FOXL2 Research Articles

Graphene oxide enhances the specificity of the polymerase chain reaction by modifying primer-template matching

Aiming at improved specificity, nanoparticle assisted polymerase chain reaction (PCR) has been widely studied and shown to improve PCR. However, the reliability and mechanism of this method are still controversial. Here, we demonstrated that 1 μg/mL of graphene oxide (GO) effectively enhances the specificity of the error-prone multi-round PCR. Mismatched primers were designed as interference to produce nonspecific products when the same amounts of matched and mismatched primers were added into semi-multiplex PCR. It was found that GO can enhance specificity by suppressing the amplification of mismatched primers. We monitored the primer-template-polymerase-GO interactions involved in the PCR using a capillary electrophoresis/laser-induced fluorescence polarization (CE-LIFP) assay. The results showed that the addition of GO promoted the formation of a matched primer-template complex, but suppressed the formation of a mismatched primer-template complex during PCR, suggesting that interactions between the primers and GO play an essential role. Furthermore, we successfully amplified the FOXL2 gene from PEGFP-N1 vectors using GO to eliminate the nonspecific products in PCR. Taken together, these results suggest that the GO can be used as an efficient additive for improving the conventional PCR system.

'Microcystic pattern' should be recognised as part of the morphological spectrum of solid-pseudopapillary neoplasm of the pancreas.

Solid pseudopapillary neoplasm (SPN) is an uncommon pancreatic tumour characterised by solid and pseudopapillary growth patterns. We have observed SPNs can show a microcystic pattern (microcystic SPN), which has been poorly described and may be confused with microcystic neoplasms. We conducted the present study to clarify the clinicopathological and immunohistochemical features of microcystic SPNs. We examined a consecutive series of 44 SPNs and 10 serous cystadenomas (SCAs), and classified them into 13 microcystic SPNs (29.5%) and 31 conventional SPNs (70.5%). Clinicopathological analysis, immunohistochemical staining and mucin histochemistry were performed. Clear cell change, hyalinised stroma and haemorrhage were observed significantly more frequently in the microcystic SPNs compared to the conventional SPNs. Immunohistochemically, the microcystic SPNs showed significantly lower frequencies of CD10 (0%) and CD56 expression (62%) compared to the conventional SPNs (87%; P<0.001, 90%; P<0.0085, respectively). There were no significant differences in other clinicopathological and immunohistochemical features between the two groups (i.e. the nuclear expression of β-catenin, E-cadherin, progesterone receptor (PgR), lack of forkhead box (Fox)L2 and occasional oestrogen receptor (ER), AE1/AE3 expression). Microcystic SCAs lack such a characteristic immunophenotype. The myxoid stroma of microcystic SPNs contained hyaluronan revealed by Alcian blue stain with hyaluronidase digestion. We thus conclude that the microcystic pattern should be recognised as a part of the morphological spectrum of SPNs. Our findings may contribute to the correct diagnosis of the pancreatic neoplasms with the microcystic pattern. In addition, we speculate that stromal change caused by an accumulation of hyaluronan may contribute to the microcystic pattern of SPN.

FOXL2 Mutation Status in Granulosa Theca Cell Tumors of the Ovary.

Ovarian sex-cord stromal tumors that have between 10% and 50% granulosa cells in a prominent fibrothecomatous background have been referred to as granulosa theca cell tumors or mixed granulosa theca cell tumors. The classification and prognosis of these tumors is not clear. Most adult granulosa cell tumors of the ovary harbor a mutation in the FOXL2 gene, whereas fibromas and thecomas lack this mutation. The aim of our study was to assess the FOXL2 mutation status of ovarian granulosa theca cell tumors and to correlate the mutation status with morphologic and clinical characteristics. A FOXL2 mutation was detected in 6 of 12 (50%) granulosa theca cell tumors. Tumors with higher cellularity of granulosa cells were more likely to harbor a FOXL2 mutation as were tumors in which the granulosa cells formed large lobules. No conclusions could be drawn regarding the clinical and prognostic significance of the presence of a mutation given the small number of cases and limited clinical follow-up. Our study shows that half of granulosa theca cell tumors harbor the same FOXL2 mutation that characterizes adult granulosa cell tumors but there is no outcome evidence to guide whether mutation status should alter the classification of the tumor or the management of the patient.

