促性腺激素对小鼠腔前卵泡及其卵母细胞体外发育的影响研究

通过使用改良的TCM199无血清培养系统,在96孔细胞培养板中,每孔放入一个腔前卵泡等条件下,探索促性腺激素(FSH、LH)对小鼠腔前卵泡卵母细胞体外生长发育的影响,旨在摸索一套适合小鼠腔前卵泡体外生长发育的培养体系.结果表明:随着浓度的增加,卵泡和卵母细胞的直径都不同程度的有所增长,在卵泡培养的同期相比,卵泡增长值最大组为FSH400+LH200组,而卵母细胞增长值最大组却是FSH400组.从添加物上看,各个实验组与对照组之间,卵泡直径和卵母细胞直径增长值都存在显著性差异(P0.05).随着培养时间的延长,FSH400+LH200组卵泡存活率、成腔率表现为最高,分别为68.6%,47%.从卵泡发生生发泡破裂率和第一极体排出率来看,FSH400+LH200组发生生发泡破裂率高于其他组,为17.6%,但第一极体排出率却低于FSH400组.因此,改良的TCM199无血清基础培养液中联合添加400mIU/mL FSH和200mIU/ML LH效果优于单独添加组的效果.     

小鼠腔前卵泡无血清体外培养的研究

目的:探索添加物维生素C和促性腺激素对小鼠腔前卵泡卵母细胞体外生长发育的影响及适合小鼠腔前卵泡体外生长发育的培养体系.方法:通过改良的TCM199无血清培养系统,在96孔细胞培养板中,每孔放入1个腔前卵泡等条件下体外培养腔前卵泡卵母细胞.结果:随着培养时间的延长,各组卵泡和卵母细胞的直径都不同程度的有所增长,其中FSH400+LH200组和VC50+FSH400+LH200影响均大于VC50和对照组,存在显著性差异(P0.05),但二者之间无显著性差异.FSH400+LH200质量浓度组卵泡存活率、发生GVBD率和第一极体排出率均最高,分别为68.6%、17.6%、9.8%.但VC50+FSH400+LH 200组卵泡成腔率最高,为48%.结论:在改良的TCM199无血清培养系统中,联合添加促性腺激素和抗氧化剂后,卵泡成腔率大有提高,卵泡存活率、发生GVBD率和第一极体排出率仅次于单独添加促性腺激素组.     

绵羊卵母细胞不同cAMP水平模型的构建

环腺苷酸(cAMP)是维持哺乳动物卵母细胞减数分裂停滞的关键分子.本试验利用ELISA和地衣红染色技术,检测了西洛酰胺(Cilostamide)与乌药醚内酯(Linderane)对绵羊卵母细胞和卵丘细胞内cAMP水平以及生发泡破裂(GVBD)的影响.结果显示:体外培养3h后,10μmol/L Cilostamide和5μmol/L Linderane分别能显著提升和抑制卵母细胞而非卵丘细胞内的cAMP水平;体外培养6h后,Cilostamide组卵母细胞GVBD率(14.91%)显著低于对照组(34.45%)与联合添加组(33.47%)(P0.05),而Linderane组与其相反,表明不同cAMP水平卵母细胞模型构建成功.     

不同诱导条件对树鼩骨髓间充质干细胞向成骨细胞分化的影响

目的 探讨不同诱导条件对树鼩骨髓间充质干细胞（BM-MSCs）体外向成骨细胞分化的影响。方法 取 P3代树鼩骨髓间充质干细胞分成7组进行诱导。A组：高糖DMEM+1μmol/L地塞米松+100μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠;B组：高糖 DMEM+50 ng/mL BMP-2;C组：高糖 DMEM+80 ng/mL BMP-2;D组：高糖DMEM+50μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠+20ng/m L BMP-2;E组：高糖 DMEM+1μmol/L地塞米松 +100μmol/L维生素C+20 mmol/Lβ-磷酸甘油钠;F组：高糖DMEM+100μmol/L地塞米松+100μmol/L维生素 C+10 mmol/Lβ-磷酸甘油钠;G组：DMEM/F12+0.1μmol/L地塞米松 +50μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠。诱导18 d后进行碱性磷酸酶和茜素红染色鉴定其诱导分化情况。结果 每一组碱性磷酸酶染色呈不同程度的阳性,茜素红染色可在A、B、C、D和E组观察到明显矿化结节。结论 A组、B组、C组、D组和 E组可以诱导树鼩BM-MSCs向成骨细胞分化,其中以A组和B组效果最为突出。     

