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1.

Background:

Ovarian tissue cryopreservation is an alternative strategy to preserve the fertility of women predicted to undergo premature ovarian failure. This study was designed to evaluate the expression of folliculogenesis-related genes, including factor in the germline alpha (FIGLA), growth differentiation factor-9 (GDF-9), follicle-stimulating hormone receptor (FSHR), and KIT LIGAND after vitrification/warming of human ovarian tissue.

Methods:

Human ovarian tissue samples were collected from five transsexual women. In the laboratory, the ovarian medullary part was removed by a surgical blade, and the cortical tissue was cut into small pieces. Some pieces were vitrified and warmed and the others were considered as non-vitrified group (control). Follicular normality was assessed with morphological observation by a light microscope, and the expression of FIGLA, KIT LIGAND, GDF-9,, and FSHR genes was examined using real-time RT-PCR in both the vitrified and non-vitrified groups.

Results:

Overall, 85% of the follicles preserved their normal morphologic feature after warming. The percentage of normal follicles and the expression of FIGLA, KIT LIGAND, GDF-9, and FSHR genes were similar in both vitrified and non-vitrified groups (P > 0.05).

Conclusion:

Vitrification/warming of human ovarian tissue had no remarkable effect on the expression of folliculogenesis-related genes. Key Words: Vitrification, Gene expression, Humans  相似文献   

2.
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)  相似文献   

3.
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  相似文献   

4.
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)  相似文献   

5.
Background:In animal models of inflammatory diseases, Mst1 facilitates the programmed cell death as a novel pro-apoptotic kinase. This research aimed to determine the expression level of Mst1 gene in a rat model of SCI treated with VPA. Methods:Severe rat model contusion was used for evaluation of the neuroprotective effect of valproic acid. The BBB test, was performed to determine locomotor functions. H&E staining and TUNEL assay were performed to detect cavity formation and apoptosis, respectively. The mRNA levels of the genes Mst1, Nrf2, and Bcl-2 were evaluated, using quantitative RT-PCR. Results:The results revealed that Mst1 gene expression and TUNEL-positive cells in the VPA-treated group were significantly reduced as compared to the untreated group (p ≤ 0.05). Conclusion:Our findings indicate that VPA has therapeutic potential and can be a candidate for the treatment of neurodegenerative disorders and traumatic injury as a promising drug. Key Words: Bcl-2, Contusion, Mst1, Nrf2, Valproic acid  相似文献   

6.
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7.
The cytotoxic effects of thirteen triterpene glycosides from Holothuria scabra Jaeger and Cucumaria frondosa Gunnerus (Holothuroidea) against four human cell lines were detected and their cytotoxicity-structure relationships were established. The apoptosis-inducing activity of a more potent glycoside echinoside A (1) in HepG2 cells was further investigated by determining its effect on the morphology, mitochondrial transmembrane potential (Δψm) and mRNA expression levels of the apoptosis-related genes. The results showed that the number of glycosyl residues in sugar chains and the side chain of aglycone could affect their cytotoxicity towards tumor cells and selective cytotoxicity. 1 significantly inhibited cell viability and induced apoptosis in HepG2 cells. 1 also markedly decreased the Δψm and Bcl-2/Bax mRNA express ratio, and up-regulated the mRNA expression levels of Caspase-3, Caspase-8 and Caspase-9 in HepG2 cells. Therefore, 1 induced apoptosis in HepG2 cells through both intrinsic and extrinsic pathway. These findings could potentially promote the usage of these glycosides as leading compounds for developing new antitumor drugs.  相似文献   

8.
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 5th 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 7th 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  相似文献   

