Cell Journal (Yakhteh)
Volume:1 Issue: 3, 1999

The Mouse embryos can successfully be vitrified using ethylene glycol as cryoprotectant, however their development differs significantly from the non-vitrified embryos. The ability of their development can be improved when they co-culture with somatic cells. In the present study, the effects of co-culturing with Vero cells on the development of vitrified two cell mouse embryos were studied. Two cell embryos were flushed from the excised uteri of superovulated mice. Morphologically normal embryos were vitrified using 10% ethylene glycol solution (EFS10), and thawed rapidly using 0.5 M sucrose solution. The survived embryos were divided to two grops: one group cultured on RPMI and the another one co-cultured with Vero cells. Control embryos was considered for each experimental groups. The survival rate was 83%. The developmental rate of embryos which could reach to morula stage for the exp. group 1 and 2 were 52% and 69% respectively which the difference between the exp. groups was significant (P<0.001). Mean while 48% of the exp. group 1 could form blastocysts, accordingly the second group showed 60% and the difference was significant (P<0.001). The difference between the two exp. groups which could reach to hatching stage was not significant. The results suggest that the co-culture with Vero cells can improve the development of vitrified two cell mouse embryos

The incidence of chromosomal abnormalities was investigated in untransfered embryos reuslted from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) procedures. A total of 238 embryos of varying morphology between the pronucleated stage and 8-cells were analysed. The cytogenetic method of Dyban was used for chromosome preparation. Embryos were kept in a medium containing 0.2mg/mL colchicine for 4-6 hrs.Then cells were transferred into watch glass contaning hypotonic solution of 1.93% sodium citrate and 0.56% KCl for 25-60 min. Cells were fixed in three different fixatives sequentially, then they were stained in 10% Giemsa and examined with a light microscope at /Far/*/Lat/1000. The cytogenetic analysis of 68 embryos resulted from IVF and 70 embryos from ICSI indicate that a total of 89.86% of embryos were cytogenetically abnormal and only 10.14% were normal. In both groups, aneuploidy was found to be the most frequent observed abnormality (43.48%). In addition to that various types of aberrations such as triploidy (2.2%), haploidy (1.5%), mosaicism (29.7%) or structural abnormalities (13.04%) were found. There is a progressive loss of the embryos with chromosomal abnormalities during preimplantation development. The maternal age may have an effect on the chromosome segregation in oocytes causing aneuploid embryos. This study also shows that both healthy and morphologically unhealthy embryos can have either normal or abnormal chromosome complements; therefore the embryo morphology is not always indicative of chromosome status.

The incidence of chromosomal abnormalities was investigated in untransfered embryos reuslted from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) procedures. A total of 238 embryos of varying morphology between the pronucleated stage and 8-cells were analysed. The cytogenetic method of Dyban was used for chromosome preparation. Embryos were kept in a medium containing 0.2mg/mL colchicine for 4-6 hrs.Then cells were transferred into watch glass contaning hypotonic solution of 1.93% sodium citrate and 0.56% KCl for 25-60 min. Cells were fixed in three different fixatives sequentially, then they were stained in 10% Giemsa and examined with a light microscope at /Far/*/Lat/1000. The cytogenetic analysis of 68 embryos resulted from IVF and 70 embryos from ICSI indicate that a total of 89.86% of embryos were cytogenetically abnormal and only 10.14% were normal. In both groups, aneuploidy was found to be the most frequent observed abnormality (43.48%). In addition to that various types of aberrations such as triploidy (2.2%), haploidy (1.5%), mosaicism (29.7%) or structural abnormalities (13.04%) were found. There is a progressive loss of the embryos with chromosomal abnormalities during preimplantation development. The maternal age may have an effect on the chromosome segregation in oocytes causing aneuploid embryos. This study also shows that both healthy and morphologically unhealthy embryos can have either normal or abnormal chromosome complements; therefore the embryo morphology is not always indicative of chromosome status.

The purpose of this study was to investigate the effect of vitrification procedure on the survival, fertilization, development and ultrastructure of metaphase (MII) mouse oocyte. Female NMRI mice 6-10 weeks old were superovulated using IP injection of 10 IU hMG and 10 IU hCG. Ovulated oocyte were collected from the ampullary portion of the oviducts at 12-13 hours after hCG injection. Cumulus cell mass were separated by 0.1% hyaluronidase. The oocyte were vitrified by solution of PBI contained 30 % (W/V) ficol 70, 0.5 M sucrose, 10.7% (W/V) acetamid and 10% (V/V) ethylene glycol and stored in liquid nitrogen. The frozen oocyte were thawed by sucrose and PBI solutions for 5 min, then the frozen and non-frozen oocytes were inseminated with epididymal sperm. For electron microscopy studies, the fertilized and unfertilized oocytes were fixed with 2.5 % glutaraldehyde and 1% osmium tetroxide. After dehydration with ethanol followed with propylene oxide, the oocytes were embeded in Araldite. Ultrathin sections were stained with uranyl acetate and leade citrate, then examined under TEM. The survival rate of vitrified oocytes were 80%. The fertilization and developmental rates of the vitrified group were not significantly different from the control. An extended subzonal space was noticed in vitrified group, morover, the cytoskeleton was disorganized and some mitochondrai were swell. In the fertilized vitrified oocytes, these changes were reversible and the ultrastructural feuture of these group was the same as fertilized control oocyte. This vitrification method is a simple and usful procedure for cryopreservation of oocytes.

