FOXL2 Research Articles

Functional Analysis of a Novel FOXL2 Indel Mutation in Chinese Families with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome Type I.

Background: Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant disease with a low incidence rate. Indel mutations in the forkhead box L2 (FOXL2) gene cause two types of BPES that are distinguished by the presence (type I) or absence (type II) of premature ovarian failure (POF). The purpose of this study was to identify a possible deletion in FOXL2 in Chinese families with BPES and to clarify its relationship with POF.Methods: An autosomal dominant Chinese BPES family with four generations was enrolled in this study. Peripheral venous blood was collected from all affected patients, and genomic DNA was extracted from leukocytes. The whole coding sequence and nearby 5' untranslated region (UTR) and 3'UTR of the FOXL2 gene were amplified using polymerase chain reaction (PCR) with three sets of overlapping primers, followed by sequencing analyses. The sequencing results were analysed using SeqMan software. Based on the patients' clinical manifestations and analysis of the identified indel mutation, we found that the mutation disturbed interactions between FOXL2 and the StAR gene. Furthermore, through subcellular localisation and functional studies, we observed significant mislocalisation of the mutant protein; the mutant protein was found in the cytoplasm, while the wild-type protein was found in the nucleus. Loss of function was confirmed by transcriptional activity assays, quantitative real-time PCR, and electrophoretic mobility shift assays.Results: All affected patients presented with clinical features of BPES type I, including small palpebral fissures, ptosis, telecanthus, and epicanthus inversus with POF. A novel FOXL2 heterozygous indel mutation, c.19_95del, a 77-bp deletion that disrupts FOXL2 protein structure, was identified in all affected members of the family. In addition, this indel mutation significantly increased StAR mRNA expression by disrupting the ability of the FOXL2 protein to bind to the StAR promoter and act as a repressor of this gene.Conclusions: A novel FOXL2 indel mutation was identified in Chinese families with BPES. Our results expand the spectrum of known FOXL2 mutations and provide additional insight into the structure-function relationships of the FOXL2 protein. Furthermore, this novel mutation resulted in the dysfunction of FOXL2 as a transcription factor, blocking its ability to bind to the promoter region of the StAR gene, resulting in POF in the affected patient.

Sex determination and maintenance: the role of DMRT1 and FOXL2

In many species, including mammals, sex determination is genetically based. The sex chromosomes that individuals carry determine sex identity. Although the genetic base of phenotypic sex is determined at the moment of fertilization, the development of testes or ovaries in the bipotential early gonads takes place during embryogenesis. During development, sex determination depends upon very few critical genes. When one of these key genes functions inappropriately, sex reversal may happen. Consequently, an individual's sex phenotype may not necessarily be consistent with the sex chromosomes that are present. For some time, it has been assumed that once the fetal choice is made between male and female in mammals, the gonadal sex identity of an individual remains stable. However, recent studies in mice have provided evidence that it is possible for the gonadal sex phenotype to be switched even in adulthood. These studies have shown that two key genes, doublesex and mad-3 related transcription factor 1 (Dmrt1) and forkhead box L2 (Foxl2), function in a Yin and Yang relationship to maintain the fates of testes or ovaries in adult mammals, and that mutations in either gene might have a dramatic effect on gonadal phenotype. Thus, adult gonad maintenance in addition to fetal sex determination may both be important for the fertility.

The Emerging Role of FOXL2 in Regulating the Transcriptional Activation Function of Estrogen Receptor β: An Insight Into Ovarian Folliculogenesis.

