Pachytene Substages Research Articles

Defects in meiotic recombination delay progression through pachytene in Tex19.1\u2212/\u2212 mouse spermatocytes

Recombination, synapsis, chromosome segregation and gene expression are co-ordinately regulated during meiosis to ensure successful execution of this specialised cell division. Studies with multiple mutant mouse lines have shown that mouse spermatocytes possess quality control checkpoints that eliminate cells with persistent defects in chromosome synapsis. In addition, studies on Trip13mod/mod mice suggest that pachytene spermatocytes that successfully complete chromosome synapsis can undergo meiotic arrest in response to defects in recombination. Here, we present additional support for a meiotic recombination-dependent checkpoint using a different mutant mouse line, Tex19.1−/−. The appearance of early recombination foci is delayed in Tex19.1−/− spermatocytes during leptotene/zygotene, but some Tex19.1−/− spermatocytes still successfully synapse their chromosomes and we show that these spermatocytes are enriched for early recombination foci. Furthermore, we show that patterns of axis elongation, chromatin modifications and histone H1t expression are also all co-ordinately skewed towards earlier substages of pachytene in these autosomally synapsed Tex19.1−/− spermatocytes. We also show that this skew towards earlier pachytene substages occurs in the absence of elevated spermatocyte death in the population, that spermatocytes with features of early pachytene are present in late stage Tex19.1−/− testis tubules and that the delay in histone H1t expression in response to loss of Tex19.1 does not occur in a Spo11 mutant background. Taken together, these data suggest that a recombination-dependent checkpoint may be able to modulate pachytene progression in mouse spermatocytes to accommodate some types of recombination defect.

Synapsis, recombination, and chromatin remodeling in the XY body of armadillos

Three xenarthrans species Chaetophractus villosus, Chaetophractus vellerosus, and Zaedyus pichiy have been used for the analysis of the structure, behavior, and immunochemical features of the XY body during pachytene. In all these species, the sex chromosomes form an XY body easily identifiable in thin sections by the special and regular packing of the chromatin fibers of the internal region of the XY body ("differential" regions) and those of the peripheral region (synaptic region). Spermatocyte spreads show a complete synapsis between the X- and the Y-axis, which lasts up to the end of pachytene. From the early pachytene substages to the late ones, the X-axis develops prominent branches, which in late pachytene span the synaptic region. Synapsis is regular as shown by SYCP1 labeling. Axial development is followed by SYCP3 labeling and in the asynaptic region of the X-axis by BRCA1. Gamma-H2AX labels exclusively the differential (asynaptic) region of the X chromosome. A single focus is labeled by MLH1 in the synaptic region. The location of this MLH1 focus spans from 0.3 to 1.6 μm from the telomere in the analyzed xenarthrans, covering approximately half of the Y-axis length. It is concluded that xenarthrans, as basal placental mammals, harbor the largest pseudoautosomal regions of presently analyzed mammals, and shows the typical features of meiotic sex chromosome inactivation (MSCI).

Chromosomes sexuels et méiose

Sex chromosome behaviour fundamentally differs between male and female meiosis. In oocyte, X chromosomes synapse giving a XX bivalent which is not recognizable in their morphology and behaviour from autosomal bivalents. In human male, X and Y chromosomes differ from one another in their morphology and their genetic content, leading to a limited pairing and preventing genetic recombination, excepted in homologous region PAR1. During pachytene stage of the first meiotic prophase, X and Y chromosomes undergo a progressive condensation and form a transcriptionally silenced peripheral XY body. The condensation of the XY bivalent during pachytene stage led us to describe four pachytene substages and to localize the pachytene checkpoint between substages 2 and 3. We also defined the pachytene index (PI=P1+P2/P1+P2+P3+P4) which is always less than 0.50 in normal meiosis. XY body undergoes decondensation at diplotene stage, but transcriptional inactivation of the two sex chromosomes or Meiotic Sex Chromosome Inactivation (MSCI) persists through to the end of spermatogenesis. Sex chromosome inactivation involves several proteins, some of them were now identified. Two isoforms of the HP1 protein, HP1β and HP1γ, are involved in the facultative heterochromatinization of the XY body, but the initiation of this process involves the phosphorylation of the protein H2AX by the kinase ATR whose recruitment depends on BRCA1. Extensive researches on the inactivation of the sex chromosomes during male meiosis will allow to a better understanding of some male infertilities.

