FOXL2 interaction with different binding partners regulates the dynamics of ovarian development.

The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing of Foxl2 deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes, suggesting different roles across development. Here, we comprehensively investigated FOXL2's role through a multi-omics approach to characterize gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in somatic cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle formation and steroidogenesis. Deletion of one interactor, ubiquitin-specific protease 7 (Usp7), results in impairment of somatic cell differentiation, germ cell nest breakdown, and ovarian development, leading to sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

Functional rewiring of G protein-coupled receptor signaling in human labor.

Current strategies to manage preterm labor center around inhibition of uterine myometrial contractions, yet do not improve neonatal outcomes as they do not address activation of inflammation. Here, we identify that during human labor, activated oxytocin receptor (OTR) reprograms the prostaglandin E2 receptor, EP2, in the pregnant myometrium to suppress relaxatory/Gαs-cAMP signaling and promote pro-labor/inflammatory responses via altered coupling of EP2 from Gαq/11 to Gαi/o. The ability of EP2 to signal via Gαi/o is recapitulated with in vitro OT and only following OTR activation, suggesting direct EP2-OTR crosstalk. Super-resolution imaging with computational modeling reveals OT-dependent reorganization of EP2-OTR complexes to favor conformations for Gαi over Gαs activation. A selective EP2 ligand, PGN9856i, activates the relaxatory/Gαs-cAMP pathway but not the pro-labor/inflammatory responses in term-pregnant myometrium, even following OT. Our study reveals a mechanism, and provides a potential therapeutic solution, whereby EP2-OTR functional associations could be exploited to delay preterm labor.

TRIM28-dependent SUMOylation protects the adult ovary from activation of the testicular pathway.

Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. In the mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying their protective effect is unknown. Here, we show that TRIM28 is required to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of Trim28, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type, different from gonadal embryonic progenitors. TRIM28 is recruited on chromatin in the proximity of FOXL2 to maintain the ovarian pathway and to repress testicular-specific genes. The role of TRIM28 in ovarian maintenance depends on its E3-SUMO ligase activity that regulates the sex-specific SUMOylation profile of ovarian-specific genes. Our study identifies TRIM28 as a key factor in protecting the adult ovary from the testicular pathway.

Long-Range Regulation of Key Sex Determination Genes.

The development of sexually dimorphic gonads is a unique process that starts with the specification of the bipotential genital ridges and culminates with the development of fully differentiated ovaries and testes in females and males, respectively. Research on sex determination has been mostly focused on the identification of sex determination genes, the majority of which encode for proteins and specifically transcription factors such as SOX9 in the testes and FOXL2 in the ovaries. Our understanding of which factors may be critical for sex determination have benefited from the study of human disorders of sex development (DSD) and animal models, such as the mouse and the goat, as these often replicate the same phenotypes observed in humans when mutations or chromosomic rearrangements arise in protein-coding genes. Despite the advances made so far in explaining the role of key factors such as SRY, SOX9, and FOXL2 and the genes they control, what may regulate these factors upstream is not entirely understood, often resulting in the inability to correctly diagnose DSD patients. The role of non-coding DNA, which represents 98% of the human genome, in sex determination has only recently begun to be fully appreciated. In this review, we summarize the current knowledge on the long-range regulation of 2 important sex determination genes, SOX9 and FOXL2, and discuss the challenges that lie ahead and the many avenues of research yet to be explored in the sex determination field.

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 (WT1) gene.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor ß-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Modeling hormonal and inflammatory contributions to preterm and term labor using uterine temporal transcriptomics.

BACKGROUND: Preterm birth is now recognized as the primary cause of infant mortality worldwide. Interplay between hormonal and inflammatory signaling in the uterus modulates the onset of contractions; however, the relative contribution of each remains unclear. In this study we aimed to characterize temporal transcriptome changes in the uterus preceding term labor and preterm labor (PTL) induced by progesterone withdrawal or inflammation in the mouse and compare these findings with human data. METHODS: Myometrium was collected at multiple time points during gestation and labor from three murine models of parturition: (1) term gestation; (2) PTL induced by RU486; and (3) PTL induced by lipopolysaccharide (LPS). RNA was extracted and cDNA libraries were prepared and sequenced using the Illumina HiSeq 2000 system. Resulting RNA-Seq data were analyzed using multivariate modeling approaches as well as pathway and causal network analyses and compared against human myometrial transcriptome data. RESULTS: We identified a core set of temporal myometrial gene changes associated with term labor and PTL in the mouse induced by either inflammation or progesterone withdrawal. Progesterone withdrawal initiated labor without inflammatory gene activation, yet LPS activation of uterine inflammation was sufficient to override the repressive effects of progesterone and induce a laboring phenotype. Comparison of human and mouse uterine transcriptomic datasets revealed that human labor more closely resembles inflammation-induced PTL in the mouse. CONCLUSIONS: Labor in the mouse can be achieved through inflammatory gene activation yet these changes are not a requisite for labor itself. Human labor more closely resembles LPS-induced PTL in the mouse, supporting an essential role for inflammatory mediators in human "functional progesterone withdrawal." This improved understanding of inflammatory and progesterone influence on the uterine transcriptome has important implications for the development of PTL prevention strategies.

