RESUMEN
Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.
Asunto(s)
Emparejamiento Cromosómico , Intercambio Genético , Meiosis , Ubiquitina-Proteína Ligasas , Animales , Ratones , Masculino , Femenino , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Ratones Noqueados , Humanos , LigasasRESUMEN
Cohesin cofactors regulate the loading, maintenance, and release of cohesin complexes from chromosomes during mitosis but little is known on their role during vertebrate meiosis. One such cofactor is PDS5, which exists as two paralogs in somatic and germline cells, PDS5A and PDS5B, with unclear functions. Here, we have analyzed their distribution and functions in mouse spermatocytes. We show that simultaneous excision of Pds5A and Pds5B results in severe defects during early prophase I while their individual depletion does not, suggesting their functional redundancy. Shortened axial/lateral elements and a reduction of early recombination nodules are observed after the strong depletion of PDS5A/B proteins. Moreover, telomere integrity and their association to the nuclear envelope are severely compromised. As these defects occur without detectable reduction in chromosome-bound cohesin, we propose that the dynamic behavior of the complex, mediated by PDS5 proteins, is key for successful completion of meiotic prophase I.
Asunto(s)
Meiosis , Telómero , Animales , Proteínas de Ciclo Celular/genética , Masculino , Meiosis/genética , Ratones , Mitosis , Espermatocitos , Complejo Sinaptonémico , Telómero/genéticaRESUMEN
The ubiquitin proteasome system regulates meiotic recombination in yeast through its association with the synaptonemal complex, a 'zipper'-like structure that holds homologous chromosome pairs in synapsis during meiotic prophase I. In mammals, the proteasome activator subunit PA200 targets acetylated histones for degradation during somatic DNA double strand break repair and during histone replacement during spermiogenesis. We investigated the role of the testis-specific proteasomal subunit α4s (PSMA8) during spermatogenesis, and found that PSMA8 was localized to and dependent on the central region of the synaptonemal complex. Accordingly, synapsis-deficient mice show delocalization of PSMA8. Moreover, though Psma8-deficient mice are proficient in meiotic homologous recombination, there are alterations in the proteostasis of several key meiotic players that, in addition to the known substrate acetylated histones, have been shown by a proteomic approach to interact with PSMA8, such as SYCP3, SYCP1, CDK1 and TRIP13. These alterations lead to an accumulation of spermatocytes in metaphase I and II which either enter massively into apoptosis or give rise to a low number of aberrant round spermatids that apoptose before histone replacement takes place.
Asunto(s)
Fertilidad/genética , Infertilidad Masculina/genética , Metafase/genética , Complejo de la Endopetidasa Proteasomal/genética , Subunidades de Proteína/genética , Animales , Apoptosis/genética , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/genética , Complejo Sinaptonémico/metabolismo , Testículo/citología , Testículo/metabolismoRESUMEN
Ubiquitin-specific protease 26 (USP26) is a deubiquitylating enzyme belonging to the USPs family with a transcription pattern restricted to the male germline. Since protein ubiquitination is an essential regulatory mechanism during meiosis, many efforts have been focused on elucidating the function of USP26 and its relationship with fertility. During the last decade, several studies have reported the presence of different polymorphisms in USP26 in patients with non-obstructive azoospermia (NOA) or severe oligozoospermia suggesting that this gene may be associated with human infertility. However, other studies have revealed the presence of these and novel polymorphisms, including nonsense mutations, in men with normal spermatogenesis as well. Thus, the results remain controversial and its function is unknown. In the present study, we describe the in vivo functional analysis of mice lacking USP26. The phenotypic analysis of two different Usp26-null mutants showed no overt-phenotype with both males and females being fertile. Cytological analysis of spermatocytes showed no defects in synapsis, chromosome dynamics, DNA repair, or recombination. Histopathological analysis revealed a normal distribution and number of the different cell types in both male and female mice. Finally, normal counts were observed in fertility assessments. These results represent the first in vivo evidence showing that USP26 is not essential for mouse gametogenesis.