Small ubiquitin-related modifier (SUMO)-1, SUMO-2/3 and SUMOylation are involved with centromeric heterochromatin of chromosomes 9 and 1 and proteins of the synaptonemal complex during meiosis in men

BACKGROUNDPost-transcriptional modification by SUMOylation is involved in numerous cellular processes including human spermatogenesis. For human male meiosis, we previously showed that the small ubiquitin-related modifier-1 (SUMO-1) protein localizes to chromatin axes in early pachytene spermatocytes, then to kinetochores as meiosis progresses. Here, we delineate possible functional roles based on subcellular localization for SUMO-1 and SUMO-2/3.METHODSWestern and immunoprecipitation analyses were conducted on proteins isolated from the testis of two normal adult fertile men. Combinatorial immunofluorescence and chromosome-specific fluorescence in situ hybridization analyses were performed on male meiocytes obtained during testicular biopsy from four patients undergoing testicular sperm extraction for assisted reproduction technologies.RESULTSThe synaptonemal complex (SC) and SC proteins (SCP)-1 and SCP2, but not SCP3, are SUMOylated by SUMO-1 during the pachytene substage. Likewise, two distinct localization patterns for SUMO-1 are identified: a linear pattern co-localized with autosomal SCs and isolated SUMO-1 near the centromeric heterochromatin of chromosomes 9 and 1. In contrast to SUMO-1, which is not detectable prior to pachytene in normal tissue, SUMO-2/3 is identified as early as leptotene and zygotene and in some, but not all, pachytene cells; no linear patterns were detected. Similar to SUMO-1, SUMO-2/3 localizes in two predominant subnuclear patterns: a single, dense signal near the centromere of human chromosome 9 and small, individual foci co-localized with autosomal centromeres.CONCLUSIONSOur data suggest that SUMO-1 may be involved in maintenance and/or protection of the autosomal SC. SUMO-2/3, though expressed similarly, may function separately and independently during pachytene in men.

Analysis of substage associations in prophase I of meiosis in floral buds of wild‐type Arabidopsis thaliana (Brassicaceae)

We developed an improved cytological protocol for producing high quality, light microscope images of plant meiotic chromosomes. Because the technique works on species with small genomes and thick microsporocyte cell walls, it should be useful for studying the wild relatives of Arabidopsis and other eudicots with small genomes. Combining this improved fixation protocol with our new analysis of associated substages in floral buds, we can unambiguously assign individual meiotic cells to particular substages of prophase I in Arabidopsis thaliana, even for difficult distinctions such as that between late zygotene or early diplotene. In this report we provide the first estimate of the individual duration of the zygotene and pachytene substages (4.8 h and 10.0 h, respectively) in A. thaliana. We also have examined the diffuse substage of prophase I and report that during this post-pachytene substage, nuclei retain the association of homologous nucleolus organizer regions and homologous centromeres, despite the generally diffuse chromatin and generally unpaired chromosome regions. Additionally, we have observed that centromeric regions of the chromosomes of diffuse-stage nuclei are highly condensed, more so than those of any other substage of prophase I.

Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line

The replication of chromosomes in meiosis is an important first step for subsequent chromosomal interactions that promote accurate disjunction in the first of two segregation events to generate haploid gametes. We have developed an assay to monitor DNA replication in vivo in mitotic and meiotic germline nuclei of the nematode Caenorhabditis elegans. Using mutants that affect the mitosis/meiosis switch, we show that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cell nuclei. Furthermore, our assay reveals that different regions of the genome replicate at different times, with the heterochromatic-like X chromosomes replicating at a distinct time from the autosomes. Finally, we have exploited S-phase labeling to monitor the timing of progression through meiotic prophase. Meiotic prophase for oocyte production in hermaphrodites lasts 54–60 h. Further, we find that the duration of the pachytene sub-stage is modulated by the presence of sperm. On the other hand, meiotic prophase for sperm production in males is completed by 20–24 h. Possible sources for the sex-specific differences in meiotic prophase kinetics are discussed.

