Isolation and Enrichment of Mouse Female Germ Line Stem Cells

Objective: The existence of female germ-line stem cells (FGSCs) has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. Materials and Methods: In this experimental study, after digesting neonate ovary from C57BI/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS) and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH) and stage-specific embryonic antigen-1 (SSEA1) markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR) (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3), alkaline phosphatase (AP) activity test and imnnunocytochemistry. Results: Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 +/- 0.49% (Mean +/- SDV) positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl) whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. Conclusion: We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries

Comparing the viability and in vitro maturation of cumulus germinal vesicle break down (GVBD) oocyte complexes using two vitrification techniques in mice

Background: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimizing. Objective: The aim of this study was to improve the single step and step-wise vitrification effects on maturing mouse GVBD oocytes by ethylene glycol (EG) in conventional straws. Materials and Methods: Oocytes with compact cumulus cells were cultured for 3hr in TCM199 supplemented with 10% fetal bovine serum (FBS) in 5% CO2 in air. GVBD oocytes were randomly allocated into three groups. (1) Control (non-vitrified group), (2) exposed to single-step vitrification (contained of EG 20%+0.5M sucrose), (3) exposed to step-wise vitrification (2%, 5%, 10%, 20%EG +0.5M sucrose). In vitrification groups,oocytes were thawed and underwent additional 21 hr maturation. Viability of oocytes and maturation to MII stage were analyzed using inverted microscope and additionally by staining of propidium iodide and Hoechst 33342. Results: All non-vitrified oocytes were viable after 24 hr; however, viability of vitrified samples in single-step group was significantly lower than that of the step-wise and control Groups. Also, the maturation rate in the step-wise group was significantly higher (p < 0.05) compared to single-step. Conclusion: These results suggest that step-wise vitrification of GVBD oocytes as compared to single step vitrification was better in the rate of survival and in vitro maturation of oocytes

The Evaluation of Epiphyseal Plate Histological Changes in Osteopetrotic op/op Mice.

This study was designed for evaluation of epiphyseal plate histological changes of femur bones in osteopetrotic op/op mice.In this study 5 osteopetrotic op/op mice which were purchased from the commercial source were used.The animals were killed by overdose of chloroform and their femur bones were extracted. The bones were fixed in 10% formaldehyde and decalcified by HCl (0.6N), and routine histological processing were performed. The sections were stained by H&amp;E methods and studied by conventional light microscopy. The results showed that, proliferative zone (PZ) and especially hypertrophic zone (HZ) were much thickened. In the ossification zone, trabecular bones were irregular and atypical osteoblast cells were observed. The osteoclast cells were not attached to trabecular bones. The bone marrow cavity was restricted and bone marrow cells were poor and scattered. Findings of the present investigation are similar to those reported about epiphyseal plate in osteosclerotic (OC) mice in which epiphyseal plate especially hypertrophic zone was thickened and chondrocytes were not substituted for osteoblasts in calcified cartilage area. Also, osteoclast cells had been inactive or absent in OC mice. For prevention of other complication due to the epiphyseal plate changes in new borne, suitable and punctually treatment protocols such as prescription of Macrophage Colony Stimulating-Factor (MCS-F) could be useful

Effects of Different Doses of Hyaloronan on Human Sperm Motility, Vitality and Morphology

Important aspect of sperm function such as motility and capacitation appear to be mediated at least partially though hyaloronic acid (HA). Present study investigated effects of different doses of HA on sperm motility and vitality in human. Sperm was obtained from 20 male from IVF clinic in Imam Khomeini Hospital. Sperm motility and vitality in human semen was analyzed according to WHO criteria before and 4 hours after treatment with different doses of HA (0.750, 1000 and 1250 &amp;micro;g/ml). The results showed that in 1000 &amp;micro;g/ml the percent of stage 3 and 4 increased compare to control group. Percent of stage 1 and 2 decreased in group with 1000 &amp;micro;g/ml HA, there was an increase in the percentage of stage 3 and 4 and decrease in percentage of stage 1 and 2 compare to control. In the group treated with 1250 &amp;micro;g/ml stage 1 and 2 increased while stage 3 and 4 decreased. Vitality in all groups decreased except of the group treated with 1000 &amp;micro;g/ml HA. The group with 1250 &amp;micro;g/ml showed significantly decrease in vitality compare to fresh group (P &amp;lt; 0.05). The present study showed that the effects of HA on sperm motility and vitality is dose dependant and 1000 &amp;micro;g/ml HA had the effective role on sperm parameters