9.
Summary Alginate coated meristems from in vitro-grown axillary buds of potato (Solanum tuberosum L.) were successfully cryopreserved by vitrification. Excised meristems were precultured on sucrose-enriched MS medium and then encapsulated. To induce dehydration tolerance (osmotolerance), encapsulated meristems were treated with a mixture of 2 M glycerol plus 0.6 M sucrose for 90 min. These encapsulated meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 hr at 0°C prior to a plunge into liquid nitrogen. Successfully vitrified meristems developed shoots within 3 weeks after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 70%. No difference was observed in RAPD analysis using 17 primers between cryopreserved and non-treated plantlets. The cryogenic protocol was successfully applied to 14 cultivars. It was also confirmed that the encapsulated vitrified meristems produced much greater shoot formation than the encapsulated dried meristems. Thus, the encapsulation vitrification protocol appears promising for cryopreservation of potato germplasm.  相似文献   

10.
Diploid interspecific hybrids from crosses betweenSolanum tuberosum haploids andS. canasense, S. multidissectum, andS. tarijense, along with tetraploid and hexaploid somatic hybrids S.tuberosum (+)S. commersonii, were screened for resistance to blackleg and tuber soft rot byErwinia carotovora subsp.atroseptica andE. carotovora subsp.carotovora. Among the diploid hybrids, those involving clone mlt la of S.multidissectum and tar 2b ofS. tarijense yielded the highest number of genotypes resistant to both blackleg and tuber soft rot. By contrast, all the hybrids involving clone tar 1lb ofS. tarijense were susceptible to both bacterial diseases. As far as resistance of somatic hybrids is concerned, the most interesting genotype was the tetraploid one which showed resistance to both blackleg and tuber soft rot Significant correlations were found betweenE. carotovora subsp.atroseptica andE. carotovora subsp.carotovora both in resistance to blackleg and in resistance to tuber soft rot, suggesting that resistance to the two subspecies is controlled by the same gene/s or that the relevant genes are linked. On the other hand, correlations between blackleg and tuber soft rot were never significant, indicating that different mechanisms may control resistance in tuber and stems. Alternatively, the resistance could be controlled by the same gene/s under a different spatial/temporal expression pattern. Twelve diploid sexual hybrids and one tetraploid somatic hybrid were selected for resistance to blackleg and/or tuber soft rot and for tuber characteristics.  相似文献   

11.
The influence of flaxseed (Linum usitatissimum L.) and its total non-digestible fraction (TNDF) on the expression of genes involved in azoxymethane (AOM)-induced colon cancer in Sprague Dawley rats was analyzed. The dose used in the animal model was two tablespoons of flaxseed per day, which is the dose recommended for humans. Flaxseed significantly decreased the crypt multiplicity (10.50?±?3.5) compared with the AOM treatment (34.00?±?11.0), which suggests that flaxseed exhibits a preventive effect against colon cancer. Both treatments (flaxseed and TNDF) influence the overexpression of genes involved in cell cycle arrest and mitochondrial apoptosis: p53, p21, bcl-2, bax and caspase-3. Flaxseed induced the expression of p53 and p21, whereas TNDF triggered the p21-independent expression of p53. This finding suggests that both of these treatments induced cell cycle arrest. In addition, TNDF induced mitochondrial apoptosis because the TNDF + AOM group exhibited the expression of caspase-3, decreased bcl-2 expression and increased bax expression. These results suggest that the expression of the analyzed genes is associated with the presence of dietary antioxidants linked to the cell wall of flaxseed.  相似文献   