Germline mutations of the fork-head transcriptional factor forkhead box L2 (FOXL2) predispose embryos to autosomal-dominant blepharophimosis-ptosis-epicanthus inversus syndrome with primary ovarian insufficiency in female patients, but the mechanisms of FOXL2 in ovarian follicular development remain elusive. Estrogens produced by ovarian granulosa cells and estrogen receptor (ER) α and ERβ play fundamental roles in ovarian pathophysiology, and a previous study revealed that ERα and ERβ physically interact with FOXL2. However, the underlying functions of these interactions have not been investigated. Herein, we report an ERβ-specific repressive function of FOXL2. Histological examination demonstrated that FOXL2 expression tends to be intense during early follicular development. Immunoprecipitation revealed that ERβ and FOXL2 interact in a ligand-independent manner. In vitro pull-down assays revealed a direct interaction between FOXL2 and the activation function (AF)-1/2 domain of ERβ. The expression of FOXL2 represses the ligand-dependent transcriptional activation of ERβ, but FOXL2 does not influence the ligand-dependent transcriptional activation of ERα. Consistent with these results, RNA interference-mediated depletion of FOXL2 stimulates the expression of the ERβ-downstream gene p450 aromatase. The convergence between FOXL2 functions and ERβ-mediated transcription in the ovary suggests the putative mechanism of FOXL2 in early-phase follicular development, which may be partially attributed to the regulation of ERβ-dependent gene expression.

Surgical Outcome of Epicanthus and Telecanthus Correction by Double Z-Plasty and Trans-Nasal Fixation with Prolene Suture in Blepharophimosis Syndrome.

Blepharophimosis Syndrome (BPES) is a complex and rare disease characterized by epicanthus inversus, telecanthus, lateral ectropion, narrowed or shortened inter-palpebral fissure distance and ptosis. It is mostly bilateral and may or may not be symmetrical. It is typically inherited as an autosomal dominant trait. In sporadic cases, the disease may occur without a prior family history as a genetic mutation from a deletion or translocation of the FOXL2 gene, which maps to chromosome 3q23. Surgical treatment of this disease poses an oculoplastic challenge due to multiple complex eyelid deformities. To evaluate the functional and cosmetic outcome of telecanthus and epicanthus correction by a Mustarde's rectangular double Z-Plasty and trans-nasal fixation using 1-0 prolene suture in BPES. This was prospective, interventional study of 16 patients over a period of three years. In this study, all patients had BPES with prominent epicanthus and telecanthus. Mustarde's double Z-plasty and trans-nasal fixation with 1-0 prolene suture was performed in the first of a two-stage operation. If ectropion was present, the lateral ectropion was corrected by a base-out flap transfer from the upper eyelid to the lower eyelid. After three months, a 2nd stage was undertaken, involving a lateral canthoplasty for horizontal widening of a short palpebral fissure and a tarso frontalis sling with silicone rod for correction of moderate to severe ptosis. Patients were followed up for six months to one year with postoperative ophthalmologic examinations and photographs. Out of 16 patients, 10 were females and six were males. All the patients had bilateral involvement. In this study preoperative Inner Intercanthal Distance (IICD) ranged from 38 mm to 42 mm and the mean IICD was 41.2±0.57 mm. Postoperative IICD ranged from 31 mm to 34 mm. Horizontal Palpebral Fissure Length (HPFL) ranged from 20 mm to 23 mm and the mean value of HPFL was 21.50 mm preoperatively. Postoperative HPFL ranged from 26 mm to 29 mm and had a mean value of 28.50 mm, which was much improved after a combined correction of telecanthus and lateral canthoplasty. The mean preoperative IICD and HPFL ratio was 1.77 and was reduced to a postoperative value of 1.2. The Marginal Reflex Distance1 (MRD-1) test value improved from +1.25 mm to +3.50 mm postoperatively after placement of a tarsofrontalis sling with silicone rod using the Fox's Pentagon technique. In this study, two eyes had minimal unequal correction but were cosmetically and functionally acceptable. Correction of IICD is possible up to 6 mm. No major complication e.g., CSF rhinorrhea was noted in this series and preoperative prominent epicanthal folds were abolished. Here we propose a two-staged procedure involving a combined Mustarde's double Z-plasty with transnasal fixation using a 1-0 prolene suture with a flap transfer from the upper lid to the lower lid in the first stage and a lateral canthoplasty with a tarsofrontalis sling and silicone rod in the second stage. This technique is effective to correct epicanthus, telecanthus, ptosis and lateral ectropion in BPES with good cosmetic and functional outcome.