HPLC法同时测定饲料添加剂中的双乙酸钠、丙酸钙、山梨酸钾和苯甲酸钠

建立了同时测定饲料添加剂中的双乙酸钠、丙酸钙、山梨酸钾和苯甲酸钠4种防腐剂的HPLC方法.使用Hypersil ODS C18反相色谱柱(5μm;250×4.6 mm),0.02 mol/L KH2PO4-甲醇(65∶35,V/V)为流动相,标准和样品的p H值为2.5,流速为1.0 m L/min,检测波长为214 nm.柱温为25℃.结果表明:双乙酸钠在30~1 000μg/m L浓度范围内峰面积与浓度线性关系良好,平均回收率为91.8%;丙酸钙在50~2 000μg/m L浓度范围内线性关系良好,平均回收率为87.5%;苯甲酸钠在3~300μg/m L浓度范围内线性关系良好,平均回收率为92.3%;山梨酸钾在4~400μg/m L浓度范围内线性关系良好,平均回收率为83.4%.双乙酸钠、丙酸钙、苯丙酸钠和山梨酸钾的检出限分别为6.3μg/m L,11.8μg/m L,0.1μg/m L和0.2μg/m L.     

三丁基氧化锡（TBTO）对太平洋牡蛎性成熟的影响

利用流水饲养法研究了不同浓度的三丁基氧化锡（0．5μg/L和1．0μg/L TBTO)太平洋牡蛎卵巢中卵母细胞直径、RNA／DNA比、蛋白质和卵黄蛋白含量的影响。卵母细胞径的增长在0．5μg/L实验组受到阻碍,并随着TBTO浓度的增大而更加明显。TBTO暴露组的RNA／DNA比在精巢中无明显变化,但在卵巢中显著低于对照组。TBTO暴露组的卵巢中蛋白质和卵黄蛋白含量显示了与RNA／DNA比相同的变化趋势,表明TBTO的积累阻碍卵黄蛋白的合成。TBTO暴露42d采集的牡蛎样品中出现3个雌雄同体,暗示TBTO可能引起牡蛎发生性转变进而产生雌雄同体。     

17β-雌二醇浸浴对小黄鱼早期生长及性腺发育的影响

17β-雌二醇(E2)是一种天然保守的性类固醇激素,被认为是一种性别决定与分化的关键因子。然而,E2对鱼类身体生长及性腺生长影响的研究较少。本研究采用浸浴的方法,探究小黄鱼早期在不同浓度E2(1、10和50μg/L)浸浴的条件下,对生长、性腺发育及性别分化的影响。结果表明,在处理后20~60 d时,E2浸浴实验组在体长和体重上均显著低于对照组,60 d时,GSI(Gonadosomatic index)水平E2浸浴实验组和雌性对照组C(0)相比无显著性差异。解剖学及组织观察学表明,E2浸浴实验组1μg/L与10μg/L性腺头端粗短,出现二时相至三时相卵母细胞,且有明显卵巢腔,50μg/L组部分性腺发育畸形。当E2浓度达到10μg/L以上时,未发现雄性及未分化性腺,且随着浓度的上升,出现畸形的比率上升。E2浸浴组雌性率分别82%,88%,63%,对照组性比近似1:1。综上所述,E2浸浴可以抑制小黄鱼的生长,且能使小黄鱼群体雌性化。     