12.
The effect ofFusarium solani (Fsc),F. sambucinum (Fs),Erwinia carotovora subsp.atroseptica (Eca), andE. carotovora subsp.carotovora (Ecc) inoculated singly and in combination (FscFs, FscEca, FscEcc, FsEca, FsEcc, and EcaEcc) on wound-healed potato seed pieces of the cultivar Kennebec was studied. Potato seed pieces wound-healed for 5 days at 13 C and approximately 100% relative humidity, inoculated with Fsc, Fs, Eca, Ecc and their combinations, and incubated for 10 days at 9 C were protected from Eca, Ecc, and EcaEcc. When wound-healed potato seed pieces were inoculated with Fsc, Fs, Eca, Ecc, and their combinations and planted in soil maintained at 7, 10, 15, 20 and 25 C, wound-healing protected seed pieces from all pathogens and their combinations in seed pieces held for 10 days at 10 and 15 C. When seed pieces of cultivars Atlantic, Kennebec, Norchip, Russet Burbank and Superior were inoculated with Fsc, Fs, Eca, Ecc, and their combinations, different levels of protection were observed: Russet Burbank had the highest level of protection, Atlantic and Kennebec were intermediate, and Norchip and Superior were slightly protected. Dusting mancozeb on wound-healed seed pieces before inoculation with Fsc, Fs, Eca, Ecc, and their combinations increased protection against all pathogens and their combinations by 39% or more compared to the nonchemical control.  相似文献   

13.
WOX基因家族是一类植物特有的转录因子基因家族,在植物的生长发育过程发挥重要作用。本研究通过同源克隆的方法,得到乌拉尔图小麦(Triticum urartu)WOX基因家族的6个成员: TuWOX1、 TuWOX2、 TuWOX3、 TuWOX4、 TuWOX5和 TuWOX3a。生物信息学分析表明, TuWOX2、 TuWOX3和 TuWOX3a属于WUS支/进化支, TuWOX4属于古老支, TuWOX5属于中间支。除这三个分支外, TuWOX1自成一支,说明在乌拉尔图小麦中可能出现了不同功能的WOX家族基因。利用qRT-PCR技术对得到的WOX基因在乌拉尔图小麦不同组织的表达模式以及幼苗在不同非生物胁迫处理下的表达情况进行了分析,结果表明,WOX基因在乌拉尔图小麦各组织中均有表达,但在不同组织中的表达量差异较大,说明WOX基因在不同的组织中发挥作用;非生物胁迫条件下WOX基因表达水平发生了变化,表明其可以响应外界的胁迫。  相似文献   

14.

Background:

Bax and Bcl-2 are the major members of Bcl-2 family whose play a key role in tumor progression or inhibition of intrinsic apoptotic pathway triggered by mitochondrial dysfunction. Therefore, the balance between pro- and anti-apoptotic members of this family can determine the cellular fate.

Methods:

In this study, the relative level of mRNA expression of Bax and Bcl-2 genes was determined using RNA extraction, cDNA synthesis and RT-qPCR technique from 22 tumoral tissues and adjacent non-tumoral tissues from adenocarcinoma colorectal cancer.

Results:

The potential prognostic and predictive significance of Bax and Bcl-2 gene expression and Bax/Bcl-2 ratio were demonstrated in colorectal cancer. The significant correlation between qPCR data and different clinicopathologic parameters of colorectal carcinoma, including age, gender, tumor size, tumor stage, tumor location, and tumor differentiation was also examined. Interestingly, no significant correlation was seen between Bax and Bcl-2 expressions and clinicopathological parameters of colorectal cancer. However, Bax/Bcl-2 ratio was statistically correlated with age and tumor location. Patients with age above 50 showed decreased levels of Bax/Bcl-2 ratio. Moreover, the Bax/Bcl-2 ratio was significantly lower in tumors resected from colon compared to sigmoid colon, rectosigmoid and rectum tumors.

Conclusion:

This study indicates a significant correlation between age and tumor location with Bax/Bcl-2 expression ratio, suggesting predictive value as a potential molecular marker of colorectal cancer.Key Words: Colorectal cancer, Bax/Bcl-2 ratio, Bax expression, Bcl-2 expression  相似文献   