Dmrt1 is necessary for male sexual development in zebrafish

The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is a key regulator of sex determination and/or gonadal sex differentiation across metazoan animals. This is unusual given that sex determination genes are typically not well conserved. The mechanisms by which zebrafish sex is determined have remained elusive due to the lack of sex chromosomes and the complex polygenic nature of sex determination in domesticated strains. To investigate the role of dmrt1 in zebrafish sex determination and gonad development, we isolated mutations disrupting this gene. We found that the majority of dmrt1 mutant fish develop as fertile females suggesting a complete male-to-female sex reversal in mutant animals that would have otherwise developed as males. A small percentage of mutant animals became males, but were sterile and displayed testicular dysgenesis. Therefore zebrafish dmrt1 functions in male sex determination and testis development. Mutant males had aberrant gonadal development at the onset of gonadal sex-differentiation, displaying reduced oocyte apoptosis followed by development of intersex gonads and failed testis morphogenesis and spermatogenesis. By contrast, female ovaries developed normally. We found that Dmrt1 is necessary for normal transcriptional regulation of the amh (anti-Müllerian hormone) and foxl2 (forkhead box L2) genes, which are thought to be important for male or female sexual development respectively. Interestingly, we identified one dmrt1 mutant allele that co-operates with a linked segregation distorter locus to generate an apparent XY sex determination mechanism. We conclude that dmrt1 is dispensable for ovary development but necessary for testis development in zebrafish, and that dmrt1 promotes male development by transcriptionally regulating male and female genes as has been described in other animals. Furthermore, the strong sex-ratio bias caused by dmrt1 reduction-of-function points to potential mechanisms through which sex chromosomes may evolve.

Mutational Characterisation and Tracking Disease Progression Using Circulating Cell-Free Tumor DNA in Multiple Myeloma Patients

