Cell Journal (Yakhteh)
Year:2003 | Volume:5 | Issue:17|Papers Count:8

Introduction: It is now generally accepted that Vero cells provide beneficial effects on improving embryo quality in many animal species. The aim of this research was to determine the effect of Vero cell co-culture on the development of vitrified 8-cell mouse embryos. Material and Methods: Eight- cell mouse embryos were provided from superovulated female NMRI mice and divided into treatment case and control groups. In group oftreatment 8-cell mouse embryos were vitrified by 40% ethylene glycol and the thawing was done in 0.5µ sucrose solution. Vitrified embryos were transfered to MEMα medium or MEMα +Vero cells. The control group which consisted of non-vitrified embryos, were also transfered to the same media. The comparison of developmental rate of both groups were evaluated in MEMα  and MEMα +Vero media. Results: Culture of vitrified embryos in  MEMα  medium indicated that there is an increase in the percentage of hatching, while culture of frozen embyros in MEMα +Vero co-culture medium had no effect on the developmental rate of embryos. Conclusion: Eight-cell stage of mouse embryo is  suitable for vitrification and Vero cell co-culture is not necessary for improving the development of embryos.

Introduction: To improve the development of embryo in vitro several attempts have been made, including the application of leukemia inhibitory factor (LIF). This factor plays an important role in embryo development and has been shown to enhance in vitro development rate in sheep and mice. There is controversy regarding the effect of LIF on the different stages of embryo development. The aim of this study was to investigate the effect of rhLIF on development of 2-cell mouse embryo. Material and Methods: Female ICR mice aged 8 to 10 weeks received intraperitoneal injection of 7.5 IU of pregnant mare serum gonadotropin (PMSG) for superovulation. This was followed by intraperitoneal administration of 7.5 IU of Human Chorionic Gonadotropin (HCG) 48 hours later. The mice were then mated with mature ICR male mice and were checked for vaginal plaque 13-14 hours later. Mice were killed 46-48 hours after HCG injection by cervical dislocation. After removing oviducts two cell embryos were collected by flushing technique and cultured in KSOM medium with different dosage of LIF (1500 IU/ml, 1000IU/ml , 500IU/ml). Also some embryos were cultured without LIF as control. All embryos were cultured in an incubator at 37ْc with %5 co2 for 120 hours. Embryo development was evaluated daily by obserunig the marphology using an invert microscope. Development of the embryos were recorded 24,48, 72, 96 and 120 hours later. Results: The rate of embryos reaching four cell, 8 cell and 6-9 cell stages were the same in all groups. But in further developmental stages such a compacted morula, blastocyst and hatching blastocyst statistically significant differences were seen between case and control groups. Conclusion: The recombinant human leukemia inhibitory factor (rhLIF) could be effective to improve in vitro development of mouse embryo.

Introduction: The frequency of sperm PCC indiction in normal and infertile men was investigated. Material and Methods: Zona – free Hamster oocyte were retrieved after super ovulation by PMSG and HCG infection. Following treatment with hyaloronidase, Zona was removed by trypsin digestion. Sperms were classified with sperms, and then transferred to fresh media containing colcemid. Slides were prepared using Tarkowskie’s standard air – drying technique. Oocytes were analyzed using  ×1000 microscope after staining in 5% Giemsa. Results: Results indicate that the frequency of sperm PCC is much higher in asthenosperm samples,compared to sperm from oligo or normal individuals and the difference is statistically significant (P<0.001). Moreover, intact sperm head was found in all samples, but the frequency of intact sperm head was higher in astheno and oligo sperm samples compared to normal. However the frequency of sperm head was not statistically different in oligo and astheno sperm samples. Conclusion: From the results it can be concluded that, formation of sperm PCC is a major cause of failed fertilization in individuals with sperm abnormalities. PCC may occur due to chromatin abnormalities, improper DNA packing, chromosomal abnormalities and penetration delay of sperm. Therefore fertilization failure in asthenosperm infertile men may be primarily due to sperm PCC formation. Also this may be the etiology of some case of idiopathic infertility.

Introduction: HeLa cell is a widely studied cell line derived from human cerzix adenocarcinoma. The cells are epithelial - like in morphology and susceptible to polio viruses and adenovirus type 5. HeLa cells are usually as monolayer for isolation and propagation of some human viruses, as well as for the expression of recombinant proteins, including hepatitis B surface antigen. The aim of this study was to multiply the HeLa cells as suspension culture, and evaluate their susceptibility polio virus types 1, 2, 3 and adenovirus type 5. Material and Methods: HeLa cells were grown in minimum essential medium supplemented with 10% heat-inactivated calf serum. HEPES buffer was used to obtain the best environmental condition for cell growth. To omit the calcium carboxy, methyl cellulose (CMC) and ethylene diamin tetraacetic (EDTA) were applied. After being adapted to suspension cell culture, cells were tested for their ability to host poliovirus types 1, 2 and 3 as well as adenovirus type 5,. Results of virus multiplication in both suspension and monolayer culture of the HeLa cells were compared. Results: It was shown that there is close relation between the number of initiatins seed cells with doubling time of the cells. The best results were obtained when the number of initial seeds were 3x105 per ml for culturing in 96 well plates, and 4x105 for suspension culture . To see the effect of different culture conditions on cell growth and doubling time it was shown that CMC inhibited the cell growth while, reduced calcium concentration, HEPES buffer, and modified culture medium resulted in doubling of cells in about 48 hours. Both monolayer and suspension cell culture were equally susceptible to the viruses. Conclusion: This study indicated that HeLa cells could be adapted to be culture as suspension, and different conditions either advantageous or disadvantageous to the cell growth and multiplication. The suspended cells maintained their susceptiblity to the test viruses. Using suspension culture of HeLa cells gives a great opportunity to use a much less volume and medium to obtain the same amount and titer of the desired virus.

