A conserved function of Human DLC3 and Drosophila Cv-c in testis development.

The identification of genes affecting gonad development is essential to understand the mechanisms causing Variations/Differences in Sex Development (DSD). Recently, a DLC3 mutation was associated with male gonadal dysgenesis in 46,XY DSD patients. We have studied the requirement of Cv-c, the Drosophila ortholog of DLC3, in Drosophila gonad development, as well as the functional capacity of DLC3 human variants to rescue cv-c gonad defects. We show that Cv-c is required to maintain testis integrity during fly development. We find that Cv-c and human DLC3 can perform the same function in fly embryos, as flies carrying wild type but not patient DLC3 variations can rescue gonadal dysgenesis, suggesting functional conservation. We also demonstrate that the StART domain mediates Cv-c's function in the male gonad independently from the GAP domain's activity. This work demonstrates a role for DLC3/Cv-c in male gonadogenesis and highlights a novel StART domain mediated function required to organize the gonadal mesoderm and maintain its interaction with the germ cells during testis development.

Estrogens: Two nuclear receptors, multiple possibilities.

Much is known about estrogen action in experimental animal models and in human physiology. This article reviews the mechanisms of estrogen activity in animals and humans and the role of its two receptors α and ß in terms of structure and mechanisms of action in various tissues in health and in relationship with human pathologies (e.g., osteoporosis). Recently, the spectrum of clinical pictures of estrogen resistance caused by estrogen receptors gene variants has been widened by our description of a woman with ß-receptor defect, which could be added to the already known descriptions of α-receptor defect in women and men and ß-receptor defect in men. The essential role of the ß-receptor in the development of the gonad stands out. We summarize the clinical pictures due to estrogen resistance in men and women and focus on long-term follow-up of two women, one with α- and the other with ß-receptor resistance. Some open questions remain on the complex interactions between the two receptors on bone metabolism and hypothalamus-pituitary-gonadal axis, which need further deepening and research.

CBX2 in DSD: The Quirky Kid on the Block.

Sex development is an intricate and crucial process in all vertebrates that ensures the continued propagation of genetic diversity within a species, and ultimately their survival. Perturbations in this process can manifest as disorders/differences of sex development (DSD). Various transcriptional networks have been linked to development of the gonad into either male or female, which is actively driven by a set of genes that function in a juxtaposed manner and is maintained through the developmental stages to preserve the final sexual identity. One such identified gene is Chromobox homolog 2 (CBX2), an important ortholog of the Polycomb group (PcG) proteins, that functions as both chromatin modifier and highly dynamic transactivator. CBX2 was shown to be an essential factor for gonadal development in mammals, as genetic variants or loss-of-function of CBX2 can cause sex reversal in mice and humans. Here we will provide an overview of CBX2, its biological functions at molecular level, and the CBX2-dependent transcriptional landscape in gonadal development and DSD.

The transcriptional regulator CBX2 and ovarian function: A whole genome and whole transcriptome approach.

The chromobox homolog 2 (CBX2) was found to be important for human testis development, but its role in the human ovary remains elusive. We conducted a genome-wide analysis based on DNA adenine methyltransferase identification (DamID) and RNA sequencing strategies to investigate CBX2 in the human granulosa cells. Functional analysis revealed that CBX2 was upstream of genes contributing to ovarian function like folliculogenesis and steroidogenesis (i.e. ESR1, NRG1, AKR1C1, PTGER2, BMP15, BMP2, FSHR and NTRK1/2). We identified CBX2 regulated genes associated with polycystic ovary syndrome (PCOS) such as TGFß, MAP3K15 and DKK1, as well as genes implicated in premature ovarian failure (POF) (i.e. POF1B, BMP15 and HOXA13) and the pituitary deficiency (i.e. LHX4 and KISS1). Our study provided an excellent opportunity to identify genes surrounding CBX2 in the ovary and might contribute to the understanding of ovarian physiopathology causing infertility in women.

CBX2-dependent transcriptional landscape: implications for human sex development and its defects.

