Cell Journal (Yakhteh)
Year:2006 | Volume:7 | Issue:4 (28)|Papers Count:9

Sperm DNA is known to contribute one half of the genomic material to the offspring. The integrity of sperm DNA is important in fertilization, embryonic and fetal development, and postnatal child well being. The nature has created multiple barriers that allow only the fittest sperm to reach and fertilize an oocyte. However, assisted reproductive techniques (ART), like IVF and ICSI, may allow sperms with abnormal genomic material to enter the oocyte with minimal effort. This article describes structure of sperm DNA and different mechanism involved in sperm chromatin anomalies and DNA damage. Furthermore, this study elaborates possible sperm selection methods that may improve the outcome of ART.

Introduction: Glycoconjugates are a class of glycoproteins or glycolipids, their terminal sugars are responsible for cell-cell and/or cell-extracellular matrix interactions. Aberrant glycosylation of these compounds are one of the most important aspects of cellular transformation, metastasis and escape of tumoral cells from immune system and resistance to antineoplastic drugs. Recent studies showed that patients with HPA (helix pomatia agglutinin) positive intraductal carcinoma cells have worse prognosis compared to patients with HPA negative cells. The aim of the present study was to define the presence of GalNac terminal sugar in glycoconjugate of different grades of intraductal breast carcinoma and to compare the degree and the pattern of reactivity of tumoral cells to HPA lectin. Materials and Methods: The paraffin blocks belonging to 20 patients of intraductal carcinoma was chosen from pathology archive of Khatam-Al-Anbia hospital in Zahedan and 5-7 micrometer sections were prepared. Two expert pathologists determined histopathological grading independently. The lectin histochemistry was performed using HPA. The same observers determined histochemical grading. Data were analyzed by NPAR (non-parametric) test of Mann Whitney. Results: Results of this study revealed that the pattern and the degree of histochemical reactivity of neoplastic cells differ in all grades of intraductal carcinoma. Histochemical staining showed significant difference between grades of intraductal carcinoma of the breast (p<0.003). The lowest reactivity was seen in grade I and the highest in grade III. Furthermore, the reaction of tumoral cells was primarily confined to apical surfaces of cells in grade I, to the Golgi zone in grade II, and to a diffuse cytoplasmic distribution in grade III. Conclusion: Our data suggest that the HPA reactivity of tumoral cells were different in all grades of intraductal carcinoma. The tumor cells showed aberrant glycosylation, which occurred in the course of anaplastic changes. It seems that our data suggest a potential and clinically important role of HPA reactivity to predict the invasive nature of malignant tumoral cells of intraductal carcinoma of the breast.

Introduction: While human endothelial progenitor cells (EPCs) have been a subject of somehow extensive investigation, EPCs from adult mouse hematopoietic system were poorly studied. Present investigation is focused on FVB mouse endothelial progenitor cells in terms of their isolation, purification, and expansion. Materials and Methods: Mononuclear cells collected from murine peripheral blood were cultured in fibronectin coated plate for two weeks, at which point, the adherent cell population were lifted and analyzed in terms of some surface markers. Using FACS Vantage equipped with one-cell deposition unit, single CD34 positive cells were plated per well already containing medium optimized for single cell growth. Several clones were then emerged, expanded, and examined in terms of some surface markers. Furthermore, the cells were investigated regarding ability to uptake DiI-ac-LDL and form capillary network on matrigel surfaces. Results: Adherent population of mononuclear cells from mouse peripheral blood was appeared morphologically heterogeneous. About 5% of the adherent cells were CD34 positive. Having optimized their culture condition, several CD34 positive clones were expanded. The cells comprising the clones were DiI-ac-LDL+ and formed capillary-like tube when being seeded on matrigel surfaces. Conclusion: The primary culture of the mononuclear cells from murine peripheral blood contains a very limited number of cells positive for endothelial lineage markers. These cells (adherent CD34 positive) could be expanded by single cell cloning technique.

Introduction: The thyroid hormones have profound effects on the development of neuromuscular system. These hormones exert their influence on both muscle fibers and related motoneurons during development. The masseter is one of the most important muscles for mastication in mammals. We attempted to evaluate the effect of thyroid hormone deficiency on the morphological characteristics of masseteric motoneurons in the period of alteration from sucking to biting and chewing in the rat. Materials and Methods: To induce hypothyroidism, timed pregnant dams received 50 ppm antithyroid drug propylthiouracil (PTU) in their drinking water and PTU was administered to the pups during suckling period. Horseradish peroxidase (HRP) was injected into the masseter (0.5-5 mlit, 40%) of normal and prenatal hypothyroid pups on postnatal days of 1, 7, 15, and 23 (n=24). After 24-48 hours, the 50 mm thick brainstem sections containing trigeminal motor nucleus were processed for TMB histochemical procedure and morphological characteristics of HRP labeled motoneurons and their HRP labeling intensity was evaluated. Student's t-test and two-way analysis of variance (ANOVA) were used for statistical analysis. Results: No significant morphological differences were observed at the end of first week of life. On day 15, hypothyroid labeled masseteric motoneurons consisted of 70% small and 30% medium neurons versus 40% and 60% in normal pups respectively (p<0.05). At the time of weaning, the number of large motoneurons dropped to 30% of normal value (p<0.001) with few, short, and disoriented dendrites. Conclusion: The alteration in particular patterns of masseteric motoneuron morphology and a severe delay in size transition could affect the development and plasticity of oral motor behavior under congenital hypothyroidism.