Ovarian Granulosa Cell Tumor: A Review of Clinical Features and Histo-Pathological Findings

Granulosa cell tumor(s) (GCT[s]) of the ovary, representing 5 to 10% of all ovarian malignancies, account for 70% of the ovarian sex-cord stromal tumors. GCTs are classified into two distinct subtypes, including the adult-type GCT and the juvenile-type GCT, and the former occurs more frequently (95%). Currently, evidence from tumor biology found that Forkhead box L2 (FOXL2), a transcription factor gene involved in ovarian development and function, whose expression is reduced and mutated in the majority of adult-type GCTs. However, FOXL2 mutant seems to be rare in juvenile-type GCTs. In addition, researchers have found the stimulatory alpha subunit of a trimeric G protein might be correlated with the prognosis of adult-type GCTs. Similar to the FOXL2 mutant, it is also unclear the precise molecular basis for juvenile-type GCTs. The recent molecular analyses of juvenile-type GCTs identified AKT1 oncogene to play an important role for the development of juvenile-type GCTs, since over 60% of the juvenile-type GCTs show overexpression of AKT1 oncogene. This provides insights into tumor biology and therapeutic leads for juvenile-type GCTs. The aim of this article is to discuss the clinico-pathology and the molecular biology of GCTs.

Testis Determination Requires a Specific FGFR2 Isoform to Repress FOXL2.

Male sex determination in mammals relies on sex determining region Y-mediated upregulation of sex determining region-box 9 (SOX9) expression in XY gonads, whereas Wnt family member (WNT)/R-spondin 1 signaling and forkhead box L2 (FOXL2) drive female sex determination in XX gonads. Fibroblast growth factor (FGF) 9 signaling ensures sustained SOX9 expression through repression of one of the ovarian pathways (WNT signaling), whereas the significance of FGF-mediated repression of the FOXL2 pathway has not been studied. Previously, we demonstrated that FGFR2 is the receptor for FGF9 in the XY gonad. Whether a specific isoform (FGFR2b or FGFR2c) is required was puzzling. Here, we show that FGFR2c is required for male sex determination. Initially, in developing mouse embryos at 12.5 to 13.5 days postcoitum (dpc), XY Fgfr2c-/- gonads appear as ovotestes, with SOX9 and FOXL2 expression predominantly localized to the posterior and anterior gonadal poles, respectively. However, by 15.5 dpc, XY Fgfr2c-/- gonads show complete male-to-female sex reversal, evident by the lack of SOX9 and ectopic expression of FOXL2 throughout the gonads. Furthermore, ablation of the Foxl2 gene leads to partial or complete rescue of gonadal sex reversal in XY Fgfr2c-/- mice. Together with previous findings, our data suggest that testis determination involves FGFR2c-mediated repression of both the WNT4- and FOXL2-driven ovarian-determining pathways.

Estrogen alters gonadal soma-derived factor (Gsdf)/Foxl2 expression levels in the testes associated with testis-ova differentiation in adult medaka, Oryzias latipes

