[Early morphological changes in the mouse testis induced by 5-fluorouracil].

OBJECTIVE: To propose a simple and practical method for preparing a mouse model of oligo-astheno-terato-spermia/azoospermia, and to offer a methodological suggestion for the studies on the related mechanism of spermatogenesis and evaluation of medication efficacy by observing the early changes of testis morphology after 5-fluorouracil treatment. METHODS: Mice were injected with a single dose of 5-flourouracil at 250 mg/kg via the tail vein, and their testes were harvested and paraffin sections prepared before and 3, 7, 11 and 14 d after the injection to be observed for the morphological changes by hematoxylin and eosin staining. RESULTS: The numbers of spermatocytes/spermatids were progressively reduced inside the testis seminiferous tubules of the mice at 3, 7 and 11 d after the 5-fluorouracil injection, and the tubule walls became thinner, which reached the nadir at 11 d, with evident swelling and crazing of the seminiferous tubules. At 14 d, the swelling almost disappeared and spermatocytes became repopulated, while the flaws still existed in the seminiferous tubules and no mature sperm were seen. CONCLUSION: One-dose injection of 5-fluorouracil via the tail vein might be a simple and effective method for preparing the animal model of reproductive function damage induced by chemotherapeutic medication.

[Interrelationship of abnormal family history in the first degree relatives and clinical phenotype of patients with polycystic ovary syndrome].

OBJECTIVE: To study the relationship of abnormal family history in the first degree relatives and the clinical phenotype of patients with polycystic ovary syndrome (PCOS). METHODS: Clinical data of first degree relatives of 139 women with PCOS were collected by questionnaires, including body mass index (BMI), waist hip ratio (WHR), and hursutism score. Follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), testosterone (T), androstenedione (A), oral glucose tolerance test (OGTT) and insulin releasing test were measured. RESULTS: (1) Compared with patients with a negative family history of diabetes mellitus, for women with a positive family history, WHR (0.99 +/- 0.10 vs 0.79 +/- 0.08) and score of hirsutism (1.9 +/- 1.2 vs 1.8 +/- 1.2) were increased, the duration of menstruation was longer [(108 +/- 10) vs (92 +/- 19) days]; A [(11 +/- 6) vs (8 +/- 5) nmol/L], homeostasis model assessment of insulin resistance (HOMA-IR, 3.5 +/- 2.0 vs 2.7 +/- 1.6), area under curve (AUC) glucose [(836 +/- 245) vs (748 +/- 139) nmol.L(-1).min(-1)], AUC insulin [(9670 +/- 4582) vs (7330 +/- 4311) mIU.L(-1).min(-1)], fasting glucose [(5.0 +/- 1.1) vs (4.8 +/- 0.5) mmol/L] and fasting insulin [(15 +/- 8) vs (11 +/- 8) mIU/L] were increased, while early insulin secretion function index (DeltaI60/DeltaG60, 32 +/- 22 vs 52 +/- 30), insulin sensitive index (ISI, 0.019 +/- 0.011 vs 0.033 +/- 0.014) and disposition index (DI, 18 +/- 10 vs 30 +/- 22; P < 0.05) were decreased. (2) For women with a positive family history of menstrual disorder, WHR and score of hirsutism (0.99 +/- 0.09 vs 0.80 +/- 0.10 and 1.9 +/- 1.0 vs 1.6 +/- 1.1) were increased respectively, the duration of menstruation [(105 +/- 28) vs (84 +/- 31) days] was longer, HOMA-IR (3.6 +/- 2.4 vs 2.5 +/- 1.7) and fasting insulin level [(15 +/- 14) vs (12 +/- 11) mIU/L] were increased, while HOMA-beta (178 +/- 134 vs 207 +/- 175), ISI (0.017 +/- 0.009 vs 0.033 +/- 0.012) and DI (23 +/- 18 vs 28 +/- 19, P < 0.05) were decreased. (3) For women with a positive family history of premature balding, BMI and the score of hirsutism [(26 +/- 4) vs (23 +/- 5) kg/m(2) and 2.1 +/- 1.1 vs 1.7 +/- 1.3] were increased respectively, while DI (20 +/- 11 vs 30 +/- 23, P < 0.05) was decreased. (4) DeltaI60/DeltaG60 (34 +/- 27 vs 50 +/- 30) was decreased and fasting insulin [FINS, (13 +/- 10) vs (10 +/- 9) mIU/L] was increased in PCOS women with a family history of hypertension (P < 0.05). (5) For women with or without a family history of coronary heart disease, they did not have any difference in every parameter mentioned before. CONCLUSIONS: The family history of diabetes mellitus has the most effect on the clinical phenotype in women with PCOS. The family history of other diseases such as menstrual disorder, premature balding and hypertension play less significant roles. A family history of positive coronary heart disease does not affect the clinical phenotype of such patients.

Neuroprotection by intravenous transplantation of bone marrow mononuclear cells from 5-fluorouracil pre-treated rats in a model of ischemic stroke.

