XX Sex Reversal Research Articles

Attempt to Rescue Sex-Reversal by Transgenic Expression of the PISRT1 Gene in XX PIS–/– Goats

The Polled Intersex Syndrome (PIS mutation) in goats leads to an absence of horn and to an early sex-reversal of the XX gonads. This mutation is a deletion of an 11.7-kb DNA fragment showing a tissue-specific regulatory activity. Indeed, in XX PIS^–/– gonads the deletion of PIS leads to the transcriptional extinction of at least 3 neighboring genes, FOXL2, PFOXic and PISRT1. Among them, only FOXL2 is a ‘classical’ gene, encoding a highly conserved transcription factor. On the other hand, knock-out of Foxl2 in mice results in an early blocking of follicle formation without sex-reversal. This phenotype discrepancy leads to two hypotheses, either FOXL2 is responsible for XX sex-reversal in goat assuming distinct functions of its protein during ovarian differentiation in different mammals, or other PIS-regulated genes are involved. To assess the second possibility, PISRT1 expression was constitutively restored in XX PIS^–/– gonads. Six transgenic fetuses were obtained by nuclear transfer and studied at 2 developmental stages, 41 and 46 days post-reconstruction. The gonads of these fetuses appear phenotypically identical to those of cloned non-transgenic controls. Conclusively, this result argues for FOXL2 being responsible for the PIS gonad-associated phenotype. Its invalidation in goat will help to better understand this complex syndrome.

Mutations in the RSPO1 Coding Region Are Not the Main Cause of Canine SRY-Negative XX Sex Reversal in Several Breeds

This report details a case of SRY-negative XX sex reversal in a mixed breed dog and surveys affected dogs of several breeds for mutations in RSPO1 coding regions. Genomic DNA from the mixed breed case was evaluated for mutations in candidate genes. Sequencing identified a homozygous G to A transition in RSPO1 exon 4 that changes a highly conserved amino acid codon in the thrombospondin domain. The possibility that this was a single nucleotide polymorphism (SNP) could not be excluded by genotyping family members. Therefore, the coding region of RSPO1 was sequenced in a survey of affected dogs, which identified a T to C transition (exon 3) in some, the above G to A transition (exon 4) in others, and no change in the remaining affected dogs. Genotypes at these base pair positions were not uniquely associated with the affected phenotype in any breed, indicating the identified transitions are most likely SNPs, not causative mutations for this canine disorder. However, the possibility that polymorphisms play a modifier role, such as changing threshold or severity of phenotypic expression in a mixed breed dog, cannot be excluded. This study emphasizes the importance of canine pedigree, breed, and population studies in evaluating candidate mutations.

Sry-negative XX true hermaphroditism in a roe deer

A two-year-old roe deer was brought down in the course of a hunt in the north of Spain (Asturias). On physical examination the individual presented well-developed bared antlers, but surprisingly a female external genitalia. Several anatomical, histological and genetic analyses were performed in order to explain the observed phenotype. Necropsy evidenced ovary-like structures with follicles on the surface; histological analyses of testes evidenced positive immunolabel against testosterone in Leydig cells; genetic analyses showed that the sex of the individual was consistent with a female individual. PCR analysis failed to detect SRY sequences; no PIS deletion, which is responsible for XX sex-reversal in goats, was detected. On the basis of its presumptive normal female sexual karyotype (XX) and the presence of two functional abdominal bilateral testes and ovaries, the roe deer was finally diagnosed as possessing an XX hermaphroditism syndrome. However, as in many other cases, the specific reason for the occurrence of this case of hermaphroditism could not be determined.