Specific Lipopolysaccharide Serotypes Induce Differential Maternal and Neonatal Inflammatory Responses in a Murine Model of Preterm Labor.

Intrauterine inflammation is recognized as a key mediator of both normal and preterm birth but is also associated with neonatal neurological injury. Lipopolysaccharide (LPS) is often used to stimulate inflammatory pathways in animal models of infection/inflammation-induced preterm labor; however, inconsistencies in maternal and neonatal responses to LPS are frequently reported. We hypothesized that LPS serotype-specific responses may account for a portion of these inconsistencies. Four different Escherichia coli LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection at gestational day 16. Although control animals delivered at term 60 ± 15 hours postinjection (p.i.), those administered with O111:B4 delivered 7 ± 2 hours p.i., O55:B5 delivered 10 ± 3 hours p.i., O127:B8 delivered 16 ± 10 hours p.i., and O128:B12 delivered 17 ± 2 hours p.i. (means ± SD). A correlation between the onset of preterm labor and myometrial activation of the inflammatory transcription factor, activator protein 1, but not NF-κB was observed. Specific LPS serotypes induced differential activation of downstream contractile and inflammatory pathways in myometrium and neonatal pup brain. Our findings demonstrate functional disparity in inflammatory pathway activation in response to differing LPS serotypes. Selective use of LPS serotypes may represent a useful tool for targeting specific inflammatory response mechanisms in these models.

Serum YKL-40 levels and chitotriosidase activity in patients with beta-thalassemia major.

BACKGROUND: YKL-40 association with human disease has been the object of many years of investigation. ß-thalassemia patients are affected by hepatic siderosis, which determines a fibrotic process and tissue remodelling. Chitotriosidase has been found to be increased in thalassemic patients returning to normal in patients submitted to bone marrow transplantation. YKL-40 is associated with macrophage activation in liver and in other tissues. The aim of the study was to analyse the level of serum YKL-40 and plasma chitotriosidase activity of patients with beta-thalassemia to assess whether their expression correlates with liver disease and degree of liver siderosis. METHODS: Expression of YKL-40 and chitotriosidase as a marker of inflammation in 69 thalassemic patients were evaluated. We sought to investigate whether these two chitinases could be considered as a significant biomarker to evaluate therapy effectiveness. RESULTS: Surprisingly we found normal value of YKL-40. We, also, analysed chitotriosidase activity in the same patients that was slightly increased as a consequence of macrophage activation. CONCLUSIONS: These data would suggest a good treatment for these patients.

Activator protein 1 is a key terminal mediator of inflammation-induced preterm labor in mice.

Activation of uterine inflammatory pathways leads to preterm labor (PTL), associated with high rates of neonatal mortality and morbidity. The transcription factors nuclear factor κB (NFκB) and activator protein 1 (AP-1) regulate key proinflammatory and procontractile genes involved in normal labor and PTL. Here we show that NFκB activation normally occurs in the mouse myometrium at gestation day E18, prior to labor, whereas AP-1 and JNK activation occurs at labor onset. Where labor was induced using the progesterone receptor antagonist RU486, NFkB and AP-1/JNK activation both occurred at the time of labor (20 h compared to 60 h in DMSO-treated controls). Using an LPS (Escherichia coli: serotype O111)-induced PTL model that selectively activates AP-1 but not NFkB, we show that myometrial AP-1 activation drives production of cytokines (Il-6, Il-8, and Il-1ß), metalloproteinases (Mmp3 and Mmp10), and procontractile proteins (Cox-2 and Cx43) resulting in PTL after 7 h. Protein levels of CX43 and IL-1ß, and IL-1ß cleavage, were increased following LPS-induced activation of AP-1. Inhibition of JNK by SP600125 (30 mg/kg) delayed PTL by 6 h (7.5 vs. 13.5 h P<0.05). Our data reveal that NFκB activation is not a functional requirement for infection/inflammation-induced preterm labor and that AP-1 activation is sufficient to drive inflammatory pathways that cause PTL.