Immunofluorescent synaptonemal complex analysis in azoospermic men.
ABSTRACT 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.
[show abstract] [hide abstract]
ABSTRACT: MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are indicated to play essential roles in spermatogenesis. However, little is known about the expression patterns or function of miRNAs in human testes involved in infertility. In this study, the miRNA expression profiles of testes of patients with non-obstructive azoospermia (NOA) and normal controls were performed by using microarray technologies. Altered microRNA expression in NOA patients was found, with 154 differentially down-regulated and 19 up-regulated miRNAs. These findings have been confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) assays on select miRNAs, including miR-302a, miR-491-3p, miR-520d-3p and miR-383. Several down-regulated miRNA clusters in patients with NOA were identified, such as the oncogenic potential of the mir-17-92 cluster and mir-371,2,3 cluster. This is the first report that the expression of miRNAs is altered in testicular tissues of patients with NOA, suggesting a role of miRNAs in regulating spermatogenesis in human males.Reproductive Biology and Endocrinology 03/2009; 7:13. · 2.05 Impact Factor
Article: Variation in MLH1 distribution in recombination maps for individual chromosomes from human males.[show abstract] [hide abstract]
ABSTRACT: Meiotic recombination is essential for the segregation of homologous chromosomes and the formation of normal haploid gametes. Little is known about patterns of meiotic recombination in human germ cells or the mechanisms that control these patterns. Documentation of the normal range of variability of recombination distribution over the genome among individuals is an essential prerequisite for understanding abnormal recombination patterns, which may be associated with non-disjunction and chromosome rearrangements. In this article, variation in recombination maps for individual chromosomes among 10 normal human males is examined for the first time. An immunocytogenetic approach allowed analysis of pachytene cells, using antibodies to detect the mature synaptonemal complex (SCP1/SCP3), the centromere (CREST) and sites of crossing over (MLH1). Individual bivalents were identified with centromere-specific multicolor fluorescence in situ hybridization. Significant heterogeneity in MLH1 focus frequency across donors was observed for larger chromosome arms (P<0.05, one-way ANOVA). Significant inter-donor variation in the overall crossover frequency per cell was also found (P<0.0001, one-way ANOVA). Furthermore, several chromosome arms showed significant differences in crossover distribution along the SCs among donors. Inter-individual variation in interference distances was observed for all chromosomes. The significance of altered recombination patterns among individuals and the role of interference are discussed.Human Molecular Genetics 09/2006; 15(15):2376-91. · 7.64 Impact Factor