foxl2l is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Association of FOXL2 and ERCC6 variants with premature ovarian insufficiency and their potential use in clinical IVF guidance.

Premature ovarian insufficiency (POI) is the main cause of infertility in women. Some cases of POI are thought to be caused by genetic defects and the clinical outcomes of these patients are unknown. Here, we performed whole-exome sequencing of the peripheral blood of a cohort of 55 subjects with POI and identified one heterozygous missense variant in FOXL2 (c.1045G>C; p.Arg349Gly) and two heterozygous missense variants in ERCC6 (c.379G>A; p.Val127Ile and c.4223A>C; p.Glu1408 Ala) in four POI patients. All of these heterozygous mutations were predicted to be deleterious and were parentally inherited from their heterozygous fathers. The mRNA and protein expression of FOXL2 and ERCC6 were absent or decreased in the patients. The patients carrying the variants of FOXL2 (c.1045G>C; p.Arg349Gly) and ERCC6 (c.379G>A; p.Val127Ile) failed to conceive in two and four assisted reproductive cycles, respectively. Another patient and her sister carrying the ERCC6 (c.4223A>C; p.Glu1408 Ala) variant achieved good clinical outcomes after assisted reproductive therapy. Our findings support the possible roles of FOXL2 and ERCC6 in POI and might contribute to the genetic counseling of POI patients.

Zebrafish Foxl2l functions in proliferating germ cells for female meiotic entry.

Zebrafish sex differentiation is a complicated process and the detailed mechanism has not been fully understood. Here we characterized a transcription factor, Foxl2l, which participates in female oogenesis. We show that it is expressed specifically in proliferating germ cells in juvenile gonads and mature ovaries. We have used CRISPR-Cas9 to generate zebrafish deficient in foxl2l expression. Zebrafish with foxl2l-/- are all males, and this female-to-male sex reversal cannot be reversed by tp53 mutation, indicating this sex reversal is unrelated to cell death. We have generated transgenic fish expressing GFP under the control of foxl2l promoter to track the development of foxl2l + -germ cells; these cells failed to enter meiosis and accumulated as cystic cells in the foxl2l-/- mutant. Our RNA-seq analysis also showed the reduced expression of genes in meiosis and oogenesis among other affected pathways. All together, we show that zebrafish Foxl2l is a nuclear factor controlling the expression of meiotic and oogenic genes, and its deficiency leads to defective meiotic entry and the accumulation of premeiotic germ cells.

pSTAT3 activation of Foxl2 initiates the female pathway underlying temperature-dependent sex determination.

Ovarian development was traditionally recognized as a "default" sexual outcome and therefore received much less scientific attention than testis development. In turtles with temperature-dependent sex determination (TSD), how the female pathway is initiated to induce ovary development remains unknown. In this study, we have found that phosphorylation of the signal transducer and activator of transcription 3 (pSTAT3) and Foxl2 exhibit temperature-dependent sexually dimorphic patterns and tempo-spatial coexpression in early embryos of the red-eared slider turtle (Trachemys scripta elegans). Inhibition of pSTAT3 at a female-producing temperature of 31 °C induces 64.7% female-to-male sex reversal, whereas activation of pSTAT3 at a male-producing temperature of 26 °C triggers 75.6% male-to-female sex reversal. In addition, pSTAT3 directly binds to the locus of the female sex-determining gene Foxl2 and promotes Foxl2 transcription. Overexpression or knockdown of Foxl2 can rescue the sex reversal induced by inhibition or activation of pSTAT3. This study has established a direct genetic link between warm temperature-induced STAT3 phosphorylation and female pathway initiation in a TSD system, highlighting the critical role of pSTAT3 in the cross talk between female and male pathways.

Insights into the role of FoxL2 in tebuconazole-induced male- biased sex differentiation of zebrafish.