R-spondin1 and FOXL2act into two distinct cellular types during goat ovarian differentiation

BackgroundUp to now, two loci have been involved in XX sex-reversal in mammals following loss-of-function mutations, PIS (Polled Intersex Syndrome) in goats and R-spondin1 (RSPO1) in humans. Here, we analyze the possible interaction between these two factors during goat gonad development. Furthermore, since functional redundancy between different R-spondins may influence gonad development, we also studied the expression patterns of RSPO2, 3 and 4.ResultsSimilarly to the mouse, RSPO1 shows a sex-dimorphic expression pattern during goat gonad development with higher levels in the ovaries. Interestingly, the PIS mutation does not seem to influence its level of expression. Moreover, using an RSPO1 specific antibody, the RSPO1 protein was localized in the cortical area of early differentiating ovaries (36 and 40 dpc). This cortical area contains the majority of germ cell that are surrounded by FOXL2 negative somatic cells. At latter stages (50 and 60 dpc) RSPO1 protein remains specifically localized on the germ cell membranes. Interestingly, a time-specific relocation of RSPO1 on the germ cell membrane was noticed, moving from a uniform distribution at 40 dpc to a punctuated staining before and during meiosis (50 and 60 dpc respectively). Interestingly, also RSPO2 and RSPO4 show a sex-dimorphic expression pattern with higher levels in the ovaries. Although RSPO4 was found to be faintly and belatedly expressed, the expression of RSPO2 increases at the crucial 36 dpc stage, as does that of FOXL2. Importantly, RSPO2 expression appears dramatically decreased in XX PIS-/- gonads at all three tested stages (36, 40 and 50 dpc).ConclusionDuring goat ovarian development, the pattern of expression of RSPO1 is in agreement with its possible anti-testis function but is not influenced by the PIS mutation. Moreover, our data suggest that RSPO1 may be associated with germ cell development and meiosis. Interestingly, another RSPO gene, RSPO2 shows a sex-dimorphic pattern of expression that is dramatically influenced by the PIS mutation.

R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling

In mammals, female development has traditionally been considered a default process in the absence of the testis-determining gene, Sry. Recently, it has been documented that the gene for R-spondin1 (RSPO1), a novel class of soluble activator for Wnt/beta-catenin signaling, is mutated in two Italian families with female-to-male (XX) sex reversal. To elucidate the role of Rspo1 as a candidate female-determining gene in a mouse model, we generated Rspo1-null (Rspo1(-/-)) mice and found that Rspo1(-/-) XX mice displayed masculinized features including pseudohermaphroditism in genital ducts, depletion of fetal oocytes, male-specific coelomic vessel formation and ectopic testosterone production in the ovaries. Thus, although Rspo1 is required to fully suppress the male differentiation program and to maintain germ cell survival during the development of XX gonads, the loss of its activity has proved to be insufficient to cause complete XX sex reversal in mice. Interestingly, these partial sex-reversed phenotypes of Rspo1(-/-) XX mice recapitulated those of previously described Wnt-4(-/-) XX mice. In accordance with this finding, the expression of Wnt-4 and its downstream genes was deregulated in early Rspo1(-/-) XX gonads, suggesting that Rspo1 may participate in suppressing the male pathway in the absence of Sry and maintaining oocyte survival through positively regulating Wnt-4 signaling.

Molecular analysis of the Y chromosome in XX sex-reversed patients

Molecular genetic analysis was performed for 26 phenotypically male patients lacking the Y chromosome in the karyotype. The sex-determining region Y (SRY) gene was found in 77% of the patients. PCR analysis of Y-specific loci in the 17 SRY-positive patients revealed Yp fragments varying in size in 16 cases and cryptic mosaicism (or chimerism) for the Y chromosome in one case. The frequencies of class I, II, and III (Yp+)XX sex reversals were 18.75, 25.25, and 56%, respectively. All of the class III (Yp+)XX sex-reversed patients had a 3.5-Mb paracentric inversion flanked by inverted repeats 3 (IR3) on the short arm of the Y chromosome.

