Substages Of Prophase Research Articles

Stage-resolved Hi-C analyses reveal meiotic chromosome organizational features influencing homolog alignment

During meiosis, chromosomes exhibit dramatic changes in morphology and intranuclear positioning. How these changes influence homolog pairing, alignment, and recombination remain elusive. Using Hi-C, we systematically mapped 3D genome architecture throughout all meiotic prophase substages during mouse spermatogenesis. Our data uncover two major chromosome organizational features varying along the chromosome axis during early meiotic prophase, when homolog alignment occurs. First, transcriptionally active and inactive genomic regions form alternating domains consisting of shorter and longer chromatin loops, respectively. Second, the force-transmitting LINC complex promotes the alignment of ends of different chromosomes over a range of up to 20% of chromosome length. Both features correlate with the pattern of homolog interactions and the distribution of recombination events. Collectively, our data reveal the influences of transcription and force on meiotic chromosome structure and suggest chromosome organization may provide an infrastructure for the modulation of meiotic recombination in higher eukaryotes.

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat

BackgroundMolecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks.ResultsWe describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed.ConclusionThe MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Centromere Dynamics in Male and Female Germ Cells.

In sexually reproducing organisms the germ line is the cellular lineage that gives rise to gametes. All germ cells originate from germline stem cells that divide asymmetrically to generate gonial pre-cursors, which are amplified in number by mitotic divisions, undergo meiosis and eventually differentiate into mature gametes (haploid eggs and sperm). Information transmitted with gametes is inherited by offspring, and potentially by subsequent generations, instructing in organismal development and beyond. Meiosis comprises one round of DNA replication, followed by two rounds of chromosome segregation; homologous chromosomes segregate in the first division (meiosis I) and sister chromatids segregate in the second division (meiosis II). Important mechanistic features of meiosis occur in substages of prophase I and are critical for genetic recombination, including pairing and synapsis of homologous chromosomes (at leptotene and zygotene), crossing-over (at pachytene), and the appearance of chiasmata (at diplotene/diakinesis). Another unique feature of meiosis is the altered centromere/kinetochore geometry at metaphase I, such that sister kinetochores face the same spindle pole (mono-orientation) and stay together at anaphase I. This chapter reviews centromere dynamics in germ cells, focusing on centromere function and assembly in meiotic cell cycles, as well as centromere inheritance in zygotes. Centromeres are functionally defined by the presence of the histone H3 variant CENP-A, the epigenetic determinant of centromere identity. In most eukaryotes, it is well established that CENP-A function is essential for chromosome segregation in mitosis. CENP-A function in meiosis is less well understood and emerging insights into the differential regulation of meiotic and mitotic CENP-A are discussed.

Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes.

BackgroundThe continuous and non-synchronous nature of postnatal male germ-cell development has impeded stage-specific resolution of molecular events of mammalian meiotic prophase in the testis. Here the juvenile onset of spermatogenesis in mice is analyzed by combining cytological and transcriptomic data in a novel computational analysis that allows decomposition of the transcriptional programs of spermatogonia and meiotic prophase substages.ResultsGerm cells from testes of individual mice were obtained at two-day intervals from 8 to 18 days post-partum (dpp), prepared as surface-spread chromatin and immunolabeled for meiotic stage-specific protein markers (STRA8, SYCP3, phosphorylated H2AFX, and HISTH1T). Eight stages were discriminated cytologically by combinatorial antibody labeling, and RNA-seq was performed on the same samples. Independent principal component analyses of cytological and transcriptomic data yielded similar patterns for both data types, providing strong evidence for substage-specific gene expression signatures. A novel permutation-based maximum covariance analysis (PMCA) was developed to map co-expressed transcripts to one or more of the eight meiotic prophase substages, thereby linking distinct molecular programs to cytologically defined cell states. Expression of meiosis-specific genes is not substage-limited, suggesting regulation of substage transitions at other levels.ConclusionsThis integrated analysis provides a general method for resolving complex cell populations. Here it revealed not only features of meiotic substage-specific gene expression, but also a network of substage-specific transcription factors and relationships to potential target genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2865-1) contains supplementary material, which is available to authorized users.

Organization and Behavior of the Synaptonemal Complex during Achiasmatic Meiosis of Four Buthid Scorpions

Testicular cells of 4 buthid scorpions, Rhopalurus agamemnon (2n = 28), R. rochai (2n = 28), Tityus bahiensis (2n = 6), and T. fasciolatus (2n = 14), which show different types of chromosomal configurations in meiosis I, were subjected to cellular microspreading in order to (1) obtain knowledge about the organization and behavior of the synaptonemal complex (SC), and (2) acquire data about the mechanisms responsible for inter- and intraindividual chromosomal variation within Buthidae. Ultrastructural analysis of microspread nuclei revealed SCs with a well-preserved structure until late substages of prophase I, but did not detect kinetochore plates and recombination nodules. Pachytene cells of R. agamemnon, R. rochai and T. bahiensis exhibited single and unsynapsed axes continuous with totally synapsed SCs, indicating the occurrence of heterozygous chromosomal rearrangements. Although chromosome chains were not observed in T. fasciolatus, the presence of gaps and interlocks points out that this species also carries heterozygous rearrangements, involving a small chromosome segment. Especially in R. rochai, the cellular microspreading analysis was useful to clarify the origin of inter- and intraindividual variation in the number of bivalent-like elements and in the number of chromosomes involved in multivalent associations. It was found that more chromosomes were involved in rearrangements than previously established through investigations using light microscopy alone.

