Spermatocyte Chromosomes Research Articles

Evidence of Non-microtubule Spindle Forces in Mesostoma ehrenbergii Spermatocytes.

We tested conclusions reached in previous experiments in which Mesostoma spermatocyte chromosomes moved rapidly to a pole in the absence of microtubules: after 10 μM nocodazole (NOC) depolymerized metaphase spindle microtubules, kinetochores from each of the 3 bivalents detached from the same pole and rapidly moved to the other pole, at speeds averaging 37.7 μm/min. with some as high as 100 μm/min. We concluded that these very fast movements were due to non-microtubule forces arising from a spindle matrix. However, since the chromosomes stretch out before detaching, there is tension in the chromosomes from the stretch. Thus the movements of detached kinetochores conceivably might be due to recoil from the tension, though we argued against this possibility (Fegaras and Forer, 2018a). In this article we test whether recoil causes the movements. We cut bivalents into 2 pieces, using a femtosecond laser, before addition of NOC. When 1 bivalent was severed, all kinetochores moved to one pole in 12/15 cells; when 2 bivalents were severed, all kinetochores moved to one pole in 4/6 cells; and when all 3 bivalents were severed all kinetochores moved to one pole in 3/9 cells. The bivalent “halves” moved rapidly, with average speeds of 47 μm/min, velocities that are not significantly different from those in cells without any laser-cut bivalents (p > 0.05). Since kinetochores move at the same speeds whether they are part of bivalents or not, NOC-induced chromosome movements are not due to recoil from tension along the full-length bivalent, strongly supporting the idea that non-microtubule forces move chromosomes in Mesostoma spermatocytes.

Elastic Tethers Between Separating Anaphase Chromosomes Regulate the Poleward Speeds of the Attached Chromosomes in Crane-Fly Spermatocytes.

Elastic “tethers” connect separating anaphase chromosomes in most (or all) animal cells. We tested whether tethers are involved in coordinating movements of separating anaphase chromosomes in crane-fly spermatocytes. In these cells the coupled movements of separating chromosomes become uncoupled after the tethers are severed by laser microbeam irradiation of the interzone region between the chromosomes (Sheykhani et al., 2017). While this strongly suggests that tethers are involved with coordinating the poleward chromosome movements, the experiments are open to another interpretation: laser irradiations that cut the tethers also might damage something else in the interzone, and those non-tether components might regulate chromosome movements. In the experiments reported herein we distinguish between those two possibilities by disabling the tethers without cutting the interzone. We cut the arms from individual chromosomes, thereby severing the mechanical connection between separating chromosomes, disconnecting them, without damaging components in the interzone. Disabling tethers in this way uncoupled the movements of the separating chromosomes. We thus conclude that tethers are involved in regulating the speeds of separating anaphase chromosomes in crane-fly spermatocytes.

The expression of histone methyltransferases and distribution of selected histone methylations in testes of yak and cattle-yak hybrid

Interspecies hybridization exists widely in nature and plays an important role in animal evolution and adaptation. It is commonly recognized that male offspring of interspecies hybrid are often sterile, which presents a crucial way of reproductive isolation. Currently, the mechanisms underlying interspecies hybrid male sterility are not well understood. Cattle-yak, progeny of yak (Bos grunniens) and cattle (Bos taurus) cross, is a unique animal model for investigating hybrid male sterility. Because histone modifications are vital for spermatogenesis, herein, we examined expressions of histone methyltransferases (HMTs) and distributions of histone methylations in the yak and cattle-yak testis. Histological examination of seminiferous tubules revealed that gonocytes and spermatocytes were established normally, however, spermatogenesis was arrested at the meiosis phase began at 10 months after birth in the hybrids. SUV420H1 was the only HMT examined showing a significant enrichment in cattle-yak testes at 3 months. Relative expressions of MLL5, SETDB1 and SUV420H1 were increased while SETDB2 and EZH2 were decreased in cattle-yak testes at 10 months. Relative concentrations of MLL5 and SUV420H1 were again increased while EHMT2 and PRDM9 expressions were decreased at 24 months. Immunofluorescent detection of selected histone methylations in cross-sections of testicular tissues or meiotic chromosomes demonstrated that depletion of H3K4me3 and significant enrichment of H3K27me3 and H4K20me3 were observed in Sertoli cells of cattle-yak. Levels and localizations of H3K4me3, H3K9me1, H3K9me3 and H4K20me3 were strikingly different in meiotic chromosomes of cattle-yak spermatocytes. These results highlighted the potential roles of histone methylations in spermatogenic failure and hybrid male sterility.