Introduction: The purpose of this study was to evaluate the effect of b-mercaptoethanol on resumption of meiosis, in vitro maturation of immature mouse oocytes and resulting embryo development with and without BSO (DL-Buthionine sulfoximine). Material and Methods: Germinal vasicle (GV) were recovered from 6-8 weeks old NMRI ovaries and cultured in maturation medium in MEM a supplemented with 7.5IU/ml hCG, 100mIU/ml rhFSH, 5% FCS (control group) and adding 100mm  b-mercaptoethanol (group 1) or with 5mM BSO + 100mm b-mercaptoethanol (group 2) for 24h. The matured oocytes then were fertilized and cultured for 5 days. Fertilization and development were accomplished in T6 medium. Results: The percentage of GV oocytes reaching to metaphase I (or undergo GVBD) were 78.5%, 85%, 86% in control group, group 1 and group 2 respectively, that no significant difference was detected between groups. The proportion of oocytes that progressed to the metaphase II (MII) stage was minimum within 5mM BSO group (group 2) and maximum within b-mercaptoetanol group (group 1) with significant difference comparing with control and each other (P£0.05). The percentage of embryos reaching to morula stage within b-mercaptoetanol group was significantly higher than the control group (5% and 12.2% respectively). None of oocytes treated with BSO could pass the 8 cell stage. Conclusion: b-mercaptoetanol enhances IVM and improves embryo development. While adding BSO into the maturation medium even with b-mercaptoetanol decreases maturation and declines the embryo development.

Introduction: Transplantation of germ cells restores the male fertility. Nevertheless, a lot of questions remain incompletely resolved. The aim of this study was to evaluate in vitro colonization efficiency of germ cells and sperm production capacity of spermatogonial cells before and after culture by sperm number assay in epididymis of recipient mice. Materials and Methods: We developed a Sertoli cell feeder in a co-culture system with spermatogonial cells and the cells were co-cultured for 2 months. The cells were isolated from mouse neonates. Colony assay was performed during culture using light microscopy. The transplanted cells were traced using BrdU incorporation. Sperm parameters were assessed 2 months after transplantation. Results: Our findings showed that spermatogonial cells created colonies during culture. Transplantation of fresh spermatogonial cells at a concentration of 2×105 cells/ml did not show significant differences. However, after transplantation of 2×105 cells/ml cultured for 2 weeks, the number of epididymal sperms in recipients increased significantly in groups with more fresh cells. Conclusion: Epididymal sperm number in recipient mice can be increased by enrichment of type A spermatogonial cells using an in vitro co-culture system. Other important factors include the source of donor cells and the number of transplanted cells.

Introduction: Shigella is a facultative intracellular pathogen that uses the host actin cytoskeleton protein for intra- and intercellular spread. The aim of this study was to determine the distribution of icsA gene and IcsA expressed protein bands among Shigella flexneri strains isolated from 3 clinical centers in Tehran. Material and Methods: Two hundred and seventy five isolated Shigella flexneri strains were identified by standard microbiological and biochemical methods. DNA isolation was performed using sodium perchlorate method. Hot start-PCR was done with 2 pairs of primers and the products were separated through agarose gel (0.8%) in TAE buffer. DNA fragments were visualized by ethidium bromide staining under UV illumination. Whole membrane preparation was used to examine the protein profiles and identification of probable IcsA (120-kda) protein band by SDS-PAGE. Results: From 100 isolated Shigella flexneri strains, both bands of 1600 bp and 1709 bp were detected in 46 isolates (46%). A 120 kDa band which seems to be related to IcsA protein was detected in 46 isolates (46%). The protein bands varied between 30 and 150 kDa. Discussion: IcsA is both necessary and sufficient for actin assembly in Shigella flexneri. Since icsA gene and IcsA protein band were not found in all Shigella strains, it seems that not all strains have the same pathogenesis. On the other hand, since the demonstration of icsA gene by PCR in all Shigella strains (46%) corresponded to the presence of a 120 kDa protein band by SDS-PAGE (46%), it seems that both tests may confirm each other. However, the PCR may be more accurate than SDS-PAGE.

Introduction: The aim of this study was to evaluate the effect of human sperm MTT viability assay on outcome of intracytoplasmic sperm injection. MTT is a tetrazolium salt, routinely used for cell proliferation and cytotoxicity assays. Materials and Methods: 50μl of processed semen was treated with MTT solution, while the remaining used as the control. Meanwhile, 109 donated human oocytes (metaphase II) obtained from 12 patients were divided into two groups. Fifty five oocytes were injected using MTT positive sperms, while 54 oocytes were injected with sperms from the control group. Then the injected oocytes were cultured and observed at 18, 42, 66, 90, and 114 hours pos- ICSI. Finally, the fertilization and embryo development rates were compared in both groups. Results: No significant differences were observed between fertilization and embryo development rates in the MTT and control groups. Conclusion: In future studies after approving that the MTT has not cytotoxic or teratogenic effects, then sperm MTT viability assay might be useful for ICSI in patients with absolute or severe asthenospermia or in patients with highly deformed sperm tails.