Genomics of sexual cell fate transdifferentiation in the mouse gonad.

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.

Developmental exposure to real-life environmental chemical mixture programs a testicular dysgenesis syndrome-like phenotype in prepubertal lambs.

Current declines in male reproductive health may, in part, be driven by anthropogenic environmental chemical (EC) exposure. Using a biosolids treated pasture (BTP) sheep model, this study examined the effects of gestational exposure to a translationally relevant EC mixture. Testes of 8-week-old ram lambs from mothers exposed to BTP during pregnancy contained fewer germ cells and had a greater proportion of Sertoli-cell-only seminiferous tubules. This concurs with previous published data from fetuses and neonatal lambs from mothers exposed to BTP. Comparison between the testicular transcriptome of biosolids lambs and human testicular dysgenesis syndrome (TDS) patients indicated common changes in genes involved in apoptotic and mTOR signalling. Gene expression data and immunohistochemistry indicated increased HIF1α activation and nuclear localisation in Leydig cells of BTP exposed animals. As HIF1α is reported to disrupt testosterone synthesis, these results provide a potential mechanism for the pathogenesis of this testicular phenotype, and TDS in humans.

Testicular Stem Cell Dysfunction Due to Environmental Insults Could Be Responsible for Deteriorating Reproductive Health of Men.

Reproductive health of men has declined over time including reduced semen quality specifically sperm count, increased incidence of infertility, and testicular cancers. Our recent findings suggest that these disease states possibly arise as a result of disruption of testicular stem cells biology by perinatal insults including exposure to endocrine disrupting chemicals. Testicular stem cells include relatively quiescent, very small embryonic-like stem cells (VSELs), and actively dividing spermatogonial stem cells (SSCs). Both VSELs and SSCs express estrogen receptors and are directly vulnerable to endocrine disruption. Exposing mice pups to estradiol (20 µg/pup/day on days 5-7) or diethylstilbestrol (2 µg/pup/day on days 1-5) affected spermatogenesis during adult life with reduced numbers of tubules in stage VIII, tetraploid cells and sperm. These mice were infertile and majority of diethylstilbestrol treated mice revealed testicular cancer-like changes. An increase in VSEL numbers, observed by both flow cytometry and qRT-PCR, was associated with marked reduction of c-KIT positive spermatogonial cells. VSELs undergo epigenetic changes due to endocrine disruption that results in blocked differentiation (impaired spermatogenesis) leading to reduced sperm count and infertility, and their excessive self-renewal initiates cancer-like changes in adult life. Thus, testicular dysgenesis syndrome (TDS) has a stem cell rather than a genetic basis.

Genome-wide identification, evolution of DNA methyltransferases and their expression during gonadal development in Nile tilapia.

DNA methyltransferases (dnmts) are responsible for DNA methylation and play important roles in organism development. In this study, seven dnmts genes (dnmt1, dnmt2, dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1, dnmt3bb.2) were identified in Nile tilapia. Comprehensive analyses of dnmts were performed using available genome databases from representative animal species. Phylogenetic analysis revealed that the dnmts family were highly conserved in teleosts. Based on transcriptome data from eight adult tilapia tissues, the dnmts were found to be dominantly expressed in the head kidney, testis and ovary. Analyses of the gonadal transcriptome data in different developmental stages revealed that all dnmts were expressed in both ovary and testis, and four de novo dnmts (dnmt3aa, dnmt3ab, dnmt3bb.1, dnmt3bb.2) showed higher expression in the testis than in the ovary. Furthermore, during sex reversal induced by Fadrozole, the expression of these four de novo dnmts increased significantly in treated group compared to female control group. By in situ hybridization, the seven dnmts were found to be expressed mainly in phase I and II oocytes of the ovary and spermatocytes of the testis. When gonads were incubated with a methyltransferase inhibitor (5-AzaCdR) in vitro, the expression of dnmts genes were down-regulated significantly, while the expression of cyp19a1a (a key gene in female pathway) and dmrt1 (a key gene in male pathway) increased significantly. Our results revealed the conservation of dnmts during evolution and indicated a potential role of dnmts in epigenetic regulation of gonadal development.

Cognitive abilities in women with complete androgen insensitivity syndrome and women with gonadal dysgenesis.

