW, late window) were excluded; the same was true for correlation between AGD and ITT. We conclude that AGD, reflecting Leydig cell function solely within the MPW, is strongly related to focal dysgenesis. Our results point to this occurring because of a common early mechanism, targeted by DBP that determines both dysgenesis and early (during the MPW) fetal Leydig cell dysfunction. The findings provide strong validation of the TDS hypothesis.

P elements inserted in the Telomere Associated Sequences (TAS) at the left end of the X chromosome are determiners of cytotype regulation of the entire P family of transposons. This regulation is mediated by Piwi-interacting (pi) RNAs derived from the telomeric P elements (TPs). Because these piRNAs are transmitted maternally, cytotype regulation is manifested as a maternal effect of the TPs. When a TP is combined with a transgenic P element inserted at another locus, this maternal effect is strengthened. However, when certain TPs are combined with transgenes that contain the small P element known as KP, stronger regulation arises from a zygotic effect of the KP element. This zygotic effect is observed with transgenic KP elements that are structurally intact, as well as with KP elements that are fused to an ancillary promoter from the hsp70 gene. Zygotic regulation by a KP element occurs only when a TP was present in the maternal germ line, and it is more pronounced when the TP was also present in the grand-maternal germ line. However, this regulation does not require zygotic expression of the TP. These observations can be explained if maternally transmitted piRNAs from TPs enable a polypeptide encoded by KP elements to repress P element transposition in zygotes that contain a KP element. In nature, repression by the KP polypeptide may therefore be facilitated by cytotype-mediating piRNAs.

BackgroundThe early gonad is bipotential and can differentiate into either a testis or an ovary. In XY embryos, the SRY gene triggers testicular differentiation and subsequent male development via its action on a single gene, SOX9. The supporting cell lineage of the bipotential gonad will differentiate as testicular Sertoli cells if SOX9 is expressed and conversely will differentiate as ovarian granulosa cells when SOX9 expression is switched off.ResultsThrough copy number variation mapping this study identified duplications upstream of the SOX9 gene in three families with an isolated 46,XX disorder of sex development (DSD) and an overlapping deletion in one family with two probands with an isolated 46,XY DSD. The region of overlap between these genomic alterations, and previously reported deletions and duplications at the SOX9 locus associated with syndromic and isolated cases of 46,XX and 46,XY DSD, reveal a minimal non-coding 78 kb sex determining region located in a gene desert 517–595 kb upstream of the SOX9 promoter.ConclusionsThese data indicate that a non-coding regulatory region critical for gonadal SOX9 expression and subsequent normal sex development is located far upstream of the SOX9 promoter. Its copy number variations are the genetic basis of isolated 46,XX and 46,XY DSDs of variable severity (ranging from mild to complete sex reversal). It is proposed that this region contains a gonad specific SOX9 transcriptional enhancer(s), the gain or loss of which results in genomic imbalance sufficient to activate or inactivate SOX9 gonadal expression in a tissue specific manner, switch sex determination, and result in isolated DSD.

Abstract XY C57BL/6J (B6) mice harboring a Mus musculus domesticus-type Y chromosome (YPOS), known as B6.YPOS mice, commonly undergo gonadal sex reversal and develop as phenotypic females. In a minority of cases, B6.YPOS males are identified and a proportion of these are fertile. This phenotypic variability on a congenic B6 background has puzzled geneticists for decades. Recently, a B6.YPOS colony was shown to carry a non-B6-derived region of chromosome 11 that protected against B6.YPOS sex reversal. Here. we show that a B6.YPOS colony bred and archived at the MRC Harwell Institute lacks the chromosome 11 modifier but instead harbors an ∼37 Mb region containing non-B6-derived segments on chromosome 13. This region, which we call Mod13, protects against B6.YPOS sex reversal in a proportion of heterozygous animals through its positive and negative effects on gene expression during primary sex determination. We discuss Mod13’s influence on the testis determination process and its possible origin in light of sequence similarities to that region in other mouse genomes. Our data reveal that the B6.YPOS sex reversal phenomenon is genetically complex and the explanation of observed phenotypic variability is likely dependent on the breeding history of any local colony.

Serum tumor markers were detected before operation and the results were as following: CA 125 34.20 U/ml, (normal range: 0-35.00 U/ml), CA 199 34.11 U/ml (normal range: 0-39.00 U/ml), alpha-fetoprotein (AFP) >1210.0 ng/ml (normal range: 0-7.0 ng/ml), beta-human chorionic gonadotropin (β-hCG) 12.81 U/L (normal range: 0-2.90 U/L), and lactic dehydrogenase (LDH) 204 U/L (normal range: 109-245 U/L). Postoperative paraffin pathology found a mixed germ cell tumor (GCT) composed of immature teratoma, dysgerminoma [Figure 1], and yolk sac tumor [Figure 2] in right ovary while a mixed tumor composed of GCT and sex cord stromal tumor (mature teratoma and gonadoblastoma) was identified in left ovary. Immunohistochemical staining results showed inhibin (+), CD99 (focal+), CD117 (focal+), Ki-67 (20%+), PLAP (focal+), OCT-4 (focal+), D2-40 (focal+), GPC3 (-), AFP (-), CD30 (-), and CK (focal+). [...]3 days after second operation, the result of karyotype analysis was out and demonstrated a genotype of pure XY. [1] It is estimated that 15% of complete sporadic 46, XY sex reversal cases (pure gonadal dysgenesis) are due to Sex-detemining Region Y (SRY) point mutations, another 15% are due to SRY deletions from aberrant X/Y recombinations, but almost 70% of all cases have unknown origin. Since the genotype result was reported later than the operation time, we only did salpingo-oophorectomy of the right side at first laparoscopy. [...]it is reasonable to perform the operation after the genotype result reported, if that was the case, bilateral salpingo-oophorectomy should be performed at the same operation and the second operation could be...

The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding/bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMPβ. The SRY-R133W mutant shows reduced IMPβ binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMPβ binding, whereas SRY-R75N and SRY-R76P show normal IMPβ binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.