15.
This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster.  相似文献   

16.
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17.
In order to apply state-of-the-art molecular breeding techniques in fibre crop it is necessary to have a good knowledge of major polymer biosynthesis gene sequences and their expression pattern. Polymerase chain reaction was employed to isolate sequences of the major genes for lignin and cellulose biosynthesis in a kenaf cultivar. CeSA, 4cl, c4h, cad, and ccr gene primers were designed according to their conservative regions; partial sequences of lignin and cellulose biosynthesis genes were obtained. One actin II gene sequence was also isolated from the kenaf genome as a housekeeping gene to be employed in qPCR analysis. Expression levels of genes c4h, cad and CeSA in bark and core from plants harvested at three different growth stages were evaluated. Using qPCR analyses it was found that the expression levels of the two biosynthesis lignin genes in bark tissues increased during plant growth, while a negative trend was recorded in core tissues. In both bark and core, the quantity of lignin was positively correlated to plant growth while cellulose content decreased.  相似文献   

18.
In recent years, there has been considerable interest in lectins from marine invertebrates. In this study, the biological activities of a lectin protein isolated from the eggs of Sea hare (Aplysia kurodai) were evaluated. The 40 kDa Aplysia kurodai egg lectin (or AKL-40) binds to D-galacturonic acid and D-galactose sugars similar to previously purified isotypes with various molecular weights (32/30 and 16 kDa). The N-terminal sequence of AKL-40 was similar to other sea hare egg lectins. The lectin was shown to be moderately toxic to brine shrimp nauplii, with an LC50 value of 63.63 µg/mL. It agglutinated Ehrlich ascites carcinoma cells and reduced their growth, up to 58.3% in vivo when injected into Swiss albino mice at a rate of 2 mg/kg/day. The morphology of these cells apparently changed due to AKL-40, while the expression of apoptosis-related genes (p53, Bax, and Bcl-XL) suggested a possible apoptotic pathway of cell death. AKL-40 also inhibited the growth of human erythroleukemia cells, probably via activating the MAPK/ERK pathway, but did not affect human B-lymphoma cells (Raji) or rat basophilic leukemia cells (RBL-1). In vitro, lectin suppressed the growth of Ehrlich ascites carcinoma and U937 cells by 37.9% and 31.8%, respectively. Along with strong antifungal activity against Talaromyces verruculosus, AKL showed antibacterial activity against Staphylococcus aureus, Shigella sonnei, and Bacillus cereus whereas the growth of Escherichia coli was not affected by the lectin. This study explores the antiproliferative and antimicrobial potentials of AKL as well as its involvement in embryo defense of sea hare.  相似文献   

19.
Wild potato (Solanum sect.Petota) germplasm has been collected in Mexico on nine major expeditions, as determined by 20 collections or more from each expedition currently at the United States potato genebank, the National Research Support Program-6 (NRSP-6). These have resulted in 609 accessions with good collection data. In addition, NRSP-6 has germplasm of approximately 90 other Mexican collections that are unspecific regarding date or place of collection. This expedition was funded to collect those remaining collections with no or little germplasm:Solanum clarum,S. xedinense, S. hintonii, S. hjertingii var.physaloides, S. leptosepalum, S. lesteri, S. macropilosum, S. xmichoacanum, S. xsambucinum, andS. stenophyllidium. In addition, some species and species groups (species groups indicated in parentheses) have unresolved taxonomic problems that needed clarification by additional field collections. These are (S. agrimonifolium and S.oxycarpum), (S. brachycarpum, S. guerreroense, S. hougasii, andS. iopetalum - theS. brachycarpum complex), (S. fendleri, S. papita, S. stoloniferum - theS. stoloniferum complex),S. leptosepalum, andS. macropilosum. We conducted a wild potato germplasm collecting expedition in Mexico from August 22 to October 31, 1997. Our 103 collections, 71 as germplasm collections, provide the first germplasm samples forS. hjertingii var.physaloides,S. leptosepalum, andS. macropilosum. They provide additional germplasm of the rare speciesS. clarum, S. xedinense, S. lesteri, S. xmichoacanum, S. xsambucinum, andS. stenophyllidium. We additionally gathered germplasm and field data to help resolve taxonomic difficulties inS. agrimonifolium andS. oxycarpum, theS. brachycarpum complex, and theS. stoloniferum complex.  相似文献   

20.
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