ABSTRACT
Rhodopsins are ubiquitous light-driven membrane proteins with diverse functions, including ion transport. Widely distributed, they are also coded in the genomes of giant viruses infecting phytoplankton where their function is not settled. Here, we examine the properties of OLPVR1 (Organic Lake Phycodnavirus Rhodopsin) and two other type 1 viral channelrhodopsins (VCR1s), and demonstrate that VCR1s accumulate exclusively intracellularly, and, upon illumination, induce calcium release from intracellular IP3-dependent stores. In vivo, this light-induced calcium release is sufficient to remote control muscle contraction in VCR1-expressing tadpoles. VCR1s natively confer light-induced Ca2+ release, suggesting a distinct mechanism for reshaping the response to light of virus-infected algae. The ability of VCR1s to photorelease calcium without altering plasma membrane electrical properties marks them as potential precursors for optogenetics tools, with potential applications in basic research and medicine.
Subject(s)
Calcium , Rhodopsin , Rhodopsin/genetics , Rhodopsin/metabolism , Light , Cell Membrane/metabolism , Phytoplankton/metabolism , Rhodopsins, Microbial/metabolismABSTRACT
By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K+ channel Inhibitor), a specific photoswitchable inhibitor of the pain-related two-pore-domain potassium TREK and TRESK channels. In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.
Subject(s)
Pain , Potassium Channels, Tandem Pore Domain , Animals , Mice , Drug Evaluation, Preclinical , Nociceptors , Nematoda , Potassium Channels, Tandem Pore Domain/antagonists & inhibitorsABSTRACT
The ability to sense pain signals is closely linked to the activity of ion channels expressed in nociceptors, the first neurons that transduce noxious stimuli into pain. Among these ion channels, TREK1, TREK2 and TRAAK from the TREK subfamily of the Two-Pore-Domain potassium (K2P) channels, are hyperpolarizing channels that render neurons hypoexcitable. They are regulated by diverse physical and chemical stimuli as well as neurotransmitters through G-protein coupled receptor activation. Here, we review the molecular mechanisms underlying these regulations and their functional relevance in pain and migraine induction.
Subject(s)
Migraine Disorders , Potassium Channels, Tandem Pore Domain , Humans , Pain , Pain Perception , PotassiumABSTRACT
Activation and sensitization of trigeminal ganglia (TG) sensory neurons, leading to the release of pro-inflammatory peptides such as calcitonin gene-related peptide (CGRP), are likely a key component in migraine-related headache induction. Reducing TG neuron excitability represents therefore an attractive alternative strategy to relieve migraine pain. Here by using pharmacology and genetic invalidation ex vivo and in vivo, we demonstrate that activating TREK1 and TREK2 two-pore-domain potassium (K2P) channels inhibits TG neuronal firing sufficiently to fully reverse the migraine-like phenotype induced by NO-donors in rodents. Finally, targeting TREK is as efficient as treatment with CGRP antagonists, which represents one of the most effective migraine therapies. Altogether, our results demonstrate that inhibiting TG excitability by pharmacological activation of TREK channels should be considered as an alternative to the current migraine treatment.
ABSTRACT
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 ß-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in ß-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 ß-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/ß-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that ß-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.
Subject(s)
Cell Differentiation , Germ Cells/cytology , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/cytology , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Female , Germ Cells/metabolism , Male , Mice , Mice, Inbred C57BL , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/metabolism , Wnt Proteins/genetics , beta Catenin/geneticsABSTRACT
Despite the importance of germ cell (GC) differentiation for sexual reproduction, the gene networks underlying their fate remain unclear. Here, we comprehensively characterize the gene expression dynamics during sex determination based on single-cell RNA sequencing of 14 914 XX and XY mouse GCs between embryonic days (E) 9.0 and 16.5. We found that XX and XY GCs diverge transcriptionally as early as E11.5 with upregulation of genes downstream of the bone morphogenic protein (BMP) and nodal/Activin pathways in XY and XX GCs, respectively. We also identified a sex-specific upregulation of genes associated with negative regulation of mRNA processing and an increase in intron retention consistent with a reduction in mRNA splicing in XY testicular GCs by E13.5. Using computational gene regulation network inference analysis, we identified sex-specific, sequential waves of putative key regulator genes during GC differentiation and revealed that the meiotic genes are regulated by positive and negative master modules acting in an antagonistic fashion. Finally, we found that rare adrenal GCs enter meiosis similarly to ovarian GCs but display altered expression of master genes controlling the female and male genetic programs, indicating that the somatic environment is important for GC function. Our data are available on a web platform and provide a molecular roadmap of GC sex determination at single-cell resolution, which will serve as a valuable resource for future studies of gonad development, function, and disease.