Testis-ova differentiation in sexually mature male medaka (Oryzias latipes) is easily induced by estrogenic chemicals, indicating that spermatogonia persist in sexual bipotentiality, even in mature testes in medaka. By contrast, the effects of estrogen on testicular somatic cells associated with testis-ova differentiation in medaka remain unclear. In this study, we focused on the dynamics of sex-related genes (Gsdf, Dmrt1, and Foxl2) expressed in Sertoli cells in the mature testes of adult medaka during estrogen-induced testis-ova differentiation. When mature male medaka were exposed to estradiol benzoate (EB; 800ng/L), testis-ova first appeared after EB treatment for 14days (observed as the first oocytes of the leptotene-zygotene stage). However, the testis remained structurally unchanged, even after EB treatment for 28days. Although Foxl2 is a female-specific sex gene, EB treatment for 7days induced Foxl2/FOXL2 expression in all Sertoli cell-enclosed spermatogonia before testis-ova first appeared; however, Foxl2 was not detected in somatic cells in control testes. Conversely, Sertoli-cell-specific Gsdf mRNA expression levels significantly decreased after EB treatment for 14days, and no changes were observed in DMRT1 localization following EB treatment, whereas Dmrt1 mRNA levels increased significantly. Furthermore, after EB exposure, FOXl2 and DMRT1 were co-localized in Sertoli cells during testis-ova differentiation, although FOXL2 localization was undetectable in Sertoli-cell-enclosed apoptotic testis-ova, whereas DMRT1 remained localized in Sertoli cells. These results indicated for the first time that based on the expression of female-specific sex genes, feminization of Sertoli cells precedes testis-ova differentiation induced by estrogen in mature testes in medaka; however, complete feminization of Sertoli cells was not induced in this study. Additionally, it is suggested strongly that Foxl2 and Gsdf expression constitute potential molecular markers for evaluating the effects of estrogenic chemicals on testicular somatic cells associated with estrogen-induced testis-ova differentiation in mature male medaka.

Identification of a novel FOXL2 mutation in a single family with both types of blepharophimosis‑-ptosis-epicanthus inversus syndrome.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare autosomal dominant disease, which has been divided into two types according to whether it involves premature ovarian failure (POF). Mutations in forkhead box L2 (FOXL2) have been identified in the majority of patients with BPES. The present study aimed to identify the causative mutation in FOXL2 in a Chinese family with both types of BPES. Clinical data and genomic DNA were collected from a single Chinese family with BPES. All the coding exons and adjacent regions of FOXL2 were screened in one affected member to detect the causative mutation using Sanger sequencing. The detected mutation was also screened in available family members and in 100 normal control chromosomes. In total, seven family members were recruited in the present study, including four affected and three unaffected members. The patient (II:5) exhibited typical features of typeII BPES, characterized by a narrowed horizontal palpehral aperture, ptosis, epicanthus inversus and telecanthus without POF, whereas the patient's three daughters (III:1,III:2 and III:3) were diagnosed with type I BPES, in which a complex eyelid malformation was accompanied with POF. A novel heterozygous mutation in FOXL2 (c.844_860dup17, p.His291Argfs*71) was found in the four affected members, which was absent in the remaining three unaffected members and in the 100 control chromosomes. This novel duplicate mutation (c.844_860dup17, p.His291Argfs*71) in FOXL2 was identified in a Chinese family with both types of BPES. These findings expand current knowledge of the mutation spectrum of the FOXL2 gene and confirmed the intra‑family phenotypic heterogeneity of BPES.

MicroRNA profiling of ovarian granulosa cell tumours reveals novel diagnostic and prognostic markers

