RESUMEN
BACKGROUND: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells. METHODS: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs. RESULTS: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts. CONCLUSIONS: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.
Asunto(s)
Respuesta al Choque Térmico , ARN Interferente Pequeño , Espermátides , Espermatocitos , Testículo , Animales , Masculino , Espermátides/metabolismo , Espermatocitos/metabolismo , ARN Interferente Pequeño/genética , Ratas , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Testículo/metabolismo , Espermatogénesis/genética , Espermatogénesis/fisiología , Fase Paquiteno/genética , Ratas Sprague-Dawley , ARN de Interacción con PiwiRESUMEN
Meiosis is essential for fertility in sexually reproducing species and this sophisticated process has been extensively studied. Notwithstanding these efforts, key factors involved in meiosis have not been fully characterized. In this study, we investigate the regulatory roles of zinc finger protein 541 (ZFP541) and its interacting protein potassium channel tetramerization domain containing 19 (KCTD19) in spermatogenesis. ZFP541 is expressed from leptotene to the round spermatid stage, while the expression of KCTD19 is initiated in pachytene. Depletion of Zfp541 or Kctd19 leads to infertility in male mice and delays progression from early to mid/late pachynema. In addition, Zfp541-/- spermatocytes show abnormal programmed DNA double-strand break repair, impaired crossover formation and resolution, and asynapsis of the XY chromosomes. ZFP541 interacts with KCTD19, histone deacetylase 1/2 (HDAC1/2), and deoxynucleotidyl transferase terminal-interacting protein 1 (DNTTIP1). Moreover, ZFP541 binds to and activates the expression of genes involved in meiosis and post-meiosis including Kctd19; in turn, KCTD19 promotes the transcriptional activation activity of ZFP541. Taken together, our studies reveal that the ZFP541/KCTD19 signaling complex, acting as a key transcription regulator, plays an indispensable role in male fertility by regulating pachytene progression.
Asunto(s)
Espermatocitos , Espermatogénesis , Masculino , Ratones , Animales , Espermatogénesis/genética , Profase Meiótica I , Fase Paquiteno/genética , Meiosis/genética , Proteínas Cromosómicas no Histona/genética , Factores de Transcripción/genéticaRESUMEN
We analyzed the synapsis and recombination between Z and W chromosomes in the oocytes of nine neognath species: domestic chicken Gallus gallus domesticus, grey goose Anser anser, black tern Chlidonias niger, common tern Sterna hirundo, pale martin Riparia diluta, barn swallow Hirundo rustica, European pied flycatcher Ficedula hypoleuca, great tit Parus major and white wagtail Motacilla alba using immunolocalization of SYCP3, the main protein of the lateral elements of the synaptonemal complex, and MLH1, the mismatch repair protein marking mature recombination nodules. In all species examined, homologous synapsis occurs in a short region of variable size at the ends of Z and W chromosomes, where a single recombination nodule is located. The remaining parts of the sex chromosomes undergo synaptic adjustment and synapse non-homologously. In 25% of ZW bivalents of white wagtail, synapsis and recombination also occur at the secondary pairing region, which probably resulted from autosome-sex chromosome translocation. Using FISH with a paint probe specific to the germline-restricted chromosome (GRC) of the pale martin on the oocytes of the pale martin, barn swallow and great tit, we showed that both maternally inherited songbird chromosomes (GRC and W) share common sequences.
Asunto(s)
Aves/genética , Emparejamiento Cromosómico/fisiología , Recombinación Genética , Cromosomas Sexuales , Animales , Pollos/genética , Femenino , Hibridación Fluorescente in Situ , Homólogo 1 de la Proteína MutL/genética , Oocitos/fisiología , Fase Paquiteno/genética , Passeriformes/genéticaRESUMEN
Meiosis is essential for the generation of gametes and sexual reproduction, yet the factors and underlying mechanisms regulating meiotic progression remain largely unknown. Here, we showed that MTL5 translocates into nuclei of spermatocytes during zygotene-pachytene transition and ensures meiosis advances beyond pachytene stage. MTL5 shows strong interactions with MuvB core complex components, a well-known transcriptional complex regulating mitotic progression, and the zygotene-pachytene transition of MTL5 is mediated by its direct interaction with the component LIN9, through MTL5 C-terminal 443-475 residues. Male Mtl5c-mu/c-mu mice expressing the truncated MTL5 (p.Ser445Arg fs*3) that lacks the interaction with LIN9 and is detained in cytoplasm showed male infertility and spermatogenic arrest at pachytene stage, same as that of Mtl5 knockout mice, indicating that the interaction with LIN9 is essential for the nuclear translocation and function of MTL5 during meiosis. Our data demonstrated MTL5 translocates into nuclei during the zygotene-pachytene transition to initiate its function along with the MuvB core complex in pachytene spermatocytes, highlighting a new mechanism regulating the progression of male meiosis.
