Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).

Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines.

Enhanced epithelial Na(+) channel (ENaC) activity in mouse endometrial epithelium by upregulation of gammaENaC subunit.

The amiloride-sensitive epithelial Na(+) channel (ENaC), which is made of three different but homologous subunits, controls the rate of transepithelial Na(+) absorption in a variety of epithelia. The present study investigated the functional role of its subunits in regulating ENaC activity, measured as amiloride sensitive short-circuit current (I(SC)), in the mouse endometrial epithelium under different culture conditions. The treatment of the cultured epithelia with aldosterone (1 microM) or culturing cells on filters coated with concentrated Matrigel resulted in an increase in the amiloride-sensitive I(SC). Semiquantitative RT-PCR demonstrated that the expression of alpha and beta subunits was not significantly altered by these treatments, but an increase in the gamma subunit expression was observed. An 11-fold increase, induced by aldosterone, in the expression of the gamma subunit, but not in the alpha and beta subunits, was confirmed by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The treatment of endometrial cells with antisense against the gammaENaC subunit abolished the aldosterone-enhanced amiloride-sensitive I(SC). The results indicated an important role of gammaENaC subunit in determining ENaC activity, and a possible role of the gammaENaC subunit in interacting with CFTR was also discussed.

Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11.

Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination. We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis. Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth. Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished. Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes. We propose that recombination initiation precedes and is required for normal synapsis in mammals. Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.

The Bradyrhizobium japonicum noeD gene: a negatively acting, genotype-specific nodulation gene for soybean.

Bradyrhizobium japonicum strain USDA 110 is restricted for nodulation by soybean genotype PI 417566. We previously reported the identification of a USDA 110 Tn5 mutant, strain D4.2-5, that had the ability to overcome nodulation restriction conditioned by PI 417566 (S. M. Lohrke, J. H. Orf, E. Martínez-Romero, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:2378-2383, 1995). In this study, we report the cloning and characterization of the negatively acting DNA region mutated in strain D4.2-5 that is involved in the genotype-specific nodulation of soybean. The Tn5 integration site was localized to a 5.2-kb EcoRI fragment isolated from wild-type USDA 110 genomic DNA. Saturation Tn5 mutagenesis of this 5.2-kb region and DNA homogenitization studies indicated that a 0.9-kb DNA region was involved in the genotype-specific nodulation of PI 417566. A single open reading frame (ORF) of 474 nucleotides, encoding a predicted protein of 158 amino acids, was identified within this region by DNA sequencing. This ORF was named noeD. Computer comparisons with available data bases revealed no significant similarities between the noeD DNA or predicted amino acid sequence and any known genes or their products. However, comparisons done with the region upstream of noeD revealed a high degree of similarity (about 76% similarity and 62% identity) to the N-terminal regions of the Rhizobium leguminosarum bv. viciae and R. meliloti nodM genes, which have been postulated to encode a glucosamine synthase. Southern hybridization analysis indicated that noeD is not closely linked to the main or auxiliary nodulation gene clusters in B. japonicum and that both nodulation-restricted and -unrestricted B. japonicum serogroup 110 strains contain a noeD homolog. High-performance liquid chromatography and fast atom bombardment-mass spectrometry analyses of the lipo-chitin oligosaccharide (LCO) nodulation signals produced by an noeD mutant showed a higher level of acetylation than that found with wild-type USDA 110. These results suggest that specific LCO signal molecules may be one of the factors influencing nodulation specificity in this symbiotic system.