Background:Multiple myeloma (MM) currently relies on bone marrow (BM) biopsy for mutational characterisation, which does not capture the putative spatial and genetic heterogeneity of this multi-focal disease. Circulating cell-free tumour DNA (ctDNA) is a powerful non-invasive biomarker, which is being utilised to monitor tumor dynamics in a number of cancers. In this study, analysis of peripheral blood plasma (PL) - derived ctDNA and contemporaneously sourced BM MM cells was undertaken to determine if ctDNA can be utilised as an adjunct to BM biopsy for mutational characterisation and non-invasive therapeutic monitoring of disease progression.Methods:Blood (30ml) from MM patients was collected into Streck Cell-Free DNA BCT tubes and DNA extracted using the QIAamp circulating nucleic acid kit (Qiagen). BM aspirates from MM patients were CD138 enriched and subject to DNA extraction (Qiagen). Paired BM MM DNA and PL samples from 54 patients (New Diagnosis (ND) = 17; Relapsed/ Refractory (RR) = 35 and monoclonal gammopathy of undetermined significance (MGUS) =2] were analysed in the high-sensitive OnTargetTM mutation detection platform (OMD, Boreal Genomics) that included 96-mutations in the KRAS, NRAS, CTNNB1, EGFR, PIK3CA, TP53, FOXL2, GNAS and BRAF genes. OMD findings were validated with ddPCR (Biorad QX200 droplet digital PCR system). Sequential ctDNA quantitation with ddPCR through longitudinal PL tracking of specific clones in two patients for the monitoring of disease was also performed.Results:OMD detected a total of 141 mutations (BM and PL=42; BM-only=63 and PL only=36) with the presence of mutations in the PL (55.3%) authenticating the existence of ctDNA harbouring mutations. The detection of PL-only mutations (25.5%) signified the existence of mutant clones predominantly or exclusively, distant to the BM biopsy site. RR patients had a higher number of PL-only mutations compared to ND (34 vs 2 mutations), with none detected in MGUS, denoting that spatial and genetic heterogeneity is more evident in patients with advanced disease.Activating RAS mutations were highly prevalent in 34/54 patients (63%) harboring at least one RAS mutation. Specifically, KRAS mutations were found in 26/54 patients (48%), with 17/35 RR (48.5%), 9/17 ND (53%) and 1/2 MGUS patients harboring at least 1 KRAS mutation, indicating that mutations in this gene occur early in MM pathogenesis. NRAS mutations were found to be present at a higher frequency in RR patients compared to ND patients (45.7% vs 35.2%). BRAF mutations were equally distributed amongst RR and ND patients, in contrast, TP53 mutations were found exclusively in RR patients. Additionally, in 2 RR patients, a total of 5 PIK3CA mutations (E542K, E545K, H1047R, C420R, E81K) were present, 4 of which were found in PL-only, denoting the presence of these mutations distant to the BM biopsy site. Two ND patients had 1 GNAS mutation each (R201H and R201C). Preliminary validation of the OMD findings was performed in 12 patients using ddPCR with 90.9% concordance between the two platforms. Furthermore, 4/13 mutations (30.7%) that were negative in OMD were detected by ddPCR, indicating a higher sensitivity for ddPCR.Sequential ddPCR of ctDNA for Patient #1 with advanced relapsed disease for previously identified TP53 R273H and KRAS G12D mutations showed that the fractional abundance of KRAS G12D rapidly increased coincident with relapse of the disease, while that of TP53 273H did not change significantly over time. For Patient #2, relapsed disease was associated with the reappearance of mutant KRAS G12V and KRAS G12D clones that had been present at diagnosis in the BM and the emergence of 2 new clones NRAS G13D and NRAS Q61K.ConclusionsThis study provides the groundwork for utilisation of PL-ctDNA as an adjunct to BM biopsy for comprehensive mutational characterisation and as a non-invasive biomarker for therapeutic monitoring in MM patients. Importantly, a higher frequency of RAS mutations and presence of mutations in PIK3CA and GNAS, which have not been previously reported in MM, provides evidence of a more complex mutational landscape in MM than previously shown with BM whole exome sequencing studies. Furthermore, sequential PL tracking of specific mutant clones in two patients enabled monitoring of tumor dynamics. In conclusion, incorporating PL ctDNA evaluation may represent a significant advance in attempts to personalize future MM treatment strategies. DisclosuresMai:Boreal Genomics: Employment. Walsh:Boreal Genomics: Employment. Broemeling:Boreal Genomics: Employment. Marziali:Boreal Genomics: Employment. Wiggin:Boreal Genomics: Employment.

Lacrimal Gland Involvement in Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome

To describe the involvement of the lacrimal gland (LG) in blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). Observational, cross-sectional study. Twenty-one patients with BPES (10 female, 11 male) aged on average 15 years (range, 2-39years), from 3 Brazilian medical centers and 1 Portuguese medical center. Patients had their ocular surface evaluated with slit-lamp biomicroscopy, and tear production quantified with the Schirmer test I. The LG volumes were measured on computed tomography (CT) scans in the BPES sample and in a group of age-matched subjects imaged for nonorbital diseases. Sixteen patients were screened for mutations in the FOXL2 gene. Lacrimal meniscus height, Schirmer test I, presence of superficial punctate keratopathy (SPK), LG volume, and molecular analysis of the FOXL2 gene. Absence of LG was detected bilaterally in 9 patients (42.8%) and unilaterally in 2 patients (9.5%). When considering only patients with measurable LG, the median volume was 0.22 cm3 in the right eye (range, 0.06-0.36 cm3) and 0.24 cm3 in the left eye (range, 0.08-0.34 cm3). These values were significantly lower than those for the age-matched controls (median= 0.54 right eye and 0.53 left eye; P < 0.05). There was a significant association between deficiency of tear production and LG volume reduction and agenesis. Molecular analysis of the FOXL2 gene revealed the presence of 8 distinct mutations, 4 of them novel ones. A significant reduction of LG size or agenesis was associated with mutations affecting protein size (due to underlying changes in the stop codon location) or the DNA-binding forkhead domain (Fisher exact test, P= 0.021). In 3 probands, the underlying genetic defect was not found. This is the first study reporting LG volumes in BPES, describing a significant number of patients with LG agenesis. The association between alacrima and BPES is not incidental, and a thorough evaluation of tear production is recommended especially if ptosis surgery is planned.