Tebuconazole (TEB) is a commonly used fungicide that inhibits the aromatase Cyp19A and downregulates the transcription factor forkhead box L2 (FoxL2), leading to male-biased sex differentiation in zebrafish larvae. However, the specific mechanism by which FoxL2 functions following TEB exposure remains unclear. In this study, the phosphorylation sites and kinase-specific residues in zebrafish FoxL2 protein (zFoxL2) were predicted. Subsequently, recombinant zFoxL2 was prepared via prokaryotic expression, and a polyclonal rabbit-anti-zFoxL2 antibody was generated. Zebrafish fibroblast (ZF4) cells were exposed to 100-µM TEB alone for 8 h, after which changes in the expression of genes involved in the foxl2 regulatory pathway (akt1, pi3k, cyp19a1b, c/ebpb and sox9a) were detected. When co-exposed to 1-µM estradiol and 100-µM TEB, the expression of these key genes tended to be restored. Interestingly, TEB did not affect the expression of the foxl2 gene or protein but it significantly suppressed the phosphorylation of FoxL2 (pFoxL2) at serine 238 (decreased by 43.64 %, p = 0.009). Co-immunoprecipitation assays showed that, following exposure to 100-µM TEB, the total precipitated proteins in ZF4 cells decreased by 17.02 % (p = 0.029) and 31.39 % (p = 0.027) in the anti-zFoxL2 antibody group and anti-pFoxL2 (ser238) antibody group, respectively, indicating that TEB suppressed the capacity of the FoxL2 protein to bind to other proteins via repression of its own phosphorylation. The pull-down assay confirmed this conclusion. This study preliminarily elucidated that the foxl2 gene functions via post-translational regulation through hypophosphorylation of its encoded protein during TEB-induced male-biased sex differentiation.

FOXL2 regulates RhoA expression to change actin cytoskeleton rearrangement in granulosa cells of chicken pre-ovulatory follicles†.

Forkhead box L2 (FOXL2) is an indispensable key regulator of female follicular development, and it plays important roles in the morphogenesis, proliferation, and differentiation of follicle granulosa cells, such as establishing normal estradiol signaling and regulating steroid hormone synthesis. Nevertheless, the effects of FOXL2 on granulosa cell morphology and the underlying mechanism remain unknown. Using FOXL2 ChIP-seq analysis, we found that FOXL2 target genes were significantly enriched in the actin cytoskeleton-related pathways. We confirmed that FOXL2 inhibited the expression of RhoA, a key gene for actin cytoskeleton rearrangement, by binding to TCATCCATCTCT in RhoA promoter region. In addition, FOXL2 overexpression in granulosa cells induced the depolymerization of F-actin and disordered the actin filaments, resulting in a slowdown in the expansion of granulosa cells, while FOXL2 silencing inhibited F-actin depolymerization and stabilized the actin filaments, thereby accelerating granulosa cell expansion. RhoA/ROCK pathway inhibitor Y-27632 exhibited similar effects to FOXL2 overexpression, even reversed the actin polymerization in FOXL2 silencing granulosa cells. This study revealed for the first time that FOXL2 regulated granulosa cell actin cytoskeleton by RhoA/ROCK pathway, thus affecting granulosa cell expansion. Our findings provide new insights for constructing the regulatory network of FOXL2 and propose a potential mechanism for facilitating rapid follicle expansion, thereby laying a foundation for further understanding follicular development.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Corrigendum: Novel FOXL2 variants in two Chinese families with blepharophimosis, ptosis, and epicanthus inversus syndrome.

[This corrects the article DOI: 10.3389/fgene.2024.1343411.].

Shifting the landscape: Dominant C-terminal rare missense FOXL2 variants in non-syndromic primary ovarian failure etiology.

