[Compression of verification of neonatal mouse ovaries using two carriers: straw and cryolock]

Vitrification of ovarian tissue is an effective method for preservation of fertility. Many attempts have been made to improve cryopreservation conditions. The aim of this study was to compare the effects of two vitrification methods using two carrier systems; straw and cryolock, on the morphology of neonatal mouse ovarian tissue. In this experimental study we used NMRI 7-day-old mice. Their ovaries were excised and divided into non-vitrified, vitrified with straw, vitrified with cryolock and toxicity test groups. In vitrification group, the ovaries were dehydrated in a mixture of 40% ethylene glycol, 30% ficoll 70 and sucrose [EGFS40] for 5 min.Then they were put in a plastic straw or cryolock and were placed into liquid nitrogen and stored for one week. Whereas in the toxicity test group the ovaries were exposed to the cryoprotectant EGFS40 and warming solution procedure without using liquid nitrogen. Then we studied the morphology of ovarian follicles by means of light and electron microscopes. No significant differences were observed between the groups in regard to the percentages of primordialand primary follicles with normal morphology. But the percentages of normal preantral follicles in the vitrified group using straw were significantly lower than those of other groups. Ultrastructural studies showed signs of degeneration and nuclear shrinkage in follicular cells in some of the preantral follicles in vitrified group using straw. Vitrification of ovarian tissue using cryolock is more effective for preserveration of the normal morphology of ovarian tissue, compared to vitrification by using straw

Temporary and permanent level shifts in distortion product otoacoustic emissions following noise exposure in an animal model

Noise-induced hearing loss [NIHL] is one of the most common occupational illnesses. Most of the studies on NIHL were conducted at high noise levels that people are rarely exposed to but in industries. The function of the outer hair cells [OHCs] is impaired after exposure to industrial noise. Distortion product otoacoustic emissions [DPOAEs] are useful in examination of noise-induced level shifts. To assess the function of OHCs by DPOAE temporary and permanent level shifts [TLSdp and PLSdp] in rabbits exposed to white noise at realistic levels typically found in industrial settings over a broad range of frequencies. 12 albino rabbits were divided into two groups: the experimental group rabbits which were exposed to 95 dB SPL white noise at 500-8000 Hz for 8 hrs/day for 5 consecutive days, and the control group rabbits with no exposure to noise. The function of OHCs was examined by DPOAE level [Ldp] in different occasions. The study groups were compared for DPOAE temporary and permanent level shifts [TLSdp and PLSdp] to assess the effect of noise on OHCs function. Noise-induced DPOAE levels [Ldp] were decreased up to 20.65 dB [on day 8] and 18.93 dB [on day 11] at 5888.50 Hz [p=0.081]. TLSdp and PLSdp were significantly decreased up to 17.99 dB and 16.27 dB, respectively in the experimental group. The most and least Ldp were significantly different [p<0.05]; they occurred at 5888.50 and 588.00 Hz, respectively. There were significant differences between temporary and permanent threshold shift at various frequencies [p<0.05]. These differences were mainly related to 5888.50 Hz compared to other frequencies in each ear [p<0.05]. DPOAEs are an attractive tool for obtaining information about small temporary or permanent threshold shifts, even when the pure tone audiogram is normal

Activity of follicular fluid phosphatases and their correlation with levels of serum esteroidal hormones and gonadotropins

The aim of this study was to evaluate the correlation between serum levels of ovarian and gonadotropin hormones, age and number of follicles with follicular alkaline and acid phosphatase activity in infertile women under controlled ovarian hyperstimulation. After collection of follicular fluid and calculation of the number of follicles, the specific activity of alkaline [ALP] and acid phosphatase [ACP] was determined according to the total protein in 19 women at the time of puncture. Also at that time, the levels of progesterone, estradiol, and follicle stimulating hormone [FSH] and leuteinizing hormone [LH] of their sera were measured. The correlation of follicular ALP and ACP with each serum hormone levels, women age and number of follicles was calculated using non-parametric analysis. The ALP has a correlation with progesterone [P=0.01] levels but doesn't have any correlation with the other factors. However, the ACP activity has a correlation not only with follicular number but also with estradiol and progesterone levels [P=0.05]. Thus ACP activity is more affected by ovarian hormone than ALP and it can affect the ovarian microenvironment and oocyte development

comparitive study of mouse ovarian alkaline phosphatase activity in normal and pseudopregnancies

The purpose of this study was to determine alkaline phosphatase [ALP] activity of ovary after ovarian induction using pregnant mare serum gonadotropin [PMSG] and human chorionic gonadotropin [hCG] during implantation periods. A total of 240 female NMRI mice aged 6-10 weeks were selected and divided into control and hyperstimulated. These mice were rendered normal or pseudopregnant. Five mice per each group were sacrificed by cervical dislocation at the first to sixth day of natural or pseudopregnancy. For biochemical assay the samples were obtained from the ovary, then were hemogenated using Tris HCl buffered salin [pH=8.3] and centrifuged with 14000 g. The activity of enzyme was determined using paranitrophenyle phosphate as substrate. Then specific activity of enzyme was calculated according to the total protein. The data were evaluated with Mann whitney test. For histochemistry the samples were cryosectioned [5mm thickness] and the ALP activity was determined by azo-coupling technique using alphanaphtole phosphate as substrate. The pattern of ALP activity in the biochemical and hisotchemical study was the same in each group. The activity of the ovarian ALP was increased during early pregnancy in the control and hyperstimulated natural pregnant groups. There were significant differences between these groups in every days except on the first and fourth day of pregnancy [p<0.05]. The ovarian enzyme activity was increased in pseudopregnancy control until 4th day and in the pseudopregnant hyperstimulation groups until 2nd day of pseudopregnancy then it was decreased. The daily patterny of these alterations were significantly different [p<0.05] comparing the above-mentioned groups. ALP activity was increased in every day of pregnancy [p<0.05] in normal pregnant hyperstimulated group in comparison with the psuedopregnant hyperstimulated group. Thus ovarion hyprstimulation alters the ovarian ALP activity during early pregnancy. These alteration may be due to esteroidogenesis activity of ovarian cells. However more investigation with complementary technique is needed

[Effect of co-culturing of mice liver cells and embryonic carcinomatous stem cells on the rate of differentiation to hematopoietic cells]

Considering the importance of co-culture in differentiation of embryonic stem cells, the aim of this study was evaluation of the effect of co-culturing fetal liver stroma cells with P19 cells on the line of differentiation. For this purpose, P19 cells were cultured directly in semisolid medium. These cells proliferated and primarily differentiated to colonies know as embryoid bodies [EBs] after 8-12 days. The Ebs cells were trypsinized and dissociated to single or double cells. Then these cells were co-cultured on the mouse fetal liver feeder layer in the absence of exogenous factors. After 14-18 days, the colonies were studied morphologically by benzidine and giemsa staining and also counted under invert microscope. The percentages of benzidine positive [or erythroid] and negative colonies were 94% and 6% respectively and also the cells of colonies were studied by Giemsa staining. Results showed that they were myeloid or lymphoid type cells. Thus, the results show that in the presence of mouse fetal liver feeder layer, the number of erythroid colonies was increased. Therefore, this technique may be effective for differentiation of stem cells from different sources into hematopoietic cells and can be used in future for human cell therapy

Quality effect of co-culturing of yolk sac cells with M2-10B4 line on their differentiation in the presence and absence of erythropoietin

The embryonic yolk sac cells have two unique characteristics: high proliferative capacity and lack of MHC associated antigen. According to the positive effects of co-culturing system on the differentiation of stem cells, in this study we evaluate the effect of M2-10B4 stromal cell line and erythropoietin [EPO] on the differentiation of embryonic yolk sac cells to erythroid cells. The yolk sacs were dissected from 10-day mice and their cells were separated using syring needles and enzyme digestions [0.1% collagenase/20 fetal calf serum [FBS] or 0.25% trypsin -0.02% EDTA at 37°C]. The M2-10B4 stromal cells were cultured in the DMEM medium and mitotically inactivated using mitomycin C. These cells were co-cultured with yolk stem cells in the medium containing stem cell factor [50 ng/ml]; EPO [IU/ml] and the colony assay of these cells [5xl0[4] cells/ml] have been done in the presence of interleukin-3 [40 ng/ml], stem cell factor [50 ng/ml] and EPO [lU/ml] in semisolid medium. After 7 days, benzidine staining on the colonies was carried out and positive benzidine colonies were considered as erythroid colonies. The colony assay showed that in the presence of EPO the growth of erythroid colonies were better than the other group and they had more benzidine colonies and cells. By using the stromal feeder layer such as bone marrow stromal cells and erythropoietin the differentiation and proliferation of YSC was improved; however, further studies are needed

[Optimization of Dendritic cells purification from mouse spleen a recommended method for purification of Dendritic cells from reproductive organs]

Dendritic cells [DC] are the principal antigen-presenting cells [APC] responsible for induction of primary immune responses by T lymphocytes. Although DCs are present in most lymphoid tissues, they occur in very low frequency accounting for 0.5% or less of nucleated cells in peripheral lymphoid organs. In the present study, we report the purification of DCs from mouse spleen with high yield and purity using a three-step purification technique including: collagenase digestion of tissue, selection of low-density cells using Optiprep density gradient medium and plastic adherence. By using techniques outlined above, we obtained 5-7x10[7] DC/spleen with purity >/= of 97%. Such large numbers of purified DCs enables us to further document their different characteristics including morphology, immunophenotype and to evaluation of their role in immune system. Finally, since DCs have been reported to be present in all reproductive organs, we suggest that this protocol be used for isolation and purification of DCs from those organs for further in vitro studies

effect of progesterone after ovarian hyperstimulation on the ultrastructure of mouse endometrium during preimplantation period

The aim of this study was to investigate the morphological and ultrastructural changes of mouse endometrium by daily injection of progesterone, after ovarian hyperstimulation during preimplantation period. Material and NMRI mice, 6-10 weeks old, were initially hyperstimulated using hMG and hCG injection and then daily progesterone injection was performed subcutaneously [1 mg/mouse], thereafter they were mated artificially. 3.5 days after hyperstimulation, the animals were killed by cervical dislocation and the samples were obtained from 13 middle part of uterine horns. Specimens were also obtained from naturally and artifically impregnated control groups. The samples were devided and processed for light [H and E, PAS] and electron microscopic studies. Our results indicated that, daily injection of progesterone after hyperstimulation decreased the height of the epithelium. In the experimental group several fat droplets were seen in the basal part of epithelium. Also intercellular spaces were decreased decidualization was not seen. Results show that daily injection of progesterone after hyperstimulation altered the ultrastructure of endometrial epithelium which resulted in inhibition of decidualization reaction and can affect the uterine receptivity during implantation