Differential expression of foxl2 and cyp19a1a mRNA during gonad developmental stages in great sturgeon Huso huso.

This study aimed to determine the sex specificity and expression pattern of foxl2 and cyp19a1a genes in great sturgeon Huso huso gonads during gonadal sex differentiation and development. The results revealed that foxl2 and cyp19a1a mainly expressed in female gonads and during gonad development the foxl2 and cyp19a1a mRNA expression is required for ovarian development.

Molecular initiating events of the intersex phenotype: Low-dose exposure to 17α-ethinylestradiol rapidly regulates molecular networks associated with gonad differentiation in the adult fathead minnow testis

Intersex, or the presence of oocytes in the testes, has been documented in fish following exposure to wastewater effluent and estrogenic compounds. However, the molecular networks underlying the intersex condition are not completely known. To address this, we exposed male fathead minnows to a low, environmentally-relevant concentration of 17alpha-ethinylestradiol (EE2) (15ng/L) and measured the transcriptome response in the testis after 96h to identify early molecular initiating events that may proceed the intersex condition. The short-term exposure to EE2 did not affect gonadosomatic index and proportion of gametes within the testes. However, the production of 11-ketotestosterone and testosterone from the testis in vitro was decreased relative to controls. Expression profiling using a 8×60K fathead minnow microarray identified 10 transcripts that were differentially expressed in the testes, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase). Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

The genetic make-up of ovarian development and function: the focus on the transcription factor FOXL2.

In a 46 XY individual, the presence of the Y chromosome harboring the testis-determining factor (SRY) triggers testis determination and differentiation. In a 46 XX individual, the absence of SRY and the activation of genes associated with the female pathway lead to ovarian development. The latter process has long been considered as a default pathway. However, recent studies have cast doubts on this dogma. Here, after a brief overview of the main steps of ovarian development, we focus on three genes WNT4, RSPO1 and FOXL2 that are essential for ovarian determination, differentiation and/or maintenance. Special attention is paid to FOXL2 whose mutations are responsible for the blepharophimosis syndrome, often associated with female infertility, and for cancer. We highlight the cooperation of WNT4, RSPO1 and FOXL2 within a regulatory network and the need for further research to better understand their role in defining and maintaining ovarian identity.

SMAD3 Activation: A Converging Point of Dysregulated TGF-Beta Superfamily Signaling and Genetic Aberrations in Granulosa Cell Tumor Development?

Ovarian granulosa cell tumors (GCTs) are rare gynecologic tumors in women. Due to the rarity and limited research efforts invested, the etiology of GCTs remains poorly defined. A landmark study has discovered the mutation of forkhead box L2 (FOXL2) as a genetic hallmark of adult GCTs in the human. However, our understanding of the role of cell signaling in GCT development is far from complete. Increasing lines of evidence highlight the importance of TGF-beta (TGFB) superfamily signaling in the pathogenesis of GCTs. This review draws on findings using genetically modified mouse models and human patient specimens and cell lines to reveal SMAD3 activation as a potentially key converging point of dysregulated TGFB superfamily signaling and genetic aberrations in GCT development. It is anticipated that deciphering the role of TGFB superfamily signaling cascades in ovarian tumorigenesis will help develop new therapeutic approaches for GCTs by targeting core signaling elements essential for tumor initiation, growth, and progression.

Identification of Multiple Gene Mutations Accounts for a new Genetic Architecture of Primary Ovarian Insufficiency.