Pathogenic germline variants in the FOXL2 gene are associated with Blepharophimosis, Ptosis, and Epicanthus Inversus syndrome (BPES) in humans, an autosomal dominant condition. Two forms of BPES have emerged: (i) type I (BPES-I), characterized by ocular signs and primary ovarian failure (POI), and (ii) type II (BPES-II) with no systemic associations. This study aimed to compare the distribution of FOXL2 variants in idiopathic POI/DOR (diminished ovarian reserve) and both types of BPES, and to determine the involvement of FOXL2 in non-syndromic forms of POI/DOR. We studied the whole coding region of the FOXL2 gene using next-generation sequencing in 1282 patients with non-syndromic POI/DOR. Each identified FOXL2 variant was compared to its frequency in the general population, considering ethnicity. Screening of the entire coding region of the FOXL2 gene allowed us to identify 10 different variants, including nine missense variants. Of the patients with POI/DOR, 14 (1%) carried a FOXL2 variant. Significantly, six out of nine missense variants (67%) were overrepresented in our POI/DOR cohort compared to the general or specific ethnic subgroups. Our findings strongly suggest that five rare missense variants, mainly located in the C-terminal region of FOXL2 are high-risk factors for non-syndromic POI/DOR, though FOXL2 gene implication accounts for approximately 0.54% of non-syndromic POI/DOR cases. These results support the implementation of routine genetic screening for patients with POI/DOR in clinical settings.

Gonadal transcriptome sequencing reveals sexual dimorphism in expression profiling of sex-related genes in Asian arowana (Scleropages formosus).

Asia arowana (Scleropages formosus) is an ornamental fish with high economic value, while its sex determination mechanism is still poorly understood. By far, no morphological evidence or molecular marker has been developed for effective distinguishment of genders, which poses a critical challenge to our captive breeding efforts. In this study, we sequenced gonadal transcriptomes of adult Asian arowanas and revealed differential expression profiling of sex-related genes. Based on the comparative transcriptomics analysis of testes (n = 3) and ovaries (n = 3), we identified a total of 8,872 differentially expressed genes (DEGs) and 18,490 differentially expressed transposable elements (TEs) between male and female individuals. Interestingly, the expression of TEs usually has been more significantly testis-biased than related coding genes. As expected, several genes related to females (such as foxl2 and cyp19a1a) are significantly transcribed in the ovary, and some genes related to male gonad development (such as dmrt1, gsdf and amh) are highly expressed in the testis. This sexual dimorphism is valuable for ascertaining the differential expression patterns of sex-related genes and enriching the genetic resources of this economically important species. These valuable genetic materials thereby provide instructive references for gender identification and one-to-one breeding practices so as to expand fish numbers for a rapid elevation of economic value.

Novel FOXL2 variants in two Chinese families with blepharophimosis, ptosis, and epicanthus inversus syndrome.

Introduction: Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is a rare inherited disorder. This study was aimed to identify and functionally validate FOXL2 variants in two Chinese families with BPES. Methods: The proband and his family members were subjected to whole-exome sequencing to identify disease-associated variants. Several bioinformatic tools were used to computationally predict altered proteins. In vitro functional assays were conducted by transfecting wild-type and mutant FOXL2 cDNAs into HEK-293 cells, followed by subcellular localization assays, luciferase reporter gene assays, and quantitative real-time polymerase chain reaction. Results: The clinical features of BPES, including small palpebral fissures, ptosis, telecanthus, and epicanthus inversus, were present in all affected patients. Two novel mutations were detected, c.292T>A and c.383G>T. Whole-exome sequencing analysis and prediction software suggested that these mutations were pathogenic. Functional studies showed that these two point mutations decreased FOXL2 protein expression, resulting in subcellular mislocalization and aberrant transcriptional activity of the steroidogenic acute regulatory protein gene promoter. Conclusion: Our results add to the current understanding of known FOXL2 variants in, and our in vitro experiments provide reference data and insights into the etiology of BPES. Further studies are needed to identify the possible mechanisms underlying the action of this mutation on the development of BPES.

Transcriptional regulation of IAG by dsx and foxl-2 in mud crab (Scylla paramamosain).