BACKGROUND: Many questions regarding the mechanisms behind sex differences in cognitive abilities are still unanswered. On a group level, men typically outperform women on certain spatial tasks, whereas women perform better on certain tests of memory and verbal ability. The prevailing theories concerning the biological predispositions for these and other differences in behaviour and brain function focus on early and prolonged exposure to sex hormones. There is, however, evidence of direct effects of sex chromosomes on sex-typical behaviour in other species. OBJECTIVES: To study the influence of sex hormones and sex chromosomes on cognition in women with Complete androgen insensitivity (CAIS) and Gonadal dysgenesis (GD). METHODS: Eighteen women with CAIS, 6 women with 46,XYGD, and 7 women with 46,XXGD were compared with age-matched male and female controls on tests of spatial and verbal abilities, memory functions, and emotion recognition. RESULTS: Women with CAIS, XYGD, and XXGD performed similar to female controls on cognitive tasks. However, on a test of emotion recognition, women with XXGD outperformed the other groups, whereas women with CAIS and XYGD performed similar to male controls. CONCLUSION: Our results support theories of androgen effects on cognitive abilities and suggest that factors related to sex chromosomes may influence emotion recognition. Implications of an atypical sex hormone situation and sex chromosome variation are discussed.

Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

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 ERß, 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.

Cytotype Regulation Facilitates Repression of Hybrid Dysgenesis by Naturally Occurring KP Elements in Drosophila melanogaster.

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.

Sex differences in ischemic stroke sensitivity are influenced by gonadal hormones, not by sex chromosome complement.

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.

MicroRNA-561 promotes acetaminophen-induced hepatotoxicity in HepG2 cells and primary human hepatocytes through downregulation of the nuclear receptor corepressor dosage-sensitive sex-reversal adrenal hypoplasia congenital critical region on the X chromosome, gene 1 (DAX-1).

One of the major mechanisms involved in acetaminophen (APAP)-induced hepatotoxicity is hepatocyte nuclear factor 4α (HNF4α)-mediated activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). In the present study, we investigated the role of miR-561 and its target gene DAX-1 encoding a corepressor of HNF4α in the process of APAP-induced hepatotoxicity. We used both human hepatocellular liver carcinoma cell line (HepG2) cells and primary human hepatocytes in this study and monitored the levels of reactive oxygen species, lactate dehydrogenase, and glutathione. Our bioinformatics study suggests an association between miR-561 and DAX-1, but not HNF4α. Treatment of HepG2 cells with APAP significantly reduced the expression of DAX-1 in a concentration-dependent manner. miR-561 was induced by APAP treatment in HepG2 cells. Transfection of HepG2 cells with an miR-561 mimic exacerbated APAP-induced hepatotoxicity. HNF4α is physically associated with DAX-1 in HepG2 cells. A decreased protein level of DAX-1 by APAP treatment was also enhanced by miR-561 mimic transfection in HepG2 cells and primary human hepatocytes. The basal and APAP-induced expression of PXR and CAR was enhanced by miR-561 mimic transfection; however, transfection of HepG2 cells or primary human hepatocytes with a miR-561 inhibitor or DAX-1 small interfering RNA reversed these effects. Additionally, the chromatin immunoprecipitation assay revealed that recruitment of DAX-1 onto the PXR promoter was inversely correlated with the recruitment of peroxisome proliferator-activated receptor-α coactivator-1α and HNF4α on APAP treatment. These results indicate that miR-561 worsens APAP-induced hepatotoxicity via inhibition of DAX-1 and consequent transactivation of nuclear receptors.

[P-M hybrid dysgenesis affects juvenile hormone metabolism in Drosophila melanogaster females].

This paper studies the metabolism of the juvenile hormone, which affects gonads functioning in Drosophila melanogasterfemales under P-M hybrid dysgenesis. It is shown that dysgenic females grown at 29°C have increased levels of the juvenile hormone (its degradation and stress reactivity are reduced), which apparently is a compensatory response to ovarian hypoplasia.

Excess DAX1 leads to XY ovotesticular disorder of sex development (DSD) in mice by inhibiting steroidogenic factor-1 (SF1) activation of the testis enhancer of SRY-box-9 (Sox9).