Asunto(s)
Meiosis/fisiología , Metalotioneína/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Proteínas de Ciclo Celular/metabolismo , Emparejamiento Cromosómico/genética , Citoplasma , Proteínas de Unión al ADN , Fertilidad/genética , Fertilidad/fisiología , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Masculino , Profase Meiótica I/fisiología , Metalotioneína/genética , Ratones , Ratones Endogámicos C57BL , Fase Paquiteno/genética , Espermatocitos/fisiología , Espermatogénesis/fisiología , Testículo , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
In most organisms, the number and distribution of crossovers that occur during meiosis are tightly controlled. All chromosomes must receive at least one 'obligatory crossover' and crossovers are prevented from occurring near one another by 'crossover interference'. However, the mechanistic basis of this phenomenon of crossover interference has remained mostly mysterious. Using quantitative super-resolution cytogenetics and mathematical modelling, we investigate crossover positioning in the Arabidopsis thaliana wild-type, an over-expressor of the conserved E3 ligase HEI10, and a hei10 heterozygous line. We show that crossover positions can be explained by a predictive, diffusion-mediated coarsening model, in which large, approximately evenly-spaced HEI10 foci grow at the expense of smaller, closely-spaced clusters. We propose this coarsening process explains many aspects of Arabidopsis crossover positioning, including crossover interference. Consistent with this model, we also demonstrate that crossover positioning can be predictably modified in vivo simply by altering HEI10 dosage, with higher and lower dosage leading to weaker and stronger crossover interference, respectively. As HEI10 is a conserved member of the RING finger protein family that functions in the interference-sensitive pathway for crossover formation, we anticipate that similar mechanisms may regulate crossover positioning in diverse eukaryotes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Cromosómicas no Histona/metabolismo , Intercambio Genético/genética , Meiosis/genética , Proteínas de Arabidopsis/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas de las Plantas/genética , Cromosomas de las Plantas/metabolismo , Simulación por Computador , Dosificación de Gen , Fase Paquiteno/genética , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/metabolismoRESUMEN
During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Nucleares/metabolismo , Fase Paquiteno/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Espermátides/citología , Espermatogénesis/genética , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Secuenciación de Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , Modelos Animales de Enfermedad , Femenino , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Humanos , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Oocitos/citología , Oocitos/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , RNA-Seq , Espermátides/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.
Asunto(s)
Infertilidad Masculina/genética , ARN Interferente Pequeño/genética , Espermatogénesis/genética , Espermatozoides/patología , Acrosoma/patología , Animales , Cromosomas/genética , Humanos , Infertilidad Masculina/patología , Masculino , Meiosis/genética , Ratones , Fase Paquiteno/genética , Espermátides/crecimiento & desarrollo , Espermátides/patología , Testículo/crecimiento & desarrollo , Testículo/patologíaRESUMEN
Piwi-interacting RNAs (piRNAs) play critical roles in protecting germline genome integrity and promoting normal spermiogenic differentiation. In mammals, there are two populations of piRNAs: pre-pachytene and pachytene. Transposon-rich pre-pachytene piRNAs are expressed in fetal and perinatal germ cells and are required for retrotransposon silencing, whereas transposon-poor pachytene piRNAs are expressed in spermatocytes and round spermatids and regulate mRNA transcript levels. MOV10L1, a germ cell-specific RNA helicase, is essential for the production of both populations of piRNAs. Although the requirement of the RNA helicase domain located in the MOV10L1 C-terminal region for piRNA biogenesis is well known, its large N-terminal region remains mysterious. Here we report a novel Mov10l1 mutation, named yama, in the Mov10l1 N-terminal region. The yama mutation results in a single amino acid substitution V229E. The yama mutation causes meiotic arrest, de-repression of transposable elements, and male sterility because of defects in pre-pachytene piRNA biogenesis. Moreover, restricting the Mov10l1 mutation effects to later stages in germ cell development by combining with a postnatal conditional deletion of a complementing wild-type allele causes absence of pachytene piRNAs, accumulation of piRNA precursors, polar conglomeration of piRNA pathway proteins in spermatocytes, and spermiogenic arrest. Mechanistically, the V229E substitution in MOV10L1 reduces its interaction with PLD6, an endonuclease that generates the 5' ends of piRNA intermediates. Our results uncover an important role for the MOV10L1-PLD6 interaction in piRNA biogenesis throughout male germ cell development.