BackgroundThe aim of this study was to explore the clinical utility of microRNAs (miRNAs) as improved markers of ovarian granulosa cell tumours (GCTs) for cancer diagnosis and prognosis prediction. Current histopathological and genetic markers, such as the presence of a FOXL2 gene mutation to distinguish between the two major subtypes are not wholly accurate and as such novel biomarkers are warranted.MethodsThe miRNA expression profiles of five formalin-fixed, paraffin-embedded (FFPE) adult-GCTs and five juvenile-GCTs were assessed using Affymetrix miRNA 3.0 Arrays and compared for differential expression. Ten miRNAs were assessed in an additional 33 FFPE tumours and four normal granulosa cell samples by quantitative RT-PCR, and their expression correlated to clinical information.ResultsMicroRNA array found 37 miRNAs as differentially expressed between the two GCT subtypes (p < 0.05, fold change ≥2 and among these, miRs -138-5p, -184, -204-5p, -29c-3p, -328-3p and -501-3p were validated by RT-qPCR as differentially expressed between the two GCT subtypes (p < 0.05). In addition, the expression of miR-184 was predictive of tumour recurrence in adult-GCTs, specifically for patients diagnosed with stage I and II and stage I only disease (p < 0.001 and p < 0.05, respectively).ConclusionsThis study is the first to report on global miRNA expression profiles of human ovarian GCTs using FFPE tumour samples. We have validated six miRNAs as novel markers for subtype classification in GCTs with low levels of miR-138-5p correlating with early tumour stage. Low miR-184 abundance was correlated with tumour recurrence in early stage adult-GCT patients as a candidate predictive biomarker. Further studies are now needed to confirm the clinical utility of these miRNAs as diagnostic and recurrence markers, and understand their possible roles in the pathogenesis of GCTs.

Abstract 3381: TERT promoter mutation in granulosa cell tumours of the ovary: Prevalence and prognostic significance

Abstract Granulosa cell tumours (GCTs) of the ovary account for 90% of sex cord-stromal tumours and have a high recurrence rate up to 50%. A missense mutation in the FOXL2 gene (c.402C&amp;gt;G; pC134W) is a defining feature of GCT and is used as a robust marker for diagnosis. However, other than the FOXL2 mutation the pathogenesis and the driving pathways remain unknown. Determining secondary genetic events in GCTs is essential to understanding and improving prognosis. In a pilot study, we completed an analysis of whole genome sequencing of ten GCTs and matched normal cases to generate a comprehensive catalogue of coding and non-coding events. We identified a TERT promoter mutation (c.228C&amp;gt;T) in 50% of these cases. TERT is normally inactivated in somatic tissues; however, this promoter mutation has been shown to re-activate transcription of TERT. We validated this TERT mutation in an international cohort of 300 GCTs and found it was present in approximately 25% of cases overall. These TERT promoter mutations have been used to revise the molecular classification of other cancer types such as gliomas. In GCT, we found that this TERT mutation was correlated with a significantly worse survival outcome in patients with primary GCT (p&amp;lt;0.005). Further, we found that this TERT mutation was present in a larger proportion of recurrent cases. Thus, this mutation may denote a novel subtype of GCT with a worse prognosis. Previous research has shown that TERT activation is evident in over 90% of cancers and is a fundamental step in tumourigenesis that enables unlimited proliferation. This TERT promoter mutation in GCT provides an explanation of how granulosa cells escape atresia and attain immortality. Thus, we hypothesize a mechanism in which the FOXL2 mutation prevents apoptosis and the TERT mutation allows limitless proliferation for oncogenes to transform granulosa cells. However, the current cell models of GCT lack relevant functional pathways and do not recapitulate the biology of these tumours. Therefore, we are developing more suitable cell models to test our hypothesis. We believe that understanding the interaction between these TERT and FOXL2 mutations may lead to novel cancer cell-specific targeted therapies. Citation Format: Jessica A. Pilsworth, Dawn R. Cochrane, Zhouchunyang Xia, Hugo M. Horlings, Winnie Yang, Melissa K. McConechy, Satoshi Yanagida, Anniina E. Färkkilä, Adele P. Wong, Genny Trigo-Gonzalez, S.W. Grace Cheng, Yikan Wang, Ali Bashashati, Gregg B. Morin, Esther Oliva, Sohrab P. Shah, David G. Huntsman. TERT promoter mutation in granulosa cell tumours of the ovary: Prevalence and prognostic significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3381. doi:10.1158/1538-7445.AM2017-3381

The anti-Müllerian hormone (AMH) induces forkhead box L2 (FOXL2) expression in primary culture of human granulosa cells in vitro.

