Human Ovarian Tissue Vitrification/Warming Has Minor Effect on the Expression of Apoptosis-Related Genes

Background: In this study, we evaluated the incidence of apoptosis at the ultrastructural levels and expression of some apoptosis-related genes in vitrified human ovarian tissue just after warming. Methods: Human ovarian tissue biopsies from 23 women after caesarean section were transported to the laboratory within 2 hours, and then they were cut into small pieces. Some pieces were vitrified and warmed and the other samples were considered as control. Apoptosis was assessed by a transmission electron microscope and also by molecular analysis of pro-apoptotic (Fas, FasL, Bax, p53, caspase8, and caspase3) and antiapoptotic (Bcl-2 and BIRC5) genem RNA levels using real-time RT-PCR before and after vitrification. Results: No sign of apoptosis was shown ultrastructurally in vitrified samples. The level of FasL, Bcl-2, Bax, p53, and caspase3 mRNA and Bax:Bcl-2 ratio were similar in non-vitrified and vitrified groups; however, the expression of Fas and caspase8 genes was higher and BIRC5 was lower in vitrified samples compared to non-vitrified group (P<0.05). Conclusion: The fine structure of human vitrified ovarian tissue was well preserved; moreover, vitrification was shown to affect the expression of some apoptosis-related genes. However, additional study is needed to confirm this observation.Key Words: Vitrification, Apoptosis, Gene expression, Ovary, Humans     

The Effect of Vitrification on Mouse Oocyte Apoptosis by Cryotop Method

Background : Oocyte cryopreservation is one of the most important topics in the field of assisted reproductive technology to preserve women fertility, but relationship between cryopreservation and apoptosis is still a matter of debate. The present study was aimed to investigate the effects of vitrification on apoptosis in mouse oocytes by Cryotop method. Method: A total of 200 germinal vesicle (GV) and 200 metaphase II (MII) oocytes were obtained from ovaries and fallopian tubes of NMRI mice, respectively and divided into control and experimental groups. Oocytes in experimental group were vitrified by Cryotop using vitrification medium and were kept in liquid nitrogen for one month. The survival rate of oocytes was evaluated after 2 hour incubation time. Then, the oocyte apoptosis was evaluated by TUNEL technique and compared with those in control group. The data was compared statistically using SPSS software and chi-square test. Results: The survival rates of vitrified GV (93%) and MII oocytes (88%) showed a significant decrease compared with the control group (P<0.05), but there was no significant difference in survival rate of both vitrified oocyte groups. The incidence of apoptosis in vitrified and control GV oocytes showed no significant difference (13% vs. 7%), but the rate of apoptosis in vitrified MII oocytes increased significantly not only in comparison with MII control group (25% vs. 5%) but also with vitrified GV oocytes (P<0.05). Conclusion: The results indicate that vitrification increases apoptosis in mouse MII oocytes and apoptosis may play a role in MII oocyte injury after vitrification. Key Words: Vitrification, Apoptosis, Oocytes     

Total Oxidative status of Mouse Vitrified Pre-Antral Follicles with Pre-Treatment of Alpha Lipoic Acid

Background: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA). Methods: Isolated pre-antral follicles (140–150 µm in diameter) were divided into vitrified–warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2'',7''-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively. Results: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA. Conclusion: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and non-vitrified samples through increasing follicular TAC level and decreasing ROS levels. Key Words: Vitrification, Pre-antral follicle, Alpha lipoic acid (ALA), Reactive oxygen species (ROS), Total antioxidant capacity (TAC)     

Detection of mistletoe infected trees using UAV high spatial resolution images

Journal of Plant Diseases and Protection - Remote detection of aerial parasitic plants in forests is imperative in precision forestry, as it can help to manage tree stands and to monitor forest...     

Characteristics of Human Endometrial Stem Cells in Tissue and Isolated Cultured Cells: An Immunohistochemical Aspect

Background:

The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage.

Methods:

Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, Sox2, and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105.

Results:

11.88±1.29% of human endometrial cells within tissue sections expressed CD146 marker vs. 28±2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma (85±2.4 and 89±3.2%) than basalis stroma (16±1.4 and 17±1.9%), respectively (P<0.05). Oct4 and Nanog-expressing cells comprise 1.43±0.08 and 0.54±0.01% of endometrial stromal cells in endometrial sections vs. 12±3.1% and 8±2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. Sox2 and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells.

Conclusion:

The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data.Key Words: Endometrium, Immunohistochemistry, Mesenchymal stem cells     

Evaluation of apoptosis in long-term culture of vitrified mouse whole ovaries

The purpose of the present study was to investigate the development of follicles and incidence of apoptosis in vitrified neonatal mouse ovaries cultured in vitro in the presence of leukemia inhibitory factor (LIF). The vitrified and non-vitrified ovaries of 1-week-old mouse were cultured in the presence or absence of LIF for 7 days. At the beginning and at the end of culture period in each ovary of all groups of study the mean area and the development of ovarian follicles were analyzed; moreover, the incidence of apoptosis was assessed by transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method, DNA laddering and caspase-3/7 activity technique. The hormonal assay was done on the conditioned media collected during culture period. The proportion of preantral follicles and the levels of hormones increased in all cultured groups and it was significantly higher in LIF treated groups than in their control (P < 0.001). The ultrastructural characteristics of cell death, DNA fragmentation and TUNEL positive signals were prominent in vitrified cultured ovaries. The level of caspase-3/7 activity was higher in vitrified cultured ovaries.     

