A change of view: homologous recombination at single-molecule resolution.

Genetic recombination occurs in all organisms and is vital for genome stability. Indeed, in humans, aberrant recombination can lead to diseases such as cancer. Our understanding of homologous recombination is built upon more than a century of scientific inquiry, but achieving a more complete picture using ensemble biochemical and genetic approaches is hampered by population heterogeneity and transient recombination intermediates. Recent advances in single-molecule and super-resolution microscopy methods help to overcome these limitations and have led to new and refined insights into recombination mechanisms, including a detailed understanding of DNA helicase function and synaptonemal complex structure. The ability to view cellular processes at single-molecule resolution promises to transform our understanding of recombination and related processes.

[Genotoxic effects of pesticide fipronil in somatic and generative cells of mice].

The pronounced genotoxic effect of fipronil in all used doses (4.75, 9.50, 19.00, and 31.70 mg/kg) at a single exposure in the liver, lungs and spleen was ascertained by the Comet assay. Organ specificity of genotoxic effects of the pesticide was revealed. The liver was the most sensitive to fipronil. Fipronil at a dose of 9.50 mg/kg in a single and repeated exposure (within 10 days) induced aberrations in mouse bone marrow cells with the frequency exceeding the spontaneous mutation rate (p < 0.01 and p < 0.001, respectively). Fipronil also showed genotoxic activity in the germ cells of the experimental animals, causing abnormalities of the structure of synaptonemal complexes in the spermatocytes.

Synaptonemal complex protein 3 as a novel prognostic marker in early stage non-small cell lung cancer.

Synaptonemal complex protein 3 is a marker for cell transformation that has prognostic significance in various cancers. However, the prognostic significance of synaptonemal complex protein 3 has not been studied in non-small cell lung cancer. To investigate the potential correlation between synaptonemal complex protein 3 and various clinicopathologic parameters, we assessed the expression of synaptonemal complex protein 3 in archival tumor tissues from 258 patients with non-small cell lung cancer by immunohistochemical staining. By immunofluorescence, synaptonemal complex protein 3 was detected in both the cytoplasmic and nuclear fractions of NCI-H1299 cell. In tumor samples, synaptonemal complex protein 3 is detected as cytoplasmic expression pattern and observed in 50 clinical samples (19.4%) by immunohistochemical staining. Synaptonemal complex protein 3 expression was correlated with T status (P = .008), lymph node metastasis (P = .010), tumor types (P = .019), and pleural invasion (P = .005). In multivariate analysis of patients with early stage disease, increased synaptonemal complex protein 3 expression predicted worse overall survival in early stage (stage I and II) with pT1 status (P = .041). These results suggest that positive synaptonemal complex protein 3 expression is a portent of poor outcome and may be a potential biomarker in the early stages of the non-small cell lung cancer for survival and may provide clues in the identification of patients for adjuvant therapy.

Chronology of meiosis & synaptonemal complex abnormalities in normal & abnormal spermatogenesis.

BACKGROUND & OBJECTIVES: The study was taken up to define criteria of normality for meiosis by assessing the frequency of meiotic prophase cell types, the frequency of pachytene substage in normal and abnormal spermatogenesis and to determine what synaptonemal complex. METHODS: A quantitative and qualitative analysis of the first meiotic prophase was performed in 10 patients presenting with non-obstructive infertility and 10 controls, using dual colour immunocytochemistry with SCP3 and BRCA1 which visualise axial elements and synaptonemal complexes (SC). The respective frequencies of the leptotene, zygotene and pachytene stages as well as the frequencies of the four substages of pachytene were evaluated. The frequencies of the main types of meiotic abnormalities at pachytene were also assessed. RESULTS: The frequencies of leptotene and zygotene stages were significantly higher in patients (7.95 and 9.75%) than in controls (2.30 and 1.45%), whereas the frequency of pachytene was significantly higher in controls than in patients (96.25 vs. 75.30%). Detailed analysis of the sex chromosomes revealed that the controls showed a presence of late pachytene substages (P3 + P4 = 64.40%), whereas the patients showed a early pachytene substages (P1 + P2 = 63.40%). From these results, a new index was defined to evaluate spermatogenesis: the Pachytene Index, or PI (PI = P1 + P2 / P1 + P2 + P3 + P4). The same abnormalities (asynapsis, fragmented SC, dotted SC, thin SC) were observed in controls and in patients, but with different frequencies. The most frequent abnormality was fragmented SC, with a significant difference between patients and controls (15.28 vs. 9.74%). There was a significant difference between patients and controls for the frequency of asynapsed nuclei (7.97 vs. 2.95%) while the difference in other abnormalities were not significant. INTERPRETATION & CONCLUSION: The accumulation of early primary spermatocytes is an indication that progression of meiosis is defective in spermatogenesis failures. The value of the PI less than 0.50 indicates that the kinetic of meiosis is normal at pachytene. There is no normal spermatogenesis when the frequency of one or several SC abnormalities is significantly higher than in controls and/ or when the PI is more than 0.50.