Sex development, a complex and indispensable process in all vertebrates, has still not been completely elucidated, although new genes involved in sex development are constantly being discovered and characterized. Chromobox Homolog 2 (CBX2) is one of these new additions and has been identified through a 46,XY girl with double heterozygous variants on CBX2.1, causing Differences of Sex Development (DSD). The mutated CBX2.1 failed to adequately regulate downstream targets important for sex development in humans, specifically steroidogenic factor 1 (NR5A1/SF1). To better place CBX2.1 in the human sex developmental cascade, we performed siRNA and CBX2.1 overexpression experiments and created a complete CRISPR/Cas9-CBX2 knockout in Sertoli-like cells. Furthermore, we deployed Next Generation Sequencing techniques, RNA-Sequencing and DamID-Sequencing, to identify new potential CBX2.1 downstream genes. The combination of these two next generation techniques enabled us to identify genes that are both bound and regulated by CBX2.1. This allowed us not only to expand our current knowledge about the influence of CBX2.1 in human sex development, but also to advance our insight in the mechanisms governing one of the most important decisions during embryonal development, the commitment to either female or male gonads.

Pluripotent Cell Models for Gonadal Research.

Sex development is a complex process involving many genes and hormones. Defects in this process lead to Differences of Sex Development (DSD), a group of heterogeneous conditions not as rare as previously thought. Part of the obstacles in proper management of these patients is due to an incomplete understanding of the genetics programs and molecular pathways involved in sex development and DSD. Several challenges delay progress and the lack of a proper model system for the single patient severely hinders advances in understanding these diseases. The revolutionary techniques of cellular reprogramming and guided in vitro differentiation allow us now to exploit the versatility of induced pluripotent stem cells to create alternatives models for DSD, ideally on a patient-specific personalized basis.

A Human Gonadal Cell Model From Induced Pluripotent Stem Cells.

Sertoli cells are main players in the male gonads development and their study may shed light on 46,XY disorders of sex development (DSD). Mature primary Sertoli cells are incapable of proliferating in prolonged in vitro cultures and the available Sertoli cell models have several limitations since they derive from mouse or human cancer tissues. We differentiated human fibroblasts (HFs)-derived induced pluripotent stem cells into Sertoli-like cells (SLC) and, in order to characterize this new Sertoli cell model, we performed gene expression analyses by NextGeneration Sequencing techniques. This approach revealed that our putative SLC have reduced expression of pluripotency markers and expressed Sertoli cell markers such as SRY-Related HMG-Box 9 (SOX9), vimentin (VIM), and claudin-11 (CLDN-11). More in detail, the transcriptional profile analysis suggested that these cells are in an early stage of Sertoli cells maturation. Harnessing the power of induced pluripotent stem cells, we were able to generate SLC that show genetic and functional similarities to human Sertoli cells (HSerCs). SLC could become an excellent source of patient-specific Sertoli cells that could be of paramount benefit for both basic research and personalized medicine in sex development and reproductive medicine.

Early-Onset Complete Ovarian Failure and Lack of Puberty in a Woman With Mutated Estrogen Receptor ß (ESR2).

Context: Estrogen resistance due to mutations in the estrogen receptor α gene (ESR1) has been described in men and women and is characterized by osteoporosis, delayed bone age and continuous growth in adulthood, and delayed puberty and multiple ovarian cysts in women. Although mutations in the estrogen receptor ß gene ESR2 were found in 46, XY patients with differences of sex development, no genetic variants of ESR2 were linked to gonadal defects in women. Settings and Patient: Here we describe a 16-year-old female patient who came to our tertiary care hospital with complete lack of estrogen action, as demonstrated by absent breast development, primary amenorrhea, and osteoporosis, resembling patients with ESR1 mutation. However, her gonads were clearly abnormal (streak), a finding not observed in ESR1-deficient patients. Design: To gain insights into the molecular consequences of the ESR2 defect, whole exome sequencing and extensive functional transactivation studies in ovarian, bone, and breast cells were conducted, with or without the natural activator of estrogen receptors, 17ß-estradiol. Results: We identified a loss-of-function heterozygous mutation of a highly conserved residue in ESR2 that disrupts estradiol-dependent signaling and has a dominant negative effect, most likely due to failure to interact with its coactivator, nuclear coactivator 1. Conclusions: This is a report of a loss-of-function mutation in the estrogen receptor ß in a young woman with complete ovarian failure, suggesting that ESR2 is necessary for human ovarian determination and/or maintenance and that ESR1 is not sufficient to sustain ovarian function in humans.