Our previous studies showed that bone marrow mononuclear cells (BMMNCs) from 5-fluorouracil (5-FU) pre-treated rats (named BMRMNCs) had a better therapeutic efficacy in ischemia/reperfusion rats as compared to BMMNCs from untreated rats. This study was undertaken to further explore the potential mechanisms underlying the neuroprotective effects of BMRMNCs in the same model. Rats were intravenously pre-treated with 5-FU, and BMRMNCs were collected 7 days later and subjected to flow cytometry for detection of CD34, CD45 and CD90. Middle cerebral artery occlusion (MCAO) was induced in rats, and BMMNCs and BMRMNCs were independently transplanted via the tail vein at 24 h after MCAO. NISSL staining was performed 14 days after cell transplantation and the viable cells in the hippocampus were counted. Stromal cell-derived factor 1 (SDF-1) mRNA expression was detected in the penumbra at 7 and 14 days after treatment. The contents of pro-inflammatory cytokines and growth factors as well as microvessel density (MVD) were determined at 14 days. Results showed more BMRMNCs were positive for CD34, CD45 and CD90. After transplantation, more viable cells were observed in the hippocampus of BMRMNCs treated rats. In addition, BMRMNCs transplantation significantly increased MVD, reduced pro-inflammatory cytokines and raised growth factors in the penumbra. However, the SDF-1 mRNA expression was comparable between BMRMNCs group and BMMNCs group. Our results indicate that BMRMNCs are likely to more effectively improve the local microenvironment to increase viable cells and elevate angiogenesis, exerting neuroprotective effects on cerebral ischemia in rats.

Total flavonoid aglycones extract in Radix scutellariae inhibits lung carcinoma and lung metastasis by affecting cell cycle and DNA synthesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Scutellariae (Scutellaria baicalensis Georgi, RS), a traditional herbal medicine commonly used to treat inflammation, hypertension, cardiovascular disease, bacterial and viral infections, is reported to treat lung cancer by supplements of modern medicine. The total flavonoid aglycones extract (TFAE) from RS is the most important composition for the pharmacodynamic effects. The present study was designed to evaluate the anti-lung tumor effect of TFAE on A549 cells and A549 cell nude mice xenografts. The aim of the study is to investigate the effect and mechanism of TFAE treating non-small cell lung cancer both in vitro and in vivo. MATERIALS AND METHODS: The anti-tumor activity of TFAE in vitro was investigated using the MTT assay. The changes of cell invasion and migration were detected by Transwell assay and tube formation experiments were used to detect the anti-angiogenic effect. The anti-tumor effects of TFAE in vivo were evaluated in A549 cell nude mice xenografts. The mechanism of TFAE was detected by flow cytometry technology, western blot assay and immuno-histochemistry assay. RESULTS: In vitro, TFAE inhibited the proliferation, invasion and migration of A549 cells in a dose- and time-dependent manner. In vivo, TFAE by oral administration at 100mg/kg for 30 days decreased the tumor volume and tumor weight in A549 cell xenograft by 25.5% with no statistical significance (P<0.05) compared to the cis-platinum positive control group (30.0%). The cell cycle and DNA synthesis experiment illustrated that TFAE could induce A549 cell cycle to arreste in S phase and DNA synthesis in A549 cells be inhibited, while TFAE had no influence on apoptosis of A549 cells. Western Blot assay demonstrated that the treatment of TFAE could make Cyclin D1 decrease and p53 increase both in vitro and in vivo. CONCLUSION: TFAE displayed the inhibition effects of non-small cell lung cancer both in vitro and in vivo and the underlying mechanism might be related to the increased p53 protein expression and decreased Cyclin D1 expression, leading to cell cycle arrested in S phase and the decrease of DNA synthesis.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes.

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Overexpression of 5-lipoxygenase increases the neuronal vulnerability of PC12 cells to Aß42.

5-Lipoxygenase (5-LOX), which is believed to be a major source of oxidative stress, participates in somatostatin-receptor transmembrane signaling in the central nervous system. We used the Tet-On inducible expression system in PC12 cells to obtain cell lines with reproducible, stable 5-LOX expression levels to study its function. Cell apoptosis rates induced by Aß(42) were determined using an apo-BrdDU kit. Lipid peroxide, antioxidant enzyme, and caspase-3 activities were evaluated with respective commercial kits. The expression of 5-LOX, bcl-2, and bax were detected by immunoblotting. A subclone of PC18 with Tet-On inducible expression of 5-LOX was selected from PC12 transfectants. Expression of 5-LOX had no significant inhibitory effect on the cell viability of the PC18 clone. In contrast, compared with the control group, the cell viability of clone PC18 was significantly reduced after the induction of 5-LOX during Aß exposure. The differences in cell viability before and after the induction of 5-LOX during Aß insult were significantly offset by AA861. Overexpression of 5-LOX only slightly improved the activities of superoxide dismutase (SOD). The levels of intracellular peroxides, SOD and caspase-3 activity, and Bax expression were significantly upregulated, and the levels of glutathione peroxidase and catalase were downregulated correspondingly in clone PC18 during Aß exposure. These results indicate that constitutive expression of 5-LOX is not detrimental per se, but overexpression of 5-LOX may become problematic during Aß exposure.

Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes.

In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, the mitochondrial DNA (mtDNA) of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos where nuclear donor and oocyte are from two distantly related species, the distribution of the mtDNA species is not known. Here we determined the levels of macaque and rabbit mtDNAs in macaque embryos reprogrammed by rabbit oocytes. Quantification using a real-time PCR method showed that both macaque and rabbit mtDNAs coexist in NT embryos at all preimplantation stages, with maternal mtDNA being dominant. Single NT embryos at the 1-cell stage immediately after fusion contained 2.6 x 10(4) copies of macaque mtDNA and 1.3 x 10(6) copies of rabbit mtDNA. Copy numbers of both mtDNA species did not change significantly from the 1-cell to the morula stages. In the single blastocyst, however, the number of rabbit mtDNA increased dramatically while macaque mtDNA decreased. The ratio of nuclear donor mtDNA to oocyte mtDNA dropped sharply from 2% at the 1-cell stage to 0.011% at the blastocyst stage. These results suggest that maternal mtDNA replicates after the morula stage.