Anti-Müllerian hormone (AMH) and forkhead box L2 (FOXL2) are two pivotal genes expressed in human granulosa cells (hGCs) where both genes share similar inhibitory functions on activation and follicular growth in order to preserve the ovarian follicle reserve. Furthermore, AMH and FOXL2 contribute to inhibit steroidogenesis, decreasing or preventing the activation of gonadotrophin-dependent aromatase CYP19A1 cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The purpose of this study is to evaluate the role of AMH in regulating the expression of FOXL2. Primary cultures of hGCs were treated with increasing concentrations of recombinant human AMH (rhAMH; range 10-100ng/ml) for 3h. Negative controls were performed using corresponding amounts of AMH vehicle. Total RNA or proteins were purified and quantified by spectrophotometry. FOXL2 and CYP19A1 gene expression, normalized by reference gene ribosomal protein S7 (RpS7), was evaluated by RT-qPCR. Each reaction was repeated in triplicate. Statistical analysis was performed. Extracted proteins were analyzed by immunoblot using anti-FOXL2 and anti-β-actin as primary antibodies. rhAMH treatments tested did not modulate the basal expression of aromatase CYP19A1 gene. rhAMH (50ng/ml) was able to increase FOXL2 gene expression and its intracellular content. This study demonstrated the existence of an AMH-FOXL2 relationship in hGCs. AMH is capable of increasing both gene and protein expression of FOXL2. Because FOXL2 induces AMH transcription, these ovarian factors could be finely regulated by a positive feedback loop mechanism to preserve the ovarian follicle reserve.

Analysis of FOXL2 gene mutations in 5 families affected with blepharophimosis, ptosis and epicanthus inversus syndrome

To screen for FOXL2 gene mutations in 6 patients with blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), and explore their genotype-phenotype correlation. Peripheral venous blood samples were collected from the patients for the extraction of genomic DNA. PCR and Sanger sequencing were employed to analyze the coding region and flanking sequences of the FOXL2 gene. Pathogenicity of the identified mutations was verified through literature review and bioinformatic analysis. A heterozygous c.672_701dup30 mutation was found in the probands from the two familial cases, while three heterozygous mutations (two were novel), namely c.462_468del (p.Pro156Argfs*113), c.251T to A (p.Ile84Asn) and c.988_989insG (p.Ala330Glyfs*204) were detected in the three sporadic cases. Literature review and bioinformatic analysis indicated that all these mutations are pathogenic. Identification of causative mutations in the BPES patients has provided a basis for genetic counseling and reproductive guidance. The novel mutations have enriched the mutation spectrum of the FOXL2 gene.

Open label phase II clinical trial of orteronel (TAK-700) in metastatic or advanced non-resectable granulosa cell ovarian tumors: The Greko II study.

5577 Background: Granulosa-cell tumors (GCT) of the ovary are a rare entity characterized by presenting a punctual mutation at the FOXL2 gene 402C→G (C134W). Such mutation leads to a disregulation and overstimulation of the steroidogenic pathway and, ultimately, hormone overproduction. A prior trial by our group (GREKO I trial-GETHI 2011-03; NCT01584297) showed promising activity of ketoconazole, a CYP17 inhibitor used to control steroidogenesis in several conditions. Thus, we aimed to assess the activity of Orteronel (TAK700), a selective inhibitor of 17, 20-lyase, in GCT. Methods: An open-label phase II single arm clinical trial was designed for women with metastatic or locally advanced non-resectable GCT who harbored the somatic mutation FOXL2 402C→G (C134W) and who had not received prior treatment with any CYP17 inhibitor. Treatment consisted on Orteronel 300mg BID, given orally, continuously in a 28-day treatment cycle. The primary objective was clinical benefit rate; secondary objectives were response rate, progression free and overall survival, assessment of the impact of Orteronel in reducing hormonal overproduction and toxicity. Sample size calculation was based on a two stage Simon´s design. A power of 80% was set to differentiate between a 5% and a 25% clinical benefit rate. 20% of losses had been assumed thus 20 patients were scheduled to be enrolled. Results: Since 30/06/2014 to 11/01/2017 10 patients have been included in 9 participating institutions members of Spanish Group for Research in Orphan and Unfrequent Tumors (GETHI). Due to a low recrutiment rate the study was terminated early. Median PFS was 3 months 95%CI (0-12) with 3 patients achieving disease stabilization longer than 12 months. 2 patients remain on treatment after 16 and 14 months. Clinical benefit rate (CR + PR + SD) was 50%, 95%CI (19%-81%). Seven patients have progressed and 2 have died. Only 6 suspected unresected adverse reactions (SUSARs) have been communicated so far (chest pain, fever, febrile neutropenia, eosinophila, neutropenia and anemia). Conclusions: Orteronel achieved a significant clinical benefit in advanced GCT with an favorable toxicity profile. Clinical trial information: NCT02101684.

