Protein synthesis and acrosome reaction-inducing activity of human cumulus cells

The acrosome reaction of human spermatozoa is a prerequisiteto fertilization. It has been hypothesized that secretions ofcumulus cells could be involved in the induction of this spermreaction. A source of variation in the in-vitro fertilizationof human oocytes could be the maturational differences in thecumulus cells which affect the acrosome reaction. In order toinvestigate this possibility, we have studied the capabilityof secretions obtained from cumulus cells of different maturationalstages to induce the acrosome reaction. Capacitated spermatozoawere exposed to the culture medium containing cumulus cell secretionsand their acrosomal status was evaluated using the triple staintechnique. The maturational status of the cumulus or fertilizationof the enclosed oocyte did not influence the percentage of acrosome-reactedlive spermatozoa. In order to further document these observations,cumulus cells were submitted to [³⁵S]methionine incorporationand total and secreted proteins were analysed by SDS-PAGE electrophoresis.Neither quantitative nor qualitative changes were detected inthe electrophoretic patterns of the proteins obtained from cumuluscells of different maturational status. Thus, variation of theacrosome reaction-inducing activity of cumulus cells does notappear to be involved in the variable fertilization of oocytesobtained from follicles of differing maturity.     

Intracerebral infusion of an EGFR-targeted toxin in recurrent malignant brain tumors

The purpose of this study is to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and intracerebral distribution of a recombinant toxin (TP-38) targeting the epidermal growth factor receptor in patients with recurrent malignant brain tumors using the intracerebral infusion technique of convection-enhanced delivery (CED). Twenty patients were enrolled and stratified for dose escalation by the presence of residual tumor from 25 to 100 ng/ml in a 40-ml infusion volume. In the last eight patients, coinfusion of (123)I-albumin was performed to monitor distribution within the brain. The MTD was not reached in this study. Dose escalation was stopped at 100 ng/ml due to inconsistent drug delivery as evidenced by imaging the coinfused (123)I-albumin. Two DLTs were seen, and both were neurologic. Median survival after TP-38 was 28 weeks (95% confidence interval, 26.5-102.8). Of 15 patients treated with residual disease, two (13.3%) demonstrated radiographic responses, including one patient with glioblastoma multiforme who had a nearly complete response and remains alive >260 weeks after therapy. Coinfusion of (123)I-albumin demonstrated that high concentrations of the infusate could be delivered >4 cm from the catheter tip. However, only 3 of 16 (19%) catheters produced intraparenchymal infusate distribution, while the majority leaked infusate into the cerebrospinal fluid spaces. Intracerebral CED of TP-38 was well tolerated and produced some durable radiographic responses at doses 相似文献   

Exposure of human spermatozoa to the cumulus oophorus results in increased relative force as measured by a 760 nm laser optical trap

Spermatozoa change their movement characteristics in responseto different environmental conditions. To investigate the relativeforce of spermatozoa exhibiting different motility patterns,a laser optical trap was used. A laser beam at 760 nm was directedthrough a microscope objective and focused above the spermatozoato create a three-dimensional optical trap. Spermatozoa weretrapped at 300 mW, and laser power was reduced until spermatozoacould escape. The force generated by the flagellar movementwas proportional to the laser power at which the spermatozoaescaped from the trap. Three motility patterns were studied:linear, hyperactivated, and cumulus-related. Mean escape powerfor spermatozoa displaying linear motility was 59.5 ±43 mW, for hyperactivated motility 122.3 ± 67 mW (P <0.0001) and for cumulus-related motility 200.6 ± 44.2mW (P < 0.0001). In this study, we showed that human spermatozoagenerated more relative force upon exposure to the cumulus mass.The combination of small-amplitude lateral head displacementand higher relative force may produce a ‘drilling’effect which is synergistic with the enzymatic digestion ofthe cumulus matrix during the fertilization process.     

