Tetraploid Spermatocytes Research Articles

Mapping post-translational modifications of mammalian testicular specific histone variant TH2B in tetraploid and haploid germ cells and their implications on the dynamics of nucleosome structure.

Histones regulate a variety of chromatin templated events by their post-translational modifications (PTMs). Although there are extensive reports on the PTMs of canonical histones, the information on the histone variants remains very scanty. Here, we report the identification of different PTMs, such as acetylation, methylation, and phosphorylation of a major mammalian histone variant TH2B. Our mass spectrometric analysis has led to the identification of both conserved and unique modifications across tetraploid spermatocytes and haploid spermatids. We have also computationally derived the 3-dimensional model of a TH2B containing nucleosome in order to study the spatial orientation of the PTMs identified and their effect on nucleosome stability and DNA binding potential. From our nucleosome model, it is evident that substitution of specific amino acid residues in TH2B results in both differential histone-DNA and histone-histone contacts. Furthermore, we have also observed that acetylation on the N-terminal tail of TH2B weakens the interactions with the DNA. These results provide direct evidence that, similar to somatic H2B, the testis specific histone TH2B also undergoes multiple PTMs, suggesting the possibility of chromatin regulation by such covalent modifications in mammalian male germ cells.

Metastasis tumor antigen 1 (MTA1) might contribute to maintain the cellular integrity of germ cells by coordination with p53 through EGF-pathway stimulation during spermatogenesis

DNA quality control is expected to be highly regulated in tetraploid spermatocytes, since chromosome crossover, which involves DNA strand break, transfer, and renaturation, is an active event at this stage of germ cell development. p53-mediated apoptosis is an effector of cellular proofreading that acts to maintain the cellular integrity of germ cells during spermatogenesis and the highest levels of testicular p53 are found in tetraploid spermatocytes. MTA1 has been previously implicated in cellular transformation through its ability to regulate the epithelial-to-mesenchymal transition and to enhance metastasis. MTA1 is a component of the Mi-2/nucleosome remodeling and deacetylating (NURD) complex that contains both histone deacetylase and nucleosome remodeling activity, and its expression and deacetylase activity could be remarkably upregulated by the stimulation of epidermal growth factor (EGF). Recent studies revealed that MTA1 inhibits p53-induced apoptosis in both cancer cells and testicular function. All these observations lent support to the hypotheses that up-regulation of MTA1 by EGF might repress testicular expression of p53 and lead to low abundance of p53 expression during adult spermatogenesis. This repression as well as p53-mediated apoptosis may play important roles in the regulation of homeostasis of germ cell proliferation and death. Further investigation in this field is urgently requested.

Triplet repeats and DNA repair: germ cell and somatic cell instability in transgenic mice.

This chapter describes methods for the isolation of specific cell types that reveal how and where expansion can occur. For the hereditary component of expansion, the male germ cell has proved useful in distinguishing processes that can contribute to expansion, as described in our article (Nature Genetics 27, 407, 2001). Mature spermatazoa (SZs) can be isolated directly from the epididymis. Haploid spermatids (STs), diploid spermatagonia (SGs), and tetraploid spermatocytes (SCs) can be removed from the testis and sorted by fluorescence-activity cell sorting (FACS); differences in DNA content and morphology allow resolution by fluorescence and light scattering. Repeat-length measurement can pinpoint the stage at which expansion occurs. Because the timing of meiosis and mitosis with respect to sperm development is known, the analysis can distinguish repair and replication processes. Furthermore, the possible contribution of Y- or X-specific factors can be evaluated by sorting X- and Y-bearing germ cells. To enable analysis of female germ cells, we describe methods for oocyte preparations and a method for the isolation of the eight-cell-stage embryo. Therefore, the methods described here can help to answer such questions as the timing during development of expansion, whether expansion is limited to a single period, whether both replication and repair contribute to instability, and the role of somatic instability in disease. If further expansion of the inherited allele contributes to the phenotype, then intervention in somatic tissue might be therapeutic.

