(1/97) Analysis of the kar3 meiotic arrest in Saccharomyces cerevisiae.
The motor protein Kar3p and its associated protein Cik1p are essential for passage through meiosis I. In the absence of either protein, meiotic cells arrest in prophase I. Experiments were performed to determine whether the arrest was caused by a structural inability to proceed through meiosis, or by a regulatory mechanism. The data demonstrate that the meiotic arrest is not structural; kar3 and cik1 mutants are able to form normal looking bipolar spindles and divide their DNA into two masses in spo11 mutant backgrounds. To identify the regulatory system necessary for the kar3/cik1 meiotic arrest, we tested whether the arrest could be bypassed by eliminating the pachytene checkpoint or the spindle checkpoint. The arrest is not solely dependent upon the pachytene checkpoint that monitors recombination and aspects of chromosome synapsis. Elimination of the spindle checkpoint failed to allow kar3 mutants to undergo meiosis I nuclear division, but phenotypes of the kar3/spindle checkpoint double mutants suggest that the kar3 meiotic arrest may be mediated by the spindle checkpoint. (+info)
(2/97) Integrating genetic linkage maps with pachytene chromosome structure in maize.
Genetic linkage maps reveal the order of markers based on the frequency of recombination between markers during meiosis. Because the rate of recombination varies along chromosomes, it has been difficult to relate linkage maps to chromosome structure. Here we use cytological maps of crossing over based on recombination nodules (RNs) to predict the physical position of genetic markers on each of the 10 chromosomes of maize. This is possible because (1). all 10 maize chromosomes can be individually identified from spreads of synaptonemal complexes, (2). each RN corresponds to one crossover, and (3). the frequency of RNs on defined chromosomal segments can be converted to centimorgan values. We tested our predictions for chromosome 9 using seven genetically mapped, single-copy markers that were independently mapped on pachytene chromosomes using in situ hybridization. The correlation between predicted and observed locations was very strong (r(2) = 0.996), indicating a virtual 1:1 correspondence. Thus, this new, high-resolution, cytogenetic map enables one to predict the chromosomal location of any genetically mapped marker in maize with a high degree of accuracy. This novel approach can be applied to other organisms as well. (+info)
(3/97) In vivo analysis of synaptonemal complex formation during yeast meiosis.
During meiotic prophase a synaptonemal complex (SC) forms between each pair of homologous chromosomes and is believed to be involved in regulating recombination. Studies on SCs usually destroy nuclear architecture, making it impossible to examine the relationship of these structures to the rest of the nucleus. In Saccharomyces cerevisiae the meiosis-specific Zip1 protein is found throughout the entire length of each SC. To analyze the formation and structure of SCs in living cells, a functional ZIP1::GFP fusion was constructed and introduced into yeast. The ZIP1::GFP fusion produced fluorescent SCs and rescued the spore lethality phenotype of zip1 mutants. Optical sectioning and fluorescence deconvolution light microscopy revealed that, at zygotene, SC assembly was initiated at foci that appeared uniformly distributed throughout the nuclear volume. At early pachytene, the full-length SCs were more likely to be localized to the nuclear periphery while at later stages the SCs appeared to redistribute throughout the nuclear volume. These results suggest that SCs undergo dramatic rearrangements during meiotic prophase and that pachytene can be divided into two morphologically distinct substages: pachytene A, when SCs are perinuclear, and pachytene B, when SCs are uniformly distributed throughout the nucleus. ZIP1::GFP also facilitated the enrichment of fluorescent SC and the identification of meiosis-specific proteins by MALDI-TOF mass spectroscopy. (+info)
(4/97) Estrogen receptor beta expression and apoptosis of spermatocytes of mice overexpressing a rat androgen-binding protein transgene.
Progression of the first meiotic division in male germ cells is regulated by a variety of factors, including androgens and possibly estrogens. When this regulation fails, meiosis is arrested and primary spermatocytes degenerate by apoptosis. Earlier studies showed that overexpression of rat androgen-binding protein (ABP) in the testis of transgenic mice results in a partial meiotic arrest and apoptosis of pachytene spermatocytes. In view of the recent localization of estrogen receptor beta (ERbeta) in primary spermatocytes and data suggesting the ability of ERbeta to repress cellular proliferation, we tested the hypothesis that variations in the testicular steroid microenvironment caused by excess ABP produce changes in ERbeta expression in this cellular type that could be associated to the meiotic arrest and, eventually, to the induction of germ cell apoptosis observed in the ABP transgenic mice. Increased levels of ERbeta mRNA and protein were demonstrated in the testis of rat ABP transgenic mice compared with nontransgenic littermates by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments, Northern blotting, and Western Blotting. The major differences were found when isolated germ cells of transgenic and nontransgenic littermates were analyzed by RT-PCR. In keeping with this finding, ERbeta was strongly immunolabeled in pachytene spermatocytes of rat ABP transgenic mice and localized in tubular stages in which TUNEL labeling was maximal. Confocal microscopy analysis of a fluorescent TUNEL assay and ERbeta immunohistochemistry revealed that degenerating pachytene spermatocytes overexpressed ERbeta. The present results are consistent with the interpretation that ERbeta is associated with the events that regulate negatively the progression of meiosis or that lead to spermatocyte apoptosis. (+info)
(5/97) RFX2 is a potential transcriptional regulatory factor for histone H1t and other genes expressed during the meiotic phase of spermatogenesis.