Asunto(s)
Infertilidad Masculina/genética , Meiosis/genética , Proteínas Mitocondriales/metabolismo , Fosfolipasa D/metabolismo , ARN Helicasas/metabolismo , ARN Interferente Pequeño/metabolismo , Retroelementos/genética , Espermatogénesis/genética , Alelos , Animales , Silenciador del Gen , Células Germinativas/metabolismo , Células Germinativas/patología , Células HEK293 , Humanos , Masculino , Ratones , Proteínas Mitocondriales/genética , Mutación , Fase Paquiteno/genética , Fosfolipasa D/genética , ARN Helicasas/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Espermátides/metabolismo , Espermatocitos/metabolismo , Testículo/metabolismoRESUMEN
In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.
Asunto(s)
Epigénesis Genética , Exones/genética , Mamíferos/genética , Fase Paquiteno/genética , ARN Interferente Pequeño/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilación , Animales , Metilación de ADN/genética , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Histonas/metabolismo , Intrones/genética , Masculino , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Regiones Promotoras Genéticas/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética , Testículo/metabolismo , Transcripción GenéticaRESUMEN
Poly (ADP-ribose) polymerase-1 (Parp1) is a member of nuclear enzymes family involved in to the response to genotoxic stresses, DNA repair, and is critical for the maintenance of genome stability. During gametogenesis, genome stability is essential for inheritance and formation of healthy gametes. The latter involves DNA double-strand break (DSB)-driven pairing of homologous chromosomes in first meiotic prophase. By analysis of DSB repair kinetics in male meiotic prophase cells of homologous recombination (HR) and nonhomologous end joining (NHEJ)-deficient mouse models, we previously demonstrated an interplay between HR and the conventional NHEJ repair pathway. In the current work, we evaluate the relative contribution of Parp1-dependent NHEJ to the repair of ectopic ionizing radiation (IR)-induced DSBs in control and Parp1-inhibited mouse pachytene spermatocytes before and after the completion of meiotic recombination in stages VI-XI. The disappearance of large, exogenous DSB-related γ-H2AX foci was quantified 1 and 8 h after 1 Gy γ-irradiation of control and 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)quinolinone (DPQ) Parp1-inhibited mice. Late pachytene control spermatocytes obtained 8 h after IR had repaired >80% of DSBs observed at 1 h after IR. However, only 64% of DSBs were repaired in late spermatocytes of DPQ-treated (Parp1-inhibited) mice. Thus, it appears that Parp1 contributes to the repair of a fraction of DSBs in late prophase I, providing further insights in DNA repair pathway choreography during spermatogenic differentiation.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Fase Paquiteno/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Espermatocitos/citología , Espermatocitos/efectos de la radiación , Animales , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Fase Paquiteno/efectos de la radiación , Espermatocitos/metabolismoRESUMEN
Pachytene PIWI-interacting RNAs (piRNAs), which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like microRNAs, cleave targets like short interfering RNAs or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 (pi6) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6-/- fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving messenger RNAs encoding proteins required for sperm function. pi6 also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10, as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.