Comparison of differentiation potential of male mouse adipose tissue and bone marrow derived-mesenchymal stem cells into germ cells

Background: Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells (ESC), it’s necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. Objective: The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells (ADMSCs) with bone marrow derived stem cells (BMMSCs). Materials and Methods: To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers (expression of CD90 and CD44 and non-expression of CD45 and CD31) were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl (Deleted in azoospermia-like), Mvh (Mouse vasa homolog gene), Stra8 (Stimulated by retinoic acid) and Scp3 (Synaptonemal complex protein 3)] flowcytometry, imunoflorescence and real time PCR were used. Results: Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers (CD90, CD44) and absence of endothelial and blood cell markers (CD31, CD45) were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers (Mvh, Dazl, Stra8, and Scp3). Conclusion: It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs

Association of serum content of 25-hydroxy vitamin D with semen quality in normozoospermic and oligoasthenoteratozoospermic men

Background: Vitamin D has multifaceted function in human reproductive physiology. It has been revealed that vitamin D is involved in spermatogenesis, and semen quality can be linked to vitamin D status in men. Objective: Evaluating the correlation of 25-hydroxy vitamin D (25-OHD) levels in serum with basic and advanced semen parameters and essential determinants of spermatozoa function. Materials and Methods: Participants were categorized, based on semen parameters, into normozoospermic (NS) and oligoasthenoteratozoospermic (OAT) men. Serum level of 25-OHD was measured. Apoptotic status of spermatozoa, mitochondrial membrane potential and reactive oxygen species content of semen were assessed. Results: Difference of 25-OHD concentration in serum of NS men versus OAT ones did not meet significance threshold. DNA fragmentation, reactive oxygen species content of semen and mitochondrial membrane potential state revealed significant difference between NS and OAT subjects. There were no significant differences in basic and functional semen parameters when men were stratified based on serum 25-OHD level. Taking both 25-OHD and semen categories (NS and OAT) into consideration did not indicate any significant difference in studied parameters. Total motility of spermatozoa was positively correlated with serum concentration of 25-OHD in all studied subjects. In addition, normal morphology of spermatozoa in NS men revealed a positive and significant correlation with levels of 25-OHD in serum. Conclusion: Vitamin D may affect motility and morphology of spermatozoa. Lower content of serum vitamin D may affect fertility of men and should be considered in examination of men with abnormal spermogram

Comparing the viability and in vitro maturation of cumulus germinal vesicle break down (GVBD) oocyte complexes using two vitrification techniques in mice

Background: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimizing. Objective: The aim of this study was to improve the single step and step-wise vitrification effects on maturing mouse GVBD oocytes by ethylene glycol (EG) in conventional straws. Materials and Methods: Oocytes with compact cumulus cells were cultured for 3hr in TCM199 supplemented with 10% fetal bovine serum (FBS) in 5% CO2 in air. GVBD oocytes were randomly allocated into three groups. (1) Control (non-vitrified group), (2) exposed to single-step vitrification (contained of EG 20%+0.5M sucrose), (3) exposed to step-wise vitrification (2%, 5%, 10%, 20%EG +0.5M sucrose). In vitrification groups,oocytes were thawed and underwent additional 21 hr maturation. Viability of oocytes and maturation to MII stage were analyzed using inverted microscope and additionally by staining of propidium iodide and Hoechst 33342. Results: All non-vitrified oocytes were viable after 24 hr; however, viability of vitrified samples in single-step group was significantly lower than that of the step-wise and control Groups. Also, the maturation rate in the step-wise group was significantly higher (p < 0.05) compared to single-step. Conclusion: These results suggest that step-wise vitrification of GVBD oocytes as compared to single step vitrification was better in the rate of survival and in vitro maturation of oocytes