大鲵胚胎发育的组织学研究

通过人工繁殖，获得了两批大鲵的受精卵及其幼苗．利用组织学方法研究了大鲵的早期胚胎发育，并描述了其主要特征．同时，就此和其他两栖动物的胚胎发育进行了比较讨论，并指出了其共性与特殊性．     

非洲爪蟾卵母细胞内源性ATP激活电流ID2成分的分析

在带有滤泡膜的非洲爪蟾卵母细胞标本上,应用双电极电压钳及胞内透析方法进行记录,外加ATP所激活的内源性膜电源包括3个成分依次为：快内向电流（early fast depolarizing current,ID1),慢内向电流（lateslow depolarizing current,ID2)和慢外向电流(slow hygerpolarrizing current,IH),本文主要分析ID2成分的特征及其产生的机制,结果如下：（1）胞内透析500μmol/L GDP-β－S,500min后被完全阻断;（2）外液中[Cl^-]从100mmol/L下降到5mmol/L时,在ATP（100μmol/L）作用下,ID2幅值随[Cl^-]的下降明显增大;（3）外液中无Na^ 时ID2幅值增高（P＜0．05）。结果表明,ID2是由P2Y受体所介导,通过G蛋白耦联及胞内信号转导而引起胸内Ca^2 依赖性Cl^-外流所致。     

Resveratrol Hinders Postovulatory Aging by Modulating Oxidative Stress in Porcine Oocytes

Postovulatory aging of the mammalian oocytes causes deterioration of oocytes through several factors including oxidative stress. Keeping that in mind, we aimed to investigate the potential of a well-known antioxidant, resveratrol (RV), to evaluate the adverse effects of postovulatory aging in porcine oocytes. After in vitro maturation (IVM), a group of (25–30) oocytes (in three replicates) were exposed to 0, 1, 2, and 4 μmol/L of RV, respectively. The results revealed that the first polar body (PB1) extrusion rate of the oocytes significantly increased when the RV concentration reached up to 2 μmol/L (p < 0.05). Considering optimum RV concentration of 2 μmol/L, the potential of RV was evaluated in oocytes aged for 24 and 48 h. We used fluorescence microscopy to detect the relative level of reactive oxygen species (ROS), while GHS contents were measured through the enzymatic method. Our results revealed that aged groups (24 h and 48 h) treated with RV (2 μmol/L) showed higher (p < 0.05) ROS fluorescence intensity than the control group, but lower (p < 0.05) than untreated aged groups. The GSH content in untreated aged groups (24 h and 48 h) was lower (p < 0.05) than RV-treated groups, but both groups showed higher levels than the control. Similarly, the relative expression of the genes involved in antioxidant activity (CAT, GPXGSH-Px, and SOD1) in RV-treated groups was lower (p < 0.05) as compared to the control group but higher than that of untreated aged groups. Moreover, the relative mRNA expression of caspase-3 and Bax in RV-treated groups was higher (p < 0.05) than the control group but lower than untreated groups. Furthermore, the expression of Bcl-2 in the RV-treated group was significantly lower than control but higher than untreated aged groups. Taken together, our findings revealed that the RV can increase the expression of antioxidant genes by decreasing the level of ROS, and its potent antiapoptotic effects resisted against the decline in mitochondrial membrane potential in aged oocytes.     

兴国红鲤卵母细胞发育中的多糖物质

以池养的兴国红鲤(Cyprius carpio var singuonensis)雌性卵巢为材料,研究了生殖季节内卵巢中卵母细胞内多糖物质的定域分布与变化规律。同时,观察了在饥饿条件下卵母细胞中多糖分布的变化。观察结果证明:1时相卵母细胞中除质膜和其周围结缔组织上有较强的PAS反应外,细胞内几乎     

高效液相色谱法测定单个蟾蜍垂体中精氨酸催产素含量

本文提出了用反相高效液相色谱结合化学衍生化和荧光检测技术测定单个蟾蜍垂体中精氨酸催产素(AVT)含量的方法。AVT经提取后,转化成荧光衍生物,然后在反相ODS术上以水和丙酮作流动相洗脱,荧光检测器检测(λ_(ex2) 340—380nm;λ_(em1) 460nm),最小检出量达1×10~(-12)mol(pmol),回收率为49.5%。     

Marinobufagenin extraction from Rhinella marina toad glands: Alternative approaches for a systematized strategy

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na⁺/K⁺‐ATPase pump by inhibiting the cardiotonic steroid dependent‐site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt‐sensitive hypertensive states such as essential hypertension, congestive heart‐failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin‐layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid‐phase extraction coupled with fractionation on high‐performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid‐phase extraction combined with high‐performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time‐consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.     

Lipid characterization of individual porcine oocytes by dual mode DESI-MS and data fusion

The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo production for porcine species.     

Rapid identification of primary constituents in parotoid gland secretions of the Australian cane toad using HPLC/MS‐Q‐TOF

Toad parotoid gland secretion or toad venom has in recent years been increasingly shown to possess potentially beneficial pharmacological effects; this speculation has drawn much interest centred on elucidating the chemical basis of its multimodal effects. For this purpose, we explored the use of a rapid and accurate analysis method for systemic investigation of the parotoid gland chemistry, when extracted from Australian cane toads. Full‐scan data of cane toad venom extract was acquired using high‐performance liquid chromatography coupled with a hybrid quadrupole–time of flight mass spectrometry system (HPLC/MS‐Q‐TOF), with multiple ionization sources (ESI and APCI) in positive and negative mixed modes. By measuring the exact mass differences between the theoretical and measured mass of each assumed compound, we confirmed the presence of 12 key constituents. The present results demonstrate that the use of HPLC/MS‐Q‐TOF with multiple ionization sources delivers exemplary selectivity and sensitivity, allowing for the rapid and accurate identification of constituents within cane toad venom. This paves the way for this technique to be used in future routine screening of components within the genus Bufo and for key analytes too, then reliably assessed for any purported beneficial (clinic) properties. Copyright © 2013 John Wiley & Sons, Ltd.     

The changing biochemical composition and organisation of the murine oocyte and early embryo as revealed by Raman spectroscopic mapping

Despite an exponential uptake in recent years of assisted reproductive techniques, such as in vitro fertilisation, much is still not fully understood about the biochemical modifications that take place during the development and maturation of the egg and embryo. As such, in order to improve the efficiency of these techniques, furthering our understanding of the processes that underpin oocyte and embryo development is necessary. Raman spectroscopic mapping as a technique enables the investigation of biochemical variation within intact cells without the need for labelling. Here, Raman maps of fixed immature and mature oocytes along with early stage embryos were collected using 785 nm excitation and a step size of 2 µm. The results were analysed using both univariate and multivariate techniques. It was found that significant macromolecular accumulation took place during oocyte maturation, while a decrease in total lipid content consistent with the formation of new cellular membranes is observed upon embryo cleavage. Furthermore, an observed asymmetrical localisation of macromolecules in the mature oocyte may indicate the existence of cytoplasmic polarisation, a phenomenon that has been observed in the eggs of lower organisms. As such, these results indicate that Raman spectroscopic mapping may present an alternative analytical tool for investigating the biochemistry of egg and embryo development. In particular, these results indicate that temporal Raman analysis may help to reveal the existence of cytoplasmic polarisation in the murine egg. Copyright © 2011 John Wiley & Sons, Ltd.