Functional analysis of Mmd2 and related PAQR genes during sex determination in mice

Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. We show that Mmd2 is expressed during the sex-determining period in XY but not XX gonads, specifically in the Sertoli cell lineage which orchestrates early testis development. Analysis of knockout mice deficient in Sox9 and Sf1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, when we used CRISPR to ablate Mmd2 in the mouse, fetal testis development appeared to progress normally. To determine if other genes might have compensated for the loss of Mmd2, we identified the closely related PAQR family members Paqr8 and Mmd as also being expressed during testis development. We used CRISPR to generate mouse strains deficient in Paqr8 and Mmd, but both knockout lines appeared phenotypically normal and fertile. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. These results may reflect functional redundancy among these factors. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes, and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

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Male mice lacking ADAMTS-16 are fertile but exhibit testes of reduced weight

Scientific Reports ◽

10.1038/s41598-019-53900-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Catherine Livermore ◽

Nick Warr ◽

Nicolas Chalon ◽

Pam Siggers ◽

Joffrey Mianné ◽

...

Keyword(s):

Zinc Binding ◽

Human Testis ◽

Testis Weight ◽

Male Mice ◽

Loss Of Function ◽

Phenotypic Data ◽

Sex Development ◽

Pathological Conditions ◽

Differences Of Sex Development ◽

Testis Determination

AbstractAdamts16 encodes a disintegrin-like and metalloproteinase with thrombospondin motifs, 16, a member of a family of multi-domain, zinc-binding proteinases. ADAMTS-16 is implicated in a number of pathological conditions, including hypertension, cancer and osteoarthritis. A large number of observations, including a recent report of human ADAMTS16 variants in cases of 46,XY disorders/differences of sex development (DSD), also implicate this gene in human testis determination. We used CRISPR/Cas9 genome editing to generate a loss-of-function allele in the mouse in order to examine whether ADAMTS-16 functions in mouse testis determination or testicular function. Male mice lacking Adamts16 on the C57BL/6N background undergo normal testis determination in the fetal period. However, adult homozygotes have an average testis weight that is around 10% lower than age-matched controls. Cohorts of mutant males tested at 3-months and 6-months of age were fertile. We conclude that ADAMTS-16 is not required for testis determination or male fertility in mice. We discuss these phenotypic data and their significance for our understanding of ADAMTS-16 function.

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Wnt Signaling in Ovarian Development Inhibits Sf1 Activation of Sox9 via the Tesco Enhancer

Endocrinology ◽

10.1210/en.2011-1347 ◽

2012 ◽

Vol 153 (2) ◽

pp. 901-912 ◽

Cited By ~ 43

Author(s):

Pascal Bernard ◽

Janelle Ryan ◽

Helena Sim ◽

Daniel P. Czech ◽

Andrew H. Sinclair ◽

...

Keyword(s):

Sertoli Cell ◽

Disorders Of Sex Development ◽

Gonadal Development ◽

Ovary Development ◽

Specific Gene ◽

Loss Of Function ◽

Sox9 Expression ◽

Protein Levels ◽

Sex Development ◽

Culture Models

Genome analysis of patients with disorders of sex development, and gain- and loss-of-function studies in mice indicate that gonadal development is regulated by opposing signals. In females, the Wnt/β-catenin canonical pathway blocks testicular differentiation by repressing the expression of the Sertoli cell-specific gene Sox9 by an unknown mechanism. Using cell and embryonic gonad culture models, we show that activation of the Wnt/β-catenin pathway inhibits the expression of Sox9 and Amh, whereas mRNA and protein levels of Sry and steroidogenic factor 1 (Sf1), two key transcriptional regulators of Sox9, are not altered. Ectopic activation of Wnt/β-catenin signaling in male gonads led to a loss of Sf1 binding to the Tesco enhancer and absent Sox9 expression that we also observed in wild-type ovaries. Moreover, ectopic Wnt/β-catenin signaling induced the expression of the female somatic cell markers, Bmp2 and Rspo1, as a likely consequence of Sox9 loss. Wnt/β-catenin signaling in XY gonads did not, however, affect gene expression of the steroidogenic Leydig cell Sf1 target gene, Cyp11a1, or Sf1 binding to the Cyp11a1 promoter. Our data support a model in ovary development whereby activation of β-catenin prevents Sf1 binding to the Sox9 enhancer, thereby inhibiting Sox9 expression and Sertoli cell differentiation.