Assembling the jigsaw puzzle: CBX2 isoform 2 and its targets in disorders/differences of sex development.

BACKGROUND: One of the defining moments of human life occurs early during embryonic development, when individuals sexually differentiate into either male or female. Perturbation of this process can lead to disorders/differences of sex development (DSD). Chromobox protein homolog 2 (CBX2) has two distinct isoforms, CBX2.1 and CBX2.2: the role of CBX2.1 in DSD has been previously established, yet to date the function of the smaller isoform CBX2.2 remains unknown. METHODS: The genomic DNA of two 46,XY DSD patients was analysed using whole exome sequencing. Furthermore, protein/DNA interaction studies were performed using DNA adenine methyltransferase identification (DamID) to identify putative binding partners of CBX2. Finally, in vitro functional studies were used to elucidate the effect of wild-type and variant CBX2.2 on selected downstream targets. RESULTS: Here, we describe two patients with features of DSD i.e. atypical external genitalia, perineal hypospadias and no palpable gonads, each patient carrying a distinct CBX2.2 variant, p.Cys132Arg (c.394T>C) and p.Cys154fs (c.460delT). We show that both CBX2.2 variants fail to regulate the expression of genes essential for sexual development, leading to a severe 46,XY DSD defect, likely because of a defective expression of EMX2 in the developing gonad. CONCLUSION: Our study indicates a distinct function of the shorter form of CBX2 and by identifying several of its unique targets, can advance our understanding of DSD pathogenesis and ultimately DSD diagnosis and management.

A novel DAX-1 (NR0B1) mutation in a boy with X-linked adrenal hypoplasia congenita.

BACKGROUND: X-linked adrenal hypoplasia congenita (AHC) is caused by mutations in DAX-1 (NR0B1) playing a key role in adrenal and reproductive development. CASE PRESENTATION: Herein we report a 2.5-year-old boy who presented with acute adrenal failure. Family history revealed unexplained death in three brothers of the patient's mother during infancy. Molecular analysis of the DAX-1 gene revealed the presence of a novel hemizygous mutation, c.870C>A in exon 1, leading to the formation of a premature stop codon. The same mutation was identified in the patient's mother. The truncated mutant protein is most likely misfolded, sequestered in the endoplasmic reticulum and therefore cannot bind to and activate its target DNA sequences in the nucleus. CONCLUSIONS: DAX-1 mutation must be considered when diagnosis of primary adrenocortical insufficiency is made, especially if there is a history of unexplained death of maternal male relatives.

Human Sex Development: from Basic Science to Clinical Practice and Back.

The process of sexual differentiation is central for the reproduction of almost all metazoan, and therefore for the maintenance of multicellular organisms. In sex development, we can distinguish two different processes¸ sex determination, the l decision directing the undifferentiated embryo into a sexually dimorphic individual. In mammals, sex determination equals gonadal development. The second process, sex differentiation, takes place once factors produced by the gonads determine the development of the phenotypic sex. Most of the knowledge on the factors involved in sexual development come from animal models and from studies of cases in which the genetic or the gonadal sex does not match the phenotypical sex, i.e. patients affected by disorders of sex development (DSD). This review focuses on the factors involved in gonadal determination and whenever possible, references on the 'prismatic' clinical cases are given. Furthermore, examples of diagnostic procedures for these complex diseases will be discussed.

Clinical follow-up of the first SF-1 insufficient female patient.

