RESUMEN
Obesity can disturb spermatogenesis and subsequently affect male fertility and reproduction. In our study, we aim to elucidate at which cellular level of adult spermatogenesis the detrimental effects of obesity manifest. We induced high fat diet (HFD) obesity in low-density lipoprotein receptor knock-out Leiden (Ldlr-/-.Leiden) mice, and studied the morphological structure of the testes and histologically examined the proportion of Sertoli cells, spermatocytes and spermatids in the seminiferous tubules. We examined sperm DNA damage and chromatin condensation and measured plasma levels of leptin, testosterone, cholesterol and triglycerides. HFD-induced obesity caused high plasma leptin and abnormal testosterone levels and induced an aberrant intra-tubular organisation (ITO) which is associated with an altered spermatids/spermatocytes ratio (2:1 instead of 3:1). Mice fed a HFD had a higher level of tubules in stages VII + VIII in the spermatogenic cycle. The stages VII + VII indicate crucial processes in spermatogenic development like initiation of meiosis, initiation of spermatid elongation, and release of fully matured spermatids. In conclusion, HFD-induced obese Ldlr-/-.Leiden mice develop an aberrant ITO and alterations in the spermatogenic cycle in crucial stages (stages VII and VII). Thereby, our findings stress the importance of lifestyle guidelines in infertility treatments.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Lipoproteínas LDL/genética , Obesidad/fisiopatología , Espermátides/crecimiento & desarrollo , Espermatogénesis , Animales , Colesterol/sangre , Modelos Animales de Enfermedad , Humanos , Leptina/sangre , Lipoproteínas LDL/deficiencia , Masculino , Meiosis , Ratones , Ratones Noqueados , Obesidad/sangre , Obesidad/etiología , Espermátides/metabolismo , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismo , Testículo/citología , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Testosterona/sangreRESUMEN
During spermatogenesis, the process in which sperm for fertilization are produced from germline cells, gene expression is spatiotemporally highly regulated. In Drosophila, successful expression of extremely large male fertility factor genes on Y-chromosome spanning some megabases due to their gigantic intron sizes is crucial for spermatogenesis. Expression of such extremely large genes must be challenging, but the molecular mechanism that allows it remains unknown. Here we report that a novel RNA-binding protein Maca, which contains two RNA-recognition motifs, is crucial for this process. maca null mutant male flies exhibited a failure in the spermatid individualization process during spermatogenesis, lacked mature sperm, and were completely sterile, while maca mutant female flies were fully fertile. Proteomics and transcriptome analyses revealed that both protein and mRNA abundance of the gigantic male fertility factor genes kl-2, kl-3, and kl-5 (kl genes) are significantly decreased, where the decreases of kl-2 are particularly dramatic, in maca mutant testes. Splicing of the kl-3 transcripts was also dysregulated in maca mutant testes. All these physiological and molecular phenotypes were rescued by a maca transgene in the maca mutant background. Furthermore, we found that in the control genetic background, Maca is exclusively expressed in spermatocytes in testes and enriched at Y-loop A/C in the nucleus, where the kl-5 primary transcripts are localized. Our data suggest that Maca increases transcription processivity, promotes successful splicing of gigantic introns, and/or protects transcripts from premature degradation, of the kl genes. Our study identified a novel RNA-binding protein Maca that is crucial for successful expression of the gigantic male fertility factor genes, spermatogenesis, and male fertility.
Asunto(s)
Drosophila melanogaster/genética , Proteínas de Unión al ARN/genética , Espermátides/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/genética , Transcriptoma , Animales , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Femenino , Fertilidad/genética , Regulación de la Expresión Génica , Ontología de Genes , Genes Reporteros , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Anotación de Secuencia Molecular , Mutación , Proteínas de Unión al ARN/metabolismo , Espermátides/citología , Espermátides/crecimiento & desarrollo , Espermatocitos/citología , Espermatocitos/crecimiento & desarrollo , Testículo/citología , Testículo/metabolismo , Cromosoma Y/químicaRESUMEN
Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 - two essential proteins for maintenance DNA methylation - are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Emparejamiento Cromosómico/genética , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADN/genética , Espermatocitos/crecimiento & desarrollo , Espermatogénesis/genética , Ubiquitina-Proteína Ligasas/genética , Células Madre Germinales Adultas/citología , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Epigénesis Genética/genética , Heterocromatina/metabolismo , Masculino , Ratones , Ratones Noqueados , Espermatocitos/fisiología , Espermatogénesis/fisiología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
In vitro spermatogenesis (IVS) using air-liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.