Spermatogenesis in the golden hamster during the first spermatogenic wave: a flow cytometric analysis.

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36-38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88-90%) in the hamster occurred in the testis and only 10-12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205-211, 2000.

Influence of oxygen on radiation-induced DNA damage in testicular cells of C3H mice

Radiation-induced DNA single-strand break (ssb) induction and rejoining were measured in murine testicular cells using the alkaline comet assay. Individual cells in different stages of differentiation were identified on the basis of DNA content. As expected, induction of DNA ssb in testis cells irradiated on ice was independent of ploidy, and the extent of damage was similar to that produced in cells from other normal tissues. However, in vivo irradiation of air-breathing mice produced more ssb in haploid than tetraploid germ cells, although their rates of rejoining were similar and comparable to repair rates of cells from other normal tissues. In addition, irradiation of testis in situ produced only half as much damage as irradiation in vitro, and this could be explained only in part by the rapid ssb rejoining occurring during irradiation and cell isolation. A lower cellular oxygenation was postulated to account for the apparent resistance of testis cells to induction of breaks and the difference in induction in relation to DNA content. This was confirmed when carbogen inhalation and treatment with nicotinamide not only increased the overall degree of ssb induction in all these cells, but also reduced differences between cells of different ploidies. Results using the hypoxic cell cytotoxin RSU 1069 confirmed that the extent of hypoxia was not as severe in the testis as in the SCCVII murine tumour. It can be concluded from these data that the oxygenation of all testis cells is low enough to confer radioresistance, and that haploid testis cells are less hypoxic than tetraploid spermatocytes.

Poly(ADPribosyl)ation System in Rat Germinal Cells at Different Stages of Differentiation

In order to study the possible functional relationship between poly(ADP-ribosyl)ation and spermatogenesis, the three main germinal cell types have been isolated and characterized as haploid spermatids and diploid and tetraploid spermatocytes. Purified germinal cell populations and rats of different age were used for activity-, immuno-, and Northern blot experiments, to determine at which level poly(ADPR)polymerase (PARP) is regulated at various stages of spermatogenesis. Poly(ADPR)glycohydrolase (PARG) activity was also determined, as was the subcellular distribution of both PARP and PARG enzymes. The results show that the maximum of both PARP amount and PARP activity can be detected on tetraploid spermatocytes which undergo meiotic division, whereas PARG activity does not differ in germinal cells; the cytoplasmic form of this enzyme is prevalent in testis. Moreover, a difference in timing was observed in maximal level between PARP expression, determined on testis from 60-day-old rats, and PARP activity, detected on testis from 30-day-old animals. It seems that different mechanisms modulate the poly(ADPribosyl)ation system during spermatogenesis. Regulation of the poly(ADPribose) turnover, variations of PARP amount, as well as changes of PARP transcription level, seem to accompany germinal cell differentiation, possibly being implicated in DNA replication, repair, and transcription.

Human meiosis V. Substages of pachytene in human spermatogenesis

The pachytene stage of human spermatogenesis has been investigated by serial sectioning of spermatocytes and three dimensional reconstruction of nuclei from electron micrographs. The analysis of 73 completely reconstructed normal nuclei from late zygotene to early diplotene revealed a continuous sequence of changes in the fine structure and morphology. The developmental sequence permits the recognition of 7 substages on the basis of morphological changes of autosomal chromatin, centromeric, chromatin, the XY bivalent, the secondary constriction on bivalents 1,9 and 16, the nucleolus and the centrioles. Of particular interest is the observation that the disappearance of the synaptonemal complex segment between the X and Y chromosomes at mid pachytene is only temporary, a distinct complex segment being present in nearly all analysed XY bivalents by the end of pachytene. The number of recombination nodules and recombination bars in nuclei of the different substages is recorded. The total number of nodules decreases from 75 at the beginning of pachytene to 1 or none at the end of this state. The bars appear at early pachytene, reach a number of 35 at mid pachytene and disappear following the nodules towards the end of pachytene. The morphology of pachytene nuclei at various stages of degeneration as well as the structural characteristics of two tetraploid spermatocytes are described.

Spermatogenesis in Sciara coprophila. II. Precocious chromosome orientation in meiosis II.

In the second meiotic division of spermatogenesis in Sciara coprophila the X dyad undergoes a directed nondisjunction appearing precociously at one pole. All other chromosomes behave in a normal fashion aligning on the metaphase plate and dividing. Crouse has determined that this directed nondisjunction is governed by a region of the X centromere heterochromatin known to contain the rDNA (Crouse et al., 1977; Crouse, 1979). In order to further characterize this system we have utilized DAPI c-banding and rDNA in situ hybridization to demonstrate that the precocious chromosome (X or translocation chromosome) orients at metaphase II with its centromere end juxtaposed to the pole. Even when the controlling region is not near the centromere as in the case of translocations T1 and T32, the precocious chromosome orients with the centromere end rather than the controlling region end adjacent to the pole. These data may argue that precocious positioning is established at telophase I and maintained throughout the second meiotic division. - We have examined the hypothesis that the controlling region is transcriptionally active at metaphase II and can find no evidence for this speculation. This argues that if an RNA product is related to precocious positioning it must be synthesized earlier in spermatogenesis. - An analysis of naturally occurring tetraploid spermatocytes demonstrates that the two independent precocious chromosomes of such cells are always associated with the same pole. This datum in conjunction with the observation that tetraploid primary spermatocytes have only one monopolar spindle and not two, further supports the notion that a precocious chromosome-pole interaction may be established in meiosis I and maintained throughout meiosis II.

Meiosis in the Djungarian hamster. II. Polyploid spermatocytes.

Polyploid spermatocytes were found in air-dried meiotic preparations of 8 of 10 adult males of the Djungarian hamster <i>(Phodopus sungorus campbelli </i>Thomas). Most of them were tetraploid spermatocytes I or diploid spermatocytes II, but cells of higher ploidy have also been found. It is argued that polyploid spermatocytes are not artifacts and that the most probable cause of their formation is fusion of cells at pachytene and soon after metaphase I.

Zoological Society of Bengal: New Journal

ZOOLOGISTS will be interested in the appearance of vol. 1, No. 1 of the Proceedings of the Zoological Society of Bengal which, it is announced, will be published twice yearly and will contain the results of original research in the various branches of zoology. The first number (obtainable from the Society at 35 Ballrgunge Circular Road, Calcutta ; price 5 Rs. or 10s.) comprises 78 pages and 4 plates ; and it contains eight papers covering a wide field, contributed mainly by the Department of Zoology of the University of Calcutta. S. P. R. Chaudhuri and I. Bose give an account of meiosis in diploid and tetraploid spermatocytes of the grasshopper Auractomorpha sp., while meiosis and chiasma formation in another grasshopper, Tristria pulvinata, is the subject of a paper by M. K. Dutt. In a study of the scales in Glossogobuis giuris, S. Mookerjee shows that the ctenoid condition is an advance on the cycloid which occurs earlier in development. The bionomics and reproduction of another fish, Ophicephalus striatus, are discussed by H. K. Mookerjee, D. N. Ganguly and R. N. Bhattacharya, and R. P. Dhar contributes a hæmatological note on a closely allied species. Some myxosporidians parasitic in fishes are described by M. Chakravarty and S. P. Basu. Termite control with various insecticides tried out in the field is the subject of an investigation by D. Mukerji and P. K. Mitra, and the final paper, concerning the development of the vertebral column in Alligator mississippiensis, is by H. K. Mookerjee and R. N. Bhattacharya. Text and figures are clearly printed on good art paper.