Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes

BACKGROUND: In vitro maturation (IVM) of mammalian oocytes has potential health benefits for patients undergoing assisted reproduction as an alternative to gonadotrophin treatment. This procedure is also useful for studying the process of oocyte and early embryo development. However, oocytes undergoing IVM have much lower competence than in vivo matured oocytes. Efforts to optimize IVM success have focused on replicating in vivo timing, hormonal milieu and cumulus cell responses associated with maturing oocytes. We have previously identified two extracellular matrix proteins, the protease Adamts1 and hyaluronan-binding proteoglycan Versican, produced by mural granulosa cells that selectively incorporate into the periovulatory cumulus-oocyte complex (COC). METHODS: Murine COC were cultured in the presence of epidermal growth factor and/or FSH. mRNA and protein were measured by real time PCR and Western blot and compared to in vivo derived COC. RESULTS: COCs from mice that underwent IVM for 6 or 20 h in the presence of epidermal growth factor, FSH or in combination had a > 10-fold reduction in mRNA (P < 0.05) for Adamts1 and Vcan when compared with in vivo matured COCs. Hyaluronan synthase 2 expression was up-regulated up to 8-fold (P < 0.05) over the unstimulated control, demonstrating successful induction of cumulus gene expression by the IVM conditions. While in vivo matured COCs showed abundant levels of these proteins, COCs that underwent IVM had neither detectable Adamts1, nor intact or Adamts1-cleaved Vcan. Human cumulus and granulosa cells matured in vivo contained abundant mRNA for Adamts1 and Vcan, demonstrating the potential relevance to human IVM. CONCLUSION: These results indicate that extensively altered COC matrix composition is present during IVM and may contribute to the observed poorer competence of the derived oocytes.     

In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system

BACKGROUND: Deficiencies remain in the ability of in vitro-matured human oocytes to acquire full developmental competence and give rise to a healthy pregnancy. A clear deficiency of current systems utilizing human oocytes has been the absence of cumulus cells. In the present study, a three-dimensional (3D) co-culture system exploiting an extracellular matrix was developed and compared to conventional methods for its ability to support maturation of human oocytes. METHODS AND RESULTS: Cumulus cells were embedded into a 3D collagen gel matrix with individual oocytes added to each gel. Oocytes from the same patient cultured in the gel matrix matured to metaphase II at rates similar to those of cumulus-free oocytes cultured in individual microdrops. Following maturation of oocytes and fixation of intact gels, chromatin and cytoskeletal elements were assessed in oocytes and cumulus cells. The activities of the key cell cycle kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were compared in oocytes matured under the two culture conditions. Compared with denuded oocytes, co-cultured oocytes exhibited increased MAPK activity, but no difference in MPF levels. CONCLUSIONS: This work characterizes a novel and efficacious culture system that takes advantage of the unique properties of the extracellular matrix, a 3D microenvironment, and the presence of cumulus cells for maturing human oocytes in vitro.     

Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection

This paper postulates that in the ovary, the close association between the cumulus cells and the oocytes permits the fertilizing ability of the oocytes to be determined indirectly through cumulus cell DNA status. The objective was to use a modified comet assay to analyse cumulus cell DNA and relate the data to oocyte fertilization after intracytoplasmic sperm injection (ICSI) procedures. Oocytes were retrieved (n = 15 cases) and free-floating cumulus cells were pooled and smeared on clear glass slides to dry. Meanwhile, the denuded oocytes were injected with spermatozoa and fertilization was assessed, followed by embryo transfer. The fixed cumulus cells were stained in acridine orange, coated with a mini-gel agarose layer, lysed in alkaline buffer and electrophoresis performed. Analyses of fluorescent cell images (n = 449) showed that the tail moment was positively correlated to the percentage of fertilization after ICSI (r = 0.567, P < 0.05). In contrast, there was no correlation between tail moment and number of oocytes retrieved, total ampoules used, endometrial thickness and age of patient. The results suggested that the competence of the oocytes was associated with the cumulus cell DNA status. A unique feature here was the comet assay for archived material with obvious advantages.     

