Inactivation of Wt1 causes pre-granulosa cell to steroidogenic cell transformation and defect of ovary development†.

Wt1 gene encodes a nuclear transcription factor which is specifically expressed in ovarian granulosa cells and testicular Sertoli cells. Our previous studies demonstrated that Wt1 is required for the lineage specification of supporting cells and inactivation of Wt1 results in Sertoli cells to Leydig-like cells transformation. To test whether Wt1 is also involved in lineage maintenance of granulosa cells during ovary development, Wt1 was specifically deleted in pre-granulosa cells using Foxl2-cre. We found that the female Wt1-/flox; Foxl2-cre mice were infertile with atrophic ovaries and no growing follicles with multiple layers of granulosa cells were observed. A large number of 3ß-HSD-positive steroidogenic cells were detected in ovaries of Wt1-/flox; Foxl2-cre mice during embryonic stage and these cells were derived from Foxl2-expressing pre-granulosa cells. The quantitative results showed the expression of granulosa cell marker genes (Foxl2, Follistatin) was downregulated and steroidogenic cell marker genes (3ß-HSD, Cyp11a1, Star and Sf1) was dramatically increased in Wt1-/flox; Foxl2-cre ovaries. We also found that the meiosis of germ cells in Wt1-/flox; Foxl2-cre ovaries was delayed but not arrested. This study demonstrates that Wt1 is required for lineage maintenance of granulosa cells and inactivation of Wt1 results in pre-granulosa cells to steroidogenic cells transformation which in turn causes the defect of ovary development.

Roles of forkhead box protein L2 (foxl2) during gonad differentiation and maintenance in a fish, the olive flounder (Paralichthys olivaceus).

As an important maricultured fish, the olive flounder Paralichthys olivaceus shows sex-dimorphic growth. Thus, the molecular mechanisms involved in sex control in P. olivaceus have attracted researchers' attention. Among the sex-related genes, forkhead box protein L2 (foxl2) exhibits significant sex-dimorphic expression patterns and plays an important role in fish gonad differentiation and development. The present study first investigated the expression levels and promoter methylation dynamics of foxl2 during flounder gonad differentiation under treatments of high temperature and exogenous 17ß-oestradiol (E2). During high temperature treatment, the expression of flounder foxl2 may be repressed via maintenance of DNA methylation. Then, flounder with differentiated testis at Stages I-II were treated with exogenous 5ppm E2 or 5ppm E2+150ppm trilostane (TR) to investigate whether exogenous sex hormones could induce flounder sex reversal. The differentiated testis exhibited phenotypic variations of gonadal dysgenesis with upregulation of female-related genes (foxl2 and cytochrome P450 family 19 subfamily A (cyp19a)) and downregulation of male-related genes (cytochrome P450 family 11 subfamily B member 2 (cyp11b2), doublesex- and mab-3 related transcription factor 1 (dmrt1), anti-Mullerian hormone (amh) and SRY-box transcription factor 9 (sox9)). Furthermore, a cotransfection assay of the cells of the flounder Sertoli cell line indicated that Foxl2 was able alone or with nuclear receptor subfamily 5 group A member 2 (Nr5a2) jointly to upregulate expression of cyp19a. Moreover, Foxl2 and Nr5a2 repressed the expression of dmrt1. In summary, Foxl2 may play an important role in ovarian differentiation by maintaining cyp19a expression and antagonising the expression of dmrt1. However, upregulation of foxl2 is not sufficient to induce the sex reversal of differentiated testis.

Clinical characterization and identification of five novel FOXL2 pathogenic variants in a cohort of 12 Mexican subjects with the syndrome of blepharophimosis-ptosis-epicanthus inversus.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant entity characterized by eyelid malformations and caused by mutations in the forkhead box L2 (FOXL2) gene. Clinical and genetic analyses of large cohorts of BPES patients from different ethnic origins are important for a better characterization of FOXL2 mutational landscape. The purpose of this study is to describe the phenotypic features and the causal FOXL2 variants in a Mexican cohort of BPES patients. A total of 12 individuals with typical facial findings were included. Clinical evaluation included palpebral measurements and levator function assessment. The complete coding sequence of FOXL2 was amplified by PCR and subsequently analyzed by Sanger sequencing. A total of 11 distinct FOXL2 pathogenic variants were identified in our cohort (molecular diagnostic rate of 92%), including 5 novel mutations. Our results broaden the BPES-related mutational spectrum and supports considerable FOXL2 allelic heterogeneity in our population.

Genetic and Functional Analyses of Two Missense Mutations in the Transcription Factor FOXL2 in Two Chinese Families with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome.

BACKGROUND: Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare autosomal-dominant inherited disease. This study was carried out to investigate the genetic and functional changes within the FOXL2 gene in two Chinese families with BPES. MATERIALS AND METHODS: DNA was extracted from the peripheral blood of 26 persons from two different Chinese BPES families (13 of which were affected), as well as 200 cataract patients to act as normal controls. FOXL2 gene mutations were detected using polymerase chain reaction (PCR) and DNA sequencing techniques. Bioinformatic analyses were performed to analyze the structures and functions of the mutant proteins. Wild-type and mutant FOXL2 genes were subcloned into pEGFP-N1 and pCDB vectors and then transfected into COS7 and HEK293T cell lines. We observed protein subcellular localization, and used quantitative real-time (qRT)-PCR and western blots to assess regulation of the target OSR2 gene. RESULTS: We detected two novel missense mutations, c.162G>T (p.Lys54Asn) and c.308G>A (p.Arg103His), in the FOXL2 gene; one in each of the study families. Bioinformatic analyses indicated no obvious differences between the wild-type and mutant protein structures. However, they did predict that the two mutations were likely damaging to protein function. We found that the two mutated proteins were both largely distributed within the nucleus and that there was little found in the cytoplasm. The OSR2 mRNA content decreased significantly when the plasmids carrying the c.162G>T and c.308G>A were transfected into COS7 and HEK293 cell lines, when compared to the empty and the wild-type FOXL2 carrier. Western blot analyses indicated, that after transfecting the c.162G>T mutation, the OSR2 protein level was relatively similar to the wild-type, but that the cells transfected with the c.308G>A mutation showed significantly decreased levels of the OSR2 protein. CONCLUSIONS: Our study broadens the BPES gene mutation spectrum and suggests a possible mechanism of action. It also provides reference data for the further studies of BPES.