Evaluation of sperm mitochondrial function using rh123/PI dual fluorescent staining in asthenospermia and oligoasthenozoospermia

Objective：The recent advent of flow cytometry（FCM）,coupled with fluorescent dyes,has been successfully applied to assess mitochondrial function.The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium（Rh123/PI）dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia.Methods：Twenty-five fertile men（with normal sperm parameters）and 230 infertile patients were examined.Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups：asthenospermia（n=30）and oligoasthenozoospermia（n=25）.Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function.Results：Significant differences were found between the normal and abnormal semen samples（P0.05）when Rh123＋/PI-,Rh123-/PI＋and Rh123-/PI-sperm were examined by FCM,but there was no significant difference between the asthenospermia（P=0.469） and oligoasthenozoospermia group（P=0.950）when Rh123＋/PI-and Rh123-/PI＋sperm were then examined;however,a significant difference was found between the 2 groups（P=0.003）when Rh123-/PI-sperm were examined.There was no correlation between Rh123-/PI-sperm and semen parameters in the normal group,but there was a significant negative correlation between the sperm concentration and Rh123-/PI-sperm in asthenospermia and oligoasthenozoospermia patients（r=-0.509,-0.660;P=0.018,0.038）.Conclusion：Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia.

In vivo histological diagnosis for gastric cancer using endocytoscopy

AIM To examine usefulness of virtual biopsy using endocytoscopy by comparing the in vivo endocytoscopic and histopathological images of gastric cancers.METHODS Endocytoscopy was performed in 30 patients with early gastric cancer. Of these, 26 patients showed well differentiated adenocarcinomas, while 4 patients showed poorly differentiated adenocarcinomas(including one signet ring cell carcinoma). Cancerous and non-cancerous areas were observed after double staining with 0.05% crystal violet and 0.1% methylene blue. The endocytoscopic images obtained were evaluated by an expert endoscopist and an expert pathologist without knowledge of patient clinical data, and endocytoscopic and histopathological diagnoses were compared.RESULTS The endocytoscopic images of the cancerous area were assessed as evaluable in 25(83.3%) and 27(90%) patients by endoscopist A and pathologist B, respectively, and those of the non-cancerous area as evaluable in 28(93.3%) and 23(76.7%) patients by the endoscopist and pathologist, respectively. The sensitivity, specificity, and diagnostic accuracy of gastric cancer diagnosis using evaluable endocytoscopic images were 88.0% and 92.9%, and 90.6% by endoscopist A, and 88.9% and 91.3%, and 90.0% by pathologist B, respectively. Evaluation of the diagnostic concordance rate between the endoscopist and the pathologist by inter-observer agreement calculation revealed no significant difference between the two observers. The inter-observer agreement(κ-value) for endocytoscopic diagnosis was 0.745. CONCLUSION Endocytoscopy is useful for the differentiation of cancerous from non-cancerous gastric mucosa, making it a promising tool for virtual biopsy.

Oligodendrocyte transcription factor 1 mRNA and protein expression in organotypic rat brain slices

Numerous studies have confirmed that oligodendrocyte transcription factor 1 （Olig-1） is vital for myelin repair. However, the effects of hypoxia and ischemia on Olig-1 expression remain unknown. In this study, Olig-1 mRNA and protein expressions were analyzed by in situ hybridization and immunohistochemistry, to determine the expression profile of Olig-1 in rat brain slices exposed to hypoxia and ischemia. Brains were obtained from 2-day-old Sprague-Dawley rats, and sections were randomly assigned to control and hypoxia/ischemia groups. Hematoxylin-eosin staining revealed karyorrhexis and karyopyknosis in cells from the hypoxia/ischemia group. Under electron microscopy, mitochondria swelling and neuropil edema were observed in the hypoxiaJischemia group. Olig-1 mRNA and protein expressions were increased at 1 day after hypoxia and ischemia treatment. These results suggest that in situ hybridization and immunohistochemistry could be used simultaneously to detect mRNA and protein expression in brain slices.

Characterization of Pro-embryogenic Calli and Somatic Embryogenesis of Byrsonima intermedia A. Juss.

