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.
- MeSH
- apoptóza genetika MeSH
- dvouřetězcové zlomy DNA MeSH
- fertilita genetika MeSH
- genetické pozadí MeSH
- kontrolní body buněčného cyklu genetika MeSH
- myši MeSH
- pachytenní stadium genetika MeSH
- počet spermií MeSH
- spermatocyty metabolismus MeSH
- testis anatomie a histologie metabolismus MeSH
- transgeny * MeSH
- variabilita počtu kopií segmentů DNA * MeSH
- velikost orgánu MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.
- MeSH
- chromozom X genetika MeSH
- chromozom Y genetika MeSH
- dvouřetězcové zlomy DNA * MeSH
- homologní rekombinace genetika MeSH
- meióza genetika MeSH
- myši MeSH
- pseudoautozomální oblasti genetika MeSH
- segregace chromozomů genetika MeSH
- spermatocyty růst a vývoj metabolismus MeSH
- transportní proteiny chemie genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In mammals, meiotic recombination occurs at 1- to 2-kb genomic regions termed hotspots, whose positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase. We show that the KRAB domain of PRDM9 forms complexes with additional proteins to allow hotspots to proceed into the next phase of recombination. By a combination of yeast-two hybrid assay, in vitro binding, and coimmunoprecipitation from mouse spermatocytes, we identified four proteins that directly interact with PRDM9's KRAB domain, namely CXXC1, EWSR1, EHMT2, and CDYL. These proteins are coexpressed in spermatocytes at the early stages of meiotic prophase I, the limited period when PRDM9 is expressed. We also detected association of PRDM9-bound complexes with the meiotic cohesin REC8 and the synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action of these proteins, ensuring the proper chromatin and spatial environment for subsequent recombination events.
- MeSH
- chromatin metabolismus MeSH
- chromozomy genetika fyziologie MeSH
- DNA metabolismus MeSH
- dvouřetězcové zlomy DNA MeSH
- genom MeSH
- histonlysin-N-methyltransferasa genetika metabolismus fyziologie MeSH
- homologní rekombinace MeSH
- meióza fyziologie MeSH
- myši MeSH
- proteinové domény MeSH
- rekombinace genetická fyziologie MeSH
- spermatocyty metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Despite similar genome sizes, a great variability in recombination rates is observed in mammals. We used antibodies against SYCP3, MLH1 and centromeres to compare crossover frequency, position along chromosome arms and the effect of crossover interference in spermatocytes of 4 species from the family Bovidae (Bos taurus, 2n = 60, tribe Bovini; Ovis aries, 2n = 54, Capra hircus, 2n = 60 and Ammotragus lervia, 2n = 58, tribe Caprini). Despite significant individual variability, our results also show significant differences in both recombination rates and the total length of autosomal synaptonemal complexes (SC) between cattle (47.53 MLH1 foci/cell, 244.59 µm) and members of the tribe Caprini (61.83 MLH1 foci, 296.19 µm) which can be explained by the length of time that has passed since their evolutionary divergence. Sheep displayed the highest number of MLH1 foci per cell and recombination density, although they have a lower diploid chromosome number caused by centric fusions corresponding to cattle chromosomes 1;3, 2;8 and 5;11. However, the proportion of MLH1 foci observed on the fused chromosomes in sheep (26.14%) was significantly lower than on the orthologous acrocentrics in cattle (27.6%) and goats (28.2%), and their distribution along the SC arms differed significantly. The reduced recombination rate in metacentrics is probably caused by interference acting across the centromere.