Sexual dimorphic expression of DMRT1 and Sox9a during gonadal differentiation and hormone‐induced sex reversal in the teleost fish Nile tilapia (Oreochromis niloticus)

We examined the expression profiles of tDMRT1 and Sox9a during gonadal sex differentiation and hormone-induced sex reversal. tDMRT1 was detected in the gonial germ-cell-surrounding cells in XY fry specifically before the appearance of any signs of morphological sex differentiation, that is, sex differences in germ cell number and histogenesis, such as differentiation into intratesticular efferent duct or ovarian cavity. The signals became localized in the Sertoli and epithelial cells comprising the efferent duct during gonadal differentiation. After the induction of XY sex reversal with estrogen, tDMRT1 decreased and then disappeared completely. In contrast, tDMRT1 was expressed in the germ-cell-surrounding cells in XX sex reversal with androgen. On the other hand, Sox9a did not show sexual dimorphism before the appearance of sex differences in histogenesis and was not expressed in the efferent duct in the testis. These results suggest that tDMRT1 is a superior testicular differentiation marker in tilapia.

Syndromic true hermaphroditism due to an R-spondin1 (RSPO1) homozygous mutation

XX true hermaphroditism, also know as ovotesticular disorder of sexual development (DSD), is a disorder of gonadal development characterized by the presence of both ovarian and testicular tissue in a 46,XX individual. The genetic basis for XX true hermaphroditism and sex reversal syndromes unrelated to SRY translocation is still mostly unclear. We report mutational analysis of the RSPO1 gene in a 46,XX woman with true hermaphroditism, palmoplantar keratoderma, congenital bilateral corneal opacities, onychodystrophy, and hearing impairment. R-spondin1 is a member of the R-spondin protein family and its pivotal role in sex determination has been recently described. We identified a homozygous splice-donor-site mutation in the RSPO1 gene in our patient. We found that the c.286+1G>A mutation led to an aberrantly spliced mRNA (r.95_286del), which is presumably translated into a partially functional protein (p.Ile32_Ile95del). Our case demonstrates for the first time, to our knowledge, that XX true hermaphroditism can be caused by a single gene mutation. The reported findings represent a further step toward a complete understanding of the complex mechanisms leading to DSDs.

Trisomy 9 mosaicism and XX sex reversal

We report on a case of a phenotypic male infant who was referred to the genetics service due to dysmorphic features and congenital cardiac anomalies. Dysmorphic features included low-set, posteriorly rotated ears with squared, simple helices, midface hypoplasia, a broad nasal root with wide nasal alae, small mouth, micrognathia, short neck, overlapping fingers, rocker-bottom feet, prominent heels, and hypoplastic toenails. The phallus was normal and no testes were palpable in the scrotal sac. Cardiac anomalies included tricuspid atresia and a ventricular septal defect. The karyotype was 46,XX. Diagnostic microarray demonstrated evidence for trisomy 9 mosaicism, metaphase FISH revealed trisomy 9 on 7% of cultured cells, and interphase FISH analysis of a peripheral blood smear showed trisomy 9 in 78% of the cells examined. This is the first reported case of XX sex reversal in a patient with trisomy 9 mosaicism.

Differential display analysis of gene expression in female-to-male sex-reversing gonads of the frog Rana rugosa

Sex steroids play pivotal roles in gonadal differentiation in many species of vertebrates. The sex can be reversed from female to male by testosterone in the Japanese wrinkled frog Rana rugosa, but it is still unclear what genes are up- or down-regulated during the XX sex-reversal in this species. To search the genes for the female-to-male sex-reversal, we employed differential display and 5′/3′-RACE. Consequently, we isolated from the gonads at day 8 after testosterone injection 24 different cDNA fragments showing a testosterone treatment-related change and then obtained three full-length cDNAs, which we termed Zfp64, Zfp112, and Rrp54. The former two cDNAs encoded different proteins with zinc-finger domains, whereas the latter cDNA encoded an unknown protein. Transcripts of the three genes were hardly detectable in the sex-reversing gonads at day 24 after the injection; at this time few growing oocytes were observed in the sex-reversing gonad. Besides, in situ hybridization analysis showed positive signals of the three genes in the cytoplasm of growing oocytes of an ovary when testosterone was injected into a tadpole. Thus, the decrease in expression of these three genes was probably due to the disappearance of growing oocytes and not to their direct involvement in the testis formation. To find the key-gene for testis formation, it will be necessary to analyze, by the differential display method, more genes showing a change in expression pattern during sex reversal.