Human DAX1 duplications cause dosage-sensitive sex reversal (DSS) whereby chromosomally XY individuals can develop as females due to gonadal dysgenesis. However, the mechanism of DSS-adrenal hypoplasia congenita on X, gene 1 (DAX1) action in the fetal testis is unknown. We show that in fetal testes from XY Dax1-overexpressing transgenic mice, the expression of the key testis-promoting gene sex-determining region on Y (SRY)-box-9 (Sox9) is reduced. Moreover, in XY Sox9 heterozygotes, in which testis development is usually normal, Dax1 overexpression results in ovotestes, suggesting a DAX1-SOX9 antagonism. The ovarian portion of the XY ovotestes was characterized by expression of the granulosa cell marker, Forkhead box-L2, with complete loss of the Sertoli cell markers, SOX9 and anti-Müllerian hormone, and the Leydig cell marker CYP17A1. However, the expression of SRY and steroidogenic factor-1 (SF1), two key transcriptional regulators of Sox9, was retained in the ovarian portion of the XY ovotestes. Using reporter mice, Dax1 overexpression reduced activation of TES, the testis enhancer of Sox9, indicating that DAX1 might repress Sox9 expression via TES. In cultured cells, increasing levels of DAX1 antagonized SF1-, SF1/SRY-, and SF1/SOX9-mediated activation of TES, due to reduced binding of SF1 to TES, providing a likely mechanism for DSS.

Inter-relationship between testicular dysgenesis and Leydig cell function in the masculinization programming window in the rat.

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 (R²â€Š= -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.

A genome-wide association study of men with symptoms of testicular dysgenesis syndrome and its network biology interpretation.

BACKGROUND: Testicular 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. METHODS: To 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. RESULTS: Markers 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 ß 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. CONCLUSIONS: The association of single-nucleotide polymorphisms in the TGFBR3 and BMP7 genes, which belong to the transforming growth factor ß 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.

Characteristics of testicular dysgenesis syndrome and decreased expression of SRY and SOX9 in Frasier syndrome.

Frasier syndrome (FS) is characterized by chronic renal failure in early adulthood, varying degrees of gonadal dysgenesis, and a high risk for gonadal germ cell malignancies, particularly gonadoblastoma. Although it is known to arise from heterozygous splice mutations in intron 9 of the Wilms' tumor gene 1 (WT1), the mechanisms by which these mutations result in gonadal dysgenesis in humans remain obscure. Here we show that a decrease in WT1 + KTS isoforms due to disruption of alternative splicing of the WT1 gene in a FS patient is associated with diminished expression of the transcription factors SRY and SOX9 in Sertoli cells. These findings provide the first confirmation in humans of the results obtained by others in mice. Consequently, Sertoli cells fail to form the specialized environment within the seminiferous tubules that normally houses developing germ cells. Thus, germ cells are unable to fully mature and are blocked at the spermatogonial-spermatocyte stage. Concomitantly, subpopulations of the malignant counterpart of primordial germ cells/gonocytes, the intratubular germ cell neoplasia unclassified type (ITGCN), are identified. Furthermore, dysregulated Leydig cells produce insufficient levels of testosterone, resulting in hypospadias. Collectively, the impaired spermatogenesis, hypospadias and ITGCN comprise part of the developmental disorder known as 'testicular dysgenesis syndrome' (TDS), which arises during early fetal life. The data presented here show that critical levels of WT1 + KTS, SRY and SOX9 are required for normal Sertoli cell maturation, and subsequent normal spermatogenesis. To further study the function of human Sertoli cells in the future, we have established a human cell line.

[Pathogenesis of neoplastic changes in germ cells as a developmental aspect]. / Patogeneza zmian nowotworowych z komórek plciowych w aspekcie rozwojowym.

Fetal germ cells transform neoplastically probably at the fetal period of life. The best place for their maintenance is a testis with the disturbed organogenesis (differentiation). These testes are called dysgenetic gonads. Besides the classical form of gonadal dysgenesis, appearing by the reversed sex features (female in genetic males), there are probably hidden forms without the disturbances in the sex differentiation. The overt and hidden disturbances of the testes' organogenesis are called testicular dysgenesis syndrome (TDS). Gonadal dysgenesis is more frequent in normal male karyotype than in numerical and structural aberrations of sex chromosomes. The cause of TDS is an inappropriate expression of genes of Y chromosome or autosomes, taking part in testicular differentiation, and/or exposition to the influence of environmental factors, mainly with estrogen-like action. It is supposed that TDS determines a cascade of events leading to germ cell tumours (GCT). This mechanism may appear as follows: 1) the inhibited organogenesis of seminal tubules (sometimes at the level of sex cords) causes 2) the delay in differentiation and development of germ and somatic Sertoli/granulosa cells; 3) disturbed somatic cells contribute to the death of most germ cells, but germ cells which retain fetal antigens have the possibility to survive and proliferate; 4) if the survived germ cells are in the undifferentiated tissue of fetal gonad (retardation of testicular development at the level of gonadal anlage) their clonal expansion leads to the creation of neoplastic lesion termed gonadoblastoma. With time gonadoblastoma undergoes atrophy and/or calcification or may transform into GCT; 5) if the survived germ cells are placed inside the seminal tubules, they have better conditions to persist in the unchanged, but developmentally delayed, form for many years as preinvasive carcinoma in situ (CIS). CIS cells can disappear but with the advancing puberty they may give rise to GCT under the influence of gonadotropins and androgens.