Asunto(s)
Cisteína Endopeptidasas/genética , Fertilidad/genética , Gametogénesis/genética , Fenotipo , Animales , Sistemas CRISPR-Cas , Femenino , Edición Génica , Marcación de Gen , Estudios de Asociación Genética , Sitios Genéticos , Células Germinativas/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Ovario/metabolismo , Testículo/metabolismoRESUMEN
The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis-specific cohesin subunits REC8 and SMC1ß. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2-PP2A.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrómero , Proteínas Cromosómicas no Histona/metabolismo , Meiosis , Complejo Sinaptonémico , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Ciclo Celular/genética , Expresión Génica , Humanos , Masculino , Ratones , Ratones Noqueados , Espermatocitos/metabolismo , CohesinasRESUMEN
Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Mutación , Proteínas Nucleares/genética , Insuficiencia Ovárica Primaria/genética , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Infertilidad Femenina/genética , Ratones , Linaje , CohesinasRESUMEN
Oligo- and azoospermia are severe forms of male infertility. However, known genetic factors account only for a small fraction of the cases. Recently, whole-exome sequencing in a large consanguineous family with inherited premature ovarian failure (POF) identified a homozygous frameshift mutation in the STAG3 gene leading to a premature stop codon. STAG3 encodes a meiosis-specific subunit of the cohesin complex, a large proteinaceous ring with DNA-entrapping ability that ensures sister chromatid cohesion and enables correct synapsis and segregation of homologous chromosomes during meiosis. The pathogenicity of the STAG3 mutations was functionally validated with a loss-of-function mouse model for STAG3 in oogenesis. However, and since none of the male members of this family was homozygous for the mutant allele, we only could hypothesized its putative involvement in male infertility. In this report, we show that male mice devoid of Stag3 display a severe meiotic phenotype that includes a meiotic arrest at zygonema-like shortening of their chromosome axial elements/lateral elements, partial loss of centromeric cohesion at early prophase and maintenance of the ability to initiate but not complete RAD51- and DMC1-mediated double-strand break repair, demonstrating that STAG3 is a crucial cohesin subunit in mammalian gametogenesis and supporting our proposal that STAG3 is a strong candidate gene for human male infertility.
Asunto(s)
Infertilidad Masculina/genética , Proteínas Nucleares/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Masculino , Meiosis/genética , Meiosis/fisiología , Ratones , Proteínas Nucleares/genética , Proteínas de Unión a Fosfato , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Complejo Sinaptonémico/metabolismo , CohesinasRESUMEN
Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Mamíferos/metabolismo , Meiosis , Anafase/efectos de los fármacos , Animales , Centrómero/efectos de los fármacos , Cromátides/efectos de los fármacos , Cromátides/metabolismo , Etopósido/farmacología , Masculino , Meiosis/efectos de los fármacos , Metafase/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitosis/efectos de los fármacos , CohesinasRESUMEN
The cohesin complex is a ring-shaped proteinaceous structure that entraps the two sister chromatids after replication until the onset of anaphase when the ring is opened by proteolytic cleavage of its α-kleisin subunit (RAD21 at mitosis and REC8 at meiosis) by separase. RAD21L is a recently identified α-kleisin that is present from fish to mammals and biochemically interacts with the cohesin subunits SMC1, SMC3 and STAG3. RAD21L localizes along the axial elements of the synaptonemal complex of mouse meiocytes. However, its existence as a bona fide cohesin and its functional role awaits in vivo validation. Here, we show that male mice lacking RAD21L are defective in full synapsis of homologous chromosomes at meiotic prophase I, which provokes an arrest at zygotene and leads to total azoospermia and consequently infertility. In contrast, RAD21L-deficient females are fertile but develop an age-dependent sterility. Thus, our results provide in vivo evidence that RAD21L is essential for male fertility and in females for the maintenance of fertility during natural aging.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Meiosis , Factores de Edad , Animales , Proteínas Cromosómicas no Histona/deficiencia , Cromosomas/metabolismo , Femenino , Histocitoquímica , Infertilidad , Masculino , Ratones , Ratones Noqueados , Ovario/patología , Subunidades de Proteína/metabolismo , Factores Sexuales , Testículo/patologíaRESUMEN
