ABSTRACT
Isolating sperms with normal chromatin content is considered vital in Intra Cytoplasmic Sperm Injection treatment. Today, researchers are trying to find new procedures to select sperms with normal chromatin content and morphology. This study examines the effectiveness of Zeta, Density Gradient Centrifugation [Pure Sperm] and combination of Pure-Zeta procedures in selecting sperms with normal chromatin structure using Sperm Chromatin Dispersion [SCD], and Chromomycin A3 [CMA3]. 28 patients participated in the study. Semen samples were analyzed according to WHO criteria. The samples were then divided into four equal portions of control, Zeta method, DGC [Pure Sperm] method and Pure-Zeta method. Sperm morphology [Diff Quick Staining], protamine deficiency [CMA3] and DNA integrity [SCD] were carried out for all the samples. The results were analyzed using ANOVA. P< 0.05 was considered significant. There was a significant reduction in the percentage of sperm protamine deficiency and DNA fragmentation in Zeta, Pure and Pure-Zeta method groups compared to the control group [p <0.05]. In terms of a normal morphology, Pure and Pure-Zeta procedures proved significantly different from the control group [p<0.05 normal morphology]. Zeta, Pure and Pure-Zeta procedures did no differ in isolating sperms with regard to normal morphology, protamine content and DNA fragmentation [p > 0.05]. Based on the result, Zeta and Pure methods improved selection of sperms with normal morphology, protamine content and DNA integrity. Yet, the combination of Pure-Zeta was more effective in terms of isolating spermatozoa with normal morphology, protamine content and DNA integrity than when Zeta and Pure methods were implemented individually
Subject(s)
Humans , Male , Chromatin/isolation & purification , Centrifugation, Density Gradient , Spermatozoa/ultrastructure , Protamines , DNA FragmentationABSTRACT
Fragile sites (FS) are chromosomal regions where the normal compactation of chromatine is not observed. FRAXA (Fra Xq27.3, X sexual chromosome) is one of the most studied FS in humans. FRAXA is an expansion of the trinucleotide CGG located in the gene FMR-1. In cattle, sites of chromosomal fragility were reported in BTAX, associated with different pathologies and fertility impairment. Chromosomal microdissection has became a valuable tool for isolating chromatine fragments. In this work, it was combined the chromosomal microdissection technique with DOP-PCR in order to carry out a molecular analysis of the fragile chromosomal region BTAXq31-34. In that region, polymorphic DNA-RAPD sequences (GC rich) are present and sequences of the gene FMR-1 are missing. The results showed the usefulness of the microdissection-DOP-PCR technique for molecular characterization of fragile chromosomal sites in cattle.
Os sítios frágeis (FS) são regiões de cromossomo onde a compactação normal da cromatina não é realizada. O FRAXA (Fra Xq27.3, cromossomo sexual X) é um dos FS mais estudados em seres humanos. O FRAXA apresenta expansão do trinucleotídeo CGG localizado no gene FMR-1. Em bovinos, existem estudos informando sobre fragilidade cromossômica em BTAX associada com diversas patologias e alterações na fertilidade. A microdissecação cromossômica é uma valiosa técnica para isolar fragmentos de cromatina. Neste trabalho, combinou-se a técnica de microdissecação de cromossomo com DOP-PCR para executar a análise molecular da região do sitio frágil cromossômico BTAXq31-34. Naquela região estão presentes seqüências do polimorfo DNA-RAPD (rico em GC), em que as seqüências do gene FMR-1 estão ausentes. Os resultados mostram a utilidade da técnica de microdissecação-DOP-PCR para a caracterização molecular de sítios frágeis cromossômicos em bovinos.
Subject(s)
Animals , Cattle , Chromosome Fragile Sites , Chromatin/isolation & purification , Microdissection/methods , Microdissection/veterinary , X ChromosomeABSTRACT
Poly-ADP-ribosylation of cellular proteins is involved with radiation induced damage and its repair. It has been observed that suspension of human kidney T1-cells in vitro attained elevated levels of poly-ADP-ribosylation due to experimental manipulations necessary for preparation of single cell suspension from monolayer cell cultures. These cells in suspension were exposed to various doses of gamma-rays with or without subsequent repair incubation. The PADPR of histones H3, H1 and H2B increased with increasing dose of radiation and decreased after 90 min or repair incubation. Concomitant with these changes, the affinity of histones to DNA in chromatin reduced immediately after irradiation. Normal affinity was reestablished after post-irradiation repair incubation. The results indicate that induction of poly-ADP-ribosylation of histone proteins by radiation and by manipulations to prepare single cell suspension involved different cellular components.