Ovarian Stimulation by Exogenous Gonadotropin Decreases the Implantation Rate and Expression of Mouse Blastocysts Integrins

Background: Integrins are heterodimeric glycoprotein receptors that regulate the interaction of cells with extracellular matrix and may have a critical role in implantation. The aim of this study was to investigate the effect of ovulation induction on the expression of α4, αv, β1, and β3 integrins in mouse blastocyst at the time of implantation. Methods: The ovarian stimulated and non-stimulated pregnant mice were sacrificed on the morning of 5^th day of pregnancy. The blastocysts were collected, and the expression of αv, α4, β1, and β3 integrins was examined using real-time RT-PCR and immunocytochemical techniques, then their ovarian hormones were analyzed at the same time. The implantation sites in uterine horns of other pregnant mice in both groups were determined under a stereomicroscope on the 7^th day of pregnancy. Results: The results showed that the expression of αv, β1, and β3 integrins in both mRNA and protein levels was significantly lower in the ovarian stimulated group than the control group, and the maximum ratio of expression was belonged to β1 molecule (P>0.05). Conclusion: The implantation rate in superovulated mice was significantly lower than control mice. It was suggested that ovulation induction decreased the expression of αv, β1, and β3 integrins of mouse blastocysts. Key Words: Blastosyst, Integrins, Implantation     

The Effect of Vitrification and in vitro Culture on the Adenosine Triphosphate Content and Mitochondrial Distribution of Mouse Pre-Implantation Embryos

Background: The mitochondria are an important source of adenosine triphosphate (ATP) production in pre-implantation embryo. Therefore, the objective of this study was to investigate the effect of vitrification and in vitro culture of mouse embryos on their mitochondrial distribution and ATP content. Methods: The embryos at 2-PN, 4-cell and blastocyst stages were collected from the oviduct of stimulated pregnant mice and uterine horns. Then, the embryos were vitrified with the cryotop method using ethylene glycol and dimethylsulphoxide. After evaluating the survival rates of vitrified embryos, their development to hatching stages were assessed. The ATP content of collected in vivo and in vitro embryos at different stages was measured by luciferin-luciferase bioluminescence assay. The distribution of mitochondria was studied using Mito-tracker green staining under a fluorescent microscope. Results: The survival rates of vitrified embryos at 2-PN, 4-cell and early blastocyst stages were 84.3, 87.87 and 89.89%, respectively. The hatching rates in previous developmental stages in vitrified group were 57.44, 66.73 and 70.89% and in non-vitrified group were 66.32, 73.25 and 75.89%, respectively (P>0.05). The ATP content of in vivo or in vitro collected embryos was not significantly different in both vitrified and non-vitrified groups (P>0.05). Mitochondrial distribution of vitrified and non-vitrified 2-PN embryos was similar, but some clampings or large aggregation of mitochondria within the vitrified 4-cell embryos was prominent. Conclusions: Vitrification method did not affect the mouse embryo ATP content. Also, the cellular stress was not induced by this procedure and the safety of vitrification was shown.Key Words: Mitochondria, Vitrification, Adenosine triphosphate (ATP)     

Effect of Satureja khuzestanica essential oil on male rat fertility

This study was undertaken to study the effect of Satureja khuzestanica essential oil (SKEO) in male rat fertility. SKEO was administered orally at doses of 75, 150, and 225 mg/kg/day for 45 days through drinking water. Treated and control rats were mated with female on day 45 of treatment. SKEO significantly improved all the parameters evaluated such as potency, fecundity, fertility index, and litter size. Moreover, concentrations of FSH and testosterone were significantly increased in SKEO-treated groups. Also the weights of testes, seminal vesicles, and ventral prostate weights were increased by SKEO (225 mg/kg). Histopathological analysis showed that in male rats treated with SKEO (150, 225 mg/kg) the number of spermatogonium, spermatid cords, Leydig cells, and spermatozoids was increased. Also in these groups, the Sertoli cells were hypertrophic.     

Growth performance,blood biochemical parameters,immune response,and antioxidant defense of Asian seabass (Lates calcarifer) fingerlings exposed to monovalent and bivalent vaccines against Streptococcus iniae and Vibrio harveyi

The present study investigated the effects of monovalent and bivalent vaccines against Streptococcus iniae and Vibrio harveyi on growth performance, blood biochemical parameters, immune system, and antioxidant defense of Asian seabass (Lates calcarifer) fingerlings. Fish were exposed to seven experimental groups containing phosphate buffer saline (control), S. iniae vaccine by injection, S. iniae vaccine by immersion method, V. harveyi vaccine by injection, V. harveyi vaccine by immersion method, S. iniae/V. harveyi vaccines by immersion method, and S. iniae/V. harveyi vaccines by injection in triplicate for 60 days. Vaccination resulted in a significant alteration of the final weight (FW), feed conversion ratio (FCR), daily weight gain (DWG), relative growth rate (RGR), glucose, lysozyme, and complement levels at 60 days post-vaccination. Also, serum glutathione activity significantly increased in group SVIM (S. iniae/V. harveyi vaccines by immersion method) compared to the control group at 60 days post-vaccination. The level of MDA in serum was significantly decreased in SIN (S. iniae vaccine by injection) and SVIM groups compared to the control group 60 days post-vaccination where the value of this parameter was significantly higher in VIM (V. harveyi vaccine by immersion method) and SVIN (S. iniae/V. harveyi vaccines by injection) groups. The results of this study suggest that exposure to monovalent and bivalent vaccines against S. iniae and V. harveyi could alter the growth performance, nonspecific immune response, and antioxidant defense of Asian seabass fingerlings.