Meiotic abnormalities in patients bearing complete AZFc deletion of Y chromosome

We studied meiosis in three infertile patients presenting complete AZFc microdeletion and three controls. Primary spermatocytes were immunolabeled with SCP3, BRCA1 and gammaH2AX. We quantified the leptotene, zygotene and pachytene stages, and pachytene abnormalities: asynapsis and fragmented and dotted synaptonemal complexes (SCs). SCP3 level was significantly higher in leptotene and zygotene (bouquet) stages in patients, suggesting AZFc may have a direct effect on early prophase. SCs were abnormal in 77.3% of pachytene nuclei of patients versus 30.8% of controls. The two groups differed significantly (P < 0.001) in asynapsed nuclei, fragmented SC and dotted SCs. In patients, asynapsis were short and limited to a few bivalents. Staging of pachytene nuclei based on the morphology of the XY pair with BRCA1 revealed a prevalence of early pachytene substages (70.7%) in patients. H2AX was normally phosphorylated. In the absence of the AZFc region, the transient zygotene stage is extended, and chromosome condensation is reduced. The low level of limited asynapsis, the normal H2AX staining and the incomplete loss of germ cells at the pachytene checkpoint indicate that the AZFc region is not critical for meiotic recombination. We suggest that the pachytene phenotype develops secondarily to a primary defect that influences meiosis.

Meiotic arrest at the midpachytene stage in a patient with complete azoospermia factor b deletion of the Y chromosome

To study the meiosis of a patient with complete azoospermia factor (AZF)b deletion of the Y chromosome. Case report. La Conception University Hospital, Marseille, France. One azoospermic patient. Yq deletion testing, testicular sperm extraction, and meiotic study with immunocytochemistry. Abnormal synapsis rates in spermatocytes. We found that most spermatocytes were at an early stage of meiosis. Half of the meiotic germ cells analyzed showed asynapsis, which was mostly extended or total. Discontinuity in the synaptonemal complex was seen in one third of the meiotic cells analyzed. An unusually small number of normal pachytene nuclei were found, all at early pachytene substages. This is the first demonstration that the synaptic process is impaired in a man with complete deletion of the AZFb interval. Our findings provide evidence that the pachytene checkpoint is situated at the midpachytene substage in humans.

Synaptonemal complexes and XY behavior in two species of argentinian armadillos: Chaetophractus villosus and Dasypus hybridus (Xenarthra, Dasypodidae)

Spermatocytes from the two armadillo species, C. villosus and D. hybridus were studied in microspreads for synaptonemal complexes (SCs) and in thin sections for electron microscopy (EM). The complete SC karyotype generally agrees with previous reports on mitotic chromosomes, except for the sex chromosomes. The X chromosome is submetacentric in both species and the Y is the shortest one in C. villosus and the second shortest in D. hybridus, and an extremely acrocentric one. A SC is formed along the total length of the Y chromosome, and this SC persists along all the pachytene substages. A single recombination nodule (RN) is located in the region of the SC nearest to the attachment to the nuclear envelope. The lateral element (LE) of the X axis in the SC shows a wavy aspect in most of the SC length distant from the nuclear envelope. Nucleoli are attached to acrocentric or submetacentric bivalents, are visibly double in some cells, and in thin sections show an elaborate nucleolonema. Some differences in the XY are species-specific, as the higher degree of tangling and stronger heteropycnosis in D. hybridus. The effective, single crossover of the XY pair is highly localized, despite the permanence of a long tract of SC.

Meiotic anomalies in infertile men with severe spermatogenic defects

This study was aimed at evaluating the rate of pairing failure in pachytene spermatocytes of patients presenting either an obstructive (O) or a non-obstructive (NO) infertility. Forty-one patients and 13 controls underwent testicular biopsy. Among the patients, 19 had an O infertility and 22 a NO infertility. Preparations of all patients and controls were Giemsa-stained, and synaptonemal complexes from nine of these patients and one control were immunostained. In all, 2931 pachytene nuclei were analysed. The mean rate of asynapsed nuclei from the NO group (25.4%) was significantly higher than that of the O group (9.8%). There was no significant difference between the O group and the controls (10.6%). Immunocytochemistry showed that the number of pachytene nuclei decreased from the early to late pachytene sub-stage in all patients. Two NO patients, one azoospermic and one oligozoospermic, had a high percentage of asynapsed nuclei (86 and 91.8% respectively); one of these patients also presented a precocious localized separation of sister chromatids. high levels of extended asynapsis could arise from a primary meiotic defect which may be responsible for 9% of the NO male infertilities at our centre. The prevalence of early pachytene substages suggests that the pachytene checkpoint is localized at the mid-pachytene stage in humans.