Impact of trisomy on fertility and meiosis in male mice.

BACKGROUND: Chromosomal abnormalities frequently are associated with impairment or arrest of spermatogenesis in mammals but are compatible with fertility in female carriers of the same anomaly. In the case of trisomy, mice have extra genomic DNA as well as the chromosomal abnormality, usually present as an extra, unpaired chromosome. Thus, impairment of spermatogenesis in trisomic males could be due to the presence of extra genomic material (i.e. triplicated genes) or due to the chromosomal abnormality and presence of an unpaired chromosome in meiosis. METHODS: In this study, fertility and chromosomal pairing configurations during meiotic prophase were analysed in male mice trisomic for different segments of the genome. Four have an extra segmental or tertiary trisomic chromosome--Ts(17(16))65Dn, Ts(10(16))232Dn, Ts(12(17))4Rk and Ts(4(17))2Lws--and one has the triplicated segment attached to another chromosome--Ts(16C-tel)1Cje. Ts(17(16))65Dn and Ts(16C-tel)1Cje have similar gene content triplication and differ primarily in whether the extra DNA is in an extra chromosome or not. RESULTS: The presence of an intact extra chromosome, rather than trisomy per se, is associated with male sterility. Additionally, sterility is correlated with a high frequency of association of the unpaired chromosome with the XY body, which contains the largely unpaired X and Y chromosomes. CONCLUSIONS: Intact extra chromosomes disrupt spermatogenesis, and unpaired chromosomes establish a unique chromatin territory within meiotic nuclei.

Immunofluorescent synaptonemal complex analysis in azoospermic men.

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.

[Schistosoma reflexum in a female bovine fetus with synaptonemal complex abnormalities]. / Schistosoma reflexum bei einem weiblichen Rinderfetus mit synaptonemalen Komplex-Störungen.

In this study we present mitotic- and meiotic investigations in an anatomical abnormal bovine fetus. The abnormality could be classified as "schistosoma reflexum", which was never described in fetuses in the literature. In the mitotical chromosome preparations from fibroblast cultures the examined fetus showed no chromosomal difference in comparison to the standard synaptonemal complexes (SC) which were prepared from the fetus at the age of 92 days post coitum. In the SCs from the abnormal fetus 43.75% of the investigated cells showed abnormalities such as "loop," "nonhomologue pairing" and "multivalency" in the pairing behavior of the chromosomes. In comparison, less than 5% of the cells in normal embryos showed such abnormalities.

Evaluation of the Stag3 gene and the synaptonemal complex in a rat model (as/as) for male infertility.

Affected males (as/as) from the mutant TT rat strain are sterile due to spermatogenesis impairment with meiotic arrest at the pachytene stage. The as locus is on rat chromosome 12, in a region that shows conserved synteny to cM 74-94 on mouse chromosome 5. Stag3, a new member of the stromalin protein family, is expressed specifically in testis and associates to the synaptonemal complex. Mouse Stag3 gene has been assigned to cM 78 on chromosome 5. In this study, we have characterized the rat Stag3 gene and examined it as a candidate for male infertility in as/as rats. The rat Stag3 cDNA is 4181 nucleotides long, contains a highly polymorphic hexanucleotide repeat in the coding region, and encodes a 1256 amino acid protein with 93 and 77% sequence identity to mouse and human Stag3 proteins, respectively. No mutations or differences in size or abundance of Stag3 mRNA were detected between as/as and control rats, suggesting that Stag3 is not responsible for the aspermic phenotype. In addition, immunohistochemistry with antibodies against SCP1 and SPC3 proteins suggest that the synaptonemal complex structures are not primarily affected in these rats.