OBJECTIVE: Steroidogenic factor 1 (SF-1/NR5A1) plays a crucial role in regulating adrenal development, gonad determination and differentiation, and in the hypothalamic-pituitary control of reproduction and metabolism. In men (46, XY), it is known that mutations in SF-1/NR5A1 gene cause a wide phenotypic spectrum with variable degrees of undervirilization. In recent years, the role of SF-1 in the ovarian function was increasingly discussed and alterations in the gene were related to primary ovarian insufficiency. We describe the follow-up of a 46, XX affected woman with a SF-1 mutation and by comparing our case with the known manifestations reported in the literature, we try to further elucidate the function of SF-1 in the ovary. RESULTS: During infancy, adrenal insufficiency was the only clinical sign of the loss-of-function as ovarian development and function seemed normal. To date, this young woman aged 16.5 years shows normal growth, normal BMI and psychomotor development, has a normal puberty and regular menstruation. CONCLUSION: This report shows one, to date uniquely described, phenotypic variant of SF-1 mutation in a 46, XX affected person with adrenocortical insufficiency but no ovarian dysfunction nor disturbance of pubertal development. To follow the natural history of SF-1 mutation in a 46, XX individual will further shed light on its role in the ovarian function and thus will help to counsel affected patients in future.

[Current SHI physician legislation: succession rules for surgical seats : Surgical vacancies can be filled with orthopedic/trauma surgery specialists only within narrow limits]. / Aktuelles Vertragsarztrecht: Nachfolge bei Chirurgensitzen : Nachbesetzung einer chirurgischen Zulassung durch Fachärzte für Orthopädie/Unfallchirurgie nur in engen Grenzen.

It is a physician's certified continuing medical education category-rather than their actual medical activity, in this case in the field of trauma surgery-that is decisive in filling statutory health insurance (SHI) practice vacancies. This evaluation arising from §16 of the requirement planning guideline applies accordingly when filling physician vacancies. Thus physician vacancies or statutory health insurance (SHI) practice places can only be filled by a physician in the same physician group in line with requirement planning. Scope for argumentation initially remains in the context of filling surgical SHI physician vacancies where the ceding physician is certified as an accident insurance consultant.

Why boys will be boys and girls will be girls: Human sex development and its defects.

Among the most defining events of an individual's life, is the development of a human embryo into male or a female. The phenotypic sex of an individual depends on the type of gonad that develops in the embryo, a process which itself is determined by the genetic setting of the individual. The development of the gonads is different from any other organ, as they possess the potential to differentiate into two functionally distinct organs, testes, or ovaries. Sex development can be divided into two distinctive processes, "sex determination," which is the commitment of the undifferentiated gonad into either a testis or an ovary, a process that is genetically programmed in a critically timed manner and "sex differentiation," which takes place through hormones produced by the gonads, once the developmental sex determination decision has been made. Disruption of any of the genes involved in either the testicular or ovarian development pathway could lead to disorders of sex development. In this review, we provide an insight into the factors important for sex determination, their antagonistic actions and whenever possible, references on the "prismatic" clinical cases are given. Birth Defects Research (Part C) 108:365-379, 2016. © 2016 Wiley Periodicals, Inc.

The Battle of the Sexes: Human Sex Development and Its Disorders.

The process of sexual differentiation is central for reproduction of almost all metazoan and therefore for maintenance of practically all multicellular organisms. In sex development we can distinguish two different processes: First, sex determination is the developmental decision that directs the undifferentiated embryo into a sexually dimorphic individual. In mammals, sex determination equals gonadal development. The second process known as sex differentiation takes place once the sex determination decision has been made through factors produced by the gonads that determine the development of the phenotypic sex. Most of the knowledge on the factors involved in sexual development came from animal models and from studies of cases in whom the genetic or the gonadal sex does not match the phenotypical sex, i.e., patients affected by disorders of sex development (DSD). Generally speaking, factors influencing sex determination are transcriptional regulators, whereas factors important for sex differentiation are secreted hormones and their receptors. This review focuses on the factors involved in gonadal determination, and whenever possible, references on the "prismatic" clinical cases are given.

Ovarian development and disease: The known and the unexpected.