We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2-/- mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/fisiología , Meiosis/fisiología , Mitosis/fisiología , Oocitos/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Testículo/enzimología , Animales , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina , Femenino , Fibroblastos , Masculino , Ratones , Ratones Noqueados , Oocitos/citología , Ovario/embriología , Pase Seriado , Espermatogénesis , Testículo/citología , Testículo/embriologíaRESUMEN
The α6 subunit of the nicotinic acetylcholine receptor (nAChR) has been proposed as the target for spinosad in insects. Point mutations that result in premature stop codons in the α6 gene of Ceratitis capitata flies have been previously associated with spinosad resistance, but it is unknown if these transcripts are translated and if so, what is the location of the putative truncated proteins. In this work, we produced a specific antibody against C. capitata α6 (Ccα6) and validated it by ELISA, Western blotting and immunofluorescence assays in brain tissues. The antibody detects both wild-type and truncated forms of Ccα6 in vivo, and the protein is located in the cell membrane of the brain of wild-type spinosad sensitive flies. On the contrary, the shortened transcripts present in resistant flies generate putative truncated proteins that, for the most part, fail to reach their final destination in the membrane of the cells and remain in the cytoplasm. The differences observed in the locations of wild-type and truncated α6 proteins are proposed to determine the susceptibility or resistance to spinosad.
RESUMEN
Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3-/- spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histonas/genética , Meiosis/fisiología , Complejo Sinaptonémico/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/genética , Epigénesis Genética , Lisina/metabolismo , Masculino , Metilación , Ratones , Espermatocitos/metabolismo , Factores de Transcripción/genéticaRESUMEN
Marsupial sex chromosomes break the rule that recombination during first meiotic prophase is necessary to ensure reductional segregation during first meiotic division. It is widely accepted that in marsupials X and Y chromosomes do not share homologous regions, and during male first meiotic prophase the synaptonemal complex is absent between them. Although these sex chromosomes do not recombine, they segregate reductionally in anaphase I. We have investigated the nature of sex chromosome association in spermatocytes of the marsupial Thylamys elegans, in order to discern the mechanisms involved in ensuring their proper segregation. We focused on the localization of the axial/lateral element protein SCP3 and the cohesin subunit STAG3. Our results show that X and Y chromosomes never appear as univalents in metaphase I, but they remain associated until they orientate and segregate to opposite poles. However, they must not be tied by a chiasma since their separation precedes the release of the sister chromatid cohesion. Instead, we show they are associated by the dense plate, a SCP3-rich structure that is organized during the first meiotic prophase and that is still present at metaphase I. Surprisingly, the dense plate incorporates SCP1, the main protein of the central element of the synaptonemal complex, from diplotene until telophase I. Once sex chromosomes are under spindle tension, they move to opposite poles losing contact with the dense plate and undergoing early segregation. Thus, the segregation of the achiasmatic T. elegans sex chromosomes seems to be ensured by the presence in metaphase I of a synaptonemal complex-derived structure. This feature, unique among vertebrates, indicates that synaptonemal complex elements may play a role in chromosome segregation.
Asunto(s)
Segregación Cromosómica/fisiología , Marsupiales/fisiología , Profase Meiótica I/fisiología , Cromosomas Sexuales/metabolismo , Complejo Sinaptonémico/fisiología , Animales , Emparejamiento Cromosómico/fisiología , Masculino , Marsupiales/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Espermatocitos/citología , Espermatocitos/fisiología , Telómero/genéticaRESUMEN
Cohesins are vital for chromosome organisation during meiosis and mitosis. In addition to the important function in sister chromatid cohesion, these complexes play key roles in meiotic recombination, DSB repair, homologous chromosome pairing and segregation. Egg-laying mammals (monotremes) feature an unusually complex sex chromosome system, which raises fundamental questions about organisation and segregation during meiosis. We discovered a dynamic and differential accumulation of cohesins on sex chromosomes during platypus prophase I and specific reorganisation of the sex chromosome complex around a large nucleolar body. Detailed analysis revealed a differential loading of SMC3 on the chromatin and chromosomal axis of XY shared regions compared with the chromatin and chromosomal axes of asynapsed X and Y regions during prophase I. At late prophase I, SMC3 accumulation is lost from both the chromatin and chromosome axes of the asynaptic regions of the chain and resolves into subnuclear compartments. This is the first report detailing unpaired DNA specific SMC3 accumulation during meiosis in any species and allows speculation on roles for cohesin in monotreme sex chromosome organisation and segregation.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Emparejamiento Cromosómico , Profase Meiótica I/genética , Ornitorrinco/genética , Cromosomas Sexuales/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Hibridación Fluorescente in Situ , Masculino , Ornitorrinco/metabolismo , CohesinasRESUMEN
Marsupials present a series of genetic and chromosomal features that are highly conserved in very distant species. One of these features is the absence of a homologous region between X and Y chromosomes. According to this genetic differentiation, sex chromosomes do not synapse during the first meiotic prophase in males, and a special structure, the dense plate, maintains sex chromosome association. In this report we present results on the process of meiotic sex chromosome pairing obtained from three different species, Thylamys elegans, Dromiciops gliroides, and Rhyncholestes raphanurus, representing the three orders of American marsupials. We have investigated the relationships between the axial structures organized along sex chromosomes and the formation of the dense plate. We found that in the three species the dense plate arises as a modification of sex chromosomal axial elements, but without the involvement of other meiotic axial structures, such as the cohesin axes. Considering the phylogenetic relationships among the marsupials studied here, our data reinforce the idea that the dense plate emerged early in marsupial evolution as an efficient mechanism to ensure the association of the nonhomologous sex chromosomes. This situation could have influenced the further evolution of sex chromosomes in marsupials.
Asunto(s)
Meiosis , Animales , Centrómero/ultraestructura , Emparejamiento Cromosómico , Didelphis/genética , Evolución Molecular , Masculino , Marsupiales , Microscopía Fluorescente , Mitosis , Zarigüeyas/genética , Fosforilación , Filogenia , Profase , Cromosomas Sexuales , Especificidad de la Especie , Cromosoma X , Cromosoma YRESUMEN
Meiotic recombination generates crossovers between homologous chromosomes that are essential for genome haploidization. The synaptonemal complex is a 'zipper'-like protein assembly that synapses homologue pairs together and provides the structural framework for processing recombination sites into crossovers. Humans show individual differences in the number of crossovers generated across the genome. Recently, an anonymous gene variant in C14ORF39/SIX6OS1 was identified that influences the recombination rate in humans. Here we show that C14ORF39/SIX6OS1 encodes a component of the central element of the synaptonemal complex. Yeast two-hybrid analysis reveals that SIX6OS1 interacts with the well-established protein synaptonemal complex central element 1 (SYCE1). Mice lacking SIX6OS1 are defective in chromosome synapsis at meiotic prophase I, which provokes an arrest at the pachytene-like stage and results in infertility. In accordance with its role as a modifier of the human recombination rate, SIX6OS1 is essential for the appropriate processing of intermediate recombination nodules before crossover formation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Fertilidad , Complejo Sinaptonémico/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Emparejamiento Cromosómico , Intercambio Genético , Proteínas de Unión al ADN , Electroporación , Femenino , Variación Genética , Genoma , Células HEK293 , Haploidia , Humanos , Masculino , Meiosis , Ratones , Proteínas Nucleares/metabolismo , Recombinación Genética , Testículo/patología , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Cohesin is a conserved multisubunit protein complex that participates in chromosome segregation, DNA damage repair, chromatin regulation, and synaptonemal complex (SC) formation. Yeast, but not mice, depleted of the cohesin subunit Rec8 are defective in the formation of the axial elements (AEs) of the SC, suggesting that, in mammals, this function is not conserved. In this paper, we show that spermatocytes from mice lacking the two meiosis-specific cohesin subunits RAD21L and REC8 were unable to initiate RAD51- but not DMC1-mediated double-strand break repair, were not able to assemble their AEs, and arrested as early as the leptotene stage of prophase I, demonstrating that cohesin plays an essential role in AE assembly that is conserved from yeast to mammals.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Meiosis , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Animales , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/deficiencia , Fosfoproteínas/deficiencia , Unión Proteica , CohesinasRESUMEN
This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes.