Molecular Genetic Analysis of FOXL2 Gene in Two Iranian Families with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome

Background: Blepharophimosis-ptosis-epicanthus syndrome (BPES) is a rare genetic disorder with autosomal dominant inheritance. There are two distinct phenotypes: BPES type I, which is associated with eyelid abnormalities as well as female infertility or premature menopause due to ovarian resistance to gonadotropins, whereas in type II only eyelid abnormalities are present. Mutations in the forkhead transcription factor 2 (FOXL2) gene are responsible for both types of BPES. Objectives: The purpose of this study was to identify mutations in FOXL2 in two Iranian families (from Tehran) with BPES who were referred to Tehran Medical Genetics laboratory. Methods: The peripheral blood was collected from the affected members of two BPES families and genomic DNA was extracted using salting out method. Then, direct sequencing of whole exon of FOXL2 genewas performed. Results: Two frameshift mutations were identified in FOXL2 gene in two familial cases including NM_023067:c.102_103insA (p.G35Rfs*61)as a novel mutation and NM_023067:c.855_871dup (p.H291Rfs*71) (17-bp insertion). Both mutations cause the protein to be truncated and are responsible for a severe phenotype (BPES type I) which was in harmony with our finding. Conclusions: Our results increased the spectrum of FOXL2 mutations and confirm the mutations associated with BPES type I.

Masculinization-Related Genes and Cell-Mass Structures During Early Gonadal Differentiation in the African Clawed Frog Xenopus laevis.

The African clawed frog Xenopus laevis has a female heterogametic ZZ/ZW-type sex-determining system. We previously discovered a W-linked female sex-determining gene dm-W that is involved in ovary formation, probably through the up-regulation of the estrogen synthesis genes cyp19a1 and foxl2. We also reported that a unique "mass-in-line structure", which disappears from ZZ gonads during early testicular development, might serve as the basis for ovary differentiation in ZW gonads. However, the molecular mechanisms underlying early masculinization are poorly understood. To elucidate the development of bipotential gonads into testes after sex determination in this species, we focused on the orthologs of five mammalian sex-related genes: three nuclear factor genes, dax1, sf1 (also known as ad4bp), and sox9, and two genes encoding members of the tumor growth factor-β (TGF-β) family, anti-Müllerian hormone (amh) and inhibin βb (inhbb). Quantitative RT-PCR analysis revealed that the expression of dax1, sox9, amh, and inhbb or sf1 was greatly or slightly higher in ZZ than in ZW gonads during early sex development. In situ hybridization analysis revealed that amh and inhbb mRNAs were expressed in somatic cells on the inner and outer sides of cell masses in the mass-in-line structure, respectively, in the developing ZZ gonads. Interestingly, estrogen exposure prevented the disappearance of the mass-in-line structure in early developing ZZ tadpoles. These findings suggest that TGF-β signaling is involved in the destruction of the mass-in-line structure, which may be maintained by estrogen.