Dissolution Rate Studies from a Stationary Disk/Rotating Fluid System

The dissolution rates for hydrocortisone alcohol and acetate were determined using a stationary disk/ rotating fluid system. The hydrocortisone was compressed in a tablet die, and the die placed in a vessel above a rotating magnetic bar. Dissolution rates were evaluated in aqueous media under conditions involving the following independent variables: solubility (C _s), diffusion coefficient (D), viscosity (v), rotational speed (), and tablet radius (r). A design equation which relates dissolution rate (R) to these variables was formulated for the system R C _s D ^2/3(v) ^–1/6() ^1/2( r)^3/2 This design equation adequately represents the system, which is related to fluid mechanics and convective diffusion models. The fluid mechanics model assumes that the fluid ideally rotates as solid-body rotation and the momentum layer is initiated at the outside radius of the tablet die. The convective diffusion model is based on the formation of a diffusion layer at the outside radius of the dissolving surface and a predictable relationship between the momentum and the mass transport quantities of bulk viscosity and diffusion coefficient. This configuration, like the rotating disk in a stationary fluid, offers the attractive attribute of being useful to study drug release mechanisms for systems of pharmaceutical interest.     

Flow and diffusion of high-stakes test scores

We apply visualization and modeling methods for convective and diffusive flows to public school mathematics test scores from Texas. We obtain plots that show the most likely future and past scores of students, the effects of random processes such as guessing, and the rate at which students appear in and disappear from schools. We show that student outcomes depend strongly upon economic class, and identify the grade levels where flows of different groups diverge most strongly. Changing the effectiveness of instruction in one grade naturally leads to strongly nonlinear effects on student outcomes in subsequent grades.     

利用低真空环境在地面模拟载人航天器内部气体对流散热的探讨

为研究相应的模拟实验方法并建立载人航天器内部环境的地面模拟装置，克服地面上模拟微重力气体对流散热的困难，本文提出了地面以低真空技术减弱自然对流模拟载人航天器内啊对流热的实验方法，建立合适的试验舱和采用合适的实验方法，以便通过实验对环境控制与生命保障系统的设备作出评价。     

不同方式连续性肾脏替代疗法对溶质清除率的影响

目的　探讨不同治疗剂量的连续性静脉 -静脉血液透析 (CVVHD)、连续性静脉 -静脉血液滤过(CVVH)及连续性静脉 -静脉血液透析滤过 (CVVHDF)对各种溶质清除率的影响。方法　采用体外试验 ,选用F6 0聚砜膜透析器 ,用DiapactCRRT机分别行模拟CVVHD、前稀释及后稀释CVVH以及前稀释及后稀释CVVHDF ,测定不同透析液和置换液流量下对尿素、肌酐、VitB12 、菊粉的血液侧清除率。结果　CVVHD对各种相对分子质量大小溶质的清除率 ,随着透析液流量的增大而升高。无论在前稀释或后稀释模式 ,CVVH和CVVHDF对各种溶质的清除率均随着置换液量的增大而相应提高 ,其中最高清除率在采用 40 0 0mL/h前稀释模式时达到。和相应置换液输注速度的前稀释模式相比 ,后稀释模式溶质清除率明显高于前稀释模式。在相同透析液和置换液流量情况下 ,CVVHDF模式溶质清除率最高 ,而CVVHD的溶液利用效率较高。结论　体外条件下各种CRRT模式对不同相对分子质量溶质清除率随透析液和置换液流量增加而上升 ,其中前稀释CVVHDF由于可采用更大置换液输注速度 ,可达到最大溶质清除率 ;CVVHD具有最高溶液利用效率     

人体气道内氧及二氧化碳的对流扩散

我们探讨了正常呼吸状态下人体气道内氧及二氧化碳对流扩散问题。通过对人体气道主气管及支气管生理结构的分析，结合Horsfield及Wanner等关于气管系统形态的最优化分析，把人体气道简化为二叉气管树。在此生理模型基础上，根据人体气道内低压降、低流速的流动特点，假定气道内气体是不可压缩粘性流体，应用LaxWendroff-三条腿有限差分格式，采用两种边界条件分别求解了非定常变截面氧及二氧化碳的一维对流扩散方程，从而得出了呼吸过程中人体气道内氧及二氧化碳的浓度分布。