The pericentromeric heterochromatin of homologous chromosomes remains associated after centromere pairing dissolves in mouse spermatocyte meiosis

In meiosis, crossovers between homologous chromosomes link them together. This enables them to attach to microtubules of the meiotic spindle as a unit, such that the homologs will be pulled away from one another at anaphase I. Homologous pairs can sometimes fail to become linked by crossovers. In some organisms, these non-exchange partners are still able to segregate properly. In several organisms, associations between the centromeres of non-exchange partners occur in meiotic prophase. These associations have been proposed to promote segregation in meiosis I. But it is unclear how centromere pairing could promote subsequent proper segregation. Here we report that meiotic centromere pairing of chromosomes in mouse spermatocytes allows the formation of an association between chromosome pairs. We find that heterochromatin regions of homologous centromeres remain associated even after centromere-pairing dissolves. Our results suggest the model that, in mouse spermatocytes, heterochromatin maintains the association of homologous centromeres in the absence crossing-over.

A segregating human allele of SPO11 modeled in mice disrupts timing and amounts of meiotic recombination, causing oligospermia and a decreased ovarian reserve†.

A major challenge in medical genetics is to characterize variants of unknown significance (VUS). Doing so would help delineate underlying causes of disease and the design of customized treatments. Infertility has presented an especially difficult challenge with respect to not only determining if a given patient has a genetic basis, but also to identify the causative genetic factor(s). Though genome sequencing can identify candidate variants, in silico predictions of causation are not always sufficiently reliable so as to be actionable. Thus, experimental validation is crucial. Here, we describe the phenotype of mice containing a non-synonymous (proline-to-threonine at position 306) change in Spo11, corresponding to human SNP rs185545661. SPO11 is a topoisomerase-like protein that is essential for meiosis because it induces DNA double stranded breaks (DSBs) that stimulate pairing and recombination of homologous chromosomes. Although both male and female Spo11P306T/P306T mice were fertile, they had reduced sperm and oocytes, respectively. Spermatocyte chromosomes exhibited synapsis defects (especially between the X and Y chromosomes), elevated apoptotic cells, persistent markers of DSBs, and most importantly, fewer Type 1 crossovers that causes some chromosomes to have none. Spo11P306T/- mice were sterile and made fewer meiotic DSBs than Spo11+/- animals, suggesting that the Spo11P306T allele is a hypomorph and likely is delayed in making sufficient DSBs in a timely fashion. If the consequences are recapitulated in humans, it would predict phenotypes of premature ovarian failure, reduced sperm counts, and possible increased number of aneuploid gametes. These results emphasize the importance of deep phenotyping in order to accurately assess the impact of VUSs in reproduction genes.

Toxicity Assessment of Chinese Herbal Medicine Cynomorium songaricum Rupr.

Cynomorium songaricum Rupr, known as Suo Yang, is most commonly used to treat fatigue, protect the liver, and invigorate kidneys in Northwest China. Given the wide medicinal utilisation and lack of safety evaluation, this work evaluated the acute toxicity, genetic toxicity, and 90-day repeated oral toxicity of Suo Yang. Twenty Kunming mice were orally given Suo Yang once and observed for 14 days in the acute toxicity test. The genetic toxicity of Suo Yang was tested using in vivo and vitro assays (bacterial reverse mutation test, mouse bone marrow micronucleus assay, and spermatocyte chromosomal aberration assay). In the 90-day repeated oral toxicity study, 80 SD rats were randomly divided into 4 groups and then orally given Suo Yang at different concentrations (1.04, 2.08 or 4.16 g/kg), while the control group was given sterile water. In the acute toxicity test, no abnormal behaviour or mortality was found in mice. The results suggest that the maximum tolerable dose of Suo Yang is greater than 15 g/kg. In the genotoxicity studies, no revertant colonies were produced in vitro. In the in vivo assays, no increased frequencies of micronuclei or structural abnormalities of spermatocyte chromosomes were found. In the 90-day repeated oral toxicity study, no significant toxicological manifestations were observed in haematological parameters or clinical and pathological examinations. In summary, these results suggest that Suo Yang at the given doses does not cause adverse effects in animals. Thus, Suo Yang can reasonably be considered a safe herbal medicine.

