Synapsis and meiotic recombination in male Chinese muntjac (Muntiacus reevesi)

The muntjacs (Muntiacus, Cervidae) have been extensively studied in terms of chromosomal and karyotypic evolution. However, little is known about their meiotic chromosomes particularly the recombination patterns of homologous chromosomes. We used immunostained surface spreads to visualise synaptonemal complexes (SCs), recombination foci and kinetochores with antibodies against marker proteins. As in other mammals pachytene was the longest stage of meiotic prophase. 39.4% of XY bivalents lacked MLH1 foci compared to less than 0.5% of autosomes. The average number of MLH1 foci per pachytene cell in M. reevesi was 29.8. The distribution of MLH1 foci differed from other mammals. On SCs with one focus, the distribution was more even in M. reevesi than in other mammals; for SCs that have two or more MLH1 foci, usually there was a larger peak in the sub-centromere region than other regions on SC in M. reevesi. Additionally, there was a lower level of interference between foci in M. reevesi than in mouse or human. These observations may suggest that the regulation of homologous recombination in M. reevesi is slightly different from other mammals and will improve our understanding of the regulation of meiotic recombination, with respect to recombination frequency and position.     

Presence of an extra chromosome alters meiotic double-stranded break repair dynamics and MLH1 foci distribution in human oocytes

Studies performed on human trisomic 21 oocytes have revealed that during meiosis, the three homologues 21 synapse and, in some cases, achieve what looks like a trivalent. This implies that meiotic recombination takes place among the three homologous chromosomes 21, and to some extent, crossovers form between them. To see how meiotic recombination is in the presence of an extra chromosome 21, we analyzed the distribution of three recombination markers (γH2AX, RPA, and MLH1) on trisomic 21 oocytes at pachynema and, in particular, on chromosomes 21. Results clearly show how the presence of an extra chromosome 21 alters meiotic recombination progression, leading to the presence of a higher number of early recombination markers at pachynema. Moreover, the distribution on these chromosomes 21 of some of these markers is different in aneuploid oocytes. Finally, there is a substantial increase in the number of MLH1 foci, a marker of most crossovers in mammals, which is related to the number of synapsed chromosomes in pachynema. Thus, bivalents 21 had fewer MLH1 foci than partial or total trivalents, suggesting a close relationship between synapsis and crossover designation. All of the data presented suggest that the presence of an extra chromosome alters meiotic recombination globally in aneuploid human oocytes.     

Patterns of meiotic recombination in human fetal oocytes

Abnormal patterns of meiotic recombination (i.e., crossing-over) are believed to increase the risk of chromosome nondisjunction in human oocytes. To date, information on recombination has been obtained using indirect, genetic methods. Here we use an immunocytological approach, based on detection of foci of a DNA mismatch-repair protein, MLH1, on synaptonemal complexes at prophase I of meiosis, to provide the first direct estimate of the frequency of meiotic recombination in human oocytes. At pachytene, the stage of maximum homologous chromosome pairing, we found a mean of 70.3 foci (i.e., crossovers) per oocyte, with considerable intercell variability (range 48-102 foci). This mean equates to a genetic-map length of 3,515 cM. The numbers and positions of foci were determined for chromosomes 21, 18, 13, and X. These chromosomes yielded means of 1.23 foci (61.5 cM), 2.36 foci (118 cM), 2.5 foci (125 cM), and 3.22 foci (161 cM), respectively. The foci were almost invariably located interstitially and were only occasionally located close to chromosome ends. These data confirm the large difference, in recombination frequency, between human oocytes and spermatocytes and demonstrate a clear intersex variation in distribution of crossovers. In a few cells, chromosomes 21 and 18 did not have any foci (i.e., were presumptively noncrossover); however, configurations that lacked foci were not observed for chromosomes 13 and X. For the latter two chromosome pairs, the only instances of absence of foci were observed in abnormal cells that showed chromosome-pairing errors affecting these chromosomes. We speculate that these abnormal fetal oocytes may be the source of the nonrecombinant chromosomes 13 and X suggested, by genetic studies, to be associated with maternally derived chromosome nondisjunction.     

人精母细胞重组频率和年龄相关性的分析

减数分裂遗传重组对同源染色体的正确分离和单倍体的正确形成起至关重要的作用,但人们对人精母细胞减数分裂遗传重组机制了解的还很少。通过免疫荧光染色技术标记减数分裂I联会复合体上的MLH1(DNA错配修复蛋白)位点可以检测人精母细胞的重组。文章对10例可育男性进行分析,发现每个细胞中重组位点数平均为49.4士4.4,范围为33~63,具有显著的个体差异,只有0.4%(1/220)的常染色体SC上缺少MLH1位点。进一步通过Spearman相关性分析,分析了年龄因素与个体间重组位点差异的相关性,结果提示年龄因素对常染色体及性染色体的重组均无影响。     

MSH4 acts in conjunction with MLH1 during mammalian meiosis.