Byrsonima intermedia A. Juss. is a species from the Brazilian Cerrado that produces edible fruits and, in common with other species from the Byrsonima genus, has pharmacological potential. Previous attempts to propagate the species through conventional methods showed difficulties. Thus, the purpose of this work was to characterize pro-embryogenic masses of Byrsonima intermedia callus, aiming for their in vitro propagation through somatic embryogenesis. Leaf segments from in vitro germinated seedlings were employed as explants for callus production. The calli were then subcultured and exposed to dyes to fulfill their embryogenic potential. Digitalizations of the cytological preparations were made in order to measure the area that was stained by both Aceto-Carmine and Evans-Blue, using image tool software. Somatic embryos were induced after treatments with l-naphthaleneacetic acid （NAA）. The percentages of double-colored areas （by Aceto-Carmine and Evans-Blue） were calculated and the data were analyzed by using the Skott-Knott test （P ≤ 0.05） and, the embryogenic callus, as well as the formation of somatic embryos were analyzed by using the Krsuskal-Wallis rank test （P ≤0.05）. The results show that double coloration is effective at identifying cells showing embryogenic potential. Early callus subculture phases show a larger percentage ofembryogenic area （83%） Somatic embryos were induced by using high auxin level.

Assembly and disassembly of mammalian chromosome pellicle

By means of indirect double immunofluorescent staining, the coordination of PI antigen and perichromonucleolin (PCN), the constituent of nuclear periphery and nucleolus respectively, in the assembly and disassembly of chromosome pellicle during mitosis was studied. It was found that in 3T3 cells, during mitosis PI antigen began to coat the condensing chromosome .surface earlier.than PCN did. However, both of them completed their coating on chromosome at approximately the same stage of mitosis, prometaphase metaphase. The dissociation of PI antigen from chromosome pellicle to participate the formation of nuclear periphery took, place also ahead of that of PCN. At early telophase PI antigen had been extensively involved in the formation of nuclear periphery, while PCN remained in association with the surface of decondensing chromosomes. At late telophase, when PI antigen was localized in an fairly well formed nuclear periphery, PCN was in a stage of forming prenucleolar bodies.

The expression and clinical significance of β-catenin and colorectal cancer stem cells marker EpCAM^(high)/CD44^+ in colorectal cancer

Objective： The aim of the study was to explore the role of Wnt βcatenin signalling pathway in the maintenance, invasion and metastasis of colorectal cancer stem cells. Methods： Double immunohistochemical staining was used to detect the expression of EpCAMhigh/CD44~ which is regarded as the marker of colorectal cancer stem cells in 80 cases of colorectal cancer and their corresponding liver metastases. The SP method of immunohistochemistry was used to detect the expression of the key protein βcatenin in the Wnt pathway in these tissue. The expression and correlation of ^-catenin and EpCAMh^gh/ CD44＋ in colorectal cancer were analyzed and their role on the biological behavior of colorectal cancer was explored. Results： The abnormal expression of βcatenin was significantly higher in colorectal cancer than in the paraneoplastic normal intes- tinal mucosa [55% （44/80） vs 10% （2/20）, P 〈 0.05]. The positive expression of EpCAMhigh/CD44＋ was significantly higher in colorectal cancer than in the paraneoplastic normal intestinal mucosa [66.25% （53/80） vs 0% （0/20）, P 〈 0.05]. In the 80 cases of colorectal cancer, the abnormal expression of ^-catenin has no correlation with gender （P = 0.079）, age （P = 0.416） and the magnitude （P = 0.816） of the tumor （P 〉 0.05）, but it was significantly correlated with degree of differentiation （P = 0.001）, depth of invasion （P = 0.001）, clinical stage （P = 0.000） and metastasis （P = 0.000）. In the colorectal cancer, the expression of EpCAMhi^h/CD44~ cells has no correlation with gender （P = 0.934） and the magnitude （P = 0.160） of the tumor （P 〉 0.05）, but was significantly correlated with age （P = 0.021）, degree of differentiation （P = 0.013）, depth of invasion （P = 0.000）, clinical stage （P = 0.000） and metastasis （P = 0.000）. In the corresponding liver metastases, we could also detecte EpCAMhih/CD44＋ cells. In cases with abnormal expression of βcatenin, the positive expression rate of EpCAMhigh/CD44＋ was significantly higher than those with normal expression of β-catenin （84.1% vs 44.4%）, and the difference was statistically significant （P 〈 0.05）. Conclusion： The abnormal activation of Wnt β-catenin signalling pathway may prompt the abnormal proliferation of the colorectal cancer stem cells, which leads to the recurrence and metastasis of the cancer.