Transcription of CYP19A1 is directly regulated by SF-1 in the theca cells of ovary follicles in chicken

Many studies have suggested the important role of estrogen in ovarian differentiation and development of vertebrates including chicken. Cytochrome P450 aromatase, encoded by CYP19A1, is a key enzyme in estrogen synthesis, but the mechanism of CYP19A1 regulation in chicken remains unknown. Here, we found that CYP19A1 was only expressed in the theca cell layers of chicken ovary follicles. Steroidogenic factor 1 (SF-1, also named as nuclear receptor subfamily 5 group A member 1, NR5A1), a potential regulators, was expressed in both the theca cell layers and granulosa cell layers. Forkheadbox L2 (FOXL2), another potential regulator, was only expressed in the granulosa cell layers. Using luciferase assays in vitro, we found that SF-1 could activate the promoter of CYP19A1 by binding to the nuclear receptor half-site (5′-TCAAGGTCA-3′) from −280 to −271 base pairs. FOXL2 did not activate the promoter of chicken CYP19A1 gene in either 293T or DF-1 cells. Overexpression of SF-1 in DF-1 cells upregulated aromatase expression, but FOXL2 could not. Taken together, our results indicated that SF-1 activates CYP19A1 mRNA expression via a conserved binding site in chicken ovary, but FOXL2 may not affect the expression of CYP19A1.

Cloning of the promoter region of a human gene, FOXL2, and its regulation by STAT3.

Forkhead box L2 (FOXL2) is a transcription factor, which is involved in blepharophimosis, ptosis, and epicanthus in versus syndrome (BPES), premature ovarian failure (POF), as well as almost all stages of ovarian development and function. FOXL2 has various target genes, which are implicated in numerous processes, including sex determination, cell cycle regulation and apoptosis and stress response regulation in mammals. However, studies regarding the upstream regulation of FOXL2 are limited. In the present study, the promoter of FOXL2 was successfully cloned and registered in Gen Bank, and a dual luciferase reporter (DLR) analysis demonstrated that the luciferase activity was significantly induced by the promoter of FOXL2. Subsequently, bioinformatics analysis indicated that FOXL2 may be regulated by STAT3, and this was confirmed by a DLR analysis and western blotting, using STAT3 inhibitors. Further study using real‑time cellular analysis indicated that the viability of He La cells was markedly suppressed by STAT3 inhibitors. The present study demonstrated novel findings regarding the upstream regulation of FOXL2 expression and provide a new perspective for future studies in the field.

Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole (Cynoglossus semilaevis)

Chinese tongue sole is a marine fish with ZW sex determination. Genome sequencing suggested that the Z-linked dmrt1 is a putative male determination gene, but direct genetic evidence is still lacking. Here we show that TALEN of dmrt1 efficiently induced mutations of this gene. The ZZ dmrt1 mutant fish developed ovary-like testis, and the spermatogenesis was disrupted. The female-related genes foxl2 and cyp19a1a were significantly increased in the gonad of the ZZ dmrt1 mutant. Conversely, the male-related genes Sox9a and Amh were significantly decreased. The dmrt1 deficient ZZ fish grew much faster than ZZ male control. Notably, we obtained an intersex ZW fish with a testis on one side and an ovary on the other side. This fish was chimeric for a dmrt1 mutation in the ovary, and wild-type dmrt1 in the testis. Our data provide the first functional evidence that dmrt1 is a male determining gene in tongue sole.

SMAD3 Regulates Follicle-stimulating Hormone Synthesis by Pituitary Gonadotrope Cells in Vivo