H1t is a novel linker histone variant synthesized in mid- to late pachytene spermatocytes. Its regulatory region is of interest because developmentally specific expression has been impressed on an otherwise ubiquitously expressed promoter. Using competitive band-shift assays and specific antisera, we have now shown that the H1t-60 CCTAGG palindrome motif region binds members of the RFX family of transcriptional regulators. The testis-specific binding complex contains RFX2, probably as a homodimer. Other DNA-protein complexes obtained from testis as well as somatic organs contain RFX1, primarily as a heterodimer. Western blots confirmed that RFX2 expression is greatly enhanced in adult testis and that RFX2 is equally prominent in highly enriched populations of late pachytene spermatocytes and round spermatids. Immunohistochemistry carried out on mouse testis showed that RFX2 is strongly expressed in pachytene spermatocytes, remains high in early round spermatids, and declines only in advance of nuclear condensation. Maximum expression correlates well with the appearance of H1t. In contrast, RFX1 immunoreactivity in germ cells was only detected in late round spermatids. RFX-specific band complexes were also identified for both the mouse lamin C2 and Sgy promoters, using either testis nuclear extracts or in vitro-synthesized RFX2. These results call attention to RFX2 as a transcription factor with obvious potential for the regulation of gene expression during meiosis and the early development of spermatids. (+info)
(6/97) BRCA1, histone H2AX phosphorylation, and male meiotic sex chromosome inactivation.
In mammalian spermatogenesis, the X and Y chromosomes are transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation, MSCI), forming a condensed chromatin domain termed the sex or XY body. The nucleosomal core histone H2AX is phosphorylated within the XY chromatin domain just prior to MSCI, and it has been hypothesized that this triggers the chromatin condensation and transcriptional repression. Here, we show that the kinase ATR localizes to XY chromatin at the onset of MSCI and that this localization is disrupted in mice with a mutant form of the tumor suppressor protein BRCA1. In the mutant pachytene cells, ATR is usually present at nonsex chromosomal sites, where it colocalizes with aberrant sites of H2AX phosphorylation; in these cells, there is MSCI failure. In rare pachytene cells, ATR does locate to XY chromatin, H2AX is then phosphorylated, a sex body forms, and MSCI ensues. These observations highlight an important role for BRCA1 in recruiting the kinase ATR to XY chromatin at the onset of MSCI and provide compelling evidence that it is ATR that phosphorylates H2AX and triggers MSCI. (+info)
(7/97) The control of Spo11's interaction with meiotic recombination hotspots.
Programmed double-strand breaks (DSBs), which initiate meiotic recombination, arise through the activity of the evolutionary conserved topoisomerase homolog Spo11. Spo11 is believed to catalyze the DNA cleavage reaction in the initial step of DSB formation, while at least a further 11 factors assist in Saccharomyces cerevisiae. Using chromatin-immunoprecipitation (ChIP), we detected the transient, noncovalent association of Spo11 with meiotic hotspots in wild-type cells. The establishment of this association requires Rec102, Rec104, and Rec114, while the timely removal of Spo11 from chromatin depends on several factors, including Mei4 and Ndt80. In addition, at least one further component, namely, Red1, is responsible for locally restricting Spo11's interaction to the core region of the hotspot. In chromosome spreads, we observed meiosis-specific Spo11-Myc foci, independent of DSB formation, from leptotene until pachytene. In both rad50S and com1Delta/sae2Delta mutants, we observed a novel reaction intermediate between Spo11 and hotspots, which leads to the detection of full-length hotspot DNA by ChIP in the absence of artificial cross-linking. Although this DNA does not contain a break, its recovery requires Spo11's catalytic residue Y135. We propose that detection of uncross-linked full-length hotspot DNA is only possible during the reversible stage of the Spo11 cleavage reaction, in which rad50S and com1Delta/sae2Delta mutants transiently arrest. (+info)
(8/97) Stage-specific expression of mouse germ cell-less-1 (mGCL-1), and multiple deformations during mgcl-1 deficient spermatogenesis leading to reduced fertility.
A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1), is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-1 in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum et al., 2003), postacrosomal sheath, and posterior ring apparently formed. In addition, MN7 and MN13, essential substances for fertilization that are located in sperm heads, were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility. (+info)