MSH4 is a meiosis-specific MutS homolog. In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast. Germ-line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1-deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals. Here we show that hMSH4 interacts with hMLH1. The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA. The domain of hMSH4 responsible for the interaction is in the amino-terminal part of the protein whereas the region that contains the ATP binding site and helix-turn-helix motif does not bind to hMLH1. Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse. The number of MSH4 foci decreases gradually as pachynema progresses. During this transition, MLH1 foci begin to appear and colocalize with MSH4. These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination.     

Sex-specific crossover patterns in Zebrafish (Danio rerio)

Some species display intersex variation in their rate of meiotic recombination, where recombination is usually suppressed in the heterogametic sex. Although no heteromorphic sex chromosomes have been detected in zebrafish (Danio rerio), genetic analysis has indicated a lower frequency of recombination in males relative to females. Our study of the meiotic recombination pattern in female zebrafish indicates that adult females have only a few meiotic oocytes that are found in groups in the ventral zone of the ovarian surface. We used antibody staining of human mutL homolog 1 (MLH1) protein to mark the sites of putative chiasmata to seek a physical basis for the pattern of recombination and its relative frequency in both sexes. We report that MLH1 foci are found mostly in distal regions of the synaptonemal complexes (SCs) in males, but tend to be more evenly distributed in females. Our cytological analysis yields a ratio of MLH1 foci per chromosome in males versus females of 1:1.55. This lower level of recombination in males is in general agreement with previously published results from linkage map analysis. However, the similar ratio of MLH1 foci per unit length of SCs in both sexes demonstrates a correlation between SC length and the frequency of recombination rather than a mechanism that suppresses recombination in males. Thus, chiasma interference seems to provide similar expression in males and females in agreement with the situation in humans, where oocytes with longer SCs display a higher level of recombination that is not a consequence of more closely spaced crossovers along the SCs.     

Meiotic recombination in normal and clone bulls and their offspring

Homologous chromosome pairing and recombination are essential components of meiosis and sexual reproduction. The reshuffling of genetic material through breakage and reunion of chromatids ensure proper segregation of homologous chromosomes in reduction division and genetic diversity in the progeny. The advent of somatic cell nuclear transfer (SCNT) as a reproductive biotechnology for use in livestock industry has made it easy to bypass these vital steps. However, few studies have been carried out on the impact of SCNT on the reproductive characteristics of cloned animals and, none to date, on the meiotic processes in animals, which were created by circumventing meiosis. In an attempt to assess the impact of cloning by SCNT on the meiotic processes, we undertook an immunocytological comparison of recombination in normal and clone bulls using antibodies raised against the synaptonemal complex protein 3 (SCP3) to label the lateral elements and the mismatch repair protein 1 (MLH1) foci on bivalents as indicators of recombination events. Our studies involving five normal bulls of proven fertility, two SCNT-derived bulls, and four mature offspring of SCNT bulls showed that the mean number of crossing over per spermatocyte for normal bulls (42 +/- 4 SD; ranging from 33 to 56), was not significantly different from that of SCNT-derived bulls (43 +/- 5 SD; ranging from 35 to 56), and the offspring of SCNT-derived bulls (43 +/- 5 SD; ranging from 37 to 58). It would appear that circumventing meiosis to produce these animals does not influence the meiotic processes revealed by MLH1 foci detected in spermatocytes.     

Meiotic Recombination in Human Oocytes

Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1) in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of “vulnerable” crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.     

Extreme heterogeneity in the molecular events leading to the establishment of chiasmata during meiosis i in human oocytes