Steroid Production by the cumulus: relationship to fertilization in vitro

Insemination media were Collected from 92 follicles of 14 patientsstimulated to progesterone and oestradiol in the inseminationdrops were assayed, corrected for carry–over from follicularfluid and volume and expressed as production per µg ofprotein in the cumulus. significantly higher progesteron productionper unit protein was associated with oocytes which fertilizedin vitro (P << 0.02). Oocytes fertilizing with subsequentfragmentation or degeneration showed progesterone levels significantlyhigher than oocytes fertilizing normaly (P << 0.05). Polyspermicoocytes ( n = 3 ) were associated with very high levels of progesteroneproduction but were not significantiy different due to the lownumbers. Oestradial production per unit protein was significantlygreater in oocytes which degenerated (P << 0.05). TheProtein content of cumuli whose oocytes fertilized appearedto be significantly lower than those which did not (P <<0.05). these results probably reflect the maturity of the folliclealthough direct actions of cumulus products upon gametes cannotbe ruled out.     

Determination of the electroactive area of graphite+polyethylene composite electrodes. Uncompensated resistance effects and convolution analysis of chronoamperograms

In this work, it is shown how the convolution analysis of chronoamperograms permits the observation of the uncompensated resistance and the natural convection effects on the electrochemical response of potassium ferrocyanide. The uncompensated resistance causes the current intensity to follow the Cottrell equation only after a certain critical time. The convolution of chronoamperograms worked out at different integration times shows a maximum when this time is long enough. The classical diffusion equations cannot explain this phenomenon themselves. The development of this maximum associated with the natural convection is discussed. If both these factors, the ohmic drop and the natural convection, together with the roughness factor, are considered, the electroactive surface for the composite can be obtained. Theoretical expressions have been derived for reversible and quasi-reversible systems to support the discussion of the experimental results.     

Effect of androgen substrates on the steroidogenic pattern of cumulus cells: Correlation with cumulus culture morphology

Background: In previous studies, higher progesterone secretion was observed in mature versus immature cumulusoocyte complexes. In mature cumulus mass that become homogeneously spread in culture (type C/D) progesterone secretion was higher than in partially (type B) or totally (type A) aggregated morphology. In sharp contrast, estradiol-17β secretion was significantly higher in type A than type C/D cumulus. Purpose: Our purpose was to assess whether the decreased estradiol-17β level in type C/D cumulus culture is caused by deficiency of substrates. Methods: The different cumulus types were incubated with or without 10⁻⁷ M dehydroepiandrosterone, 4-androstane-3, 17-dione, or testosterone. The levels of estradiol-17β, testosterone, and progesterone, were measured after 24 hr of culture. Results: The addition of dehydroepiandrosterone or 4-androstane-3,17-dione significantly increased the estradiol-17β levels in all types of cumulus cells, whereas the addition of testosterone was less effective. In all types of cumulus cells the testosterone levels increased significantly on adding these androgen substrates. In the type C/D cumulus, the testosterone increased to lower levels compared to type A cumulus cells. In the presence of these androgens progesterone secretion is significantly reduced in type A cumulus cells. In type C/D cumulus cells, however, progesterone levels were significantly higher than in type A. The estradiol-17β/testosterone and progesterone/estradiol-17β ratios, which partially resemble the degree of aromatase activity and the degree of selectivity for progesterone secretion, respectively, were higher in type C/D than in type A cumulus cells. Conclusions: In type C/D cumulus the significant increase in estradiol-17β secretion in the presence of various androgens suggests that, under basal conditions, androgen is less available for estradiol-17β biosynthesis compared to type A cumulus. Furthermore, the higher progesterone secretion in type C/D cumulus may suggest that the follicles yielding type C/D cumulus cells are more mature than the follicles yielding type A cumulus.     