Zygotene-pachytene substaging and synaptonemal complex karyotyping of boar spermatocytes.

Synaptonemal complex analysis, by electron microscopy of spread spermatocytes after phosphotungstic acid staining, made possible description of the chromosomal synaptic patterns and the synaptonemal complex karyotype of the pig (Sus scrofa domestica). The autosomal synaptic pattern in conjunction with the sex chromosome morphology and pairing behaviour can serve as a reference for the meiotic cell progression from the zygotene stage to the pachytene. The autosomes started terminal synapsis at early zygotene (Z1) and at mid-zygotene (Z2) some of the small bivalents were completely paired. The extension of pairing between the X and the Y chromosome, and the differentiation of their axes, disclosed seven types of sex bivalent (Types 0-VI). The Type 0 sex bivalent occurred in late zygotene (Z3), at which the X and Y axes began terminal synapsis by their short arms. Each one of the pachytene substages, early, mid-, and late, included two sex bivalent types. By early pachytene (P1-P2) the entire Y chromosome was paired with the X axis. Subsequently, progressive desynapsis and differentiation of the sex chromosome axes defined the mid- (P3-P4) and late pachytene substages (P5-P6). At mid-pachytene, the unpaired XY segments were associated end-to-end and showed differing degrees of complexity (thickening, splitting, despiralization and strandedness). These axial complexities were replaced at late pachytene by fine excrescences along the sex chromosome axes, which still formed a short SC stretch. Additionally, an attempt to construct an SC karyotype for the pig is presented. There was general agreement between the autosomal SC karyotype and the autosomal somatic karyotype when comparisons were made, considering the relative lengths and arm ratios of individual chromosome pairs.

The XY pair of the mink (Mustela vision) during different periods of testicular activity.

Synaptonemal complexes of the mink (Mustela vison) were examined during different stages of testicular activity to determine whether the distribution of prophase substages and the configuration of the sex complement are altered during pre-quiescent and regenerative phases compared to those detected during the breeding period. Spermatocytes obtained during pre-quiescence showed no differences from those of breeding season in terms of substage distribution, whereas those from regenerating testes were mainly in zygotene and early pachytene substages, reflecting the high mitotic activity of spermatogonia and their subsequent transit to meiosis. Based on the location of kinetochores on the sex complement, the synapsed segments were identified as the short arm of the X (Xp) and the long arm of the Y (Yq), although pairing of the X and Y beyond the "pseudoautosomal region" was frequently observed. In some spermatocytes, the entire Y chromosome synapsed with the X or split into two strands with only one strand "paired" with the X while the other remained unpaired. It is not clear at present whether the Y chromosome splitting is part of the mechanisms that prevent crossing over in the non-homologous segments of the sex complement that often undergo synapsis or a post-crossover phenomenon unrelated to pairing mechanisms.

HP1beta and HP1gamma, but not HP1alpha, decorate the entire XY body during human male meiosis.

During meiosis in male mammals, the X and Y chromosomes become heterochromatic and transcriptionally silent, and form the XY body. Although the HP1 proteins are known to be involved in the packaging of chromosomal DNA into repressive heterochromatin domains, their involvement in facultative heterochromatinization has not been precisely determined. Here, we analyse, for the first time in humans, the subcellular distribution of the heterochromatin protein HP1alpha, HP1beta and HP1gamma isoforms, in male pachytene spermatocytes, and the XY body facultative heterochromatin in particular. Our results demonstrate that HP1beta and HP1gamma, but not the HP1alpha isoforms, decorate the entire XY body in half the pachytene nuclei observed. In some nuclei, the XY body appears to be only partially labelled. In these cases, the HP1beta and HP1gamma signals are adjacent to the Yq12 constitutive heterochromatin and signal appears to originate in this region before spreading over the entire XY body. This distribution suggests that HP1beta and HP1gamma proteins, which are components of the constitutive heterochromatin, may also be involved in the facultative heterochromatinization of the XY body. Nevertheless, their absence from the early pachytene substage, even though the XY body is already condensed, suggests that these proteins are not involved in the initiation of this process.