Tomographical maps from the serial sections for light microscopy were prepared and reconstructed by autocad software. In this study, the three-dimensional reconstruction method was used and the following results were obtained (see below). 1) The surface ectodermal epithelium was thick and invaginated. 2) The invagination of surface ectodemal epithelium was enveloped especially in ventral ridge. 3) The lens vesicle was formed by asymmetrical invagination of the surface ectodermal epithelium and the lens pit was closed. The superior region of the primary lens was formed first which then followed by the formation of the inferior region. It seems that the invagination of the central lens placode was formed together with evagination of the lateral regions of the lens placode.

L.Pneumophila has been known as a facultative intracellular bacterium which could proliferate within mammalian macrophages and a wide spectrum of free-living protozoa such as A. castellianii. Its pathgenicity for both host cell types could be mostly dependent on the intracellular living conditions. L. pneumophila-infected acanthamoeba were examined by a flour rescent microscopy after lysosomal labeling with acridine orange (AO) to determine whether intracellular survival of the bacteria is associated with the failure of legionella containing phagosomes to fuse with the primary lysosomes. L. pneumophila acrively inhibited phagosome-lysosome-fusion and appeared to preferentially divide within unfused phagocytic vesicles. Interestingly, most of the E.coli containing phagosomes can fuse with amoebae lysosomes. Free-living amoebae usually engulf different ubiquitous bacteria in the environment and ingest them as food particles. Legionaries, disease bacterium however, overcome nasty intra-amoebae phagosomes by inhibition of phago-lysosome. This simply explains how the efforts for legionella eradiation from the water sources have had unsuccessful results

The purpose of this study is to evaluated the effect of low power laser on the survival of a skin flap on the dorsum of the rats. Fifty male rats were divided into five groups. The surgery was done under general anesthesia where a full thickness skin flap (including panniculus carnosus) from the dorsum of the rats were randomly dissected. The flap size was 20 mm/100 mm. The day of the surgery was day zero. The rats in the first group were irradiated daily from day 1 to day 7 post-surgery. In the second group, the rats were irradiated daily from day zero to day 7 every 6 hours. The rats of the third group were irradiated 5 days, before surgery, then the treatment was continued every day until the day of the surgery. On day zero, the animals were irradiated every 6 hours, then the animals were treated once a day from day 1 to day 7. The animals of the fourth group were treated as in third group 3 except, the treatment on the day of the surgery was omitted. The fifth group was a negative control group where all the animal were not irradiated after surgery. The energy density of the laser was 0.2 j/cm2. Immediately after surgery, the size of the flap was recorded a on sterile transparent sheet and on day 7, the survived part of the flap was also recorded, then the animals were killed and the flaps were sampled, fixed, processed in paraffin and stained with Hematoxylin and Eosin. Also Toulidine blue was used to stain additional section. The number of the vessels, the number of points (in eye-peice graticule) on the vessels were counted and the number of the mast cells were also counted. The data were analyzed by the analysis of the variance. The size of the flaps at day zero were not significantly difference from the controls. The size of the flap and the point which were counted on the vessels on day 7 in the third group were significantly different from other groups. The low power helium-neon laser radiation can produce vasodilation of blood vessels which may result in an increase in the flap survival.

Electrophysiologycal histological methods were for studing the anatomical distribution of group I and II muscle afferent in the lumbosacral segments of wistar rat spinal cord. Quadriceps, Deep proneal, Hamestring, Gastrocnemius-Soleus, Tibialis posterior, Plantaris, Flexor digitorum longus and popliteus muscle nerves were dissected and mounted on surface electodes. Using graded stimuli, the afferent volley and Cord Dorsum potentials (CDPs) were recorded throughout the lumbosacral segments of spinal cord. Several sets of electrophysiological and histological evidences from this study indicate that in the rat spinal cord, anatomical organization of group I and II muscle afferents from lower limb is similar in principal to that of the cat. Accordingly, it is highly likely that cat results could be extended to the rat. These observations raise the possibility that this aspect of spinal cord organization may be a fundamental feature of the mammalian spinal cord. This information will be of particular value for experiments involving motor control, gait and balance in mammalian.