Asunto(s)
Técnicas de Cultivo de Órganos , Espermátides/crecimiento & desarrollo , Espermatogénesis , Animales , Animales Modificados Genéticamente , Antioxidantes , Medios de Cultivo , Hormonas , Masculino , Meiosis , Oxígeno/análisis , Ratas , Espermátides/citología , Espermatocitos/crecimiento & desarrolloRESUMEN
Many specialized cells use unconventional strategies of cytoskeletal control. Nematode spermatocytes discard their actin and tubulin following meiosis, and instead employ the regulated assembly/disassembly of the Major Sperm Protein (MSP) to drive sperm motility. However, prior to the meiotic divisions, MSP is sequestered through its assembly into paracrystalline structures called fibrous bodies (FBs). The accessory proteins that direct this sequestration process have remained mysterious. This study reveals SPE-18 as an intrinsically disordered protein that is essential for MSP assembly within FBs. In spe-18 mutant spermatocytes, MSP forms disorganized cortical fibers, and the cells arrest in meiosis without forming haploid sperm. In wild-type spermatocytes, SPE-18 localizes to pre-FB complexes and functions with the kinase SPE-6 to localize MSP assembly. Changing patterns of SPE-18 localization uncover previously unappreciated complexities in FB maturation. Later, within newly individualized spermatids, SPE-18 is rapidly lost, yet SPE-18 loss alone is insufficient for MSP disassembly. Our findings reveal an alternative strategy for sequestering cytoskeletal elements, not as monomers but in localized, bundled polymers. Additionally, these studies provide an important example of disordered proteins promoting ordered cellular structures.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Espermatocitos/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Puntos de Control del Ciclo Celular , Citoesqueleto/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Masculino , Meiosis , Mutagénesis , Alineación de Secuencia , Espermátides/metabolismo , Espermatocitos/citología , Espermatocitos/crecimiento & desarrollo , EspermatogénesisRESUMEN
BACKGROUND: Mutation in S-phase cyclin A-associated protein rin the endoplasmic reticulum (SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies. METHODS: Human clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis of short spindle 3 (ssp3) (SCAPER orthologue) Drosophila CAS9-knockout lines. In vitro microtubule (MT) binding assayed by total internal reflection fluorescence microscopy. RESULTS: We show that patients homozygous for a SCAPER mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells. Interestingly, Drosophila null mutants for the ubiquitously expressed ssp3 gene are viable and female fertile but male sterile. We further show that male sterility in ssp3 null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and Ssp3 directly bind MTs. CONCLUSIONS: Our results show that SCAPER null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in ssp3 specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and Drosophila meiosis.
Asunto(s)
Proteínas Portadoras/genética , Infertilidad Masculina/genética , Microtúbulos/genética , Serina Endopeptidasas/genética , Animales , Segregación Cromosómica/genética , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Predisposición Genética a la Enfermedad , Humanos , Infertilidad Masculina/patología , Masculino , Meiosis/genética , Mutación/genética , Espermatocitos/crecimiento & desarrollo , Espermatocitos/patología , Huso Acromático/genética , Huso Acromático/patología , Testículo/crecimiento & desarrollo , Testículo/patologíaRESUMEN
Sexually reproducing organisms use meiosis to generate haploid gametes and faithfully transmit their genome to the next generation. In comparison to oogenesis in many organisms, spermatogenesis is particularly sensitive to small temperature fluctuations, and spermatocytes must develop within a very narrow isotherm [1-4]. Although failure to thermoregulate spermatogenetic tissue and prolonged exposure to elevated temperatures are linked to male infertility in several organisms, the mechanisms of temperature-induced male infertility have not been fully elucidated [5]. Here, we show that upon exposure to a brief 2°C temperature increase, Caenorhabditis elegans spermatocytes exhibit up to a 25-fold increase in double-strand DNA breaks (DSBs) throughout meiotic prophase I and a concurrent reduction in male fertility. We demonstrate that these heat-induced DSBs in spermatocytes are independent of the endonuclease SPO-11. Further, we find that the production of these heat-induced DSBs in spermatocytes correlate with heat-induced mobilization of Tc1/mariner transposable elements, which are known to cause DSBs and alter genome integrity [6, 7]. Moreover, we define the specific sequences and regions of the male genome that preferentially experience these heat-induced de novo Tc1 insertions. In contrast, oocytes do not exhibit changes in DSB formation or Tc1 transposon mobility upon temperature increases. Taken together, our data suggest spermatocytes are less tolerant of higher temperatures because of an inability to effectively repress the movement of specific mobile DNA elements that cause excessive DNA damage and genome alterations, which can impair fertility.