Subject(s)
Gene Expression Profiling/methods , Sex Determination Processes , Animals , Female , Gene Expression Regulation, Developmental , Germ Cells , Male , Mice , Mice, Transgenic , Single-Cell Analysis , Time Factors , X Chromosome , Y ChromosomeABSTRACT
In mammals, the timing of meiosis entry is regulated by signals from the gonadal environment. All-trans retinoic acid (ATRA) signaling is considered the key pathway that promotes Stra8 (stimulated by retinoic acid 8) expression and, in turn, meiosis entry. This model, however, is debated because it is based on analyzing the effects of exogenous ATRA on ex vivo gonadal cultures, which not accurately reflects the role of endogenous ATRA. Aldh1a1 and Aldh1a2, two retinaldehyde dehydrogenases synthesizing ATRA, are expressed in the mouse ovaries when meiosis initiates. Contrary to the present view, here, we demonstrate that ATRA-responsive cells are scarce in the ovary. Using three distinct gene deletion models for Aldh1a1;Aldh1a2;Aldh1a3, we show that Stra8 expression is independent of ATRA production by ALDH1A proteins and that germ cells progress through meiosis. Together, these data demonstrate that ATRA signaling is dispensable for instructing meiosis initiation in female germ cells.
Subject(s)
Meiosis , Ovary , Animals , Female , Germ Cells/metabolism , Mammals/metabolism , Mice , Ovary/metabolism , Proteins/metabolism , Tretinoin/pharmacologyABSTRACT
R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/ß-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility.
Subject(s)
Intercellular Signaling Peptides and Proteins/physiology , Oocytes , Ovary , Thrombospondins/physiology , Wnt Signaling Pathway , Animals , Cell Communication , Cell Proliferation , Embryo, Mammalian , Female , Humans , Infant , Mice , Mice, Inbred C57BL , Oocytes/cytology , Oocytes/metabolism , Ovary/cytology , Ovary/metabolismABSTRACT
Testis differentiation requires high levels of proliferation of progenitor cells that give rise to two cell lineages forming the testis, the Sertoli and the Leydig cells. Hence defective cell cycling leads to testicular dysgenesis that has profound effects on androgen production and fertility. The growth factor NRG1 has been implicated in adult Leydig cell proliferation, but a potential function in the fetal testis has not been analysed to date. Here we show that Nrg1 and its receptors ErbB2/3 are already expressed in early gonadal development. Using tissue-specific deletion, we further demonstrate that Nrg1 is required in a dose-dependent manner to induce proliferation of Sertoli progenitor cells and then differentiated Sertoli cells. As a result of reduced numbers of Sertoli cells, Nrg1 knockout mice display a delay in testis differentiation and defects in sex cord partitioning. Taken together Nrg1 signalling is essential for the establishment of the stock of Sertoli cells and thus required to prevent testicular hypoplasia.
Subject(s)
Neuregulin-1/metabolism , Sertoli Cells/cytology , Sertoli Cells/metabolism , Signal Transduction , Testis/cytology , Animals , Cell Count , Cell Differentiation , Cell Proliferation , ErbB Receptors/metabolism , Female , Gene Deletion , Male , Mice, Inbred C57BL , Sex Determination Processes , Stem Cells/cytology , Stem Cells/metabolism , Testis/embryology , Thrombospondins/metabolismABSTRACT
The differentiation of germ cells into oogonia or spermatogonia is the first step that eventually gives rise to fully mature gametes. In the female fetal gonad, the RSPO1/WNT/CTNNB1 signalling pathway is involved in primordial germ cell proliferation and differentiation into female germ cells, which are able to enter meiosis. In the postnatal testis, the WNT/CTNNB1 pathway also mediates proliferation of spermatogonial stem cells and progenitor cells. Here we show that forced activation of the WNT/CTNNB1 pathway in fetal gonocytes using transgenic mice leads to deregulated spermatogonial proliferation, and exhaustion of the spermatocytes by apoptosis, resulting in a hypoplastic testis. These findings demonstrate that a finely tuned timing in WNT/CTNNB1 signalling activity is required for spermatogenesis.