Micromanipulation of Chromosomes in Insect Spermatocytes

Micromanipulation of Chromosomes in Insect Spermatocytes

Micromanipulation of Chromosomes in Insect Spermatocytes.

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase chromosome movements, and has been key to understanding what controls chromosome movements during a cell division. A skilled biologist can use a micromanipulator to detach chromosomes from the spindle, to reposition chromosomes within the cell, and to apply forces to chromosomes using a small glass needle with a very fine tip. While perturbations can be made to chromosomes using other methods such as optical trapping and other uses of a laser, to date, no other method allows the repositioning of cellular components on the scale of tens to hundreds of microns with little to no damage to the cell. The selection and preparation of appropriate cells for the micromanipulation of chromosomes, specifically describing the preparation of grasshopper and cricket spermatocyte primary cultures for the use in live-cell imaging and micromanipulation, are described here. In addition, we show the construction of a needle to be used for moving chromosomes within the cell, and the use of a joystick-controlled piezoelectric micromanipulator with a glass needle attached to it to reposition chromosomes within dividing cells. A sample result shows the use of a micromanipulator to detach a chromosome from a spindle in a primary spermatocyte and to reposition that chromosome within the cell.

Anaphase Chromosomes in Crane-Fly Spermatocytes Treated With Taxol (Paclitaxel) Accelerate When Their Kinetochore Microtubules Are Cut: Evidence for Spindle Matrix Involvement With Spindle Forces

Various experiments have indicated that anaphase chromosomes continue to move after their kinetochore microtubules are severed. The chromosomes move poleward at an accelerated rate after the microtubules are cut but they slow down 1–3 min later and move poleward at near the original speed. There are two published interpretations of chromosome movements with severed kinetochore microtubules. One interpretation is that dynein relocates to the severed microtubule ends and propels them poleward by pushing against non-kinetochore microtubules. The other interpretation is that components of a putative “spindle matrix” normally push kinetochore microtubules poleward and continue to do so after the microtubules are severed from the pole. In this study we distinguish between these interpretations by treating cells with taxol. Taxol eliminates microtubule dynamics, alters spindle microtubule arrangements, and inhibits dynein motor activity in vivo. If the dynein interpretation is correct, taxol should interfere with chromosome movements after kinetochore microtubules are severed because it alters the arrangements of spindle microtubules and because it blocks dynein activity. If the “spindle matrix” interpretation is correct, on the other hand, taxol should not interfere with the accelerated movements. Our results support the spindle matrix interpretation: anaphase chromosomes in taxol-treated crane-fly spermatocytes accelerated after their kinetochore microtubules were severed.

Telomeric TERB1-TRF1 interaction is crucial for male meiosis.

During meiotic prophase, the meiosis-specific telomere-binding protein TERB1 regulates chromosome movement required for homologous pairing and recombination by interacting with the telomeric shelterin subunit TRF1. Here, we report the crystal structure of the TRF1-binding motif of human TERB1 in complex with the TRFH domain of TRF1. Notably, specific disruption of the TERB1-TRF1 interaction by a point mutation in the mouse Terb1 gene results in infertility only in males. We find that this mutation causes an arrest in the zygotene-early pachytene stage and mild telomere abnormalities of autosomes but unpaired X and Y chromosomes in pachytene, leading to massive spermatocyte apoptosis. We propose that the loss of telomere structure mediated by the TERB1-TRF1 interaction significantly affects homologous pairing of the telomere-adjacent pseudoautosomal region (PAR) of the X and Y chromosomes in mouse spermatocytes. Our findings uncover a specific mechanism of telomeres that surmounts the unique challenges of mammalian X-Y pairing in meiosis.

Segregation of the amphitelically attached univalent X chromosome in the spittlebug Philaenus spumarius.