Linkage to CFA29 Detected in a Genome-Wide Linkage Screen of a Canine Pedigree Segregating Sry-Negative XX Sex Reversal

Canine Sry-negative XX sex reversal is a disorder of gonadal development wherein individuals having a female karyotype develop testes or ovotestes. In this study, linkage mapping was undertaken in a pedigree derived from one proven carrier American cocker spaniel founder male and beagle females. All affected dogs in the analysis were XX true hermaphrodites and confirmed to be Sry negative by polymerase chain reaction. A genome-wide linkage screen conducted using 245 microsatellite markers revealed highest LOD score of 3.4 (marker CPH9) on CFA29. Fine mapping with additional microsatellites in the region containing CPH9 localized the Sry-negative XX sex reversal locus to a 5.4-Mb candidate region between markers CPH9 and FH3003 (LOD score 3.15). Insignificant LOD scores were found at genome-wide screen or fine mapping markers that were within 10 Mb of 45 potential candidate genes reported to have a role in mammalian sex determination or differentiation. Together, these results suggest that a novel locus on CFA29 may be responsible for sex reversal in this pedigree.

The regulation of Sox9 gene expression by the GATA4/FOG2 transcriptional complex in dominant XX sex reversal mouse models

We have previously established an in vivo requirement for GATA4 and FOG2 transcription factors in sexual differentiation. Fog2 null mouse fetuses or fetuses homozygous for a targeted mutation in Gata4 ( Gata4 ki ), which cripples the GATA4–FOG2 interaction, exhibit a profound and early block in testis differentiation in both sexes. Others have shown that XX mice with the Ods transgenic insertion or the Wt1-Sox9 YAC transgene overexpress the testis differentiation gene, Sox9. Thus, these XX animals undergo dominant sex reversal by developing into phenotypically normal, but sterile, males. Now we have determined that Fog2 haploinsufficiency prevents (suppresses) this dominant sex reversal and Fog2 +/− Wt1-Sox9 or Ods XX animals develop normally—as fertile females. The suppression of sex reversal in Fog2 heterozygous females results from approximately 50% downregulation of the expression from the transgene-associated allele of Sox9. The GATA4/FOG2-dependent sex reversal observed in the transgenic XX gonads has to rely on gene targets other than the Y chromosome-linked Sry gene. Importantly, Fog2 null or Gata4 ki/ki embryos (either XX or XY) fail to express detectable levels of Sox9 despite carrying the Ods mutation or Wt1-Sox9 transgene. Fog2 haploinsufficiency leads to a decreased amount of SOX9-positive cells in XY gonads. We conclude that FOG2 is a limiting factor in the formation of a functional GATA4/FOG2 transcription complex that is required for Sox9 expression during gonadogenesis.

DMY gene induces male development in genetically female (XX) medaka fish

Although the sex-determining gene SRY/Sry has been identified in mammals, homologues and genes that have a similar function have yet to be identified in nonmammalian vertebrates. Recently, DMY (the DM-domain gene on the Y chromosome) was cloned from the sex-determining region on the Y chromosome of the teleost fish medaka (Oryzias latipes). DMY has been shown to be required for the normal development of male individuals. In this study, we show that a 117-kb genomic DNA fragment that carries DMY is able to induce testis differentiation and subsequent male development in XX (genetically female) medaka. In addition, overexpression of DMY cDNA under the control of the CMV promoter also caused XX sex reversal. These results demonstrate that DMY is sufficient for male development in medaka and suggest that the functional difference between the X and Y chromosomes in medaka is a single gene. Our data indicate that DMY is an additional sex-determining gene in vertebrates.