Subject(s)
Adult Germline Stem Cells/cytology , Enzyme Activation/physiology , Spermatogenesis/physiology , Spermatogonia/cytology , Wnt Signaling Pathway/physiology , beta Catenin/metabolism , Animals , Apoptosis , Cell Differentiation , Cell Proliferation/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Spermatocytes/cytology , Testis/pathologyABSTRACT
Retinoic acid (RA) is a potent inducer of cell differentiation and plays an essential role in sex-specific germ cell development in the mammalian gonad. RA is essential for male gametogenesis and hence fertility. However, RA can also disrupt sexual cell fate in somatic cells of the testis, promoting transdifferentiation of male Sertoli cells to female granulosa-like cells when the male sexual regulator Dmrt1 is absent. The feminizing ability of RA in the Dmrt1 mutant somatic testis suggests that RA might normally play a role in somatic cell differentiation or cell fate maintenance in the ovary. To test for this possibility we disrupted RA signaling in somatic cells of the early fetal ovary using three genetic strategies and one pharmaceutical approach. We found that deleting all three RA receptors (RARs) in the XX somatic gonad at the time of sex determination did not significantly affect ovarian differentiation, follicle development, or female fertility. Transcriptome analysis of adult triple mutant ovaries revealed remarkably little effect on gene expression in the absence of somatic RAR function. Likewise, deletion of three RA synthesis enzymes (Aldh1a1-3) at the time of sex determination did not masculinize the ovary. A dominant-negative RAR transgene altered granulosa cell proliferation, likely due to interference with a non-RA signaling pathway, but did not prevent granulosa cell specification and oogenesis or abolish fertility. Finally, culture of fetal XX gonads with an RAR antagonist blocked germ cell meiotic initiation but did not disrupt sex-biased gene expression. We conclude that RA signaling, although crucial in the ovary for meiotic initiation, is not required for granulosa cell specification, differentiation, or reproductive function.
Subject(s)
Ovary/embryology , Ovary/metabolism , Signal Transduction/drug effects , Tretinoin/pharmacology , Aldehyde Dehydrogenase 1 Family , Animals , Cell Lineage/drug effects , Female , Fetus/embryology , Fetus/metabolism , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Gene Knockout Techniques , Genes, Dominant , Isoenzymes/metabolism , Male , Mammals , Meiosis/drug effects , Mesonephros/drug effects , Mesonephros/embryology , Mesonephros/metabolism , Mice , Ovary/drug effects , Receptors, Retinoic Acid/metabolism , Retinal Dehydrogenase/metabolism , Retinoids/pharmacology , Sex Determination Processes/drug effects , Tissue Culture TechniquesABSTRACT
Wnt-mediated signals are involved in many important steps in mammalian regeneration. In multiple cell types, the R-spondin (Rspo) family of secreted proteins potently activates the canonical Wnt/ß-catenin pathway. Here, we identify Rspo1 as a mediator of skeletal muscle tissue repair. First, we show that deletion of Rspo1 results in global alteration of muscle regeneration kinetics following acute injury. We find that muscle progenitor cells lacking Rspo1 show delayed differentiation due to reduced activation of Wnt/ß-catenin target genes. Furthermore, muscle cells lacking Rspo1 have a fusion phenotype leading to larger myotubes containing supernumerary nuclei both in vitro and in vivo. The increase in muscle fusion was dependent on downregulation of Wnt/ß-catenin and upregulation of non-canonical Wnt7a/Fzd7/Rac1 signaling. We conclude that reciprocal control of antagonistic Wnt signaling pathways by Rspo1 in muscle stem cell progeny is a key step ensuring normal tissue architecture restoration following acute damage.
Subject(s)
Myoblasts/cytology , Myoblasts/metabolism , Thrombospondins/metabolism , Wnt Signaling Pathway , Animals , Cell Differentiation , Cell Fusion , Cell Proliferation , Cells, Cultured , Mice, Inbred C57BL , Muscle Development , PAX7 Transcription Factor/metabolism , Regeneration , Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/metabolism , beta Catenin/metabolismABSTRACT
In mammals, sex determination is a process through which the gonad is committed to differentiate into a testis or an ovary. This process relies on a delicate balance between genetic pathways that promote one fate and inhibit the other. Once the gonad is committed to the female pathway, ovarian differentiation begins and, depending on the species, is completed during gestation or shortly after birth. During this step, granulosa cell precursors, steroidogenic cells, and primordial germ cells start to express female-specific markers in a sex-dimorphic manner. The germ cells then arrest at prophase I of meiosis and, together with somatic cells, assemble into functional structures. This organization gives the ovary its definitive morphology and functionality during folliculogenesis. Until now, 2 main genetic cascades have been shown to be involved in female sex differentiation. The first is driven by FOXL2, a transcription factor that also plays a crucial role in folliculogenesis and ovarian fate maintenance in adults. The other operates through the WNT/CTNNB1 canonical pathway and is regulated primarily by R-spondin1. Here, we discuss the roles of FOXL2 and RSPO1/WNT/ CTNNB1 during ovarian development and homeostasis in different models, such as humans, goats, and rodents.