Asunto(s)
Caenorhabditis elegans/fisiología , Elementos Transponibles de ADN/genética , Respuesta al Choque Térmico/genética , Espermatocitos/crecimiento & desarrollo , Espermatogénesis/genética , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Fertilidad/genética , Calor/efectos adversos , Masculino , Oocitos/fisiología , Espermatocitos/enzimología , Transposasas/genética , Transposasas/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) have been shown to execute key roles in spermatogenesis. However, little is known about how lncRNAs gene expression is itself regulated in the germ cells of testis. We previously demonstrated that high expression of lncRNA-Gm2044 exists in spermatocytes and can regulate male germ cell proliferation. Here, the transcriptional regulation of lnRNA-Gm2044 expression in spermatocytes and the downstream signaling were further explored. A bioinformatics assessment predicted two potential binding-sites for the spermatocyte-specific transcription factor A-MYB in the promoter region of lncRNA-Gm2044. Our results proved that the transcription factor A-MYB promotes the expression of lncRNA-Gm2044 in mouse spermatocyte-derived GC-2spd(ts) cells. ChIP and luciferase assays verified that A-MYB mainly binds to the distal promoter region (-819 bp relative to the transcription start site) of lncRNA-Gm2044 and regulates lncRNA-Gm2044 expression through the -819 bp binding-site. In addition, we confirmed that lncRNA-Gm2044 functions as a miR-335-3p sponge to enhance the levels of miR-335-3p's direct target protein, Sycp1. Furthermore, A-MYB can up-regulate Sycp1 expression and down-regulate GC-2spd(ts) cell proliferation by activating its target, lncRNA-Gm2044. Overexpression of lncRNA-Gm2044 or knockdown of miR-335-3p can, at least partially, rescue the effects of A-MYB on Sycp1 expression and GC-2spd(ts) cell proliferation.Taken together, our results provide new information on the mechanistic roles of lncRNA-miRNA in transcription factor A-MYB regulation of spermatocyte function.
Asunto(s)
Proteínas de Unión al ADN/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas c-myb/genética , ARN Largo no Codificante/genética , Espermatocitos/citología , Transactivadores/genética , Animales , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones , Espermatocitos/crecimiento & desarrollo , Transcripción Genética/genética , Activación Transcripcional/genéticaRESUMEN
Lamin C2 (LMN C2) is a short product of the lamin a gene. It is a germ cell-specific lamin and has been extensively studied in male germ cells. In this study, we focussed on the expression and localization of LMN C2 in fully-grown germinal vesicle (GV) oocytes. We detected LMN C2 in the fully-grown germinal vesicle oocytes of various mammalian species with confirmation done by immunoblotting the wild type and Lmnc2 gene deleted testes. Expression of LMN C2 tagged with GFP showed localization of LMN C2 to the nuclear membrane of the oocyte. Moreover, the LMN C2 protein notably disappeared after nuclear envelope breakdown (NEBD) and the expression of LMN C2 was significantly reduced in the oocytes from aged females and ceased altogether during meiotic maturation. These results provide new insights regarding LMN C2 expression in the oocytes of various mammalian species.
Asunto(s)
Laminina/genética , Oocitos/crecimiento & desarrollo , Oogénesis/genética , Ovario/crecimiento & desarrollo , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Células Germinativas/crecimiento & desarrollo , Masculino , Meiosis/genética , Ratones , Ratones Noqueados , Membrana Nuclear/genética , Oocitos/metabolismo , ARN Mensajero/genética , Espermatocitos/crecimiento & desarrollo , Testículo/crecimiento & desarrolloRESUMEN
Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the ribosomal DNA (rDNA). Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y) Using fluorescence in situ hybridization to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis, while the autosomal-specific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.
Asunto(s)
Emparejamiento Cromosómico/genética , Eucromatina/genética , Meiosis/genética , Cromosomas Sexuales/genética , Animales , Centrómero/genética , Segregación Cromosómica/genética , ADN Ribosómico/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Hibridación Fluorescente in Situ , Masculino , Espermatocitos/crecimiento & desarrollo , Cromosoma X/genética , Cromosoma Y/genéticaRESUMEN
Tandemly-repeated DNAs, or satellites, are enriched in heterochromatic regions of eukaryotic genomes and contribute to nuclear structure and function. Some satellites are transcribed, but we lack direct evidence that specific satellite RNAs are required for normal organismal functions. Here, we show satellite RNAs derived from AAGAG tandem repeats are transcribed in many cells throughout Drosophila melanogaster development, enriched in neurons and testes, often localized within heterochromatic regions, and important for viability. Strikingly, we find AAGAG transcripts are necessary for male fertility, and that AAGAG RNA depletion results in defective histone-protamine exchange, sperm maturation and chromatin organization. Since these events happen late in spermatogenesis when the transcripts are not detected, we speculate that AAGAG RNA in primary spermatocytes 'primes' post-meiosis steps for sperm maturation. In addition to demonstrating essential functions for AAGAG RNAs, comparisons between closely related Drosophila species suggest that satellites and their transcription evolve quickly to generate new functions.
Asunto(s)
Drosophila melanogaster/genética , Fertilidad/genética , Regulación del Desarrollo de la Expresión Génica , Repeticiones de Microsatélite , ARN Mensajero/genética , Maduración del Esperma/genética , Espermatogénesis/genética , Animales , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Evolución Molecular , Heterocromatina/metabolismo , Heterocromatina/ultraestructura , Histonas/metabolismo , Masculino , Protaminas/metabolismo , ARN Mensajero/biosíntesis , Espermatocitos/citología , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismo , Transcripción GenéticaRESUMEN
The DAZ family genes, including boule, dazl, and daz, play pivotal roles in germ cell development and differentiation during gametogenesis in organisms, which have been widely studied in mammals, reptiles, or fishes. Dazl was bisexual expressed in both mitotic and meiotic germ cells, daz was male premeiotic expressed, whereas boule exhibits largely in unisexual meiotic germ cells but bisexual expression in several fishes, however, there is lack of report on boule gene and the evolutionary conservation and divergence of dazl and boule in reptile. Here, both boule and dazl genes were characterized in Pelodiscus sinensis. The quantitative real-time polymerase chain reaction analysis showed that boule and dazl were abundantly expressed in adult ovary and testis but barely in somatic tissues, such as heart, brain, liver, spleen, and kidney. Moreover, through fluorescent in situ hybridization, bisexual and germline-specific expression profiles of boule and dazl messenger RNAs (mRNAs) were demonstrated. Boule mRNA exhibited a maximal meiotic expression in spermatocytes, and a relatively low, but distinct expression in oocytes at meiotic stages in P. sinensis, similar to the expression profile of human boule in ovary. However, dazl mRNA was richly distributed in male germ cells at almost all stages during spermatogenesis, and predominantly expressed in most of stages of oocytes including premeiotic and meiotic stages. These findings imply that boule and dazl would play distinct roles in the sexual differentiation of germ cells during turtle gametogenesis, and the major functions of daz family members involved in germ cell differentiation would be conserved across species including P. sinensis.
Asunto(s)
Proteínas de Unión al ARN/genética , Tortugas/genética , Tortugas/metabolismo , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Meiosis , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Reptiles/genética , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismoRESUMEN
During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata close to those observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.
Asunto(s)
Proteínas de Unión al ADN/genética , Endonucleasas/genética , Profase Meiótica I/genética , Proteínas MutL/genética , Animales , Emparejamiento Cromosómico/genética , Intercambio Genético , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Recombinación Homóloga/genética , Masculino , Meiosis/genética , Ratones , Homólogo 1 de la Proteína MutL/genética , Proteínas MutS/genética , Recombinasa Rad51/genética , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismoRESUMEN
A number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. This requires alterations in the germline (sperm or egg) epigenome. Previously, the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis. Male control and vinclozolin lineage F3 generation rats were used as a source of embryonic day 13 (E13) primordial germ cells, embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus vinclozolin lineage samples were determined for each developmental stage. The top 100 statistically significant DMRs for each stage were compared. The developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were investigated. In addition, the DMR associated genes were identified. Previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs). Interestingly, the majority of the DMRs identified in the current study for the caudal sperm originated during the spermatogenic process in the testis. A cascade of epigenetic alterations initiated in the PGCs appears to be required to alter the epigenetic programming during spermatogenesis to modify the sperm epigenome involved in the transgenerational epigenetic inheritance phenomenon.
Asunto(s)
Metilación de ADN/genética , Oxazoles/farmacología , Espermatogénesis/genética , Espermatozoides/efectos de los fármacos , Animales , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/genética , Epigenoma/efectos de los fármacos , Epigenoma/genética , Células Germinativas/efectos de los fármacos , Masculino , Ratas , Espermátides/efectos de los fármacos , Espermátides/crecimiento & desarrollo , Espermátides/metabolismo , Espermatocitos/efectos de los fármacos , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismo , Espermatogénesis/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Espermatogonias/crecimiento & desarrollo , Espermatogonias/metabolismo , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismo , Testículo/efectos de los fármacos , Testículo/crecimiento & desarrolloRESUMEN
Intron gigantism, where genes contain megabase-sized introns, is observed across species, yet little is known about its purpose or regulation. Here we identify a unique gene expression program utilized for the proper expression of genes with intron gigantism. We find that two Drosophila genes with intron gigantism, kl-3 and kl-5, are transcribed in a spatiotemporal manner over the course of spermatocyte differentiation, which spans ~90 hours. The introns of these genes contain megabases of simple satellite DNA repeats that comprise over 99% of the gene loci, and these satellite-DNA containing introns are transcribed. We identify two RNA-binding proteins that specifically localize to kl-3 and kl-5 transcripts and are needed for the successful transcription or processing of these genes. We propose that genes with intron gigantism require a unique gene expression program, which may serve as a platform to regulate gene expression during cellular differentiation.
Asunto(s)
ADN Satélite/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Intrones , Espermatocitos/metabolismo , Espermatogénesis/genética , Animales , ADN Satélite/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Exones , Regulación del Desarrollo de la Expresión Génica , Masculino , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Espermatocitos/citología , Espermatocitos/crecimiento & desarrollo , Transcripción Genética , Cromosoma Y/químicaRESUMEN
Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.
Asunto(s)
Proteínas Portadoras/genética , Roturas del ADN de Doble Cadena , Recombinación Homóloga/genética , Meiosis/genética , Animales , Proteínas Portadoras/química , Segregación Cromosómica/genética , Masculino , Ratones , Regiones Pseudoautosómicas/genética , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismo , Cromosoma X/genética , Cromosoma Y/genéticaRESUMEN
Male gametes are generated through a specialised differentiation pathway involving a series of developmental transitions that are poorly characterised at the molecular level. Here, we use droplet-based single-cell RNA-Sequencing to profile spermatogenesis in adult animals and at multiple stages during juvenile development. By exploiting the first wave of spermatogenesis, we both precisely stage germ cell development and enrich for rare somatic cell-types and spermatogonia. To capture the full complexity of spermatogenesis including cells that have low transcriptional activity, we apply a statistical tool that identifies previously uncharacterised populations of leptotene and zygotene spermatocytes. Focusing on post-meiotic events, we characterise the temporal dynamics of X chromosome re-activation and profile the associated chromatin state using CUT&RUN. This identifies a set of genes strongly repressed by H3K9me3 in spermatocytes, which then undergo extensive chromatin remodelling post-meiosis, thus acquiring an active chromatin state and spermatid-specific expression.
Asunto(s)
Histonas/metabolismo , Espermatocitos/crecimiento & desarrollo , Espermatogénesis/fisiología , Transcripción Genética/fisiología , Cromosoma X/metabolismo , Animales , Separación Celular/métodos , Cromatina/metabolismo , Mapeo Cromosómico/métodos , Cromosomas Humanos Par 21/genética , Epigénesis Genética/fisiología , Femenino , Citometría de Flujo/métodos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Histonas/genética , Humanos , Masculino , Profase Meiótica I/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Espermatocitos/metabolismo , Testículo/citologíaRESUMEN
Germ cells manifest a unique gene expression program and regain totipotency in the zygote. Here, we perform Hi-C analysis to examine 3D chromatin organization in male germ cells during spermatogenesis. We show that the highly compartmentalized 3D chromatin organization characteristic of interphase nuclei is attenuated in meiotic prophase. Meiotic prophase is predominated by short-range intrachromosomal interactions that represent a condensed form akin to that of mitotic chromosomes. However, unlike mitotic chromosomes, meiotic chromosomes display weak genomic compartmentalization, weak topologically associating domains, and localized point interactions in prophase. In postmeiotic round spermatids, genomic compartmentalization increases and gives rise to the strong compartmentalization seen in mature sperm. The X chromosome lacks domain organization during meiotic sex-chromosome inactivation. We propose that male meiosis occurs amid global reprogramming of 3D chromatin organization and that strengthening of chromatin compartmentalization takes place in spermiogenesis to prepare the next generation of life.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Meiosis/fisiología , Espermátides/crecimiento & desarrollo , Espermatocitos/crecimiento & desarrollo , Espermatogénesis/fisiología , Animales , Cromatina/metabolismo , Cromosomas/metabolismo , Interfase/fisiología , Masculino , Profase Meiótica I/fisiología , Ratones , Ratones Endogámicos C57BL , Dominios Proteicos/fisiologíaRESUMEN
Repression of retrotransposons is essential for genome integrity during germ cell development and is tightly controlled through epigenetic mechanisms. In primordial germ cells, protein arginine N-methyltransferase (Prmt5) is involved in retrotransposon repression by methylating Piwi proteins, which is part of the piRNA pathway. Here, we show that in mice, genetic inactivation of coprs (which is highly expressed in testis and encodes a histone-binding protein required for the targeting of Prmt5 activity) affects the maturation of spermatogonia to spermatids. Mass spectrometry analysis revealed the presence of Miwi in testis protein lysates immunoprecipitated with an anti-Coprs antibody. The observed deregulation of Miwi and pachytene pre-piRNAs levels and the derepression of LINE1 repetitive sequences observed in coprs-/- mice suggest that Coprs is implicated in genome surveillance mechanisms.
Asunto(s)
Chaperonas de Histonas/genética , Elementos de Nucleótido Esparcido Largo/genética , Espermatocitos/crecimiento & desarrollo , Espermatogénesis/genética , Animales , Masculino , Ratones , Ratones Noqueados , Proteína-Arginina N-Metiltransferasas/metabolismo , Espermatocitos/metabolismoRESUMEN
Meiosis is a cellular program that generates haploid gametes for sexual reproduction. While chromosome events that contribute to reducing ploidy (homologous chromosome pairing, synapsis, and recombination) are well conserved, their execution varies across species and even between sexes of the same species. The telomere bouquet is a conserved feature of meiosis that was first described nearly a century ago, yet its role is still debated. Here we took advantage of the prominent telomere bouquet in zebrafish, Danio rerio, and super-resolution microscopy to show that axis morphogenesis, synapsis, and the formation of double-strand breaks (DSBs) all take place within the immediate vicinity of telomeres. We established a coherent timeline of events and tested the dependence of each event on the formation of Spo11-induced DSBs. First, we found that the axis protein Sycp3 loads adjacent to telomeres and extends inward, suggesting a specific feature common to all telomeres seeds the development of the axis. Second, we found that newly formed axes near telomeres engage in presynaptic co-alignment by a mechanism that depends on DSBs, even when stable juxtaposition of homologous chromosomes at interstitial regions is not yet evident. Third, we were surprised to discover that ~30% of telomeres in early prophase I engage in associations between two or more chromosome ends and these interactions decrease in later stages. Finally, while pairing and synapsis were disrupted in both spo11 males and females, their reproductive phenotypes were starkly different; spo11 mutant males failed to produce sperm while females produced offspring with severe developmental defects. Our results support zebrafish as an important vertebrate model for meiosis with implications for differences in fertility and genetically derived birth defects in males and females.