In humans, ~50% of conceptuses are chromosomally aneuploid as a consequence of errors in meiosis, and most of these aneuploid conceptuses result in spontaneous miscarriage. Of these aneuploidy events, 70% originate during maternal meiosis, with the majority proposed to arise as a direct result of defective crossing over during meiotic recombination in prophase I. By contrast, <1%-2% of mouse germ cells exhibit prophase I-related nondisjunction events. This disparity among mammalian species is surprising, given the conservation of genes and events that regulate meiotic progression. To understand the mechanisms that might be responsible for the high error rates seen in human females, we sought to further elucidate the regulation of meiotic prophase I at the molecular cytogenetic level. Given that these events occur during embryonic development in females, samples were obtained during a defined period of gestation (17-24 weeks). Here, we demonstrate that human oocytes enter meiotic prophase I and progress through early recombination events in a similar temporal framework to mice. However, at pachynema, when chromosomes are fully paired, we find significant heterogeneity in the localization of the MutL homologs, MLH1 and MLH3, among human oocyte populations. MLH1 and MLH3 have been shown to mark late-meiotic nodules that correlate well with--and are thought to give rise to--the sites of reciprocal recombination between homologous chromosomes, which suggests a possible 10-fold variation in the processing of nascent recombination events. If such variability persists through development and into adulthood, these data would suggest that as many as 30% of human oocytes are predisposed to aneuploidy as a result of prophase I defects in MutL homolog-related events.     

Recombination correlates with synaptonemal complex length and chromatin loop size in bovids—insights into mammalian meiotic chromosomal organization

Homologous chromosomes exchange genetic information through recombination during meiosis, a process that increases genetic diversity, and is fundamental to sexual reproduction. In an attempt to shed light on the dynamics of mammalian recombination and its implications for genome organization, we have studied the recombination characteristics of 112 individuals belonging to 28 different species in the family Bovidae. In particular, we analyzed the distribution of RAD51 and MLH1 foci during the meiotic prophase I that serve, respectively, as proxies for double-strand breaks (DSBs) which form in early stages of meiosis and for crossovers. In addition, synaptonemal complex length and meiotic DNA loop size were estimated to explore how genome organization determines DSBs and crossover patterns. We show that although the number of meiotic DSBs per cell and recombination rates observed vary between individuals of the same species, these are correlated with diploid number as well as with synaptonemal complex and DNA loop sizes. Our results illustrate that genome packaging, DSB frequencies, and crossover rates tend to be correlated, while meiotic chromosomal axis length and DNA loop size are inversely correlated in mammals. Moreover, axis length, DSB frequency, and crossover frequencies all covary, suggesting that these correlations are established in the early stages of meiosis.     

Discontinuities and unsynapsed regions in meiotic chromosomes have a trans effect on meiotic recombination of some chromosomes in human males

During meiosis, homologous chromosome pairing and synapsis are essential for subsequent meiotic recombination (crossing-over). Discontinuous regions (gaps) and unsynapsed regions (splits) were most frequently observed in the heterochromatic regions of bivalent synaptonemal complex (SC) 9, and we have previously demonstrated that gaps and splits significantly altered the distribution of MLH1 recombination foci on SC 9. Here, immunofluorescence techniques (using antibodies against SC proteins and the crossover-associated MLH1 protein) were combined with a centromere-specific fluorescence in situ hybridization technique that allows identification of every individual chromosome. The effect of gaps/splits on meiotic recombination patterns in autosomes other than chromosome 9 during the pachytene stage of meiotic prophase was then examined in 6,026 bivalents from 262 pachytene cells from three human males. In 64 analyzed cells with a gapped SC 9, the frequency of MLH1 foci in SCs 5 and 10 and in SC arms 10q, 11p and 16q was decreased compared to 168 analyzed cells with a normally-synapsed SC 9 (controls). In 24 analyzed cells with splits in SC 9, there was a significant reduction in MLH1 focus frequency for SC 5q and the whole SC5 bivalent. The positioning of MLH1 foci on other SCs in cells with gapped/split SC 9 was not altered. These studies suggest that gaps and splits not only have a cis effect, but may also have a trans effect on meiotic recombination in humans.     

Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.     

Intra and Interspecific Variation in Cranial Morphology on the Southernmost Distributed Cebus (Platyrrhini,Primates) Species

Capuchin monkeys (Cebus) are one of the genera with the widest distribution among Neotropical primates (New World Monkeys, Platyrrhini), accompanied by an elevated genetic, phenotypic, behavioral, morphological, and ecological diversity, both at the interspecific and population levels. Despite being one of the most studied primate genera, this high diversity has led to a particularly complex and controversial taxonomy. In this contribution, we explored the patterns of skull size and shape variation among the southernmost distributed populations of Cebus using three-dimensional geometric morphometric techniques. Results showed a marked morphological differentiation (in size and shape) between previously recognized species (C. nigritus and southern C. libidinosus), and also among C. libidinosus populations, which were quantitatively related with the geographic distance between them. This pattern supports a differentiation between the northwestern Argentina and southern Bolivia and Paraguay forms. Other taxonomic implications are also discussed.     