Scylla paramamosain is an important cultured crab species on the southeast coast of China. However, the molecular regulation mechanism of its gonadal development still has not been thoroughly studied. Dsx (doublesex) and foxl-2 (forkhead transcription factor gene 2) are important transcription factors involved in gonadal development. So far, studies on the functions of dsx and foxl-2 in crustaceans are very limited. Insulin-like androgenic gland hormone (IAG) is an effector molecule that regulates the differentiation, development and sex maintenance of testes in crustaceans. In this study, the promoter region of Sp-IAG was predicted, and several potential binding sites of dsx and foxl-2 were found. Site-directed mutagenesis was performed on the predicted potential binding sites, and their promoter activity was analyzed. The results showed that there was a dsx and a foxl-2 binding site, respectively, that could regulate the expression of Sp-IAG. In order to verify the regulatory effect of these two transcription factors on Sp-IAG, we constructed the expression plasmids of dsx and foxl-2 and co-transfected them into HEK293T cell lines with the promoter of Sp-IAG, respectively. The results showed that dsx could significantly promote the expression of Sp-IAG, while foxl-2 could inhibit its expression substantially. Then we carried out in vivo RNA interference experiment on mud crabs. The expression of dsx and foxl-2 in crabs was interfered respectively. The results of qRT-PCR showed that the expression of Sp-IAG was significantly inhibited after interfering with dsx, while significantly increased after interfering with foxl-2, which was consistent with the cell experiment. In conclusion, dsx and foxl-2 transcription factors play opposite roles in regulating the expression of Sp-IAG.

Conservation and contrast in cell states of echinoderm ovaries.

Echinoderms produce functional gametes throughout their lifespan, in some cases exceeding 200 years. The histology and ultrastructure of echinoderm ovaries has been described but how these ovaries function and maintain the production of high-quality gametes remains a mystery. Here, we present the first single cell RNA sequencing data sets of mature ovaries from two sea urchin species (Strongylocentrotus purpuratus [Sp] and Lytechinus variegatus [Lv]), and one sea star species (Patiria miniata [Pm]). We find 14 cell states in the Sp ovary, 16 cell states in the Lv ovary and 13 cell states in the ovary of the sea star. This resource is essential to understand the structure and functional biology of the ovary in echinoderms, and better informs decisions in the utilization of in situ RNA hybridization probes selective for various cell types. We link key genes with cell clusters in validation of this approach. This resource also aids in the identification of the stem cells for prolonged and continuous gamete production, is a foundation for testing changes in the annual reproductive cycle, and is essential for understanding the evolution of reproduction of this important phylum.

Relevance of Molecular Pathology for the Diagnosis of Sex Cord-Stromal Tumors of the Ovary: A Narrative Review.

Ovarian sex cord-stromal tumors (SCSTs) account for 8% of all primary ovarian neo-plasms. Accurate diagnosis is crucial since each subtype has a specific prognostic and treatment. Apart from fibrosarcomas, stromal tumors are benign while sex cord tumors may recur, sometimes with a significant time to relapse. Although the diagnosis based on morphology is straightforward, in some cases the distinction between stromal tumors and sex cord tumors may be tricky. Indeed, the immunophenotype is usually nonspecific between stromal tumors and sex cord tumors. Therefore, molecular pathology plays an important role in the diagnosis of such entities, with pathognomonic or recurrent alterations, such as FOXL2 variants in adult granulosa cell tumors. In addition, these neoplasms may be associated with genetic syndromes, such as Peutz-Jeghers syndrome for sex cord tumors with annular tubules, and DICER1 syndrome for Sertoli-Leydig cell tumors (SLCTs), for which the pathologist may be in the front line of syndromic suspicion. Molecular pathology of SCST is also relevant for patient prognosis and management. For instance, the DICER1 variant is associated with moderately to poorly differentiated SLCTS and a poorer prognosis. The present review summarizes the histomolecular criteria useful for the diagnosis of SCST, using recent molecular data from the literature.

Cloning, Identification, and Functional Analysis of the Foxl2 Gene in Procambarus clarkii.