Pituitary follicle-stimulating hormone (FSH) is an essential regulator of fertility in females and of quantitatively normal spermatogenesis in males. Pituitary-derived activins are thought to act as major stimulators of FSH synthesis by gonadotrope cells. In vitro, activins signal via SMAD3, SMAD4, and forkhead box L2 (FOXL2) to regulate transcription of the FSHβ subunit gene (Fshb). Consistent with this model, gonadotrope-specific Smad4 or Foxl2 knock-out mice have greatly reduced FSH and are subfertile. The role of SMAD3 in vivo is unresolved; however, residual FSH production in Smad4 conditional knock-out mice may derive from partial compensation by SMAD3 and its ability to bind DNA in the absence of SMAD4. To test this hypothesis and determine the role of SMAD3 in FSH biosynthesis, we generated mice lacking both the SMAD3 DNA binding domain and SMAD4 specifically in gonadotropes. Conditional knock-out females were hypogonadal, acyclic, and sterile and had thread-like uteri; their ovaries lacked antral follicles and corpora lutea. Knock-out males were fertile but had reduced testis weights and epididymal sperm counts. These phenotypes were consistent with those of Fshb knock-out mice. Indeed, pituitary Fshb mRNA levels were nearly undetectable in both male and female knock-outs. In contrast, gonadotropin-releasing hormone receptor mRNA levels were significantly elevated in knock-outs in both sexes. Interestingly, luteinizing hormone production was altered in a sex-specific fashion. Overall, our analyses demonstrate that SMAD3 is required for FSH synthesis in vivo.

Parabens Accelerate Ovarian Dysfunction in a 4-Vinylcyclohexene Diepoxide-Induced Ovarian Failure Model.

Parabens are widely used preservatives in basic necessities such as cosmetic and pharmaceutical products. In previous studies, xenoestrogenic actions of parabens were reported in an immature rat model and a rat pituitary cell line (GH3 cells). The relationship between parabens and ovarian failure has not been described. In the present study, the influence of parabens on ovarian folliculogenesis and steroidogenesis was investigated. A disruptor of ovarian small pre-antral follicles, 4-vinylcyclohexene diepoxide (VCD, 40 mg/kg), was used to induce premature ovarian failure (POF). Methylparaben (MP, 100 mg/kg), propylparaben (PP, 100 mg/kg), and butylparaben (BP, 100 mg/kg) dissolved in corn oil were treated in female 8-week-old Sprague-Dawley rat for 5 weeks. Estrus cycle status was checked daily by vaginal smear test. Ovarian follicle development and steroid synthesis were investigated through real-time PCR and histological analyses. Diestrus phases in the VCD, PP, and BP groups were longer than that in the vehicle group. VCD significantly decreased mRNA level of folliculogenesis-related genes (Foxl2, Kitl and Amh). All parabens significantly increased the Amh mRNA level but unchanged Foxl2 and Kitlg acting in primordial follicles. VCD and MP slightly increased Star and Cyp11a1 levels, which are related to an initial step in steroidogenesis. VCD and parabens induced an increase in FSH levels in serum and significantly decreased the total number of follicles. Increased FSH implies impairment in ovarian function due to VCD or parabens. These results suggest that VCD may suppress both formation and development of follicles. In particular, combined administration of VCD and parabens accelerated inhibition of the follicle-developmental process through elevated AMH level in small antral follicles.

Transcriptome analysis of the potential roles of FOXL2 in chicken pre-hierarchical and pre-ovulatory granulosa cells

Forkheadbox L2 (FOXL2) is a transcription factor involved in mammalian ovarian development, especially in granulosa cell differentiation. However, this factor's function in mature chicken ovary is unclear. To explore the function of FOXL2 in chicken granulosa cells, we performed RNA-seq to compare the transcriptomes of pre-hierarchical (phGCs) and pre-ovulatory granulosa cells (poGCs) by FOXL2 overexpression. We observed that focal adhesion might be one of the key pathways activated during the differentiation of granulosa cells, and FOXL2 might be involved in follicle selection by regulating the expression of cytokines and the concentration of cyclic adenosine monophosphate (cAMP). Interestingly, we observed that FOXL2 played different roles in phGCs and poGCs, which might contribute to homeostasis in the chicken follicle by inducing differentiation of granulosa cells in pre-hierarchal follicles and preventing premature ovulation in pre-ovulatory follicles. Taken together, the results of our study establish a framework for understanding the potential functions of FOXL2 in the chicken granulosa cell.