Idiopathic primary ovarian insufficiency (POI) is a major cause of amenorrhea and infertility. POI affects 1% of women before age 40 years, and several genetic causes have been reported. To date, POI has been considered a monogenic disorder. The aim of this study was to identify novel gene variations and to investigate if individuals with POI harbor mutation in multiple loci. One hundred well-phenotyped POI patients were systematically screened for variants in 19 known POI loci (and potential candidate genes) using next-generation sequencing. At least one rare protein-altering gene variant was identified in 19 patients, including missense mutations in new candidate genes, namely SMC1β and REC8 (involved in the cohesin complex) and LHX8, a gene encoding a transcription factor. Novel or recurrent deleterious mutations were also detected in the known POI candidate genes NOBOX, FOXL2, SOHLH1, FIGLA, GDF9, BMP15, and GALT. Seven patients harbor mutations in two loci, and this digenicity seems to influence the age of symptom onset. Genetic anomalies in women with POI are more frequent than previously believed. Digenic findings in several cases suggest that POI is not a purely monogenic disorder and points to a role of digenicity. The genotype-phenotype correlations in some kindreds suggest that a synergistic effect of several mutations may underlie the POI phenotype.

A comparison of biomarker responses in juvenile diploid and triploid African catfish, Clarias gariepinus, exposed to the pesticide butachlor

Influence of waterborne butachlor (BUC), a commonly used pesticide, on morphometric, biochemical, and molecular biomarkers was evaluated in juvenile, full sibling, diploid and triploid African catfish (Clarias gariepinus). Fish were exposed for 21 days to one of three concentrations of BUC [mean measured µg/L: 22, 44 or 60]. Unexposed (control) triploids were heavier and longer and had higher visceral-somatic index (VSI) than diploids. Also, they had lighter liver weight (HSI) and showed lower transcript levels of brain gonadotropin-releasing hormone (GnRH), aromatase (cyp191b) and fushi tarazu-factor (ftz-f1), and plasma testosterone levels than diploids. Butachlor treatments had no effects, in either diploid or triploid fish, on VSI, HSI, weight or length changes, condition factor (CF), levels of plasma testosterone, 17-β estradiol (E2), cortisol, cholesterol, or mRNA levels of brain tryptophan hydroxylase (tph2), forkhead box L2 (foxl2), and 11 β-hydroxysteroid dehydrogenase type 2 (11β-hsd2). Expressions of cyp191b and ftz-f1 in triploids were upregulated by the two highest concentrations of BUC. In diploid fish, however, exposures to all BUC concentrations decreased GnRH transcription and the medium BUC concentration decreased ftz-f1 transcription. Substantial differences between ploidies in basal biomarker responses are consistent with the reported impaired reproductive axis in triploid C. gariepinus. Furthermore, the present study showed the low impact of short term exposure to BUC on reproductive axis in C. gariepinus.

Virgin microplastics cause toxicity and modulate the impacts of phenanthrene on biomarker responses in African catfish (Clarias gariepinus)

Despite the ubiquity of microplastics (MPs) in aquatic environments and their proven ability to carry a wide variety of chemicals, very little is known about the impacts of virgin or contaminant-loaded MPs on organisms. The primary aim of this study was to investigate the impacts of virgin or phenanthrene (Phe)-loaded low-density polyethylene (LDPE) fragments on a suite of biomarker responses in juvenile African catfish (Clarias gariepinus). Virgin LDPE (50 or 500µg/L) were preloaded with one of two nominal Phe concentrations (10 or 100µg/L) and were exposed to the fish for 96h. Our findings showed one or both Phe treatments significantly increased the degree of tissue change (DTC) in the liver while decreased the transcription levels of forkhead box L2 (foxl2) and tryptophan hydroxylase2 (tph2) in the brain of C. gariepinus. Exposure to either levels of virgin MPs increased the DTC in the liver and plasma albumin: globulin ratio while decreased the transcription levels of tph2. Moreover, MPs modulated (interacted with) the impact of Phe on the DTC in the gill, plasma concentrations of cholesterol, high-density lipoprotein (HDL), total protein (TP), albumin, and globulin, and the transcription levels of fushi tarazu-factor 1 (ftz-f1), gonadotropin-releasing hormone (GnRH), 11 β-hydroxysteroid dehydrogenase type 2 (11β-hsd2), and liver glycogen stores. Results of this study highlight the ability of virgin LDPE fragments to cause toxicity and to modulate the adverse impacts of Phe in C. gariepinus. Due to the wide distribution of MPs and other classes of contaminants in aquatic environments, further studies are urgently needed to elucidate the toxicity of virgin or contaminant-loaded MPs on organisms.