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The Desert Hedgehog Signalling Pathway in Human Gonadal Development and Differences of Sex Development

Sexual Development ◽

10.1159/000518308 ◽

2021 ◽

pp. 1-14

Author(s):

Svenja Pachernegg ◽

Elizabeth Georges ◽

Katie Ayers

Keyword(s):

Peripheral Neuropathy ◽

Gonadal Dysgenesis ◽

Gonadal Development ◽

Signalling Pathway ◽

Hedgehog Signalling ◽

Sex Development ◽

Xy Gonadal Dysgenesis ◽

Desert Hedgehog ◽

Differences Of Sex Development ◽

Hedgehog Signalling Pathway

While the Hedgehog signalling pathway is implicated in numerous developmental processes and maladies, variants in the Desert Hedgehog (DHH) ligand underlie a condition characterised by 46,XY gonadal dysgenesis with or without peripheral neuropathy. We discuss here the role and regulation of DHH and its signalling pathway in the developing gonads and examine the current understanding of how disruption to this pathway causes this difference of sex development (DSD) in humans.

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Heterozygous deletion of Sox9 in mouse mimics the gonadal sex reversal phenotype associated with campomelic dysplasia in humans

Human Molecular Genetics ◽

10.1093/hmg/ddaa259 ◽

2020 ◽

Author(s):

Stefan Bagheri-Fam ◽

Alexander N Combes ◽

Cheuk K Ling ◽

Dagmar Wilhelm

Keyword(s):

Genetic Background ◽

Digital Pcr ◽

Sex Reversal ◽

Campomelic Dysplasia ◽

Skeletal Malformation ◽

Testis Development ◽

Mixed Genetic Background ◽

Sex Development ◽

Differences Of Sex Development ◽

Human Disorders

Abstract Heterozygous mutations in the human SOX9 gene cause the skeletal malformation syndrome campomelic dysplasia which in 75% of 46,XY individuals is associated with male-to-female sex reversal. While studies in homozygous Sox9 knockout mouse models confirmed that SOX9 is critical for testis development, mice heterozygous for the Sox9-null allele were reported to develop normal testes. This led to the belief that the SOX9 dosage requirement for testis differentiation is different between humans, which often require both alleles, and mice, in which one allele is sufficient. However, in prior studies, gonadal phenotypes in heterozygous Sox9 XY mice were assessed only by either gross morphology, histological staining or analyzed on a mixed genetic background. In this study, we conditionally inactivated Sox9 in somatic cells of developing gonads using the Nr5a1-Cre mouse line on a pure C57BL/6 genetic background. Section and whole-mount immunofluorescence for testicular and ovarian markers showed that XY Sox9 heterozygous gonads developed as ovotestes. Quantitative droplet digital PCR confirmed a 50% reduction of Sox9 mRNA as well as partial sex reversal shown by an upregulation of ovarian genes. Our data show that haploinsufficiency of Sox9 can perturb testis development in mice, suggesting that mice may provide a more accurate model of human disorders/differences of sex development (DSD) than previously thought.

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Ovotesticular disorders of sex development in FGF9 mouse models of human synostosis syndromes

Human Molecular Genetics ◽

10.1093/hmg/ddaa100 ◽

2020 ◽

Vol 29 (13) ◽

pp. 2148-2161

Author(s):

Anthony D Bird ◽

Brittany M Croft ◽

Masayo Harada ◽

Lingyun Tang ◽

Liang Zhao ◽

...

Keyword(s):

Disorders Of Sex Development ◽

Gonadal Development ◽

Testis Development ◽

Sex Development ◽

Mouse Mutants ◽

Skeletal Defects ◽

Xy Sex Reversal

Abstract In mice, male sex determination depends on FGF9 signalling via FGFR2c in the bipotential gonads to maintain the expression of the key testis gene SOX9. In humans, however, while FGFR2 mutations have been linked to 46,XY disorders of sex development (DSD), the role of FGF9 is unresolved. The only reported pathogenic mutations in human FGF9, FGF9S99N and FGF9R62G, are dominant and result in craniosynostosis (fusion of cranial sutures) or multiple synostoses (fusion of limb joints). Whether these synostosis-causing FGF9 mutations impact upon gonadal development and DSD etiology has not been explored. We therefore examined embryonic gonads in the well-characterized Fgf9 missense mouse mutants, Fgf9S99N and Fgf9N143T, which phenocopy the skeletal defects of FGF9S99N and FGF9R62G variants, respectively. XY Fgf9S99N/S99N and XY Fgf9N143T/N143T fetal mouse gonads showed severely disorganized testis cords and partial XY sex reversal at 12.5 days post coitum (dpc), suggesting loss of FGF9 function. By 15.5 dpc, testis development in both mutants had partly recovered. Mitotic analysis in vivo and in vitro suggested that the testicular phenotypes in these mutants arise in part through reduced proliferation of the gonadal supporting cells. These data raise the possibility that human FGF9 mutations causative for dominant skeletal conditions can also lead to loss of FGF9 function in the developing testis, at least in mice. Our data suggest that, in humans, testis development is largely tolerant of deleterious FGF9 mutations which lead to skeletal defects, thus offering an explanation as to why XY DSDs are rare in patients with pathogenic FGF9 variants.

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Generation and mutational analysis of a transgenic mouse model of human SRY

10.1101/2021.03.04.433906 ◽

2021 ◽

Author(s):

Ella Thomson ◽

Liang Zhao ◽

Yen-Shan Chen ◽

Enya Longmuss ◽

Ee Ting Ng ◽

...

Keyword(s):

Mouse Model ◽

Sex Determination ◽

Mutational Analysis ◽

Testis Development ◽

Cellular Effects ◽

Sex Development ◽

Genetic Modifications ◽

Variation Database ◽

Male Sex

AbstractSRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive male sex determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRY’s three domains (N-terminal, HMG box, and C-terminal) and performed detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and the causes of DSD, and present a versatile and powerful system in which to demonstrate causality of SRY variations in DSD, to functionally study the SRY variation database, and to characterize new pathogenic SRY variations found in DSD.

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Regulation of the gonadal transcriptome during sex determination and testis morphogenesis: comparative candidate genes

Reproduction ◽

10.1530/rep-06-0341 ◽

2007 ◽

Vol 134 (3) ◽

pp. 455-472 ◽

Cited By ~ 14

Author(s):

Tracy M Clement ◽

Matthew D Anway ◽

Mehmet Uzumcu ◽

Michael K Skinner

Keyword(s):

Gene Expression ◽

Sex Determination ◽

Candidate Genes ◽

Expression Profiles ◽

Gonadal Development ◽

Developmental Period ◽

Fold Change ◽

Ovary Development ◽

Testis Development

Gene expression profiles during sex determination and gonadal differentiation were investigated to identify new potential regulatory factors. Embryonic day 13 (E13), E14, and E16 rat testes and ovaries were used for microarray analysis, as well as E13 testis organ cultures that undergo testis morphogenesis and develop seminiferous cordsin vitro. A list of 109 genes resulted from a selective analysis for genes present in male gonadal development and with a 1.5-fold change in expression between E13 and E16. Characterization of these 109 genes potentially important for testis development revealed that cytoskeletal-associated proteins, extracellular matrix factors, and signaling factors were highly represented. Throughout the developmental period (E13–E16), sex-enriched transcripts were more prevalent in the male with 34 of the 109 genes having testis-enriched expression during sex determination. In ovaries, the total number of transcripts with a 1.5-fold change in expression between E13 and E16 was similar to the testis, but none of those genes were both ovary enriched and regulated during the developmental period. Genes conserved in sex determination were identified by comparing changing transcripts in the rat analysis herein, to transcripts altered in previously published mouse studies of gonadal sex determination. A comparison of changing mouse and rat transcripts identified 43 genes with species conservation in sex determination and testis development. Profiles of gene expression during E13–E16 rat testis and ovary development are presented and candidate genes for involvement in sex determination and testis differentiation are identified. Analysis of cellular pathways did not reveal any specific pathways involving multiple candidate genes. However, the genes and gene network identified influence numerous cellular processes with cellular differentiation, proliferation, focal contact, RNA localization, and development being predominant.

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The Natural History of a Man With Ovotesticular 46,XX DSD Caused by a Novel 3-Mb 15q26.2 Deletion Containing NR2F2 Gene

Journal of the Endocrine Society ◽

10.1210/js.2019-00241 ◽

2019 ◽

Vol 3 (11) ◽

pp. 2107-2113 ◽

Cited By ~ 4

Author(s):

Gianna Carvalheira ◽

Andrea M Malinverni ◽

Mariana Moysés-Oliveira ◽

Renata Ueta ◽

Leonardo Cardili ◽

...

Keyword(s):

Natural History ◽

Sex Determination ◽

De Novo ◽

External Genitalia ◽

Molecular Study ◽

Sex Development ◽

Differences Of Sex Development ◽

Number Variation ◽

History Of ◽

Male External Genitalia

Abstract Gonadal sex determination is a complex genetic process by which an embryonic primordium is driven to form an ovary or a testis, which requires a delicate dosage balance involving many genes. Disruption in this molecular pathway can lead to differences of sex development (DSD). Although some genetic mechanisms leading to 46,XY DSD have been elucidated, little is known about copy-number variation (CNV) causing testicular or ovotesticular 46,XX DSD. We describe a 20-year natural history of a man with SRY-negative 46,XX who was born with atypical male external genitalia, aortic coarctation, and bilateral blepharophimosis-ptosis. The molecular study identified a de novo heterozygous 3-Mb 15q26.2 deletion, a gene-poor locus containing NR2F2, which encodes the nuclear receptor COUP-TFII that is highly expressed in ovary and cardiac arteries. Immunohistochemistry confirmed the low COUP-TFII expression on his ovotestis tissue. Monosomy of 15q26.2, encompassing the NR2F2 gene, may act as a Z-factor regulating the male sex determination negatively. This finding supports a novel type of CNV resulting in DSD in an individual who developed male puberty spontaneously.

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The molecular genetics of Sry and its role in mammalian sex determination

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1995.0153 ◽

1995 ◽

Vol 350 (1333) ◽

pp. 205-214 ◽

Cited By ~ 32

Keyword(s):

Sex Determination ◽

Target Genes ◽

Gene Action ◽

Human Gene ◽

Cell Lineage ◽

Supporting Cell ◽

Gonadal Development ◽

Genital Ridge ◽

Testis Differentiation ◽

Testicular Differentiation

The process of sex determination, by which is meant the decision as to whether an embryo develops as a male or a female, is considered as a paradigm of how gene action can influence developmental fate. In mammals the decision is dependent on the action of the testis determining gene present on the Y chromosome, now known to be the gene Sry . Sry is expressed for only a brief period in the mouse embryo and must act to initiate rather than maintain the pathway of gene activity required for testis differentiation. It probably acts within cells of the supporting cell lineage to direct their differentiation into Sertoli cells, rather than the granulosa cells characteristic of the ovary. Other lineages in the gonad then follow the male pathway. The nature of the Sry transcript in the genital ridge of mice has been determined and compared with that from the human gene which is dramatically different. The expression of Sry has been carefully examined during the critical stages of genital ridge development and compared to the expression of a number of other genes involved in gonadal development and male development such as that for anti-Mullerian hormone. This has defined the period in which Sry must act to between 11 and 11.5 days post coitum . The expression of Sry has also been examined in cases of sex reversal in the mouse. There is a dependence on level of expression and extent of testicular differentiation that suggests thresholds for both the amount of SRY per cell and the number of cells expressing the gene. The SRY protein interacts with DNA through an HMG box type of DNA binding domain, however at present no definite target genes have been found. Progress on strategies to find such genes is discussed.

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RIP140 Represses Intestinal Paneth Cell Differentiation and Interplays with SOX9 Signaling in Colorectal Cancer

Cancers ◽

10.3390/cancers13133192 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3192

Author(s):

Antoine Gleizes ◽

Mouna Triki ◽

Sandrine Bonnet ◽

Naomi Baccari ◽

Gabriel Jimenez-Dominguez ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Differentiation ◽

Paneth Cell ◽

Wnt Signaling Pathway ◽

Cell Lineage ◽

Human Colon ◽

Luciferase Reporter ◽

Loss Of Function ◽

Sox9 Expression ◽

Human Colon Cancer

RIP140 is a major transcriptional coregulator of gut homeostasis and tumorigenesis through the regulation of Wnt/APC signaling. Here, we investigated the effect of RIP140 on Paneth cell differentiation and its interplay with the transcription factor SOX9. Using loss of function mouse models, human colon cancer cells, and tumor microarray data sets we evaluated the role of RIP140 in SOX9 expression and activity using RT-qPCR, immunohistochemistry, luciferase reporter assays, and GST-pull down. We first evidence that RIP140 strongly represses the Paneth cell lineage in the intestinal epithelium cells by inhibiting Sox9 expression. We then demonstrate that RIP140 interacts with SOX9 and inhibits its transcriptional activity. Our results reveal that the Wnt signaling pathway exerts an opposite regulation on SOX9 and RIP140. Finally, the levels of expression of RIP140 and SOX9 exhibit a reverse response and prognosis value in human colorectal cancer biopsies. This work highlights an intimate transcriptional cross-talk between RIP140 and SOX9 in intestinal physiopathology.

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