Asunto(s)
Eimeria tenella/citología , Eimeria tenella/metabolismo , Meiosis/fisiología , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Animales , Proteínas de Ciclo Celular/fisiología , Pollos , Regulación de la Expresión Génica/fisiología , Transporte de Proteínas , Organismos Libres de Patógenos EspecíficosRESUMEN
Meiosis is a fundamental process that generates new combinations between maternal and paternal genomes and haploid gametes from diploid progenitors. Many of the meiosis-specific events stem from the behavior of the cohesin complex (CC), a proteinaceous ring structure that entraps sister chromatids until the onset of anaphase. CCs ensure chromosome segregation, participate in DNA repair, regulate gene expression, and also contribute to synaptonemal complex (SC) formation at meiosis by keeping long-range distant DNA interactions through its conserved structure. Studies from yeast to humans have led to the assumption that Scc1/RAD21 is the α-kleisin that closes the tripartite CC that entraps two DNA molecules in mitosis, while its paralog REC8 is essential for meiosis. Here we describe the identification of RAD21L, a novel mammalian CC subunit with homology to the RAD21/REC8 α-kleisin subfamily, which is expressed in mouse testis. RAD21L interacts with other cohesin subunits such as SMC1α, SMC1b, SMC3 and with the meiosis-specific STAG3 protein. Thus, our results demonstrate the existence of a new meiotic-specific CC constituted by this α-kleisin and expand the view of REC8 as the only specific meiotic α-kleisin.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Proteínas de Unión al ADN/química , Meiosis , Proteínas Nucleares/química , Fosfoproteínas/química , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Secuencia Conservada , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Masculino , Meiosis/genética , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Fosfoproteínas/genética , Estructura Terciaria de Proteína/genética , Homología de Secuencia de Aminoácido , Testículo/química , Testículo/fisiología , CohesinasRESUMEN
In Eimeria tenella, meiotic division occurs exclusively in oocysts within the first 8h of sporulation. Difficulties with the wall-oocyst breakage in gaining access to chromosomes during meiosis have resulted in a scarcity of morphological data on Eimeria chromosomes. This study tracks the general behaviour of telomeres, attachment plaques and synaptonemal complexes in the nucleus of the meiotic oocyst of E. tenella. Fluorescence microscopy methods, in combination with immunoelectron microscopy techniques, were applied to obtain a series of time-lapse images during oocyst sporulation. Antibodies to Structural Maintenance of Chromosome proteins SMC1 and SMC3, and lamin were labelled with either fluorescence or colloidal gold to visualise the telomeres, central elements of the synaptonemal complex (SC) and nuclear periphery, respectively, at both the structural and ultrastructural levels. Using oocyst spreads and ultrathin sections of fixed oocysts it was possible to study telomere dynamics at stages during meiosis. The stages of the meiotic prophase I are delineated on the basis of the telomere position and the SC synapsis and desynapsis. During the leptotene stage, at 4h following the start of sporulation, meiotic chromosomes attached to the nuclear envelope. At that stage, chromosome synapsis was initiated in the telomeric regions but no interstitial synapsis pairing was observed. In the zygotene stage, telomere signals were clustered in a limited area of the nuclear envelope. Bouquet formation occurred at 5h after the start of sporulation, whereas chromosomes did not appear completely synapsed until the pachytene stage at 6h of sporulation. Desynapsis was observed at 8h of sporulation during the diplotene stage. This study provides the first morphological description of both the behaviour of the chromosomes and the timing of the prophase I stages in the meiotic nucleus of E. tenella.