Hypermethylation of CDH13, DKK3 and FOXL2 promoters and the expression of EZH2 in ovary granulosa cell tumors.

Aberrant epigenetic modification is associated with the development and progression of cancer. Hypermethylation of tumor suppressor gene promoters and cooperative histone modification have been considered to be the primary mechanisms of epigenetic modification. Ovary granulosa cell tumors (GCTs) are relatively rare, accounting for ~3% of all ovarian malignancies. The present study assessed hypermethylation of the cadherin13 (CDH13), dickkopf WNT signaling pathway inhibitor 3 (DKK3) and forkhead boxL2 (FOXL2) promoters in 30 GCT tissues and 30healthy control tissues using methylation-specific polymerase chain reaction analysis. The data showed that the frequencies of CDH13, DKK3 and FOXL2 promoter methylation were significantly higher in the GCT tissues, compared with the healthy control tissues (86.67,vs.23.33%; 80, vs. 26.67% and 66.67, vs. 20%, respectively; P<0.001). Immunostaining of enhancer of zeste homolog2 (EZH2), a histone H3K27 methyltransferase, showed that the EZH2 protein was expressed in 11 of the 30GCT tissue samples, whereas no EZH2 protein was expressed in the30 healthy control tissues (P<0.01). These data suggested that hypermethylation of the CDH13, DKK3 and FOXL2 gene promoters, and overexpression of the EZH2 protein were involved in the development of GCT.

Uterine Tumor Resembling Ovarian Sex Cord Tumor (UTROSCT) Commonly Exhibits Positivity With Sex Cord Markers FOXL2 and SF-1 but Lacks FOXL2 and DICER1 Mutations.

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare neoplasm which morphologically and immunohistochemically exhibits overlap with an ovarian sex cord tumor. Although many of these neoplasms are positive with markers of ovarian sex cord-stromal tumors, staining is often limited and the pathogenesis of UTROSCT is unknown. To further explore the sex cord lineage of UTROSCT, we studied 19 of these neoplasms and examined the expression of 2 recently described markers of ovarian sex cord-stromal tumors, FOXL2, and steroidogenic factor-1. We also undertook FOXL2 and DICER1 mutation analysis in these cases; a somatic missense mutation in codon C134W (402C→G) of FOXL2 gene has been demonstrated in the vast majority (>95%) of ovarian adult granulosa cell tumors and somatic DICER1 mutations are found in approximately 60% of ovarian Sertoli-Leydig cell tumors. Ten of 19 cases (53%) exhibited nuclear immunoreactivity with FOXL2 and 11 of 19 (58%) exhibited nuclear staining with steroidogenic factor-1. Neither FOXL2 nor DICER1 mutations were identified in any case where there was sufficient tumor tissue for analysis (18 and 9 cases, respectively). Despite exhibiting an immunophenotype characteristic of a sex cord-stromal tumor, mutations in FOXL2 and DICER1, the 2 most common mutations hitherto reported in ovarian sex cord-stromal tumors, are not a feature of UTROSCT.

Daily Rhythms of the Expression of Key Genes Involved in Steroidogenesis and Gonadal Function in Zebrafish.

Fish present daily and seasonal rhythms in spawning and plasmatic levels of steroids that control reproduction. However, the existence of the rhythms of expression of the genes that underlie the endocrine mechanisms responsible for processes such as steroidogenesis and reproduction in fish have still been poorly explored to date. Here we investigated the daily pattern of the expression of key genes involved in sex steroid production that ultimately set the sex ratio in fish. Adult zebrafish were maintained under a 12:12 h light-dark cycle at a constant temperature of 27°C and were sampled every 4 h during a 24-hour cycle. The expression of key genes in the gonads and brains of female and male individuals were analyzed. In gonads, the expression of aromatase (cyp19a1a, ovarian aromatase) and the antimüllerian hormone (amh, testis) was rhythmic, with almost opposite acrophases: ZT 5:13 h (in the light phase) and ZT 15:39 h (at night), respectively. The expression of foxl2 (forkhead box L2) was also rhythmic in the ovary (acrophase located at ZT 5:02 h) and the expression of dmrt1 (doublesex and mab-3-related transcription factor 1) was rhythmic in testes (acrophase at ZT 18:36 h). In the brain, cyp19a1b (brain aromatase) and cyp11b (11beta-hydroxylase) presented daily differences, especially in males, where the expression peaked at night. These results provide the first evidence for marked time-of-the-day-dependent differences in the expression of the genes involved in sex ratio control, which should be considered when investigating processes such as reproduction, sex differentiation and steroidogenesis in fish.

FOXL2 molecular status in adult granulosa cell tumors of the ovary: A study of primary and metastatic cases.

Granulosa cell tumors (GCTs) of the ovary are uncommon neoplasms, accounting for ~5% of all malignant ovarian tumors. GCTs are a relatively homogeneous group of tumors, categorized into two distinct subtypes, juvenile GCT and adult GCT (AGCT), likely arising from a limited set of molecular events usually involving the disruption of pathways that regulate granulosa cell proliferation. In the present study, the presence of forkheadbox L2 (FOXL2) c.402C>G mutation was investigated in a series of 42 samples of primary and metastatic AGCT of the ovary. The samples consisted of 37 primary and 5 metastatic ovarian AGCTs from 37 patients. FOXL2 mutational status was evaluated using a pyrosequencing approach on 2.5-µm sections of formalin-fixed paraffin-embedded tissue. FOXL2 c.402C>G mutation was found in 33/37 (89.2%) primary AGCTs and in 4/5 (80.0%) metastases, with the molecular status of the metastases recapitulating that of the primary tumors (4 mutated cases and 1 wild-type case). Overall, FOXL2 mutation is present in the majority of primary and metastatic AGCTs, and could be used as a valid tool in the diagnosis of the disease and in cases of metastatic lesions from an unknown primary origin. Moreover the concordance of FOXL2 molecular status in primary and associated metastases suggests its early appearance and genomic stability in AGCT tumorigenesis.

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

5577Background: 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 a...

Molecular cloning, computational analysis and expression pattern of forkhead box l2 (Foxl2) gene in catfish

Foxl2 belongs to forkhead/HNF-3-related family of transcription factors which is involved in ovarian differentiation and development. In present study, the Foxl2 mRNA was cloned from ovary of C. batrachus. The full length cDNA sequence of the Foxl2 was 1056bp which consists of 5′ (41bp) and 3′ (106bp) non-coding regions, as well as a 909bp of open reading frame (ORF) that encodes 302 amino acids. The putative protein was having the theoretical molecular weight (MW) of 34.018kD and a calculated isoelectric point (pI) of 9.38. There were 11 serine (Ser), 5 threonine (Thr), and 5 tyrosine (Tyr) phosphorylation sites and 2 putative N-glycosylation sites on the predicted protein. The ligand binding sites were predicted to be present on amino acids 42, 49, 50, 91, 92 and 95 respectively. The signal peptide analysis predicted that C. batrachus Foxl2 is a non-secretory protein. The hydropathy profile of Foxl2 protein revealed that this protein is hydrophilic in nature. Protein-protein interaction demonstrated that Foxl2 protein chiefly interacts with cytochrome P450 protein family. The mRNA transcript analysis of various tissues indicated that the C. batrachus Foxl2 mRNA was more expressed in the brain, pituitary and ovary in female while, the former two tissues and testis showed low expression in male. This study provides a basis for further structural and functional exploration of the Foxl2 from C. batrachus, including its deduced protein and its signal transduction function.