In meiosis I, homologous chromosomes combine to form bivalents, which align on the metaphase plate. Homologous chromosomes then separate in anaphase I. Univalent sex chromosomes, on the other hand, are unable to segregate in the same way as homologous chromosomes of bivalents due to their lack of a homologous pairing partner in meiosis I. Here, we studied univalent segregation in a Hemipteran insect: the spittlebug Philaenus spumarius. We determined the chromosome number and sex determination mechanism in our population of P. spumarius and showed that, in male meiosis I, there is a univalent X chromosome. We discovered that the univalent X chromosome in primary spermatocytes forms an amphitelic attachment to the spindle and aligns on the metaphase plate with the autosomes. Interestingly, the X chromosome remains at spindle midzone long after the autosomes have separated. In late anaphase I, the X chromosome initiates movement towards one spindle pole. This movement appears to be correlated with a loss of microtubule connections between the kinetochore of one chromatid and its associated spindle pole.

Elastic tethers between separating anaphase chromosomes in crane-fly spermatocytes coordinate chromosome movements to the two poles.

Separating anaphase chromosomes in crane-fly spermatocytes are connected by elastic tethers, as originally described by LaFountain et al. (2002): telomere-containing arm fragments severed from the arms move backwards to the partner telomeres. We have tested whether the tethers coordinate the movements of separating partner chromosomes. In other cell types anaphase chromosomes move faster, temporarily, when their kinetochore microtubules are severed. However, in crane-fly spermatocytes the chromosomes move at their usual speed when their kinetochore microtubules are severed. To test whether the absence of increased velocity is because tethers link the separating chromosomes and coordinate their movements, we cut tethers with a laser microbeam and then cut the kinetochore microtubules. After this procedure, the associated chromosome sped up, as in other cells. These results indicate that the movements of partner anaphase chromosomes in crane-fly spermatocytes are coordinated by elastic tethers connecting the two chromosomes and confirm that chromosomes speed up in anaphase when their kinetochore microtubules are severed. © 2016 Wiley Periodicals, Inc.

Distinct Roles of Meiosis-Specific Cohesin Complexes in Mammalian Spermatogenesis.

Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1β, we generated Smc1β-/-Rec8-/- and Smc1β-/-Rad21L-/- mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1β complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1β and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in Smc1β-/-Rad21L-/- cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion.

FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis.

Fanconi anemia (FA) is a recessive X-linked and autosomal genetic disease associated with bone marrow failure and increased cancer, as well as severe germline defects such as hypogonadism and germ cell depletion. Although deficiencies in FA factors are commonly associated with germ cell defects, it remains unknown whether the FA pathway is involved in unique epigenetic events in germ cells. In this study, we generated Fancb mutant mice, the first mouse model of X-linked FA, and identified a novel function of the FA pathway in epigenetic regulation during mammalian gametogenesis. Fancb mutant mice were infertile and exhibited primordial germ cell (PGC) defects during embryogenesis. Further, Fancb mutation resulted in the reduction of undifferentiated spermatogonia in spermatogenesis, suggesting that FANCB regulates the maintenance of undifferentiated spermatogonia. Additionally, based on functional studies, we dissected the pathway in which FANCB functions during meiosis. The localization of FANCB on sex chromosomes is dependent on MDC1, a binding partner of H2AX phosphorylated at serine 139 (γH2AX), which initiates chromosome-wide silencing. Also, FANCB is required for FANCD2 localization during meiosis, suggesting that the role of FANCB in the activation of the FA pathway is common to both meiosis and somatic DNA damage responses. H3K9me2, a silent epigenetic mark, was decreased on sex chromosomes, whereas H3K9me3 was increased on sex chromosomes in Fancb mutant spermatocytes. Taken together, these results indicate that FANCB functions at critical stages of germ cell development and reveal a novel function of the FA pathway in the regulation of H3K9 methylation in the germline.

Key mediators of somatic ATR signaling localize to unpaired chromosomes in spermatocytes.

Meiotic silencing of unpaired chromatin (MSUC) occurs during the first meiotic prophase, as chromosomes that fail to pair are sequestered into a transcriptionally repressive nuclear domain. This phenomenon is exemplified by the heterologous sex chromosomes of male mammals, where the ATR DNA damage response kinase is crucial for this silencing event. However, the mechanisms underlying the initiation of MSUC remain unknown. Here, we show that essential components of ATR signaling in murine somatic cells are spatially confined to unpaired chromosomes in spermatocytes, including the ATR-dependent phosphorylation of the single-stranded DNA (ssDNA)-binding complex replication protein A (RPA) and the checkpoint kinase CHK1. These observations support a model in which ssDNA plays a central role in the recruitment of ATR during MSUC, and provide a link to meiotic progression through activation of CHK1.

Key mediators of somatic ATR signaling localize to unpaired chromosomes in spermatocytes

Key mediators of somatic ATR signaling localize to unpaired chromosomes in spermatocytes

Distribution of constitutive heterochromatin and nucleolar organizer regions in two species of family Gomphidae (Odonata: Anisoptera)

Spermatogonial and primary spermatocyte chromosomes of Paragomphus lineatus (Selys) and Nepogomphus modestus (Selys) of family Gomphidae have been described on the basis of C-banding and NOR staining. Both the species possess 2n (♂) =23, n (♂) =12 without m chromosomes with XO(♂)/XX(♀) type sex determining mechanism. The sex chromosome is the largest element in the whole complement in Paragomphus lineatus but it is medium sized in Nepogomphus modestus. In Paragomphus lineatus, all the autosomal bivalents possess terminal C-bands while X chromosome is C-positive throughout the length but lightly stained in the centre. Similarly, terminal NOR bands are present on all the autosomal bivalents and X chromosome shows terminal NOR bands with large interstitial band. In Nepogomphus modestus, all the autosomal bivalents except one bivalent possess terminal C-bands while X chromosome represents large C-positive region on one end and small C-negative region on the other end. NOR staining shows terminal / non terminal NOR bands on some autosomal bivalents while X chromosome possesses dark NOR band only on one end.

Tumor suppressor properties and a role in maintaining genome integrity for the Meig1 gene (736.3)

Meig1 is a highly conserved gene first identified in mouse pachytene spermatocytes undergoing meiotic recombination. In these cells the MEIG1 protein enters the nucleus and forms foci along the meiotic chromosomes, reminiscent of foci obtain by proteins known to be involved in the DNA damage response. Meig1 knockout (KO) male mice are infertile. These mice exhibit increased apoptosis of post recombination spermatogenic cells and significantly fragmented DNA in the few epididymal sperm cells that could be obtained. Given these findings the objectives of this study were to examine the hypothesis that MEIG1 plays a role in the system responsible for maintaining genome integrity. Immunocytochemistry analysis of γH2AX signaling in meiotic chromosomes of late pachytene spermatocytes showed that in KO mice they were highly “decorated” with γH2AX foci, compared to WT chromosomes. Moreover, analysis of post‐recombination meiotic chromosomes revealed the existence of numerous chromosomal fragments in KO meiotic preparations whereas no such fragments could be seen in preparations from WT spermatocytes. Regarding somatic cells, KO MEFs exposed to etoposide treatment exhibited significantly slower disappearance of the γH2AX foci compare to WT MEFs, suggesting delayed or aberrant repair. Interestingly, most Meig1 KO mice (more than 30 mice to date), and about one third of the Meig1 +/‐ heterozygotes developed huge and aggressive tumors between 6‐18 months of age, whereas none of the age‐matched wild‐type (WT) siblings developed tumors. These tumors were identified as being mainly malignant adeno‐carcinomas and lymphomas. These results imply that Meig1 has tumor suppressive properties and suggest that it is involved in maintaining chromatin integrity not only in spermatogenic cells but in somatic cells as well, rendering it as a new player in the general DNA damage response, especially the response to DSB.This study was supported by grant No. 20072019B from the Israel Cancer Association.

Strong nucleosomes reside in meiotic centromeres of C. elegans

Recently discovered strong nucleosomes (SNs) are characterized by strongly periodical DNA sequence, with visible rather than hidden sequence periodicity. In a quest for possible functions of the SNs, it has been found that the SNs concentrate within centromere regions of A. thaliana chromosomes . They, however, have been detected in Caenorhabditis elegans as well, although the holocentric chromosomes of this species do not have centromeres. Scrutinizing the SNs of C. elegans and their distributions along the DNA sequences of the chromosomes, we have discovered that the SNs are located mainly at the ends of the chromosomes of C. elegans. This suggests that, perhaps, the ends of the chromosomes fulfill some function(s) of centromeres in this species, as also indicated by the cytogenetic studies on meiotic chromosomes in spermatocytes of C. elegans, where the end-to-end association is observed. The centromeric involvement of the SNs, also found in A. thaliana, opens new horizons for the chromosome and centromere structure studies.

The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse

In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIβ-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.