Male pseudohermaphroditism in dogs: three case reports

Three Cocker Spaniel dogs, 2−3 months old, weighing 3−4 kg, were presented to the Chonbuk Animal Medical Centre, Chonbuk National University, with intersex anomalies. Physical, radiological, gross, histological, hormonal and cytogenetic studies were performed. Physical examination of the external genitalia revealed that the dogs possessed the vulva with an enlarged clitoris protruding from the vulvar juncture and the scrotum with an undescended testis in Case 1 and 2, and both testes remained undescended in Case 3. Hyperoestrogenaemia and low testosterone serum concentrations were found. Laparotomy revealed persistent Mullerian ducts (PMD) in Case 1 and 2, and abdominally located testicle(s) in all the cases. Histological examinations of the gonads revealed inactive seminiferous tubules. Cytogenetic analysis showed 78XY male karyotype in Case 1 and 2, whereas Case 3 showed 79XX female karyotype. The congenital defects were diagnosed as male pseudohermaphroditism (MPH) and PMD in Case 1 and 2, and XX sex reversal MPH in Case 3.

R-spondin1 is essential in sex determination, skin differentiation and malignancy

R-spondins are a recently characterized small family of growth factors. Here we show that human R-spondin1 (RSPO1) is the gene disrupted in a recessive syndrome characterized by XX sex reversal, palmoplantar hyperkeratosis and predisposition to squamous cell carcinoma of the skin. Our data show, for the first time, that disruption of a single gene can lead to complete female-to-male sex reversal in the absence of the testis-determining gene, SRY.

Influence of sex chromosome constitution on the genomic imprinting of germ cells.

Germ cells in XY male mice establish site-specific methylation on imprinted genes during spermatogenesis, whereas germ cells in XX females establish their imprints in growing oocytes. We showed previously that in vitro, sex-specific methylation patterns of pluripotent stem cell lines derived from germ cells were influenced more by the sex chromosome constitution of the cells themselves than by the gender of the embryo from which they had been derived. To see whether the same situation would prevail in vivo, we have now determined the methylation status of H19 expressed from the maternal allele, and the expression and methylation status of a paternally expressed gene Peg3, in germ cells from sex-reversed and control embryos. For these imprinted genes, we conclude that the female imprint is a response of the germ cells to undergoing oogenesis, rather than to their XX chromosome constitution. Similarly, both our XY and our sex-reversed XX male germ cells clearly showed a male rather than a female pattern of DNA methylation; here, however, the sex chromosome constitution had a significant effect, with XX male germ cells less methylated than the XY controls.

513: Correlation between Clinical Findings and Expression of Transcription Factors Implicated in Sexual Differentiations and Steroidogenesis in Testicular Tissue of XX Sex Reversal Boys with Hypospadias

You have accessJournal of UrologyModerated Poster, Sunday, May 21, 2006, 3:30 - 5:30 pm1 Apr 2006513: Correlation between Clinical Findings and Expression of Transcription Factors Implicated in Sexual Differentiations and Steroidogenesis in Testicular Tissue of XX Sex Reversal Boys with Hypospadias Yoshiyuki Kojima, Yutaro Hayashi, Shoichi Sasaki, Kenjiro Kohri, Ken-lchiro Morohashi, and Peter Koopman Yoshiyuki KojimaYoshiyuki Kojima More articles by this author , Yutaro HayashiYutaro Hayashi More articles by this author , Shoichi SasakiShoichi Sasaki More articles by this author , Kenjiro KohriKenjiro Kohri More articles by this author , Ken-lchiro MorohashiKen-lchiro Morohashi More articles by this author , and Peter KoopmanPeter Koopman More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)32759-9AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "513: Correlation between Clinical Findings and Expression of Transcription Factors Implicated in Sexual Differentiations and Steroidogenesis in Testicular Tissue of XX Sex Reversal Boys with Hypospadias." The Journal of Urology, 175(4S), pp. 166–167 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 175Issue 4SApril 2006Page: 166-167 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Yoshiyuki Kojima More articles by this author Yutaro Hayashi More articles by this author Shoichi Sasaki More articles by this author Kenjiro Kohri More articles by this author Ken-lchiro Morohashi More articles by this author Peter Koopman More articles by this author Expand All Advertisement PDF downloadLoading ...