The idea that the female sexual development happens by default was born in the middle of the last century after Jost carried out his innovative experiments to study the bases of differentiation of the reproductive tract, and found that the female reproductive tract develops even in the absence of any gonad. The term default (passive) attributed to the whole female developmental pathway therefore established itself, even if it was not originally so intended. However, recent developments have demonstrated that ovarian development is an active process. WNT4, one of a few factors with a demonstrated function in the ovarian-determination pathway, has been found to be involved in sexual differentiation by suppressing male sexual differentiation, promoting Müllerian ducts differentiation and maintaining oocyte health. WNT4 expression in the ovary seems to be regulated by R-spondin 1 (RSPO1), a thrombospondin family member protein. The role and interactions of WNT4, RSPO1 and other factors, such as FOXL2 as well as the possible role of chromatin modifiers such as the polycomb protein CBX2 in ovarian development and function will be discussed.

Effects of Steroid Hormones on Sex Differences in Cerebral Perfusion.

Sex differences in the brain appear to play an important role in the prevalence and progression of various neuropsychiatric disorders, but to date little is known about the cerebral mechanisms underlying these differences. One widely reported finding is that women demonstrate higher cerebral perfusion than men, but the underlying cause of this difference in perfusion is not known. This study investigated the putative role of steroid hormones such as oestradiol, testosterone, and dehydroepiandrosterone sulphate (DHEAS) as underlying factors influencing cerebral perfusion. We acquired arterial spin labelling perfusion images of 36 healthy adult subjects (16 men, 20 women). Analyses on average whole brain perfusion levels included a multiple regression analysis to test for the relative impact of each hormone on the global perfusion. Additionally, voxel-based analyses were performed to investigate the sex difference in regional perfusion as well as the correlations between local perfusion and serum oestradiol, testosterone, and DHEAS concentrations. Our results replicated the known sex difference in perfusion, with women showing significantly higher global and regional perfusion. For the global perfusion, DHEAS was the only significant predictor amongst the steroid hormones, showing a strong negative correlation with cerebral perfusion. The voxel-based analyses revealed modest sex-dependent correlations between local perfusion and testosterone, in addition to a strong modulatory effect of DHEAS in cortical, subcortical, and cerebellar regions. We conclude that DHEAS in particular may play an important role as an underlying factor driving the difference in cerebral perfusion between men and women.

Genome-wide identification of CBX2 targets: insights in the human sex development network.

Chromobox homolog 2 (CBX2) is a chromatin modifier that plays an important role in sexual development and its disorders (disorders of sex development [DSD]), yet the exact rank and function of human CBX2 in this pathway remains unclear. Here, we performed large-scale mapping and analysis of in vivo target loci of the protein CBX2 in Sertoli-like NT-2D1 cells, using the DNA adenine methyltransferase identification technique. We identified close to 1600 direct targets for CBX2. Intriguingly, validation of selected candidate genes using qRT-PCR in cells overexpressing CBX2 or in which CBX2 has been knocked down indicated that several CBX2-responsive genes encode proteins that are involved in DSD. We further validated these effects on the candidate genes using a mutated CBX2 causing DSD in human patient. Overall, our findings suggest that CBX2 role in the sex development cascade is to stimulate the male pathway and concurrently inhibit the female pathway. These data provide fundamental insights into potential etiology of DSD.

Identification of a SIRT1 mutation in a family with type 1 diabetes.

Type 1 diabetes is caused by autoimmune-mediated ß cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to ß cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes.

Of marsupials and men: "Backdoor" dihydrotestosterone synthesis in male sexual differentiation.

Following development of the fetal bipotential gonad into a testis, male genital differentiation requires testicular androgens. Fetal Leydig cells produce testosterone that is converted to dihydrotestosterone in genital skin, resulting in labio-scrotal fusion. An alternative 'backdoor' pathway of dihydrotestosterone synthesis that bypasses testosterone has been described in marsupials, but its relevance to human biology has been uncertain. The classic and backdoor pathways share many enzymes, but a 3α-reductase, AKR1C2, is unique to the backdoor pathway. Human AKR1C2 mutations cause disordered sexual differentiation, lending weight to the idea that both pathways are required for normal human male genital development. These observations indicate that fetal dihydrotestosterone acts both as a hormone and as a paracrine factor, substantially revising the classic paradigm for fetal male sexual development.