Presence of TSPY transcript and absence of transcripts of other Y chromosomal genes in a case of microscopic gonadoblastoma.

BACKGROUND: Gonadoblastoma is found almost exclusively in people with gonadal dysgenesis and Y chromosomal DNA fragment. Accordingly, GBY (gonadoblastoma locus on the Y chromosome) has been mapped to the Y chromosome. Testis-specific protein Y-encoded (TSPY) gene may participate in the oncogenesis of gonadoblastoma expression of TSPY in the tumor tissue. This might suggest TSPY as a candidate gene for gonadoblastoma. CASE REPORT: A 14-year-old phenotypic girl with typical features of gonadal dysgenesis and a normal male karyotype. She underwent prophylactic bilateral gonadectomy to prevent future malignant changes of streak gonads. Histopathologic examination revealed microscopic foci of gonadoblastoma on the left side of ovary. We tested transcripts of 14 Y chromosomal genes by RT-PCR (TSPY, DAZ, BPY1 and BPY2, PRY, XKRY, CDY1 and CDY2, TTY1 and TTY2, PRKY, RBMY1, DBY and USP9Y), and only transcript of TSPY was detectable in the tumor tissue. CONCLUSION: Expression of TSPY in microscopic gonadoblastoma might suggest important roles of TSPY, but not other Y chromosomal genes, in the very early stage of oncogenesis.

Insulin-like factor 3 gene mutations in testicular dysgenesis syndrome: clinical and functional characterization.

Insulin-like factor 3 (INSL3) plays a crucial role in testicular descent. Genetic ablation of Insl3 or its G protein-coupled receptor, leucine-rich repeat-containing G-protein-coupled receptor (Lgr8), causes cryptorchidism in mice. Mutation analyses of INSL3 in humans showed an association with cryptorchidism but led to non-conclusive data about a causative role. In this study, we explored the hypothesis that mutations in INSL3 may be associated with the signs of testicular dysgenesis syndrome (TDS). We screened for mutations in INSL3 gene in 967 subjects with a history of maldescended testes and/or infertility and/or testicular cancer and in 450 controls. Furthermore, we carried out in vitro functional analysis of three novel mutations by analysis of INSL3-dependent cAMP increase in cells expressing LGR8. We found six INSL3 mutations in 18 of 967 patients (1.9%) and no mutations in controls. Prevalence of mutations was similar in the different groups of patients (cryptorchidism and/or infertility and/testicular cancer). Three mutations were novel findings (R4H, W69R, and R72K); however, their analysis showed normal cAMP increase after the activation of LGR8 receptor. In conclusion, we found a significant association of INSL3 gene mutations in men presenting one or more signs of TDS syndrome. However, a causative role for some of these mutations is not clearly supported by functional analyses. Although a role for mutations of INSL3 and LGR8 genes in cryptorchidism is reasonable, additional studies are needed to establish an association between the disruption of INSL3 pathway and higher risk of infertility or testicular cancer.

Gonadoblastoma arising in undifferentiated gonadal tissue within dysgenetic gonads.