Genetics, genomics, and molecular biology of sex determination in small animals

The genomic revolution is beginning to facilitate advances in canine and feline medicine, as illustrated in our research. Our studies are focused upon identifying the gene mutation that causes canine Sry-negative XX sex reversal, a disorder of sex determination in which chromosomal females (78,XX) develop testicular tissue, becoming either XX true hermaphrodites with ovotestes, or XX males with bilateral testes. A genome-wide screen, using mapped markers in our pedigree of Sry-negative XX sex reversed dogs founded upon the American cocker spaniel, identified five chromosomal regions in which the causative gene may be located. The canine genome was used to identify the canine homologue of goat Pisrt1 and so determine that canine and caprine Sry-negative XX sex reversal are genetically heterogeneous. A second goal of our research is to determine the molecular mechanism by which the mutation causes testis induction. Thus far, we have reported gonadal Sry and Sox9 expression patterns in normal embryos, which have temporal and spatial patterns similar to those reported in humans, sheep, and pigs. Once gene mutations causing such inherited disorders are identified, DNA tests will become a part of general veterinary practice, advancing both diagnostic techniques and preventative medicine.

Exclusion of Candidate Genes for Canine SRY-Negative XX Sex Reversal

In mammals, the Y-linked SRY gene is normally responsible for testis induction, yet testis development can occur in the absence of Y-linked genes, including SRY. The canine model of SRY-negative XX sex reversal could lead to the discovery of novel genes in the mammalian sex determination pathway. The autosomal genes causing testis induction in this disorder in dogs, humans, pigs, and horses are presently unknown. In goats, a large deletion is responsible for sex reversal linked to the polled (hornless) phenotype. However, this region has been excluded as being causative of the canine disorder, as have WT1 and DMRT1 in more recent studies. The purpose of this study was to determine whether microsatellite marker alleles near or within five candidate genes (GATA4, FOG2, LHX1, SF1, SOX9) are associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal. Primer sequences flanking nucleotide repeats were designed within genomic sequences of canine candidate gene homologues. Fluorescence-labeled polymorphic markers were used to screen a subset of the multigenerational pedigree, and marker alleles were determined by software. Our results indicate that the mutation causing canine SRY-negative XX sex reversal in this pedigree is unlikely to be located in regions containing these candidates.

XX sex reversal, palmoplantar keratoderma, and predisposition to squamous cell carcinoma: Genetic analysis in one family

We describe a large inbred Sicilian family that includes four 46, XX (SRY-) brothers. Palmoplantar hyperkeratosis (PPK) and an associated predisposition to squamous cell carcinoma (SCC) of the skin, segregates as a recessive trait within the family. Interestingly, all the PPK-affected members of the family are phenotypic males (46,XY or 46,XX) while seven XX sibs are healthy phenotypic females with no signs of PPK. We propose that homozygosity for a single mutational event, possibly including contiguous genes, may cause PPK/SCC in both XY or XX individuals and sex reversal in XX individuals. The family is informative for linkage analysis for the PPK trait and allows linkage exclusion for the sex reversal trait. Here we show that 15 loci involved in PPK etiology, skin differentiation, function or malignancy, and nine loci involved in sex determination/differentiation are not implicated in the phenotype of this family.