- MeSH
- hybridizace in situ fluorescenční MeSH
- jaderné proteiny metabolismus MeSH
- kozy genetika MeSH
- meióza genetika MeSH
- ovce genetika MeSH
- rekombinace genetická * MeSH
- skot genetika MeSH
- spermatocyty metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot genetika MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The captive bred animal populations showing centric fusion polymorphism can serve as a model for analysis of the impact of the rearrangement on meiosis and reproduction. The synapsis of homologous chromosomes and the frequency and distribution of meiotic recombination events were studied in pachytene spermatocytes of captive bred male impalas (Aepyceros melampus) polymorphic for der(14;20) by immunofluorescent analysis and fluorescence in situ hybridization. The chromosomes 14 and 20 involved in the centric fusion were significantly shorter due to the loss of sat I repeats indicating ancient origin of the rearrangement. The fused chromosome and the normal acrocentric chromosomes 14 and 20 formed trivalent in pachynema which showed either protruding proximal ends of the acrocentric chromosomes or single axis with synaptic adjustment in the pericentromeric region. There was no significant difference in the number of recombination events per cell between the group of translocation heterozygotes and the animals with normal karyotype. A significant reduction in the number of recombination events was observed in the trivalent chromosomes compared to the normal chromosomes 14 and 20. The level of the recombination reduction was related to the trivalent configuration. The centric fusion der(14;20) was not apparently demonstrated by any spermatogenic defects or reproductive impairment in heterozygous impalas. However, the high incidence of the chromosomal polymorphism within the captive bred population shows the importance of cytogenetic examinations in captive breeding and wildlife conservation programs, especially in the case of reintroduction of the endangered species.
- MeSH
- hybridizace in situ fluorescenční MeSH
- jaderné proteiny genetika MeSH
- lymfocyty metabolismus MeSH
- meióza genetika MeSH
- metafáze genetika MeSH
- modely u zvířat MeSH
- pachytenní stadium MeSH
- přežvýkavci genetika MeSH
- rekombinace genetická genetika MeSH
- rozmnožování genetika MeSH
- savčí chromozomy genetika MeSH
- spermatocyty cytologie metabolismus MeSH
- synaptonemální komplex genetika MeSH
- translokace genetická * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.
- MeSH
- akrozom imunologie fyziologie MeSH
- akrozomální reakce MeSH
- benzoáty farmakologie MeSH
- exocytóza MeSH
- fertilizace * účinky léků MeSH
- fosfotyrosin imunologie MeSH
- furany farmakologie MeSH
- glykoproteiny analýza imunologie MeSH
- interakce spermie a vajíčka * MeSH
- kapacitace spermií * MeSH
- prasata metabolismus fyziologie MeSH
- proteiny semenné plazmy analýza imunologie MeSH
- protilátky imunologie MeSH
- pyrazoly farmakologie MeSH
- spermatocyty metabolismus MeSH
- spermatogonie metabolismus MeSH
- spermie metabolismus MeSH
- ubikvitin aktivující enzymy metabolismus MeSH
- ubikvitin imunologie MeSH
- ubikvitinace MeSH
- zona pellucida metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Heterozygosity for certain mouse and human chromosomal rearrangements is characterized by the incomplete meiotic synapsis of rearranged chromosomes, by their colocalization with the XY body in primary spermatocytes, and by male-limited sterility. Previously, we argued that such X-autosomal associations could interfere with meiotic sex chromosome inactivation. Recently, supporting evidence has reported modifications of histones in rearranged chromosomes by a process called the meiotic silencing of unsynapsed chromatin (MSUC). Here, we report on the transcriptional down-regulation of genes within the unsynapsed region of the rearranged mouse chromosome 17, and on the subsequent disturbance of X chromosome inactivation. The partial transcriptional suppression of genes in the unsynapsed chromatin was most prominent prior to the mid-pachytene stage of primary spermatocytes. Later, during the mid-late pachytene, the rearranged autosomes colocalized with the XY body, and the X chromosome failed to undergo proper transcriptional silencing. Our findings provide direct evidence on the MSUC acting at the mRNA level, and implicate that autosomal asynapsis in meiosis may cause male sterility by interfering with meiotic sex chromosome inactivation.
- MeSH
- chromatin genetika MeSH
- chromozom X genetika MeSH
- down regulace MeSH
- financování organizované MeSH
- genová přestavba MeSH
- inaktivace chromozomu X MeSH
- lidé MeSH
- meióza genetika MeSH
- mužská infertilita genetika MeSH
- myši inbrední C57BL MeSH
- myši kongenní MeSH
- myši MeSH
- spermatocyty cytologie metabolismus MeSH
- spermatogeneze genetika MeSH
- translokace genetická MeSH
- umlčování genů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
