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1.
Development ; 150(7)2023 04 01.
Article in English | MEDLINE | ID: mdl-36975404

ABSTRACT

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications - principally increased intron retention - resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biology.


Subject(s)
RNA Isoforms , Testis , Animals , Mice , Male , Testis/metabolism , RNA Isoforms/metabolism , Spermatogenesis/genetics , Spermatids/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism
2.
PLoS Genet ; 15(8): e1008316, 2019 08.
Article in English | MEDLINE | ID: mdl-31437213

ABSTRACT

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.


Subject(s)
Fertility/genetics , Infertility, Male/genetics , Metaphase/genetics , Proteasome Endopeptidase Complex/genetics , Protein Subunits/genetics , Animals , Apoptosis/genetics , Disease Models, Animal , Female , Male , Mice , Mice, Knockout , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Subunits/metabolism , Spermatocytes/metabolism , Spermatogenesis/genetics , Synaptonemal Complex/metabolism , Testis/cytology , Testis/metabolism
3.
Cell Rep ; 23(1): 143-155, 2018 04 03.
Article in English | MEDLINE | ID: mdl-29617656

ABSTRACT

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.


Subject(s)
Azoospermia/genetics , Erythropoiesis , Muscle Contraction , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , RNA Splicing , Ribonucleoproteins/genetics , Spermatogenesis , Animals , Azoospermia/pathology , Cells, Cultured , Male , Mice , Mutation , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Ribonucleoproteins/metabolism , Splicing Factor U2AF
4.
Reproduction ; 151(3): 239-51, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26667018

ABSTRACT

In sexually reproducing organisms, accurate gametogenesis is crucial for the transmission of genetic material from one generation to the next. This requires the faithful segregation of chromosomes during mitotic and meiotic divisions. One of the main players in this process is the kinetochore, a large multi-protein complex that forms at the interface of centromeres and microtubules. Here, we analyzed the expression profile and function of small kinetochore-associated protein (SKAP) in the mouse. We found that two distinct SKAP isoforms are specifically expressed in the germline: a smaller isoform, which is detected in spermatogonia and spermatocytes and localized in the outer mitotic and meiotic kinetochores from metaphase to telophase, and a larger isoform, which is expressed in the cytoplasm of elongating spermatids. We generated SKAP-deficient mice and found that testis size and sperm production were severely reduced in mutant males. This phenotype was partially caused by defects during spermatogonia proliferation before entry into meiosis. We conclude that mouse SKAP, while being dispensable for somatic cell divisions, has an important role in the successful outcome of male gametogenesis. In germ cells, analogous to what has been suggested in studies using immortalized cells, SKAP most likely stabilizes the interaction between kinetochores and microtubules, where it might be needed as an extra safeguard to ensure the correct segregation of mitotic and meiotic chromosomes.


Subject(s)
Cell Cycle Proteins/metabolism , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Spermatogenesis , Spermatozoa/metabolism , Animals , Apoptosis , Female , Fertility , HeLa Cells , Humans , Male , Meiosis , Mice , Mitosis , Spermatozoa/growth & development
5.
EMBO J ; 30(15): 3091-105, 2011 Jul 08.
Article in English | MEDLINE | ID: mdl-21743440

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Meiosis , Age Factors , Animals , Chromosomal Proteins, Non-Histone/deficiency , Chromosomes/metabolism , Female , Histocytochemistry , Infertility , Male , Mice , Mice, Knockout , Ovary/pathology , Protein Subunits/metabolism , Sex Factors , Testis/pathology
6.
Cell Cycle ; 10(9): 1477-87, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21527826

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA-Binding Proteins/chemistry , Meiosis , Nuclear Proteins/chemistry , Phosphoproteins/chemistry , Amino Acid Sequence , Animals , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Conserved Sequence , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , Male , Meiosis/genetics , Mice , Molecular Sequence Data , Nuclear Proteins/genetics , Phosphoproteins/genetics , Protein Structure, Tertiary/genetics , Sequence Homology, Amino Acid , Testis/chemistry , Testis/physiology , Cohesins
7.
Chromosoma ; 118(5): 575-89, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19495784

ABSTRACT

We have analyzed in a true bug, Graphosoma italicum (Pentatomidae, Hemiptera), the temporal and functional relationships between recombination events, synapsis progression, and SMC1alpha and SMC3 cohesin axis maturation throughout the male first meiotic prophase. The localization of the histone variant histone H3 trimethylated at lysine 9 at chromosome ends has allowed us to determine the association of these heterochromatic domains through prophase I stages. Results highlighted that cohesins provide to be good markers for synapsis progression since the formation, morphology, and development of the SMC1alpha and SMC3 cohesin axes resemble the synaptonemal complex dynamics and, also, that in this species the initiation of recombination precedes synapsis. In addition, we have carried out an accurate cytological characterization of the diffuse stage, which takes place after pachytene, and also analyzed the presence of the cohesin subunits, SMC1alpha and SMC3, and the recombinase RAD51 at this stage. The mechanisms underlying the absence of SMC1alpha and SMC3 axes from the diffuse stage onwards are discussed.