Introduction: In this research the effects of ultrasound therapy with 1MHz frequency were studied on bone regeneration by descriptive histologic and mean descriptive rank method. Material and Methods: A total of 40 white male Dutch-Poland rabbits underwent dental hole partial osteotomy (DHPO) of the tibia bone in both Lower extremities under general anesthesia and sterile conditions. The right lower extremity of each rabbit was considered as experimental limb and at the same time the left lower extremity was considered as control. Rabbits were randomly divided into four weeks for assessment (10 rabbits for each week). All rabbits were treated by 1MHz frequency ultrasound, pulse rate 1:3, 0.1 W/Cm2 and 5 minutes per day, from first day after surgery. After a predetermined period (7th., 14th., 21st. and 28th days after surgery), rabbits were killed by ether and histological specimens were obtained and placed in formal-saline. The specimens were embedded by parafin subsequently. In the later stage, they were sectioned with a 7َµm thickness and were stained by Heamatoxyline & Eosine and Massons Trichrome methods. Descriptive rank (grades) and qualitative methods were used for histological study. Results: Findings of this research revealed that Ultrasound therapy with 1MHz frequency caused a significant increase in mean descriptive rank of the experimental extremity, 28 days after surgery. Conclusion: Ultrasound therapy with 1MHz frequency can accelerate bone regeneration in rabbits after partial osteotomy.

Introduction: To investigate the role of borrelia infections in pathogenesis of multiple sclerosis (MS) the presence of antibody against Borrelia persica (the most common species of borrelia in Iran) in the serum and CSF of MS patients was studied. Material and Methods: The presence of antibodies against Borrelia persica antigens was studied in the serum and CSF of 50 MS patients and 30 age and sex matched normal controls by immunoblotting. The two most important surface lipoproteins of Borrelia burgdorferi (OspA & OspD), which were produced by recombinant technology, were also included in the study. Results: The obtained results showed that 33 of 50 patients who were tested and some of normal controls, had antibodies in their serum against Borrelia protein bands. Fisher’s exact test indicated a significant difference between MS patients and normal controls regarding 43 KDa protein band, which became positive in 7 MS patient but none of our controls. We could not detect positive reaction with Borrelia surface lipoproteins (OspA & OspD) in serum and CSF of any of our patients or normal controls. Conclusion: Considering our results and a lot of similarities between pathogenesis and clinical manifestation of MS and spirochetal lyme disease there are some evidences for spirochetal basis of MS but its confirmation needs further investigations.

Introduction: Due to the relation between structure and function of renal glomeruli, the study of glomeruli is very important to the understanding of initiation and progression of diabetic glomerulopathy. It seems that glomeruli disappear during the course of diabetic nephropathy without leaving recognizable corpuscles. This study was aimed to show diabetes mellitus effects on changes in renal glomeruli number in male diabetic rats. Material and Methods: Forty-eight male Wistar rats were randomly divided into eight groups. Diabetes was induced by an intravenous injection of streptozotocin 90 mg/kg in 1.0 ml of acetate buffer in four groups and the other four groups were taken as control. The animals were anesthetized and dissected three months, six months, nine months and twelve months after administration of streptozotocin respectively. Then fixation, sampling, processing, embedding in paraplast, sectioning and staining were performed followed by stereological glomerular counting by means of an unbiased new method of stereology, Physical Disector/Fractionator. Data were analysed by Mann-Whitney U Test. Results: The counting of glomeruli in the group of cases in comparison with their controls showed that three, six and nine months streptozotocin induced diabetes had no statistically significant effect on glomerular number (P> 0.05) while in long terms, twelve-months, reduction of glomeruli was significant (P= 0.030). Conclusion: Induction of diabetes mellitus by Streptozotocin in a period less than 9 months has no effect on renal glomeruli number, whilst during 12 months diabetes mellitus may cause a significant reduction in glomerular number.

Introduction: The acrosome is a sac shaped organelle contaning lysosomal enzyme that is located on the anterior of sperm head. During the acrosome reaction its content is released in the vicinity of the egg. The acrosome reacted sperm then penetrates through the egg barriers. There are several methods for evaluating the acrosomal status; some of those are complex, expensive and/or non-accurate methods. The aim of this study was comparison between three of these methods, quinacrine fluorescent and current histochemical double & triple staining methods. Material and Methods: Ten norm al ejaculate semen samples were selected according to the WHO, 1999 criteria. The motile sperm were liberated from seminal plasma using percoll preparation. The acrosome reaction was then evaluated before and after capacitation, following induction by calcium ionophore for 1 to 2 hrs using three different quinacrine, double and triple staining methods. Results: There was no significant difference between the three different staining methods (P> 0.05), while after capacitation and induction using calcium ionophore the acrosome reaction increased significantly (P< 0.01). In all experiments, the percentage of acrosome reaction detected in  quinacrine was significantly higher than the other methods (P< 0.01). There was a direct correlation among different methods (P< 0.001, r=7). Conclusion: The acrosome reaction occurred in sperm following capacitation and induction by calcium ionophore. The quinacrine method is a rapid and simple method for detecting the acrosomal status of human spermatozoa.