The dissection of meiotic chromosome movement in mice using an in vivo electroporation technique.

During meiosis, the rapid movement of telomeres along the nuclear envelope (NE) facilitates pairing/synapsis of homologous chromosomes. In mammals, the mechanical properties of chromosome movement and the cytoskeletal structures responsible for it remain poorly understood. Here, applying an in vivo electroporation (EP) technique in live mouse testis, we achieved the quick visualization of telomere, chromosome axis and microtubule organizing center (MTOC) movements. For the first time, we defined prophase sub-stages of live spermatocytes morphologically according to GFP-TRF1 and GFP-SCP3 signals. We show that rapid telomere movement and subsequent nuclear rotation persist from leptotene/zygotene to pachytene, and then decline in diplotene stage concomitant with the liberation of SUN1 from telomeres. Further, during bouquet stage, telomeres are constrained near the MTOC, resulting in the transient suppression of telomere mobility and nuclear rotation. MTs are responsible for these movements by forming cable-like structures on the NE, and, probably, by facilitating the rail-tacking movements of telomeres on the MT cables. In contrast, actin regulates the oscillatory changes in nuclear shape. Our data provide the mechanical scheme for meiotic chromosome movement throughout prophase I in mammals.

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.

FISHing for acrocentric associations between chromosomes 14 and 21 in human oogenesis

Objective The purpose of this study was to search for cytologic evidence of robertsonian translocation formation that involves chromosomes 14q and 21q in human oogenesis with the use of dual color fluorescent in situ hybridization with whole chromosome paints. Study design The oocytes from a chromosomally normal fetus at 23.5 weeks of gestation underwent cohybridization with chromosome specific DNA libraries from chromosomes 14 and 21. The nuclei were scored for the proportion of meiosis I prophase substages and for hybridization efficiency and were evaluated for the presence of hybridization signals that were suggestive of heterologous associations between chromosomes 14q and 21q in zygotene, pachytene, and diplotene. Results A total of 1769 meiotic nuclei were analyzed. Of 272 informative nuclei at zygotene, pachytene, and diplotene, 1 nucleus at pachytene demonstrated hybridization signals for chromosomes 14 and 21 that could be consistent with a robertsonian translocation. Conclusion A heterologous association between chromosomes 14q and 21q that possibly represent robertsonian translocation formation was observed cytologically with the use of fluorescent in situ hybridization.

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.

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.

Synaptic adjustment of inversion loops in Neurospora crassa.

Heterozygotes for three long inversions on chromosome 1 were analyzed by serial reconstruction from electron micrographs. Measurements of loop lengths at different meiotic prophase substages revealed that the homologous synapsis of the inverted region was gradually replaced by nonhomologous synapsis as loops were eliminated during pachytene. This synaptic adjustment was apparently not affected by crossovers which occurred within the 150- and 160-cM long loops.

Interaction of tetracycline (TC) with chromosomes in Allium cepa

Tetracycline (TC), a widely used antibiotic, exhibited a marked effect on microsporogenesis when administered to cell at early prophase sub-stages (zygotene, pachytene, diplotene and diakinesis). Meiotic aberrations induced by tetracycline consisted of stickness, terminal deletion heteromorphic bivalents, fragments, bridges, laggards and polyads. At a 20 μg/ml TC concentration pollen-mother-cells failed to undergo meiosis and showed degeneration of nuclei into small chromatin droplets, anthers remained in metastasis for about 10 days and then shrivelled up without forming pollen grains. Chromosome aberrations were also observed in bulbs and seedlings obtained from seeds of treated inflorescences. Hyprodiploid cells were observed in 16.2% of mitotic cells in seedlings obtained from plants treated with 16 μg/ml TC. Root tio cells of seedlings as well as that of bulbs were devoid of breakages.

Interaction of daunomycin with meiotic chromosomes in Vicia faba.

Daunomycin interferes with the meiotic cycle and structural integrity of chromosmes if administered to microsporocytes of Vicia faba during the meiotic prophase substages prior to diplotene. The principal cytological consequences of daunomycin treatment are as follows: induced achromatic lesions, chromosome fragmentation, terminal and interstitial deletions, loose or non-pairing regions, multivalent associations, reduced chromosome condensation, chromosome stickiness, dicentric and side-arm bridges and chromatid loops. A correlation between inhibition of DNA or RNA and the cytological manifestation of structural anomalies or unfolding of meiotic events is discussed.