1995 Whitaker lecture: Delivery of molecules,particles, and cells to solid tumors

To reach cancer cells in a tumor, a blood-borne therapeutic agent must make its way into the blood vessels of the tumor and across the vessel wall into the interstitium, and finally migrate through the interstitium. Unfortunately, tumors often develop in ways that hinder each of these steps. Our research goals are to analyze each of these steps experimentally and theoretically, and then to integrate the resulting information in a unified theoretical framework. This paradigm of analysis and synthesis has allowed us to obtain a better understanding of physiological barriers in solid tumors, and to develop novel strategies to exploit and/or to overcome these barriers for improved cancer detection and treatment.     

Longitudinal and radial gradients of PO(2) in the hamster cheek pouch microcirculation

OBJECTIVES: The aim of this study was to determine longitudinal and radial gradients in oxygen tension (PO(2)) in microvessels of the hamster cheek pouch. METHODS: We measured PO(2) using the phosphorescence-quenching method in two orders of arterioles (45.8 +/- 5.5 and 19.9 +/- 1.8 micro m diameter), capillaries, and two orders of venules (50.5 +/- 3.4 and 21.4 +/- 2.0 micro m diameter) in order to determine the longitudinal PO(2) gradient. At the arteriolar and venular sites, we also measured PO(2) at four different sites for an analysis of radial PO(2) gradients: centerline, inside wall (larger arteriole and venule only), outside wall, and interstitium. We used 10 hamsters weighing 115 +/- 27 g anesthetized with pentobarbital intraperitoneally and maintained with alpha-chloralose intravenously. The cheek pouch was everted and a single-layered preparation was studied by intravital microscopy. Albumin-bound Pd-porphyrin was infused into the circulation and excited by flash illumination at 10 Hz, with a rectangular diaphragm limiting the excitation field to 5 x 25 micro m. RESULTS: In the longitudinal direction, intravascular PO(2) decreased significantly (P < 0.01) from large arterioles (39.5 +/- 2.3 mmHg) to small arterioles (32.2 +/- 0.3 mmHg), then to capillaries (30.2 +/- 1.8 mmHg), and on to small venules (27.3 +/- 2.1 mmHg) and large venules (25.5 +/- 2.2 mmHg). In the radial direction, PO(2) decreased significantly (P < 0.01) in and around larger arterioles, and to a lesser extent, around the smaller ones (P < 0.05). There was no significant PO(2) gradient, longitudinal or radial, associated with venules. The PO(2) difference from the centerline to the outside wall in large arterioles was 8.3 +/- 1.4 mmHg, and most of the decline in PO(2) in the radial direction was contributed by the intravascular difference (4.7 +/- 2.1 mmHg) and only about 1.0 +/- 2.7 mmHg by the transmural difference. CONCLUSIONS: Our data show that there are large intra-arteriolar radial PO(2) gradients, but no large transmural PO(2) differences, suggesting that the oxygen consumption of the microvessel wall is not exceptionally high.     

人卵丘颗粒细胞和壁层颗粒细胞体外培养方法比较

目的:建立有效分离、提纯及培养人卵丘颗粒细胞的方法,并与人壁层颗粒细胞的体外培养相比较。方法:收集卵胞浆内单精子显微注射取卵时的卵泡液和直接机械分离卵母细胞所得的卵丘颗粒细胞复合物,直接将卵丘颗粒细胞复合物接种于培养皿中培养。用密度梯度离心法分离卵泡液中的人壁层颗粒细胞。用免疫荧光法检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)的表达;用CCK-8法检测细胞生长曲线;用ELISA检测这2种颗粒细胞分泌雌激素的能力。结果:直接法所得人卵丘颗粒细胞培养24 h后可贴壁,体外培养生长状态与人壁层颗粒细胞相似;免疫荧光检测显示两者均表达FSHR;CCK-8实验结果表明,两者体外培养生长曲线相似;ELISA结果显示两者分泌雌激素能力相当。结论:利用机械切割获得人卵子周围卵丘颗粒细胞复合物直接培养的方法操作简单,获得的人卵丘颗粒细胞具有与人壁层颗粒细胞相似的生长状态、生长曲线以及雌激素分泌能力,可作为人颗粒细胞亚群体外培养方法的补充。