Procambarus clarkii is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 (Foxl2) gene has been found to be involved in the reproductive development of many crustaceans. To understand the role of the Foxl2 gene in the gonad development of P. clarkii, we designed CDS-specific primers for the P. clarkii Foxl2 (PcFoxl2) gene and cloned its CDS sequence using RT-PCR. The nucleotide and protein sequence information was then analyzed through bioinformatics analysis. The expression and subcellular localization of PcFoxl2 in various tissues were detected using qRT-PCR and in situ hybridization. The effects of PcFoxl2 knockdown on gonad development were investigated using RNA interference. The results showed that the CDS length of the PcFoxl2 gene was 1614 bp and encoded 537 amino acids. Protein sequence comparison and phylogenetic analysis showed that PcFoxl2 was the closest relative to Crayfish. qRT-PCR analysis indicated that the expression level of PcFoxl2 in the testis was significantly higher (>40 fold) than that in the ovary (p < 0.01). The in situ hybridization results showed that PcFoxl2 was expressed in both the cytoplasm and the nucleus of egg cells, and that the expression was strongest in egg cells at the early stage of yolk synthesis, while weak in the secondary oocytes. The positive signal was strongest in the spermatocyte nucleolus, while only a trace signal was observed in the cytoplasm. After interfering with the PcFoxl2 gene using dsRNA, the expression of PcFoxl2 in the RNA interference group was significantly lower than that in the control group, and this interference effect lasted for one week. Moreover, the gonad index of the experimental group was significantly lower than that of the control group (p < 0.05) after 10 days of P. clarkii cultivation following PcFoxl2 knockdown. The expression levels of the nanos and S3a genes, which are related to gonad development, decreased significantly after PcFoxl2 gene interference. The results suggest that the Foxl2 gene is involved in the growth and development of gonads, particularly in the development of testis, and is related to the early development of oocytes. This study provides a theoretical basis for the artificial breeding of P. clarkii.

Expanded phenotypic spectrum of FOXL2 Variant c.672_701dup revealed by whole-exome sequencing in a rare blepharophimosis, ptosis, and epicanthus inversus syndrome family.

INTRODUCTION: Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is a rare genetic disease with diverse ocular malformations. This study aimed to investigate the disease-causing gene in members of a BPES pedigree presenting with the rare features of anisometropia, unilateral pathologic myopia (PM), and congenital cataracts. METHODS: The related BPES patients underwent a comprehensive ocular examination. Next, whole-exome sequencing (WES) was performed to screen for the disease-causing genetic variants. A step-wise variant filtering was performed to select candidate variants combined with the annotation of the variant's pathogenicity, which was assessed using several bioinformatic approaches. Co-segregation analysis and Sanger sequencing were then conducted to validate the candidate variant. RESULTS: The variant c.672_701dup in FOXL2 was identified to be the disease-causing variant in this rare BPES family. Combined with clinical manifestations, the two affected individuals were diagnosed with type II BPES. CONCLUSION: This study uncovered the variant c.672_701dup in FOXL2 as a disease causal variant in a rare-presenting BPES family with anisometropia, unilateral pathogenic myopia, and/or congenital cataracts, thus expanding the phenotypic spectrum of FOXL2.

Transcriptome Analyses Reveal Expression Profiles of Morphologically Undifferentiated and Differentiated Gonads of Yangtze Sturgeon Acipenser dabryanus.

Sturgeon is known as a primitive fish with the ZZ/ZW sex determination system and is highly prized for its valuable caviar. Exploring the molecular mechanisms underlying gonadal differentiation would contribute to broadening our knowledge on the genetic regulation of sex differentiation of fish, enabling improved artificial breeding and management of sturgeons. However, the mechanisms are still poorly understood in sturgeons. This study aimed to profile expression patterns between female and male gonads at morphologically undifferentiated and early differentiated stages and identify vital genes involved in gonadal sex differentiation of sturgeons. The sexes of Yangtze sturgeon (Acipenser dabryanus) juveniles were identified via the sex-specific DNA marker and histological observation. Transcriptome analyses were carried out on female and male gonads at 30, 80 and 180 days post-hatching. The results showed that there was a total of 17 overlapped DEGs in the comparison groups of between female and male gonads at the three developmental stages, in which there were three DEGs related to ovarian steroidogenesis, including hsd17b1, foxl2 and cyp19a1. The three DEGs were highly expressed in the female gonads, of which the expression levels were gradually increased with the number of days after hatching. No well-known testis-related genes were found in the overlapped DEGs. Additionally, the expression levels of hsd17b1 and cyp19a1 mRNA were decreased with the knockdown of foxl2 mRNA via siRNA. The results further suggested that foxl2 should play a crucial role in the ovarian differentiation of sturgeons. In conclusion, this study showed that more genes involved in ovarian development than testis development emerged with sexually dimorphic expression during early gonadal sex differentiation, and it provided a preliminary understanding of the molecular regulation on gonadal differentiation of sturgeons.

Comparative Studies on Duplicated foxl2 Paralogs in Spotted Knifejaw Oplegnathus punctatus Show Functional Diversification.

As a member of the forkhead box L gene family, foxl2 plays a significant role in gonadal development and the regulation of reproduction. During the evolution of deuterostome, whole genome duplication (WGD)-enriched lineage diversifications and regulation mechanisms occurs. However, only limited research exists on foxl2 duplication in teleost or other vertebrate species. In this study, two foxl2 paralogs, foxl2 and foxl2l, were identified in the transcriptome of spotted knifejaw (Oplegnathus punctatus), which had varying expressions in the gonads. The foxl2 was expressed higher in the ovary, while foxl2l was expressed higher in the testis. Phylogenetic reconstruction, synteny analysis, and the molecular evolution test confirmed that foxl2 and foxl2l likely originated from the first two WGD. The expression patterns test using qRT-PCR and ISH as well as motif scan analysis revealed evidence of potentially functional divergence between the foxl2 and foxl2l paralogs in spotted knifejaw. Our results indicate that foxl2 and foxl2l may originate from the first two WGD, be active in transcription, and have undergone functional divergence. These results shed new light on the evolutionary trajectories of foxl2 and foxl2l and highlights the need for further detailed functional analysis of these two duplicated paralogs.

Detection of FOXL2 C134W Mutation Status by a Novel BaseScope In Situ Hybridization Assay is Highly Sensitive and Specific for Adult Granulosa Cell Tumors.

Adult granulosa cell tumors (AGCTs) are a molecularly distinct group of malignant ovarian sex cord-stromal tumors (SCSTs) characterized by a nearly ubiquitous c.402C>G/p.C134W mutation in FOXL2 (hereafter referred to as "C134W"). In some cases, AGCT exhibits marked morphologic overlap with other SCSTs and has an identical immunophenotype, and molecular testing may be necessary to help confirm the diagnosis. However, molecular testing is time consuming, relatively expensive, and unavailable in many pathology laboratories. We describe the development and validation of an in situ hybridization (ISH) custom BaseScope assay for the detection of the FOXL2 C134W mutation. We evaluated 106 ovarian SCSTs, including 78 AGCTs, 9 juvenile granulosa cell tumors, 18 fibromas (cellular and conventional), and 1 SCST, not otherwise specified, as well as 53 epithelial ovarian tumors (42 endometrioid carcinomas and 11 carcinosarcomas) and 1 STK11 adnexal tumor for the presence or absence of FOXL2 wild-type and FOXL2 C134W RNA expression via BaseScope-ISH. Fifty-one tumors had previously undergone DNA sequencing of the FOXL2 gene. Across the entire cohort, the FOXL2 C134W probe staining was positive in 77 of 78 (98.7%) AGCTs. Two of 81 (2.5%) non-AGCTs also showed positive staining, both of which were epithelial ovarian tumors. The assay worked in tissue from blocks >20 years old. There was 100% concordance between the FOXL2 sequencing and BaseScope-ISH results. Overall, assessment of FOXL2 mutation status by custom BaseScope-ISH demonstrated 98.7% sensitivity and 97.5% specificity for the diagnosis of AGCT. BaseScope-ISH for FOXL2 C134W represents a reasonable alternative to sequencing, is quicker and less expensive, and is more easily incorporated than molecular testing into many pathology laboratories. It also has the advantage of requiring less tissue, and the neoplastic cells can be directly visualized on stained sections.

Partial male-to-female reprogramming of mouse fetal testis by Sertoli cell ablation.

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions: near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3ßHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.