PURPOSE: The purpose of the study was to define the histological origin of gonadoblastomas, allowing the identification of high-risk patients. EXPERIMENTAL DESIGN: Sixty paraffin-embedded gonadectomy or gonadal biopsy samples of 43 patients with gonadal dysgenesis were selected from our archives. We studied the morphology and immunohistochemical properties of the germ cells in 40 samples without neoplastic transformation and compared these findings with the morphological and immunohistochemical characteristics of 20 samples containing gonadoblastoma/dysgerminoma. RESULTS: The overall incidence of germ cell tumors in our patient series was 35%. In dysgenetic gonads without germ cell neoplasia, besides the presence of areas with testicular and/or ovarian differentiation, areas of undifferentiated gonadal tissue were identified in 13 of 40 samples (32.5%). A subpopulation of germ cells within these undifferentiated areas stained positive for octamer binding transcription factor (OCT)3/4, the stem cell factor receptor, placental-like alkaline phosphatase, and testis-specific protein-Y encoded. Gonadoblastoma germ cells display identical staining results. Moreover, in gonads containing gonadoblastoma, adjacent to this lesion, areas of undifferentiated gonadal tissue with identical immunohistochemical characteristics were identified in 10 of 20 samples (50%). No adjacent tissue was available in five cases, whereas in the five remaining cases, it consisted of streak tissue. In three cases, an accumulation of OCT3/4-positive germ cells in the proximity of the malignant lesions was found, suggesting clonal expansion and final organization into gonadoblastoma nests. CONCLUSIONS: Based on these observations, we hypothesize that gonadoblastomas originate from surviving OCT3/4-positive germ cells in areas of undifferentiated gonadal tissue within the dysgenetic gonad. Supportive evidence was obtained that carcinoma in situ arises in regions with testicular differentiation.

Gonadoblastoma: evidence for a stepwise progression to dysgerminoma in a dysgenetic ovary.

Gonadoblastomas are neoplasms of dysgenetic gonads which may undergo regression or become overgrown by malignant germ cell tumors (mGCTs). Since little is known about their relationship to normal gonadal development and mGCTs, we studied the phenotype and antigenic profile of gonadoblastomas in comparison with adjacent dysgerminomas and fetal gonads. Three cases of gonadoblastomas and fetal gonads of both sexes were analyzed using oncofetal markers to M2A-antigen (M2A), germ cell alkaline phosphatase (PLAP/GCAP), receptor tyrosine kinase c-kit (c-kit), and somatic angiotensin converting enzyme (sACE) as well as the proliferation marker MIB-1. Morphologically, microfollicular pattern of gonadoblastomas showed a fetal germ cell organization reminiscent of oocytic clusters of fetal ovaries. They contained both cell types, similar to oocytes (M2A-, GCAP-, c-kit+/-, sACE-) and oogonia (M2A+, GCAP+, c-kit+, sACE+). The percentage of germ cells immunoreactive for oncofetal markers and the proliferation index increased from microfollicular over coronary patterns to adjacent dysgerminomas. Supportive cells of gonadoblastomas showed a uniform phenotype (CK18+, vimentin+, sACE+, alpha-inhibin+, M2A-) but in contrast to fetal germ cells lacked a clear equivalence to fetal tissues. Our results show that gonadoblastomas mimic female fetal ovary and exhibit a stepwise progression from follicular pattern to coronary pattern and finally to dysgerminomas.

Urogenital and caudal dysgenesis in adrenocortical dysplasia (acd) mice is caused by a splicing mutation in a novel telomeric regulator.

Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutant with developmental defects in organs derived from the urogenital ridge. In surviving adult mutants, adrenocortical dysplasia and hypofunction are predominant features. Adults are infertile due to lack of mature germ cells, and 50% develop hydronephrosis due to ureteral hyperplasia. We report the identification of a splice donor mutation in a novel gene, which is the mouse ortholog of a newly discovered telomeric regulator. This gene (Acd) has recently been characterized as a novel component of the TRF1 protein complex that controls telomere elongation by telomerase. Characterization of Acd transcripts in mutant animals reveals two abnormal transcripts, consistent with a splicing defect. Expression of a wild-type Acd transgene in acd mutants rescues the observed phenotype. Most mutants die within 1-2 days of life on the original genetic background. Analysis of these mutant embryos reveals variable, yet striking defects in caudal specification, limb patterning and axial skeleton formation. In the tail bud, reduced expression of Wnt3a and Dll1 correlates with phenotypic severity of caudal regression. In the limbs, expression of Fgf8 is expanded in the dorsal-ventral axis of the apical ectodermal ridge and shortened in the anterior-posterior axis, consistent with the observed loss of anterior digits in older embryos. The axial skeleton of mutant embryos shows abnormal vertebral fusions in cervical, lumbar and caudal regions. This is the first report to show that a telomeric regulator is required for proper urogenital ridge differentiation, axial skeleton specification and limb patterning in mice.