A non-sister act: Recombination template choice during meiosis

Meiotic recombination has two key functions: the faithful assortment of chromosomes into gametes and the creation of genetic diversity. Both processes require that meiotic recombination occurs between homologous chromosomes, rather than sister chromatids. Accordingly, a host of regulatory factors are activated during meiosis to distinguish sisters from homologs, suppress recombination between sister chromatids and promote the chromatids of the homologous chromosome as the preferred recombination partners. Here, we discuss the recent advances in our understanding of the mechanistic basis of meiotic recombination template choice, focusing primarily on developments in the budding yeast, Saccharomyces cerevisiae, where the regulation is currently best understood.     

Variation in meiotic recombination frequencies among human males

Meiotic recombination is essential for the segregation of homologous chromosomes and the formation of normal haploid gametes. Little is known about patterns of meiotic recombination in human germ cells or the mechanisms that control these patterns. Here, newly developed immunofluorescence techniques, based on the detection of MLH1 (a DNA mismatch repair protein) foci on synaptonemal complexes (SCs) at prophase I of meiosis, were used to examine recombination in human spermatocytes. The mean number of MLH1 foci per cell in all donors was 48.0 with range from 21 to 65. Remarkable variation in the recombination frequency was noted among 11 normal individuals: the mean frequencies of chromosomal recombination foci ranged from a low of 42.5 to a high of 55.0 exchanges. Donor age did not contribute to this variation. There was no correlation between this variation and the frequency of gaps (discontinuities) or splits (unpaired chromosome regions) in the SCs. The mean percentage of cells with gaps was 35% (range: 20% to 58%) and with splits was 7% (range: 0% to 37%). Bivalents without a recombination focus were rare, with a frequency of only 0.3%. Thus, achiasmate chromosomes appear to be rare in human male meiosis.     

Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse

Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies.     

Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein

We have used immunofluorescent localization to examine the distribution of MLH1 (MutL homolog) foci on synaptonemal complexes (SCs) from juvenile male mice. MLH1 is a mismatch repair protein necessary for meiotic recombination in mice, and MLH1 foci have been proposed to mark crossover sites. We present evidence that the number and distribution of MLH1 foci on SCs closely correspond to the number and distribution of chiasmata on diplotene-metaphase I chromosomes. MLH1 foci were typically excluded from SC in centromeric heterochromatin. For SCs with one MLH1 focus, most foci were located near the middle of long SCs, but near the distal end of short SCs. For SCs with two MLH1 foci, the distribution of foci was bimodal regardless of SC length, with most foci located near the proximal and distal ends. The distribution of MLH1 foci indicated interference between foci. We observed a consistent relative distance (percent of SC length in euchromatin) between two foci on SCs of different lengths, suggesting that positive interference between MLH1 foci is a function of relative SC length. The extended length of pachytene SCs, as compared to more condensed diplotene-metaphase I bivalents, makes mapping crossover events and interference distances using MLH1 foci more accurate than using chiasmata.     

标记减数分裂遗传重组的免疫荧光染色方法

联会复合体免疫荧光技术在全基因减数分裂遗传重组研究中具有精确和直观的优势.本研究通过免疫荧光染色方法制备小鼠精母细胞联会复合体,研究其形态组成与遗传重组特征,展示雄性小鼠遗传重组图谱并分析其重组位点(MLH1位点)的分布特征.4只小鼠共145个精母细胞在平均每个细胞的MLH1位点数为23.3±2.4;在常染色体联会复合体中,未发现有3个MLH1位点的联会复合体,具有1个MLH1位点的联会复合体较多,平均为14.2;无XY联会复合体的细胞占所有细胞的4.1%,XY联会复合体上有MLH1位点的细胞占30.2%;联会复合体上有裂缝的细胞占0.7%.通过联会复合体免疫荧光染色可以清晰地分辨出联会复合体(红色)、着丝粒(蓝色)和MLH1位点(绿色),是遗传重组分析的一种强有力工具.     

Immunocytological analysis of meiotic recombination in the American mink (Mustela vison)

Using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites along synaptonemal complexes, we estimated the total length of the genetic map, the recombination rate and crossover distribution in the American mink ( Mustela vison ). We prepared spreads from 130 spermatocytes of five male minks and mapped 3320 MLH1 foci along 1820 bivalents. The total recombination length of the male mink genome, based on the mean number of MLH1 foci for all chromosomes, was 1327 cM. The overall recombination rate was estimated to be 0.48 cM/Mb. In all bivalents, we observed prominent peaks of MLH1 foci near the distal ends and a paucity of them near the centromeres. This indicates that genes located at proximal regions of the chromosomes should display much tighter genetic linkage than physically equidistant markers located near the telomeres.