RESUMO
Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.
Assuntos
Infertilidade Masculina , Mutação de Sentido Incorreto , Humanos , Masculino , Camundongos , Animais , Reprodução , Alelos , Infertilidade Masculina/genética , Modelos Animais de Doenças , Septinas/genéticaRESUMO
Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share an evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that displayed remarkable conservation in placental mammals and were sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR-Cas9 approach severely impaired stem cell pluripotency. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.
Assuntos
Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes , Animais , Proteínas de Ciclo Celular/metabolismo , Elementos Facilitadores Genéticos/genética , Eutérios/genética , Feminino , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Placenta/metabolismo , Células-Tronco Pluripotentes/metabolismo , Gravidez , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Approximately 7% of men worldwide suffer from infertility, with sperm abnormalities being the most common defect. Though genetic causes are thought to underlie a substantial fraction of idiopathic cases, the actual molecular bases are usually undetermined. Because the consequences of most genetic variants in populations are unknown, this complicates genetic diagnosis even after genome sequencing of patients. Some patients with ciliopathies, including primary ciliary dyskinesia and Bardet-Biedl syndrome, also suffer from infertility because cilia and sperm flagella share several characteristics. Here, we identified two deleterious alleles of RABL2A, a gene essential for normal function of cilia and flagella. Our in silico predictions and in vitro assays suggest that both alleles destabilize the protein. We constructed and analyzed mice homozygous for these two single-nucleotide polymorphisms, Rabl2L119F (rs80006029) and Rabl2V158F (rs200121688), and found that they exhibit ciliopathy-associated disorders including male infertility, early growth retardation, excessive weight gain in adulthood, heterotaxia, pre-axial polydactyly, neural tube defects and hydrocephalus. Our study provides a paradigm for triaging candidate infertility variants in the population for in vivo functional validation, using computational, in vitro and in vivo approaches.
Assuntos
Ciliopatias/etiologia , Infertilidade Masculina/etiologia , Polimorfismo de Nucleotídeo Único , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Ciliopatias/patologia , Feminino , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , FenótipoRESUMO
Primordial germ cells (PGCs) are the founder cells of the germline. The ability to generate PGC-like cells (PGCLCs) from pluripotent stem cells has advanced our knowledge of gametogenesis and holds promise for developing infertility treatments. However, generating an ample supply of PGCLCs for demanding applications such as high-throughput genetic screens has been a limitation. Here, we demonstrated that simultaneous overexpressing 4 transcriptional factors - Nanog and three PGC master regulators Prdm1, Prdm14 and Tfap2c - in suspended mouse epiblast like cells (EpiLCs) and formative embryonic stem cells (ESCs) results in efficient and cost-effective production of PGCLCs. The overexpression of Nanog enhances the PGC regulatory network and suppresses differentiation of somatic lineages, enabling a significant improvement in the efficiency of PGCLC production. Transcriptomic analysis reveals that differentiated PGCLCs exhibit similarities to in vivo PGCs and are more advanced compared to cytokine-induced PGCLCs. These differentiated PGCLCs could be sustained over prolonged periods of culture and could differentiate into spermatogonia-like cells in vitro. Importantly, the ability to produce PGCLCs at scale, without using costly cytokines, enables biochemical and functional genomic screens to dissect mechanisms of germ cell development and infertility.
RESUMO
Certain environmental factors can impact fertility and reproductive parameters such as the number and quality of sperm and eggs. One possible mechanism is the perturbation of epigenetic landscapes in the germline. To explore this possibility, we conducted a CRISPRi screen of epigenetic-related genes to identify those that specifically perturb the differentiation of embryonic stem cells (ESCs) into primordial germ cell-like cells (PGCLCs), exploiting a highly scalable cytokine-free platform. Of the 701 genes screened, inhibition of 53 decreased the efficiency of PGCLC formation. NCOR2, a transcriptional repressor that acts via recruitment of Class I and Class IIa histone deacetylases (HDACs) to gene targets, was particularly potent in suppressing PGCLC differentiation. Consistent with evidence that histone deacetylation is crucial for germline differentiation, we found that the HDAC inhibitors (HDACi) valproic acid (VPA; an anti-convulsant) and sodium butyrate (SB; a widely-used dietary supplement) also suppressed ESC>PGCLC differentiation. Furthermore, exposure of developing mouse embryos to SB or VPA caused hypospermatogenesis. Transcriptome analyses of HDACi-treated, differentiating ESC>PGCLC cultures revealed suppression of germline-associated pathways and enhancement of somatic pathways. This work demonstrates the feasibility of conducting large-scale functional screens of genes, chemicals, or other agents that may impact germline development.