Damaging Effect of Antibiotics on the Structure of Synaptonemal Complexes of Meiotic Mouse Chromosomes

The structure of synaptonemal complexes (SCs) of chromosomes of mouse primary spermatocytes were studied using electron microscopy on days 1, 10, and 36 after the completion of per os administration of drugs belonging to three groups of antibiotics: tetracyclins, macrolides, and fluoroquinolones. The antibiotics were administered to mice during ten days. At the substages of early and middle pachytene, heteromorphic SC bivalents and fragments of chromosome-core elements were detected in spermatocytes at all times studied after the administration of the antibiotics of three groups. As cells passed through the period from early to middle pachytene, the number of cells containing heteromorphic SC bivalents and the fragments of axial cores gradually decreased, which could be an indication of selection of cells with chromosomal aberrations. A high level of associations between the X chromosome and autosome bivalents (including heteromorphic ones) also favors this suggestion. A gradual decrease in the number of chromosomal aberrations was detected, as time elapsed from the completion of antibiotics administration. The study of sperm obtained from epididymises of males did not reveal significant differences in both morphology and motility of sperm between males of the control and experimental groups.

Sequential meiotic prophase development in the pubertal Indian pygmy field mouse: Synaptic progression of the XY chromosomes, autosomal heterochromatin, and pericentric inversions

Sequential meiotic prophase development has been followed in the pubertal male pygmy mouse Mus terricolor, with the objective to identify early meiotic prophase stages. The pygmy mouse differs from the common mouse by having large heterochromatic blocks in the X and Y chromosomes. These mice also show various chromosomal mutations; for example, fixed variations of autosomal short arms heterochromatin among different chromosomal species and pericentric inversion polymorphism. Identification of prophase stages was crucial to analyzing effects of heterozygosity for these chromosomal changes on the process of homologous synapsis. Here we describe identification of the prophase stages in M. terricolor, especially the pachytene substages, on the basis of morphology of the XY bivalent. Based on this substaging, we show delayed pairing of the heterochromatic short arms, which may be the reason for their lack of chiasmata. The identification of precise pachytene substages also reveals an early occurrence of "synaptic adjustment" in the pericentric inversion heterobivalents, a mechanism that would prevent chiasma formation in the inverted segment and thereby would abate adverse effects of such heterozygosity. The identification of pachytene substages would serve as the basis to analyze the nature of synaptic anomalies met in M. terricolor hybrids (which will be the basis of a subsequent paper).

Synaptonemal complex investigations on lamas (Lama glama) with differing fertility recordings

The present study involved three male lamas (Lama glama). Mitotic preparations from fibroblast culture and an electron microscopic observation on the synaptonemal complexes (Scs) were reported. Special attention was given to the morphology and behaviour of the chromosomes at the zygotene and pachytene substages of prophase I in primary spermatocytes from lamas. Analysis of mitotic preparations show diploid and triploid cells, with a relatively high frequency of polyploidy. Analyses of synaptonemal complexes in primary spermatocytes were carried out on 89 cells. Pairing abnormalities were only recorded in an average of 63% of the cells of the animal Tabasco. The other two animals were normal. The photographed cells give an upper limit estimate of the existing abnormalities, as there was a deliberate tendency towards selecting abnormal cells for photography. The presence of degenerating primary spermatocytes in SC preparations as well as in testicular sections, and the absence of spermatozoa in ejaculates confirm the chromosomally derived male sterility of one animal.

Studies of synaptonemal complexes in farm mammals--a review.

For the last 10 years extensive studies of synaptonemal complexes in farm mammals were carried out. In this article a survey of the obtained results is presented. Studies on synaptic behavior of chromosomes at pachytene substage of meiotic prophase I in carriers of centric fusions (cattle, sheep, goat, pig, and blue fox) showed that the trivalent is rarely involved in an association with the sex bivalent. In carriers of reciprocal translocations (pig and cattle) a wide range of synaptic configurations were found. Besides the expected cross-shaped quadrivalent, the following configurations were observed: open quadrivalent, trivalent plus univalent, and two heteromorphic bivalents. The latter configurations were quite frequently involved in the association with XY bivalent. Studies of synaptonemal complexes in interspecies hybrids (cattle x zebu, river x swamp buffalo, blue fox x silver fox) revealed a wide range of synaptic disturbances. Cases of pericentric inversions, aneuploidy, and chromosomal polymorphisms are also reviewed. This survey shows that synaptonemal complex analysis is a very useful tool in studies on causes of altered fertility in carriers of abnormal chromosome complement.