Subject(s)
Forkhead Transcription Factors/metabolism , Ovary/metabolism , Sex Determination Processes/physiology , Thrombospondins/metabolism , Animals , Female , Forkhead Transcription Factors/genetics , Germ Cells/metabolism , Germ Cells/physiology , Humans , Male , Mammals , Ovary/physiology , Sex Determination Processes/genetics , Testis/metabolism , Testis/physiology , Thrombospondins/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
Sex differentiation is a unique developmental process. Starting from a bipotential gonad, it gives rise to the ovary and the testis, two highly specialized organs that differ morphologically and physiologically despite sharing common reproductive and endocrine functions. This highlights the specific plasticity of the gonadal precursors and the existence of complex antagonistic genetic regulation. Mammalian sex determination is controlled by paternal transmission of the Y-linked gene, sex-determining region Y (SRY). Using mouse models, it has been shown that the main role of Sry is to activate the expression of the transcription factor Sox9; either one of these two genes is necessary and sufficient to allow testicular development through Sertoli cell differentiation. Thus, defects in SRY/Sry and/or SOX9/Sox9 expression result in male-to-female sex reversal of XY individuals. Molecular mechanisms governing ovarian differentiation remained unknown for a long time, until the discovery of the roles of R-spondin1 (RSPO1) and WNT4. In XX individuals, activation of the ß-catenin signaling pathway by the secreted proteins RSPO1 and WNT4 is required to allow granulosa cell differentiation and, in turn, ovarian differentiation. Thus, mutations in RSPO1 result in female-to-male sex reversal of XX patients, and mouse models have allowed the identification of genetic cascades activated by RSPO1 and WNT4 to regulate ovarian development. In this review, we will discuss the respective roles of RSPO1, WNT4, and the ß-catenin signaling pathway during ovarian differentiation in mice.
Subject(s)
Cell Differentiation/physiology , Morphogenesis/physiology , Ovary/cytology , Signal Transduction/physiology , Thrombospondins/physiology , Wnt4 Protein/physiology , beta Catenin/physiology , 46, XX Testicular Disorders of Sex Development/physiopathology , Animals , Biological Evolution , Female , Gene Expression Regulation, Developmental/physiology , Germ Cells/cytology , Male , Mice , Mice, Knockout , Models, Animal , Sex Determination Processes/physiology , Thrombospondins/deficiency , Thrombospondins/genetics , Wnt4 Protein/deficiency , Wnt4 Protein/geneticsABSTRACT
The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.
Subject(s)
Fetus/cytology , Gene Expression Regulation, Developmental/physiology , Genetic Markers/genetics , Ovary/cytology , Ovary/embryology , Receptors, G-Protein-Coupled/metabolism , Animals , COUP Transcription Factor II/metabolism , Cell Lineage/physiology , Female , Fluorescent Antibody Technique , Forkhead Box Protein L2 , Forkhead Transcription Factors/metabolism , In Situ Hybridization , Mice , Mice, Inbred C57BL , Ovary/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
Mammalian sex determination is controlled by antagonistic pathways that are initially co-expressed in the bipotential gonad and subsequently become male- or female-specific. In XY gonads, testis development is initiated by upregulation of Sox9 by SRY in pre-Sertoli cells. Disruption of either gene leads to complete male-to-female sex reversal. Ovarian development is dependent on canonical Wnt signaling through Wnt4, Rspo1 and ß-catenin. However, only a partial female-to-male sex reversal results from disruption of these ovary-promoting genes. In Wnt4 and Rspo1 mutants, there is evidence of pregranulosa cell-to-Sertoli cell transdifferentiation near birth, following a severe decline in germ cells. It is currently unclear why primary sex reversal does not occur at the sex-determining stage, but instead occurs near birth in these mutants. Here we show that Wnt4-null and Rspo1-null pregranulosa cells transition through a differentiated granulosa cell state prior to transdifferentiating towards a Sertoli cell fate. This transition is preceded by a wave of germ cell death that is closely associated with the disruption of pregranulosa cell quiescence. Our results suggest that maintenance of mitotic arrest in pregranulosa cells may preclude upregulation of Sox9 in cases where female sex-determining genes are disrupted. This may explain the lack of complete sex reversal in such mutants at the sex-determining stage.