RESUMO
Assisted reproduction procedures often encounter an issue called oocyte maturation arrest (OMA), which is manifested as failed IVF/ICSI attempts using oocytes from some infertile women. In this issue of EMBO Molecular Medicine, Wang et al identify infertile women bearing novel DNA sequence variants in a gene called PABPC1L, which is essential for translation of maternal mRNAs. By conducting a series of in vitro and in vivo experiments, they demonstrated certain variants as being causal for OMA, confirming a conserved requirement for PABPC1L in human oocyte maturation. This study offers a promising therapeutic target for treating OMA patients.
Assuntos
Infertilidade Feminina , Humanos , Feminino , Infertilidade Feminina/genética , OócitosRESUMO
Genetic causes are thought to underlie about half of infertility cases, but understanding the genetic bases has been a major challenge. Modern genomics tools allow more sophisticated exploration of genetic causes of infertility through population, family-based, and individual studies. Nevertheless, potential therapies based on genetic diagnostics will be limited until there is certainty regarding the causality of genetic variants identified in an individual. Genome modulation and editing technologies have revolutionized our ability to functionally test such variants, and also provide a potential means for clinical correction of infertility variants. This review addresses strategies being used to identify causative variants of infertility.
Assuntos
Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Variação Genética , Infertilidade/diagnóstico , Infertilidade/genética , Sistemas CRISPR-Cas , Biologia Computacional/métodos , Gerenciamento Clínico , Edição de Genes/métodos , Testes Genéticos/métodos , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Ensaios de Triagem em Larga Escala , Humanos , Infertilidade/terapia , Técnicas de Reprodução AssistidaRESUMO
Introduction: Fate determination of germline stem cells remains poorly understood at the chromatin structure level. Objectives: Our research hopes to develop successful offspring production of ovarian organoids derived from spermatogonial stem cells (SSCs) by defined factors. Methods: The offspring production from oocytes transdifferentiated from mouse SSCs with tracking of transplanted SSCs in vivo, single cell whole exome sequencing, and in 3D cell culture reconstitution of the process of oogenesis derived from SSCs. The defined factors were screened with ovarian organoids. We uncovered extensive chromatin reorganization during SSC conversion into induced germline stem cells (iGSCs) using high throughput chromosome conformation. Results: We demonstrate successful production of offspring from oocytes transdifferentiated from mouse spermatogonial stem cells (SSCs). Furthermore, we demonstrate direct induction of germline stem cells (iGSCs) differentiated into functional oocytes by transduction of H19, Stella, and Zfp57 and inactivation of Plzf in SSCs after screening with ovarian organoids. We uncovered extensive chromatin reorganization during SSC conversion into iGSCs, which was highly similar to female germline stem cells. We observed that although topologically associating domains were stable during SSC conversion, chromatin interactions changed in a striking manner, altering 35% of inactive and active chromosomal compartments throughout the genome. Conclusion: We demonstrate successful offspring production of ovarian organoids derived from SSCs by defined factors with chromatin reorganization. These findings have important implications in various areas including mammalian gametogenesis, genetic and epigenetic reprogramming, biotechnology, and medicine.
Assuntos
Células-Tronco Germinativas Adultas , Espermatogônias , Animais , Técnicas de Cultura de Células em Três Dimensões , Cromatina/genética , Feminino , Masculino , Camundongos , OrganoidesRESUMO
Perfluorooctane acid (PFOA), a persistent organic pollutant, is ubiquitously present in the environment and may detrimentally affect male reproductive health. In this study, mature human sperm were in vitro exposed to different concentrations of PFOA (0.25, 2.5 or 25⯵g/ml) alone or in combination with progesterone (P4) to evaluate the toxicity and the potential mechanism of action. Exposure to high-dose PFOA (25⯵g/ml) alone for 4â¯h caused a decline in capacity of human spermatozoa to penetrate synthetic mucus, with an increased production of reactive oxygen species (ROS). Furthermore, PFOA treatment (2.5 and 25⯵g/ml) evoked a transient rise in intracellular calcium concentration [Ca2+]i by activating the sperm-specific CatSper channel. However, preincubation with PFOA (2.5-25⯵g/ml) for 4â¯h significantly suppressed P4-stimulated extracellular Ca2+ influx in human spermatozoa. Moreover, PFOA pretreatment at all concentrations evaluated markedly compromised P4-induced acrosome reaction and sperm penetration into viscous medium. Taken together, these results suggest that PFOA exposure may impair human sperm function through inducing oxidative stress and disturbing P4-induced Ca2+ signaling.
Assuntos
Canais de Cálcio/metabolismo , Fluorocarbonos/toxicidade , Substâncias Perigosas/toxicidade , Reação Acrossômica , Cálcio/metabolismo , Humanos , Masculino , Progesterona/farmacologia , Espermatozoides/metabolismoRESUMO
Perfluorooctane acid (PFOA), a typical perfluorinated chemical, has been suggested to interfere with male reproductive function. In this study, mouse spermatogonial GC-1 cells were in vitro treated with PFOA (250, 500 or 750 µM) for 24 h to investigate the cytotoxicity of PFOA and its underlying mechanisms. Our results indicated that exposure to intermediate and high doses of PFOA suppressed the viability of GC-1 cells in a concentration-dependent manner. Furthermore, PFOA treatment markedly enhanced the generation of reactive oxygen species and malondialdehyde, with diminished activity of superoxide dismutase. Particularly, PFOA exposure evoked a decline in mitochondrial membrane potential and ATP production. Furthermore, the apoptotic index and caspase-3 activity were significantly elevated after treatment with PFOA. In addition, PFOA incubation caused an increase in LC3B-II/LC3B-I ratio. Meanwhile, PFOA resulted in an excessive accumulation of autophagosomes in the cytoplasm. Taken together, exposure to PFOA can elicit cytotoxicity to spermatogonial GC-1 cells in vitro, which may be link to the mitochondrial oxidative damage and induction of apoptosis and autophagy.
Assuntos
Caprilatos/toxicidade , Fluorocarbonos/toxicidade , Acetatos , Animais , Apoptose , Autofagia , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Fenóis , Espécies Reativas de Oxigênio , Superóxido DismutaseRESUMO
Stem cells are unique cell types with the ability of self-renewal and differentiation, which mainly include embryonic stem cells, induced pluripotent stem cells, and adult stem cells. Recently, several research groups have reported that stem cells can differentiate into germ cells under appropriate conditions in vitro. Advances in this field have revealed new perspectives for reproductive and regenerative medicine. Here, we review the progress of in vitro gamete production from stem cells.
Assuntos
Diferenciação Celular , Células Germinativas/citologia , Células-Tronco/citologia , Células-Tronco Adultas/citologia , Animais , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
Stem cells have great value in clinical application because of their ability to self-renew and their potential to differentiate into many different cell types. Mammalian gonads, including testes for males and ovaries for females, are composed of germline and somatic cells. In male mammals, spermatogonial stem cells maintain spermatogenesis which occurs continuously in adult testis. Likewise, a growing body of evidence demonstrated that female germline stem cells could be found in mammalian ovaries. Meanwhile, prior studies have shown that somatic stem cells exist in both testes and ovaries. In this chapter, we focus on mammalian gonad stem cells and discuss their characteristics as well as differentiation potentials.
Assuntos
Mamíferos/fisiologia , Ovário/citologia , Células-Tronco/fisiologia , Testículo/citologia , Animais , Diferenciação Celular , Feminino , Humanos , Masculino , Modelos BiológicosRESUMO
Human female germline stem cells (FGSCs) have opened new opportunities for understanding human oogenesis, delaying menopause, treating infertility, and providing a new strategy for preserving fertility. However, the shortage of adult human ovaries tissues available impedes their future investigations and clinical applications. Here, we have established FGSC lines from scarce ovarian cortical tissues that exist in follicular aspirates (faFGSCs), which are produced and discarded in in vitro fertilization centers worldwide. The faFGSCs have characteristics of germline stem cells involved in the gene expression profile, growth characteristics, and a normal karyotype consistent with that of FGSCs obtained from ovarian cortexes surgically removed from patients (srFGSCs). Furthermore, faFGSCs have developmental potentials including spontaneous differentiation into oocytes under feeder-free conditions, communicating with granulosa cells by gap junctions and paracrine factors, entering meiosis after RA induction, as well as forming follicles after injection into human ovarian cortical tissues xenografted into adult immunodeficient female mice. Lastly, we developed a strategy guiding FGSCs differentiated into germinal vesicle (GV) stage oocytes in vitro and revealed their developmental mechanisms. Our study not only provides a new approach to obtain human FGSCs for medical treatment, but also opens several avenues to investigate human oogenesis in vitro.