Asunto(s)
ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiología , Espermatogénesis/genética , Animales , Evolución Biológica , Núcleo Celular , Desarrollo Embrionario , Femenino , Fertilidad , Eliminación de Gen , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Fase Paquiteno/genética , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Capacitación Espermática/genética , Capacitación Espermática/fisiología , Interacciones Espermatozoide-Óvulo/fisiologíaRESUMEN
Long transgenes are often used in mammalian genetics, e.g., to rescue mutations in large genes. In the course of experiments addressing the genetic basis of hybrid sterility caused by meiotic defects in mice bearing different alleles of Prdm9, we discovered that introduction of copy-number variation (CNV) via two independent insertions of long transgenes containing incomplete Prdm9 decreased testicular weight and epididymal sperm count. Transgenic animals displayed increased occurrence of seminiferous tubules with apoptotic cells at 18 days postpartum (dpp) corresponding to late meiotic prophase I, but not at 21 dpp. We hypothesized that long transgene insertions could cause asynapsis, but the immunocytochemical data revealed that the adult transgenic testes carried a similar percentage of asynaptic pachytene spermatocytes as the controls. These transgenic spermatocytes displayed less crossovers but similar numbers of unrepaired meiotic breaks. Despite slightly increased frequency of metaphase I spermatocytes with univalent chromosome(s) and reduced numbers of metaphase II spermatocytes, cytological studies did not reveal increased apoptosis in tubules containing the metaphase spermatocytes, but found an increased percentage of tubules carrying apoptotic spermatids. Sperm counts of subfertile animals inversely correlated with the transcription levels of the Psmb1 gene encoded within these two transgenes. The effect of the transgenes was dependent on sex and genetic background. Our results imply that the fertility of transgenic hybrid animals is not compromised by the impaired meiotic synapsis of homologous chromosomes, but can be negatively influenced by the increased expression of the introduced genes.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Variaciones en el Número de Copia de ADN , Fertilidad/genética , Fase Paquiteno/genética , Transgenes , Animales , Apoptosis/genética , Roturas del ADN de Doble Cadena , Femenino , Antecedentes Genéticos , Masculino , Ratones , Tamaño de los Órganos , Recuento de Espermatozoides , Espermatocitos/metabolismo , Testículo/anatomía & histología , Testículo/metabolismoRESUMEN
The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allow us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fase Paquiteno/genética , Complejo Sinaptonémico/genética , Cromosoma X/genética , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Recombinación Genética/genética , Eliminación de Secuencia/genéticaRESUMEN
Spermatogenesis is tightly regulated by ubiquitination and proteasomal degradation, especially during spermiogenesis, in which histones are replaced by protamine. However, the functions of proteasomal activity in meiosis I and II remain elusive. Here, we show that PSMA8-associated proteasomes are essential for the degradation of meiotic proteins and the progression of meiosis I during spermatogenesis. PSMA8 is expressed in spermatocytes from the pachytene stage, and assembles a type of testis-specific core proteasome. Deletion of PSMA8 decreases the abundance of proteasome in testes. Meiotic proteins that are normally degraded at late prophase I, such as RAD51 and RPA1, remain stable in PSMA8-deleted spermatocytes. Moreover, PSMA8-null spermatocytes exhibit delayed M-phase entry and are finally arrested at this stage, resulting in male infertility. However, PSMA8 is neither expressed nor required for female meiotic progression. Thus, meiosis I progression in spermatogenesis, particularly entry into and exit from M-phase, requires the proteasomal activity of PSMA8-associated proteasomes.
Asunto(s)
Profase Meiótica I/genética , Complejo de la Endopetidasa Proteasomal/genética , Espermatogénesis/genética , Testículo/enzimología , Animales , División Celular/genética , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Fase Paquiteno/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Espermatocitos/enzimología , Espermatocitos/metabolismoRESUMEN
An imbalance in copper (Cu) tissue homeostasis has a degenerative effect on spermatogenesis and male fertility. The high-affinity Cu transporter 1 (CTR1; SLC31A1) is the major protein responsible for Cu acquisition in eukaryotes and is highly expressed in mouse testes. Studies on yeast and Drosophila have demonstrated the conserved essential function of Cu and CTR1 for meiosis and fertility, implying that CTR1 may play an essential function in mammalian spermatogenesis. In mice, spermatogenesis takes place within the seminiferous epithelium, where tight junctions between somatic Sertoli cells (SCs) create a specialized microenvironment for the development of meiotic germ cells (GCs) by tightly regulating the free transport of metabolites and ions to reach these cells. Here, it is demonstrated that within the seminiferous epithelium, CTR1 is expressed on the membrane of primary pachytene spermatocytes and SCs. To examine the physiological significance of CTR1 in spermatogenesis, mice with a GC-specific (Ctr1ΔGC) and SC-specific (Ctr1ΔSC) disruption of the Ctr1 gene were generated. The testis of Ctr1ΔGC mice exhibits a severe progressive loss of GCs starting at postnatal day (PND) 28 leading to testis hypoplasia by adulthood. No spermatogenic recovery was observed in Ctr1ΔGC testis beyond PND 41, despite the presence of FOXO-1 expressing undifferentiated spermatogonial cells. However, Ctr1ΔSC mice displayed functional spermatogenesis and were fertile, even though testicular Cu levels and Cu-dependent cellular activities were significantly reduced. These results reveal, for the first time, the importance of CTR1 expression by GCs for maintaining functional spermatogenesis.
Asunto(s)
Transportador de Cobre 1/genética , Expresión Génica , Células de Sertoli/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/genética , Testículo/metabolismo , Animales , Cobre/metabolismo , Transportador de Cobre 1/metabolismo , Fertilidad/genética , Masculino , Meiosis/genética , Ratones Noqueados , Ratones Transgénicos , Fase Paquiteno/genética , Células de Sertoli/citología , Espermatocitos/citología , Testículo/citologíaRESUMEN
Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms can also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid-pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated initially by NHEJ, involving Ku70 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early "meiotic" repair pathway that acts by default at the beginning of meiosis is replaced from mid-pachytene onwards by a "somatic-like" repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, changes in the gene expression profile and acquisition of competence to proceed to metaphase.
Asunto(s)
Proteínas de Ciclo Celular/genética , Reparación del ADN por Unión de Extremidades/genética , Recombinación Homóloga/genética , Proteínas Nucleares/genética , Recombinasa Rad51/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Animales , Cromosomas/genética , Cromosomas/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Rayos gamma , Histonas/genética , Autoantígeno Ku/genética , Meiosis/genética , Ratones , Fase Paquiteno/genética , Proteínas de Unión a Fosfato , Complejo Sinaptonémico/genéticaRESUMEN
Several developmental stages of spermatogenesis are transcriptionally quiescent which presents major challenges associated with the regulation of gene expression. Here we identify that the zygotene to pachytene transition is not only associated with the resumption of transcription but also a wave of programmed mRNA degradation that is essential for meiotic progression. We explored whether terminal uridydyl transferase 4- (TUT4-) or TUT7-mediated 3' mRNA uridylation contributes to this wave of mRNA degradation during pachynema. Indeed, both TUT4 and TUT7 are expressed throughout most of spermatogenesis, however, loss of either TUT4 or TUT7 does not have any major impact upon spermatogenesis. Combined TUT4 and TUT7 (TUT4/7) deficiency results in embryonic growth defects, while conditional gene targeting revealed an essential role for TUT4/7 in pachytene progression. Loss of TUT4/7 results in the reduction of miRNA, piRNA and mRNA 3' uridylation. Although this reduction does not greatly alter miRNA or piRNA expression, TUT4/7-mediated uridylation is required for the clearance of many zygotene-expressed transcripts in pachytene cells. We find that TUT4/7-regulated transcripts in pachytene spermatocytes are characterized by having long 3' UTRs with length-adjusted enrichment for AU-rich elements. We also observed these features in TUT4/7-regulated maternal transcripts whose dosage was recently shown to be essential for sculpting a functional maternal transcriptome and meiosis. Therefore, mRNA 3' uridylation is a critical determinant of both male and female germline transcriptomes. In conclusion, we have identified a novel requirement for 3' uridylation-programmed zygotene mRNA clearance in pachytene spermatocytes that is essential for male meiotic progression.
Asunto(s)
Profase Meiótica I/genética , Fase Paquiteno/genética , Procesamiento Postranscripcional del ARN/fisiología , Espermatogénesis/genética , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Estabilidad del ARN/genética , ARN Mensajero/genética , UDP-Glucosa-Hexosa-1-Fosfato Uridiltransferasa/metabolismoRESUMEN
PIWI-interacting RNAs (piRNAs) engage PIWI proteins to silence transposons and promote germ cell development in animals. In diverse species, piRNA biogenesis occurs near the mitochondrial surface, and involves mitochondrial membrane-anchored factors. In mice, two cytoplasmic PIWI proteins, MIWI and MILI, receive processed pachytene piRNAs at intermitochodrial cement (IMC). However, how MIWI and MILI are initially recruited to the IMC to engage multiple steps of piRNA processing is unclear. Here, we show that mitochondria-anchored TDRKH controls multiple steps of pachytene piRNA biogenesis in mice. TDRKH specifically recruits MIWI, but not MILI, to engage the piRNA pathway. It is required for the production of the entire MIWI-bound piRNA population and enables trimming of MILI-bound piRNAs. The failure to recruit MIWI to the IMC with TDRKH deficiency results in loss of MIWI in the chromatoid body, leading to spermiogenic arrest and piRNA-independent retrotransposon LINE1 de-repression in round spermatids. Our findings identify a mitochondrial surface-based scaffolding mechanism separating the entry and actions of two critical PIWI proteins in the same piRNA pathway to drive piRNA biogenesis and germ cell development.
Asunto(s)
Proteínas Argonautas/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Animales , Masculino , Ratones , Miosis/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Fase Paquiteno/genética , Retroelementos/genética , Espermatogénesis/genética , Testículo/crecimiento & desarrollo , Testículo/metabolismoRESUMEN
Meiotic recombination plays a critical role in sexual reproduction by creating crossovers between homologous chromosomes. These crossovers, along with sister chromatid cohesion, connect homologs to enable proper segregation at Meiosis I. Recombination is initiated by programmed double strand breaks (DSBs) at particular regions of the genome. The meiotic recombination checkpoint uses meiosis-specific modifications to the DSB-induced DNA damage response to provide time to convert these breaks into interhomolog crossovers by delaying entry into Meiosis I until the DSBs have been repaired. The meiosis-specific kinase, Mek1, is a key regulator of meiotic recombination pathway choice, as well as being required for the meiotic recombination checkpoint. The major target of this checkpoint is the meiosis-specific transcription factor, Ndt80, which is essential to express genes necessary for completion of recombination and meiotic progression. The molecular mechanism by which cells monitor meiotic DSB repair to allow entry into Meiosis I with unbroken chromosomes was unknown. Using genetic and biochemical approaches, this work demonstrates that in the presence of DSBs, activated Mek1 binds to Ndt80 and phosphorylates the transcription factor, thus inhibiting DNA binding and preventing Ndt80's function as a transcriptional activator. Repair of DSBs by recombination reduces Mek1 activity, resulting in removal of the inhibitory Mek1 phosphates. Phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, then results in fully activated Ndt80. Ndt80 upregulates transcription of its own gene, as well as target genes, resulting in prophase exit and progression through meiosis.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Meiosis/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Puntos de Control del Ciclo Celular , Secuencia Conservada , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Genes Fúngicos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MAP Quinasa Quinasa 1/genética , Meiosis/genética , Modelos Biológicos , Modelos Moleculares , Mutación , Fase Paquiteno/genética , Fase Paquiteno/fisiología , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Reparación del ADN por Recombinación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Accurate quantification of DNA double strand breaks (DSB) in testicular germ cells is difficult because of cellular heterogeneity and the presence of endogenous γH2AX. Here, we used confocal microscopy to quantify DNA damage and repair kinetics following γ-irradiation (0.5-4 Gy) in three major mouse male germ cell stages, early and late pachytene spermatocytes and round spermatids (RSs), following a defined post irradiation time course. Dose-response curves showing linear best fit validated γH2AX focus as a rapid biodosimetric tool in these substages in response to whole body in vivo exposure. Stage specific foci yield/dose and repair kinetics demonstrated differential radiosensitivity and repair efficiency: early pachytenes (EP) repaired most rapidly and completely followed by late pachytene (LP) and RSs. Repair kinetics for all three stages followed 'exponential decay' in response to each radiation dose. In pachytenes immediate colocalisation of γH2AX and 53BP1, which participates in non-homologous end-joining repair pathway, was followed by dissociation from the major focal area of γH2AX by 4 h demonstrating ongoing DSB repair. These results confirm the differential radiosensitivity and repair kinetics of DSBs in male germ cells at different stages. Taken together, our results provide a simple and accurate method for assessing DNA damage and repair kinetics during spermatogenesis.