Subject(s)
Cell Cycle Checkpoints , Cell Differentiation , Cell Transdifferentiation , Granulosa Cells/cytology , Mitosis , Mutation/genetics , Wnt4 Protein/metabolism , Animals , Animals, Newborn , Anti-Mullerian Hormone/metabolism , Cell Death , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Female , Forkhead Box Protein L2 , Forkhead Transcription Factors/metabolism , Germ Cells/cytology , Germ Cells/metabolism , Granulosa Cells/metabolism , Male , Meiosis , Mice , Sertoli Cells/cytology , Sertoli Cells/metabolism , Thrombospondins/metabolismABSTRACT
The gonad arises from the thickening of the coelomic epithelium and then commits into the sex determination process. Testis differentiation is activated by the expression of the Y-linked gene Sry, which promotes cell proliferation and differentiation of Sertoli cells, the supporting cells of the testis. In absence of Sry (XX individuals), activation of WNT/CTNNB1 signalling, via the upregulation of Rspo1 and Wnt4, promotes ovarian differentiation. However, Rspo1 and Wnt4 are expressed in the early undifferentiated gonad of both sexes, and Axin2-lacZ, a reporter of canonical WNT/CTNNB1 signalling, is expressed in the coelomic region of the E11.5 gonadal primordium, suggesting a role of these factors in early gonadal development. Here, we show that simultaneous ablation of Rspo1 and Wnt4 impairs proliferation of the cells of the coelomic epithelium, reducing the number of progenitors of Sertoli cells in XY mutant gonads. As a consequence, in XY Wnt4(-/-); Rspo1(-/-) foetuses, this leads to the differentiation of a reduced number of Sertoli cells and the formation of a hypoplastic testis exhibiting few seminiferous tubules. Hence, this study identifies Rspo1 and Wnt4 as two new regulators of cell proliferation in the early gonad regardless of its sex, in addition to the specific role of these genes in ovarian differentiation.
Subject(s)
Gonads/embryology , Sex Determination Processes , Thrombospondins/metabolism , Wnt4 Protein/metabolism , beta Catenin/metabolism , Animals , Cell Differentiation/genetics , Cell Proliferation , Female , Gonads/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovary/embryology , SOX9 Transcription Factor/biosynthesis , Sertoli Cells/metabolism , Signal Transduction , Testis/embryology , Thrombospondins/genetics , Wnt4 Protein/geneticsABSTRACT
In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1(KO)Sox9(cKO) ()), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1(KO)Sox9(cKO) mice. Moreover, since testis development occurs in XY Rspo1(KO)Sox9(cKO) mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9(cKO) mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised.
Subject(s)
SOX9 Transcription Factor/genetics , Sex Determination Processes/genetics , Sex Differentiation/genetics , Testis , Thrombospondins/genetics , Animals , Cell Differentiation , Female , Gene Expression Regulation, Developmental , Genes, sry , Humans , Male , Mice , Ovary/growth & development , Ovary/metabolism , SOX9 Transcription Factor/metabolism , Signal Transduction , Testis/cytology , Testis/growth & development , Thrombospondins/metabolism , Wnt Signaling PathwayABSTRACT
Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/ß-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/ß-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that ß-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/ß-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.
Subject(s)
Cell Differentiation , Meiosis , Oogonia/cytology , Ovary/embryology , Signal Transduction , Thrombospondins/metabolism , beta Catenin/metabolism , Adaptor Proteins, Signal Transducing , Animals , Cell Differentiation/genetics , Cell Lineage , Cell Proliferation , Female , Fetus/cytology , Fetus/metabolism , Fibroblast Growth Factor 9/metabolism , Gene Expression Regulation, Developmental , Male , Meiosis/genetics , Mice , Mutation/genetics , Oogonia/metabolism , Ovary/cytology , Ovary/metabolism , Proteins/metabolism , Sertoli Cells/cytology , Sertoli Cells/metabolism , Thrombospondins/deficiency , Transcription, Genetic , Up-Regulation/genetics , beta Catenin/geneticsABSTRACT
The sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9(Tg/+) gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads and is able to induce the expression of EGFP, knocked into the 3' UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9-EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX fetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth.