Cell Journal (Yakhteh)
Year:2004 | Volume:6 | Issue:23|Papers Count:12

Introduction: Pulsatile injection of GnRH in hypothalamic amenorrhea is known to result in ovulation. The aim of this research was to study the effect of non pulsatile GnRH agonist administration on the ovaries of immature rats. Material and Methods: In this research the immature rats was divided to 7,17 and 27 daysand each were subdivided to four subgroups. Subcutaneous injection every 12 hours for 3 consecutivedays was carried out, and mature rats were used as a second control group. After spinal transetion, the ovaries were excised and processed for histolgical study using 5µm serial section. Results: The results were as follows, 1- In the 7 day rats high dose injection significantly decreasedthe number of vesicular follicles compared to control group. No significant difference was observed in the other groups with the same dosage. 2- In the 17 day rats high dose injection significantly increased the number of primary follicles but decreased the primary and secondary vesicular follicles. In contrast,the middle dose in comparison to high dose administration resulted in significant increase in the number of primary and secondary vesicular follicles, but not the primary follicles. Low doseadministration in the same group, showed no significant change compared with the control group.3- In the 27 day group the high dose did not result in any change in the number of primary follicles andvesicular follicles, but decreased the number of secondary vesicular and graffian follicles, and the number of corpus luteums obviously increased, similar to middle dose administration. This was incontrast to high dose which the number of secondary vesicular and graffian follicles increased and the number of corpus luteum, as compared to former group also significantly increased. 4- Comparisonbetween immature 27 day and 60 day mature rats showed decrease in the primary follicles andvesicular follicles while increase in the number of secondary and graffian follicles and corpus luteum,but the middle dose administration compared to mature rats showed almost a similar pattern.Conclusion: Finally it can be concluded that the non pulsatile administration can result in maturation of follicle and may be similar to the mature group. High dose stimulates the maturation of primary follicles but suppresses maturation of graffian follicles and low dose administration dose not have any effect on maturation of follicles.

Introduction: Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. These cells are appropriate for creation of animal models of human genetic diseases, the study of gene functions in vivo and differentiation into specific types as potential therapeutic agents for several diseases. We describe here production of transgenic chimeric mouse blastocysts by aggregation of tetraploid embryos and embryonic stem cells expressing histone variant (H2A.Z) fused with EGFP.Materials and Methods: To optimize electrofusion, two-cell mouse (NMRI) embryos were fused together by different voltages and durations. In optimumstate, tetraploid embryos were aggregated by genetically manipulated mouse embryonic stem cells (Royan B1) derived from C57BL/6. Electroporation used for insertion of vector included H2A.Z-EGFP. The aggregated embryos were cultured to blastocycts in vitro.Results: Mouse blastomere fusion and tetraploid blastocyst development occurred by 100v and 25µs. 82% of tetraploid embryos reached blastocyst and hatching blastocyst stages. Also 75% of embryos aggregated together and 42% of them expressed H2A.Z-EGFP. Conclusion: It is possible to use genetic manipulated ES cells and tetraploid embryos to study gene function.

Introduction: The aim of this study was to evaluate the effects of high frequency electromagnetic radiation (27.12 MHz) on ultrastructure of bone inthe rat embryos.Material and Methods: Three experimental groups were formed: Experimental group 1 was exposed continuously during the pregnancy periodfrom day 0 through day 6, Experimental group 2 from day 7 through day 13 and Experimental group 3 from day 14 through day 20 of gestation. These experimental groups were exposed to 10 W/cm2 at 27.12 MHz radiation for 15minutes twice daily for 7 days. Total exposure time for each rat was 210 minutes. Three Sham exposure groups were also exposed to 0 W/cm2 for 15minutes twice daily for 7 days. The Control group had no exposure.Ultrastructure of mid-diaphysis of tibia from twenty one 21 day old rat embryoswere studied by transmission electron microscope.Results: The results showed that colonic temperature of experimental ratswere increased. Ultrastructure of mid-diaphysial portion of tibia in the experimental rats revealed cytoplasmic vacuolization and shrinkage,degeneration of some organelles, nuclear condensation in the osteoblasts and decreased of amount of bony trabecula in the extracellular matrix. These degenerative changes were increased in experimental groups especially inexperimental group 2 that received radiation during 7 through 13 days ofgestation.Conclusion: The results showed that high frequency radiation caused structural changes in exposed osteoblasts and it retarded bone formation in the rat embryos

Introduction: Activity of the paragigantocellularis (PGi) nucleus located in the rostral ventrolateral medulla is the mediator of dependence, tolerance and withdrawal syndrome. The most important systems which change their activity during morphine consumption include glutaminergic, opioidergicand purinergic (especially adenosine) receptors in the PGi nucleus. However, it has been shown that the main adaptive changes in the PGi nucleus concerns the adenosine system and especially A1 adenosine receptors.The aim of this study was to investigate whether the different patterns of activity of PGi neurons in tolerance or withdrawal periods could be attributed to adaptive changes in Aladenosine receptors after chronic morphine consumption.Material and Methods: Animals were allotted randomly to control and dependent groups (in each group n=36). Rats in the dependent group received morphine sulfate in their drinking water for 21 days. The animals were prepared for recording of PGi neuronal firing by micromanipulation of a glassmicropipette into the PGi nucleus near a unit. The unit activity of each neuron was conducted to anextracellular preamplifier and then to an oscilloscope. A window discriminator isolated neuronal firingrate from oscilloscope background activity and the data were recorded and analysed by a PSTH computer program. A1 adenosine receptor agonist and antagonist were administered after a stable recording period (30 min) and the fluctuation of firing rates (spikes/s) was considered as the drug effect.Results: The spontaneous activity of PGi neurons was significantly decreased by microinjection of the A1 adenosine receptor selective agonist, cyclohexyladenosine (200 µM, Lp), into the PGi nucleus in both control and morphine-dependent rats, but the amount of decrement in morphine-dependent rats(52.5±3.4%) was higher than (P<0.05) that in control rats (35.2±3.1 %).There was also a significant enhancement of spontaneous activity of PGi neurons 10-18 min after the injection of A1 receptor selective antagonist, 8-phenyltheophilline (10 mg/kg; i.p.), in both control and morphine-dependent rats (P<0.05). However, the effect of the antagonist in morphine-dependent rats(39.3±2.5%) was more marked than in control animals (27.2±2.2%). In complementary experiment scyclohexyl adenosine (CHA) was administered in caffeine (50 mg/kg, i.p) pretreated rats. Results show that CHA could not affected PGi neuronal firing rate significantly in both control and dependent rats in this condition.Conclusion: The data suggest that there was an increase in the sensitivity of A1 adenosine receptors in morphine-dependent rats that may be associated with the PGi neuronal activity patternsduring tolerance and withdrawal syndrome.

Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major worldwide health problem. The only TB vaccine currently available is anattenuated strain of mycobacterium bovis termed Bacillus Calmette -Guerin (BCG).The efficacy of BCG remains controversial. Mycobacterium secretory proteins are generally considered important antigens for protection against TB. A major protein component of mycobacterial culture filtrate is Antigen 85 complex which is apromising potential vaccine candidate.Material and Methods: Antigen 85 complex was purified from Mycobacterium bovis (BCG) culture filtrate by sequential chromatography on phenyl Sepharose 4B, DEAE-Sephacel and Superdex G75. Purification was confirmed by SDS-PAGE and immunoblotting with a specific monoclonal antibody. The in vitro ability of Ag 85 complex to stimulate cell proliferation was compared with that of Purified Protein Derivative (PPD) and the polyclonal T cell mitogen PHA in a whole blood assay in which the target cells were derived from 25 healthy PPD-positive and 25 healthy PPD-negative subjects.Results: Antigen 85 complex was found to have a molecular weight of 30-32 KDa by SDS-polyacrylamide gel electrophoresis and reacted strongly by immunoblotting with the monoclonal antibody specified against Ag 85. The responses to Ag 85 and PPD were significantly higher in cells of PPD- positive than in cells of PPD-negative donors. Eighty eight percent (22/25) of the PPD- positive cells responded to Ag 85 whereas only 16% (4/25) of the PPD-negative cells responded.Conclusion: The cell proliferation response to Ag 85 complex is significantly different between cells of skin-test positive and skin- test negative subjects and may suggest an immuno-protective role for Ag 85 complex against M. tuberculosis infection.

Introduction: The aim of this investigation was to study the developmental potential of human fragmented embryos to blastocyst stage and to evaluate thedeveloped blastocysts in the term of size, cellularity, the type of inner cell mass(ICM), trophoetoderm (TE) and the number of dead cells.Material and Methods: Four to eight cell fragmented human embryos wereobtained from the embryology lab of Royan Institute. Embryos were scoredaccording to the degree of fragmentation, using invert microscope, into four groups(group I: embryos with least fragmentation, group II: embryos with small fragmentamong blastomers, group Ill: embryos with large fragment approximately in the sizeof blastomers and group IV: embryos with necrotic fragments). Each group wascultured in rS2 medium and their development was recorded to the day six (embryoswere cultured in rS1 for first 3 days).In this study the size and the quality of developed blastocyst and number ofblastomer in the ICM and TE were evaluated .The number of dead cells in eachblastocyst (apoptotic or necrotic) were counted using TUNEL staining and theresults were analyzed statistically by X2 and ANOVA.Results: High percentage of embryos from group IV had developmental block at 2- 4 cell stage. In addition, a high percentage of embryos from all groups stoppedtheir development at 8-cell to morolla stage. The rate of blastocyst formation ofembryos from group I and II was higher than group III and IV. The results showedthat size of embryos from group IV was statistically lower than groups I and II.Blastocyst quality (the number of blastomers in ICM) of embryos from group I and II was better than group III and IV. Differential staining results suggested that the ratioof ICM to TE in embryos from each group is not statistically different compared toother groups. But the mean total number of blastomer and the mean number of ICMand TE of embryos from groups I and II was higher than the other two groups. The results of TUNEL staining showed that embryos with high number and size offragmentation at 4-cell stage had a high number of apoptotic and necrotic cell.Conclusion: Embryos with high fragmentation have reduced developmental potential, blastocyst size, low quality blastocyst, low quality ICM and TE, and lownumber of total cell number. In addition the number of dead cell was higher.

Introduction: Clostridium difficile is the etiologic agent of several infections such as pseudomembranous colitis in human. These diseases are frequently associated with antibiotic useage but many infections with this organisms are seen without antibiotic administration. In addition it is believed that Clostridium difficile is one of the most important bacteria involved in acquired hospital infections. Therefore it is important to consider physical and chemical factors affecting growth and toxin production of Clostridium difficile for control purposes. The aim of this study was to determine different dosages of two kinds of electromagnetic waves: Short- wave and macro- wave on Clostridium difficile growth and toxin production.Material and Methods: In this study, cultures of two strains of Clostndium difficile (one of them toxigenic and the other nontoxigenic) were exposed to different doses of short-wave and macro-wave radiation for different time periods. The cultures were then incubated for 4 days to study the effect of irradiation on growth and toxin production of the bacteria. The rate of bacterial growth was measured by colony count. Toxin production and toxicity assays were performed by cell culture and inoculation of rabbit ilealligated loops.Results: Macro-wave irradiation had no effect on bacterial growth but inhibited toxin production and induced sporolation of organisms after 10 hours ofincubation. The effect of 30 - 60 minute short- wave irradiation was increased toxin production and bacterial growth. Furthermore short- wave irradation of the nontoxigenic Clostridium difficile strain changed it into a toxigenic form. Conclusion: The results of this study showed that macro- wave irradiation inhibited toxin production but induced sporolation of the organism, while short-wave irradiation increased toxigenic potency of the toxigenic strain and inducedtoxin production by the nontoxigenic strain.

Introduction: Extracellular matrix (ECM) components significantly influence the growth characteristics of cell, development of spontaneous contractile activity, andmorphologic differentiation. The present study evaluates the effect of ECM ongenotypic and physiologic characteristics of cardiomyocytes derived from embryonic stem (ES) cells.Material and Methods: Embryoid bodies derived from mouse ES cells line Royan B1 (C57BL/6) were cultured on commercial ECM (matrigel), a cardiac fibroblast-derived extracellular matrix (cardiogel), and control media (without ECM)for 21 days. The growth characteristics of cardiomyocytes were assessed by immunocytochemistery, RT-PCR, and chronotropic drugs.Results: The beating frequency of cardiomyocytes cultured on cardiogel increasedin comparison with cardiomyocytes cultured on matrigel and/or control during days 7-9(p<0.05) The spontaneously beating cells expressed markers characteristic ofcardiomyocytes including α-actinin, desmin, tropnin-I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin 43, cardiac α-MHC, cardiacβ-MHC, myosin light chain isoform-2V, and atrial natriuretic factor. In addition,spontaneousness of cardiomyocytes on both ECMs was enhanced by treatment of cells with isopernaline, while reduced more on matrigel and carbacol when compared with control and cardiogel. However, the change in beating was similar in all groups upon treatment with phenylephrine, propranolol and for Bay-K.Conclusions: We conclude that the ECM influences ES-derived cardiomyocytes, in terms of chronotropic characteristics.