The role of anti-WNT ZNRF3 is central to determining gonadal fate: XY mice lacking functional ZNRF3 exhibit a highly variable gonadal sex reversal phenotype in the fetal period, characterised by appearance of ovarian tissue. To investigate this sex reversal further, we used single-cell RNA-seq to examine the transcriptomes of XY Znrf3-deficient gonads during the mouse sex-determining period. Analyses of cell trajectories in mutant gonads reveal the failure of pre-supporting cells to commit to the Sertoli cell fate, XY granulosa cell development, unstable commitment in those cells that reach the Sertoli path and enhanced contribution to a supporting- like cell fate. By developing a machine learning-based score for transcriptomic similarity to Sertoli and granulosa, we show pervasive disruption to acquisition of testicular cell fate in the mutant supporting cell lineage, with large numbers of cells co-expressing pro-Sertoli and pro-granulosa markers. These data reveal that loss of Znrf3 results in transcriptomic and cellular heterogeneity, with shifts in cellular sex identity that undermine a simple binary model in which mutant supporting cell precursors achieve either Sertoli or granulosa cell differentiation.

There is a thriving, worldwide, biomedical research community working to understand the molecular bases of diseases of all types, continuously driving improved diagnostics and therapies. Developments in genetics and experimental medicine are yielding novel genetic therapies that were hardly dreamt of 40 years ago. But along with these scientific achievements, there exist challenges in ensuring that 21st century medical interventions are accessible to all who need them. This perspective will discuss how preclinical research, with a focus on rare diseases, can better contribute to healthcare ecosystems that are oriented towards greater health equity. This contribution may require changes to the prevailing scientific research culture that will need support from relevant institutions and the wider community.

Purpose Disorders/differences of sex development (DSD) result from variants in many different human genes but, frequently, have no detectable molecular cause. Methods Detailed clinical and genetic phenotyping was conducted on a family with three children. A Sec31a animal model and functional studies were used to investigate the significance of the findings. Results By trio whole-exome DNA sequencing we detected a heterozygous de novo nonsense SEC31A variant, in three children of healthy non-consanguineous parents. The children had different combinations of disorders that included complete gonadal dysgenesis and multiple pituitary hormone deficiency. SEC31A encodes a component of the COPII coat protein complex, necessary for intracellular anterograde vesicle-mediated transport between the endoplasmic reticulum (ER) and Golgi. CRISPR-Cas9 targeted knockout of the orthologous Sec31a gene region resulted in early embryonic lethality in homozygous mice. mRNA expression of ER-stress genes ATF4 and CHOP was increased in the children, suggesting defective protein transport. The pLI score of the gene, from gnomAD data, is 0.02. Conclusions SEC31A might underlie a previously unrecognised clinical syndrome comprising gonadal dysgenesis, multiple pituitary hormone deficiencies, dysmorphic features and developmental delay. However, a variant that remains undetected, in a different gene, may alternatively be causal in this family.

Sex determination in mammals is controlled by the dominance of either pro-testis (SRY-SOX9-FGF9) or pro-ovary (RSPO1-WNT4-FOXL2) genetic pathways during early gonad development in XY and XX embryos, respectively. We have previously shown that early, robust expression of mouse Sry is dependent on the nuclear protein GADD45g. In the absence of GADD45g, XY gonadal sex reversal occurs, associated with a major reduction of Sry levels at 11.5 dpc. Here, we probe the relationship between Gadd45g and Sry further, using gain- and loss-of-function genetics. First, we show that transgenic Gadd45g overexpression can elevate Sry expression levels at 11.5 dpc in the B6.Y POS model of sex reversal, resulting in phenotypic rescue. We then show that the zygosity of pro-ovarian Rspo1 is critical for the degree of gonadal sex reversal observed in both B6.Y POS and Gadd45g -deficient XY gonads, in contrast to that of Foxl2 . Phenotypic rescue of sex reversal is observed in XY gonads lacking both Gadd45g and Rspo1 , but this is not associated with rescue of Sry expression levels at 11.5 dpc. Instead, Sox9 levels are rescued by around 12.5 dpc. We conclude that Gadd45g is absolutely required for timely expression of Sry in XY gonads, independently of RSPO1-mediated WNT signalling, and discuss these data in light of our understanding of antagonistic interactions between the pro-testis and pro-ovary pathways.

Abstract Introduction XYdisorders of sex development (DSD) result from variants in many different human genes but frequently have no detectable molecular cause. In approximately 25% of cases of XY DSD, the index case may have associated malformations. Genetic disorders of endoplasmic reticulum (ER) function are increasingly being recognised but have not been associated with DSD or pituitary disorders. Clinical case Three siblings (with unaffected non-consanguineous parents) were reviewed at the tertiary endocrine clinic. Child I was noted at birth to have cliteromegaly. Imaging and examination under anaesthetic revealed a normal vagina and uterus but gonads of indeterminate origin. She was 46,XY and basal endocrine investigations at the age of 4 years showed a low AMH for male but otherwise normal gonadal and thyroid function and normal IGF-1. She had a laparoscopic bilateral gonadectomy aged 5 years. Pathology demonstrated bilateral testicular tissue, with substantial fibrotic atrophic change and occasional placental alkaline phosphatase (PLAP) positive cells, suggestive of germ cell tumours. Aged 8 years she developed obesity and later hypertension. Child II was reviewed due to short stature and diagnosed with GH deficiency aged 2 years. She has normal adrenal and thyroid function and gonadotrophins. MRI demonstrated an ectopic posterior pituitary. Child III presented with perineal hypospadias, a small phallus, bilateral undescended testes and craniofacial abnormalities. Endocrine investigations revealed hypogonadotrophic hypogonadism, with no testosterone response to hCG stimulation, a low normal AMH and no response of LH or FSH on LHRH stimulation. He has panhypopituitarism with an ectopic posterior pituitary gland on MRI and is currently on treatment with GH, hydrocortisone and levothyroxine. His BP is on the 98th centile for age and height. Child I and Child III have mild developmental delay but are in mainstream school with additional educational support. High-throughput DNA sequencing revealed, in all three siblings, a heterozygous truncating variant in the SEC31A gene that encodes a component of the COPII-complex that coats the vesicles mediating ER to Golgi transport. CRISPR-Cas9 targeted knockout of the corresponding Sec31a region resulted in embryonic lethality in homozygous mice. mRNA phenotyping of ER-related genes demonstrated increased mRNA expression of ATF4 and CHOP in the affected children, genes encoding key ER stress-related proteins, associated with defective protein transport. Conclusions Dysregulation ofanterograde and retrograde COPII-coated-vesicle ER-Golgi transport is increasingly recognised to underlie human developmental disorders, including Craniolenticulosutural dysplasia (OMIM 607812) and Saul-Wilson syndrome (OMIM 618150). The de novo SEC31A nonsense variant in all three affected siblings, the ER stress response, plus reported developmental syndromes with dysfunction of this transport mechanism and evidence from the preclinical mouse model suggest that SEC31A might underlie a previously unrecognised clinical syndrome comprising DSD, endocrine abnormalities, dysmorphic features and developmental delay. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Context Homozygous and heterozygous variants in PPP2R3C are associated with syndromic 46,XY complete gonadal dysgenesis (Myo-Ectodermo-Gonadal Dysgenesis (MEGD) syndrome), and impaired spermatogenesis, respectively. This study expands the role of PPP2R3C in the aetiology of gonadal dysgenesis (GD). Method We sequenced the PPP2R3C gene in four new patients from three unrelated families. The clinical, laboratory, and molecular characteristics were investigated. We have also determined the requirement for Ppp2r3c in mice (C57BL6/N) using CRISPR/Cas9 genome editing. Results A homozygous c.578T>C (p.L193S) PPP2R3C variant was identified in one 46,XX girl with primary gonadal insufficiency, two girls with 46,XY complete GD, and one undervirilised boy with 46,XY partial GD. The patients with complete GD had low gonadal and adrenal androgens, low anti-Müllerian hormone, and high follicle-stimulating hormone and luteinizing hormone concentrations. All patients manifested characteristic features of MEGD syndrome. Heterozygous Ppp2r3c knockout mice appeared overtly normal and fertile. Inspection of homozygous embryos at 14.5, 9.5, and 8.5 days post coitum(dpc) revealed evidence of dead embryos. We conclude that loss of function of Ppp2r3c is not compatible with viability in mice and results in embryonic death from 7.5 dpc or earlier. Conclusion Our data indicate the essential roles for PPP2R3C in mouse and human development. Germline homozygous variants in human PPP2R3C are associated with distinctive syndromic GD of varying severity in both 46,XY and 46,XX individuals.

Background The human INHA gene encodes the inhibin subunit alpha protein, which is common to both inhibin A and B. The functional importance of inhibins in male sex development, sexual function, and reproduction remain largely unknown. Objective We report for the first time two male siblings with homozygous INHA mutations. Methods The medical files were examined for clinical, biochemical, and imaging data. Genetic analysis was performed using next-generation and Sanger sequencing methods. Results Two brothers complained of gynecomastia, testicular pain, and had a history of hypospadias. Biochemistry revealed low serum testosterone, high gonadotropin and anti-Mullerian hormone, and very low/undetectable inhibin concentrations, where available. Both patients had azoospermia in the spermiogram. We have identified a homozygous 2 bp deletion (c.208_209delAG, R70Gfs*3) variant, which leads to a truncated INHA protein in both patients, and confirmed heterozygosity in the parents. The external genital development, pubertal onset and progression, reproductive functions, serum gonadotropins, and sex hormones of mother and father, who were heterozygous carriers of the identified mutation, were normal. Conclusion Homozygosity for INHA mutations causes decreased prenatal and postnatal testosterone production and infertility in males, while the heterozygous female and male carriers of INHA mutations do not have any abnormality in sex development and reproduction.

Gonadal sex determination represents a unique model for studying cell fate decisions. However, a complete understanding of the different cell lineages forming the developing testis and ovary remains elusive. Here, we investigated the origin, specification, and subsequent sex-specific differentiation of a previously uncharacterized population of supporting-like cells (SLCs) in the developing mouse gonads. The SLC lineage is closely related to the coelomic epithelium and specified as early as E10.5, making it the first somatic lineage to be specified in the bipotential gonad. SLC progenitors are localized within the genital ridge at the interface with the mesonephros and initially coexpress Wnt4 and Sox9 . SLCs become sexually dimorphic around E12.5, progressively acquire a more Sertoli- or pregranulosa-like identity and contribute to the formation of the rete testis and rete ovarii. Last, we found that WNT4 is a crucial regulator of the SLC lineage and is required for normal development of the rete testis.

The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.

Genome editing, particularly the use of CRISPR-Cas9-based methodologies, is revolutionizing biology through its impacts on research and the translation of these into applications in biomedicine. Somatic genome editing aimed at treating individuals with disease raises some significant ethical issues, but proposed heritable interventions, through the use of genome editing in gametes or embryos, raise a number of distinct social, ethical and political issues. This review will consider some proposed uses of heritable human genome editing (HHGE) and several of the objections to these that have been raised. Making sense of such proposed uses requires viewing HHGE as an assisted reproductive technology (ART) that, like preimplantation genetic testing (PGT) and mitochondrial replacement techniques (MRT), aims to prevent disease transmission during sexual reproduction, rather than acting as a therapy for an existing individual. Applications beyond the paradigm of disease prevention raise even more difficult scientific and ethical questions. Here, I will discuss various themes that are prominent in discussions of the science and ethics of HHGE, including impacts on human dignity and society, the language of HHGE used for public dialogue and the governance of HHGE.

Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell-intrinsic beta-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in beta-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of beta-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3, a negative regulator of WNT/beta-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that beta-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.

The birth of Dolly the sheep in 1996 elicited a tsunami of commentaries, both in the popular media and academic journals, including responses to the prospect of human reproductive cloning. Much of the anxiety expressed over this imagined consequence of Dolly’s genesis revealed fundamental concerns about our losing our commitments to certain ethical goods, such as human dignity, or even ‘what it means to be human’. Over the last 25 years, the focus of much of the ethical debate over human biotechnology has slowly shifted towards other genetic technologies that aim to influence inheritance, such as mitochondrial replacement techniques (MRT) and heritable genome editing. Genome editing, in particular, is a technology with multiple fields of application, actual and potential, in research and innovation. In this review, I suggest that many of the fundamental concerns about the possibility of human reproductive cloning that were precipitated by Dolly persist today in the arguments of those who oppose MRT and any use of heritable human genome editing (HHGE). Whilst I do not accept that an understanding of human nature and dignity alone can demonstrate the ethical unacceptability of such assisted reproductive technologies, there are themes of justice, which extend into our relationships with animals, that demand continued wide-ranging examination and public deliberation. Dolly has cast a long shadow over such discussions, but I suggest that the general existential angst over human uses of biotechnology that she came to symbolise is neither compulsory, nor a reliable guide for how to think about biotechnologies today.

The granulosa cell is the somatic supporting cell of the ovary and plays critical roles in follicle development and germ cell development. In the adult female, granulosa cells continue to support the oocyte, allowing it to be ovulated and, ultimately, fertilised. This review will describe the cellular origins of granulosa cells in the fetal gonad, and specific genes and signalling pathways that are required for specifying and maintaining granulosa cell identity throughout the life of an individual. Relying on recent studies, particularly from the mouse but also humans, it will emphasise new transcriptomic and epigenomic approaches to investigating how the granulosa cell lineage emerges in the bipotential fetal gonad during sex determination and develops subsequently throughout the fetal and perinatal period.

XY C57BL/6J (B6) mice harboring a Mus musculus domesticus-type Y chromosome (YPOS), known as B6.YPOS mice, commonly undergo gonadal sex reversal and develop as phenotypic females. In a minority of cases, B6.YPOS males are identified and a proportion of these are fertile. This phenotypic variability on a congenic B6 background has puzzled geneticists for decades. Recently, a B6.YPOS colony was shown to carry a non-B6-derived region of chromosome 11 that protected against B6.YPOS sex reversal. Here. we show that a B6.YPOS colony bred and archived at the MRC Harwell Institute lacks the chromosome 11 modifier but instead harbors an ∼37 Mb region containing non-B6-derived segments on chromosome 13. This region, which we call Mod13, protects against B6.YPOS sex reversal in a proportion of heterozygous animals through its positive and negative effects on gene expression during primary sex determination. We discuss Mod13's influence on the testis determination process and its possible origin in light of sequence similarities to that region in other mouse genomes. Our data reveal that the B6.YPOS sex reversal phenomenon is genetically complex and the explanation of observed phenotypic variability is likely dependent on the breeding history of any local colony.

Mitochondria are organelles that generate energy required for biochemical reactions in cells. They contain a small genome, mitochondrial DNA (mtDNA), which encodes a variety of protein and RNA molecules required for energy production. Individuals inherit mtDNA only from their mother, and if this maternal mtDNA contains a disruptive mutation, it may result in diseases in offspring, which are frequently devastating and life-limiting. Mitochondrial donation (MD), or mitochondrial replacement, comprises a group of related embryological techniques that can be used to prevent or reduce transmission of mtDNA from mother to offspring in order to avoid disease transmission. It does so by replacing the mtDNA of an egg or embryo with that of a donor egg or embryo. The clinical use of two of these techniques, maternal spindle transfer (MST) and pronuclear transfer (PNT), for the prevention of transmission of mitochondrial diseases is lawful in the United Kingdom. This chapter describes the role of mtDNA in mitochondrial disease, the basics of the methodologies of MST and PNT, and issues that must be addressed in their technical refinement. MD has attracted much attention due to the fact that it modifies the inheritance of mtDNA by altering constituents of the germ line (eggs or embryos). Such germ line interventions are opposed by some people, for a variety of reasons. Here, several arguments that have been deployed against the clinical use of mitochondrial donation are evaluated. Finally, mitochondrial donation is considered in the context of assisted reproductive technologies more broadly and comparisons are made with other lawful and nonlawful germ line interventions.

Primary sex determination is the decision by which the bipotential embryonic gonad commits to either the testicular or ovarian fate. The developing gonad constitutes a unique paradigm for the study of lineage specification, cell fate commitment and the exploration of how distinct cell populations diverge from multipotent progenitors. After the separation of the adreno-gonadal primordium into two distinct primordia, somatic progenitor cells of the gonadal primordium undergo several cell fate decisions and sex-specific cell differentiation. The specification of the supporting and steroidogenic cell lineages into either Sertoli and Leydig cells in the testis, or granulosa and theca cells in the ovary is essential for germ cell development and endocrine function of the gonads. In this review, we focus on the early events leading to gonad formation, including the identity of gonadal progenitors, the genetic networks involved in cell lineage specification, the mutual antagonism of the pro-testis and pro-ovary networks and the importance of timing of developmental events orchestrating testis and ovary development. We discuss, and put into perspective, a number of experiments performed in mice or humans that have shed light on sex-determining mechanisms and, where possible, the clinical significance and limitations of such model organism data.