Subject(s)
Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/physiology , Meiotic Prophase I/physiology , Rad51 Recombinase/physiology , Animals , Chromosomes/ultrastructure , Hemiptera , Histones/metabolism , Male , Rad51 Recombinase/genetics , Recombination, Genetic/physiology , Spermatocytes/metabolism , Synaptonemal Complex/physiology , Cohesins
8.
PLoS Genet ; 3(11): e198, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17983272

ABSTRACT

In most eutherian mammals, sex chromosomes synapse and recombine during male meiosis in a small region called pseudoautosomal region. However in some species sex chromosomes do not synapse, and how these chromosomes manage to ensure their proper segregation is under discussion. Here we present a study of the meiotic structure and behavior of sex chromosomes in one of these species, the Mongolian gerbil (Meriones unguiculatus). We have analyzed the location of synaptonemal complex (SC) proteins SYCP1 and SYCP3, as well as three proteins involved in the process of meiotic recombination (RAD51, MLH1, and gamma-H2AX). Our results show that although X and Y chromosomes are associated at pachytene and form a sex body, their axial elements (AEs) do not contact, and they never assemble a SC central element. Furthermore, MLH1 is not detected on the AEs of the sex chromosomes, indicating the absence of reciprocal recombination. At diplotene the organization of sex chromosomes changes strikingly, their AEs associate end to end, and SYCP3 forms an intricate network that occupies the Y chromosome and the distal region of the X chromosome long arm. Both the association of sex chromosomes and the SYCP3 structure are maintained until metaphase I. In anaphase I sex chromosomes migrate to opposite poles, but SYCP3 filaments connecting both chromosomes are observed. Hence, one can assume that SYCP3 modifications detected from diplotene onwards are correlated with the maintenance of sex chromosome association. These results demonstrate that some components of the SC may participate in the segregation of achiasmate sex chromosomes in eutherian mammals.


Subject(s)
Chromosome Pairing/genetics , Chromosome Segregation/genetics , Gerbillinae/genetics , Nuclear Proteins/metabolism , Sex Chromosomes/genetics , Animals , Centromere/metabolism , Chromatin/metabolism , Histones/metabolism , Male , Models, Genetic , Rad51 Recombinase/metabolism , Recombination, Genetic , Spermatocytes/cytology , Spermatocytes/enzymology , Synaptonemal Complex/metabolism
9.
PLoS Genet ; 2(8): e136, 2006 Aug 25.
Article in English | MEDLINE | ID: mdl-16934004

ABSTRACT

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.


Subject(s)
Chromosome Segregation/physiology , Marsupialia/physiology , Meiotic Prophase I/physiology , Sex Chromosomes/metabolism , Synaptonemal Complex/physiology , Animals , Chromosome Pairing/physiology , Male , Marsupialia/genetics , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Spermatocytes/cytology , Spermatocytes/physiology , Telomere/genetics
10.
Chromosoma ; 115(3): 250-9, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16544151

ABSTRACT

During first meiotic prophase, homologous chromosomes are held together by the synaptonemal complex, a tripartite proteinaceous structure that extends along the entire length of meiotic bivalents. While this feature is applicable for autosomes, sex chromosomes often escape from this rule. Many species present sex chromosomes that differ between them in their morphology, length, and gene content. Moreover, in some species, sex chromosomes appear in a single dose in one of the sexes. In all of these cases, the behavior of sex chromosomes during meiosis is conspicuously affected, and this includes the assembly and dynamics of the synaptonemal complex. We review in this study the structure of the synaptonemal complex in the sex chromosomes of three groups of organisms, namely: mammals, orthopterans, and hemipterans, which present different patterns of sex chromosome structure and behavior. Of special interest is the analysis of the organization of the axial/lateral elements of the synaptonemal complex in relation to other axial structures organized along meiotic chromosomes, mainly the cohesin axis. The differences found in the behavior of both axial structures reveal that while the organization of a cohesin axis along sex chromosomes is a conserved feature in most organisms and it shows very little morphological variations, the axial/lateral elements of the synaptonemal complex present a wide range of structural modifications on these chromosomes.


Subject(s)
Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/physiology , Chromosome Pairing/physiology , Nuclear Proteins/physiology , Sex Chromosomes/physiology , Animals , BRCA1 Protein/physiology , Cell Cycle Proteins/ultrastructure , Chromosomal Proteins, Non-Histone/ultrastructure , Humans , Nuclear Proteins/ultrastructure , Protein Serine-Threonine Kinases/physiology , Sex Chromosomes/chemistry , Sex Chromosomes/genetics , Cohesins
11.
Genetics ; 170(2): 793-9, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15802509

ABSTRACT

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.


Subject(s)
Meiosis , Animals , Centromere/ultrastructure , Chromosome Pairing , Didelphis/genetics , Evolution, Molecular , Male , Marsupialia , Microscopy, Fluorescence , Mitosis , Opossums/genetics , Phosphorylation , Phylogeny , Prophase , Sex Chromosomes , Species Specificity , X Chromosome , Y Chromosome
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