Crossing over analysis at pachytene in man.

The distribution of anti-MLH1 (MutL homologue 1) antibody labelling was studied in human prophase meiocytes. A labelling pattern consisting of distinct foci, always associated with the synaptonemal complex (SC) and never in closely juxtaposed pairs, was observed. Comparison of the number and general positions of autosomal foci with previous studies of the number and positions of autosomal chiasmata indicates that the anti-MLH1 antibody marks sites of crossing over in human pachytene spermatocytes. A mean number of 50.9 autosomal foci was observed from 46 human pachytene spermatocytes corresponding to a genetic length of 2545 cm. Division of these spermatocytes into sub-stages revealed that the number of foci remains stable throughout pachytene. A focus was found on the XY bivalent in 56.5% of the nuclei. The presence or absence of foci from the XY bivalent could not be correlated to pachytene sub-stage.

Pairing and Crossing-over within a Paracentric Inversion Associated to a Heterozygous Robertsonian Translocation in Dichroplus pratensis (Melanoplinae, Acrididae).

The meiotic behaviour of a spontaneous paracentric inversion was analyzed in a male of Dichroplus pratensis from a population of the Sierra de la Ventana hybrid zone (Argentina), characterized by four Robertsonian polymorphisms. The mutant was 2n=15, homozygous for the 3/4 tanslocation and heterozygous for translocations 1/2 and 5/6. The inversion was located in arm 2 of the 1-1.2-2 trivalent and comprised the first half of its distal end, involving about 25% of the chro-mosome length. Analysis of pairing at three pachytene substages showed that the main mode of synapsis was homologous, with a high frequency of complete loop formation at early pachytene. No synaptic adjustment seems to occur, and homosynapsis is significantly correlated with crossing over within the inverted region. As a result, high frequencies of bridge and fragment configurations occur at first anaphase/telophase stages which produce significant levels of unreduced cells at second meiotic division and later, macrospermatids. Microspermatids are also abundant because of micronuclei formation by lagging acentric fragments. These results suggest a deletereous effect of paracentric inversions in D. pratensis due to production of aberrant recombinant chromosomes and formation of increased levels of abnormal sperm. Furthermore, since the paracentric inversion induces a chiasma repatterning in the involved chromosome arm, a strong relationship between homologous pairing and crossing over is evident. This is discussed in the light of current models of chromosome synapsis.

HSP70-2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes.

Spermatogenic cells synthesize a unique 70-kDa heat shock protein (HSP70-2) during prophase of meiosis I, and targeted disruption of the Hsp70-2 gene has shown that this protein is required for spermatogenic cell differentiation in adult mice. HSP70-2 is associated with synaptonemal complexes formed between paired homologous chromosomes during meiotic prophase. The present study focuses on the nearly synchronous first wave of spermatogenesis in 12- to 28-day old juvenile mice to determine more precisely when HSP70-2 is required and what meiotic processes are affected by its absence. Spermatogenesis in homozygous mutant mice (Hsp70-2[-/-]) proceeded normally until day 15 when increasing numbers of pachytene spermatocytes became apoptotic and differentiation of cells beyond the pachytene stage began to falter. Synaptonemal complexes assembled in Hsp70-2(-/-) mice and spermatocytes developed through the final pachytene substage. However, synaptonemal complexes failed to desynapse and normal diplotene spermatocytes were not observed. Metaphase spermatocytes were not seen in tissue sections from testes of Hsp70-2(-/-) mice, and expression of mRNAs and antigens characteristic of late pachytene spermatocytes (e.g., cyclin A1) and development of spermatids did not occur. Thus, HSP70-2 is required for synaptonemal complex desynapsis, and its absence severely impairs the transition of spermatogenic cells through the late meiotic stages and results in apoptosis beginning with the first wave of germ cell development in juvenile mice.