猪卵母细胞体外成熟及电激活后发育能力的研究

探讨了不同的成熟培养液及外源激素对猪卵母细胞体外成熟培养及电激活后孤雌胚胎发育能力的影响. 结果表明：（1）改良M199（mM199）组猪卵母细胞成熟率显著高于M199组，且这两组又较NCSU-23组能极显著提高卵母细胞的成熟率. （2）孕马血清（PMSG）+人绒毛膜促性腺激素（hCG）+促卵泡素（FSH）组卵母细胞成熟率略高于FSH+促黄体素（LH）组，两者卵母细胞成熟率极显著高于尿促性腺激素（hMG）组. （3）含胎牛血清（FBS），猪卵泡液（PFF），表皮生长因子（EGF）的培养液较对照组在卵母细胞成熟率上无显著差异，但均能显著提高电激活后的卵裂率.添加EGF组桑囊率明显高于不添加组.但分别添加FBS和PFF组较对照组在桑囊率上均无显著差异. （4）卵丘细胞扩散与卵母细胞第一极体排出之间无直接相关性，但扩散程度好能提高电激活后的卵裂率.     

分离方法和卵巢发育状况对猪腔前卵泡分离及体外培养的影响

以直径在200～300μm的腔前卵泡为研究对象，研究了不同分离方法及卵巢不同发育状况对猪腔前卵泡分离及体外培养的影响。结果表明，显微分离法每个卵巢可采集的卵泡数极显著高于剪碎过滤法（24．75±2．99vs2．60±0．35，P〈0．01），显微分离法分离每个腔前卵泡所用的时间极显著低于剪碎过滤法（5．40±0．44vs15．52±1．18，P〈0．01）；有黄体和无黄体的卵巢对卵泡分离的影响差异不显著（26．10±2．02vs24．35±2．32，P〈0．05），但有黄体的卵巢分离的卵泡数稍多于无黄体的卵巢。此外，对两种分离方法分离出的腔前卵泡进行培养研究表明，两种分离方法对卵泡体外生长和存活，以及正常卵母细胞比率均没有显著影响（P〉0．05）。     

大黄素对辣椒素诱导的TRPV1通道电流的抑制作用

本研究通过显微注射技术将TRPV1 cRNA(1ng/nL)注射至非洲爪蟾卵母细胞(20nL/个)体内,放置于G-ORi培养液中,并在(19±1)℃的恒温培养箱内表达.同时利用灌流技术将不同浓度梯度的大黄素、辣椒素按照设计顺序依次灌流进入流动腔,同时控制流速保证非洲爪蟾卵母细胞完全浸没于流动腔内.使用双电极电压钳技术记录0.1,1.0,10.0,50.0μmol/L浓度梯度的大黄素对500nmol/L浓度的辣椒素激活TRPV1电流的影响.得到大黄素作用辣椒素激活TRPV1通道的抑制作用表现出浓度依耐性和电压非依赖性,IC50=38μmol/L,Hill系数n=0.5.结果表明了大黄素对TRPV1位点的相互作用是负协同的,在天然药物里面是一类比较弱的拮抗剂,且大黄素在ORi溶液中开始析出沉淀的浓度在50~60μmol/L之间.我们首次发现了大黄素能够抑制TRPV1通道电流,这可能为开发新的镇痛药物提供理论基础.     

Follicular growth,differentiation and atresia

Only limited numbers of primordial follicles in mammalian ovary grow and differentiate to reach the stage of dominate follicles and ovulate. 99% of the follicles in the ovary undergo atresia at various stages of development.Regulation of follicular growth, development and atresia is a complex process and involves interactions between endocrine factors and intraovarian regulators. This review summarized: i ) FSH may not be a survival factor in regulating slow-growing preantral follicles. Some locally produced growth factors, activin and orphan receptors might play a more important role at this stage, ii ) Estrogen, activin/inhibin and follistatin coordinate with FSH to regulate and control follicle differentiation, iii) There are two types of follicular atresia induced by apoptosis which originates from GC or oocyte, respectively. Early translation of tPA mRNA into tPA protein in oocyte may be associated with oocytea apoptosis.     

四氯化碳对雌性小鼠卵母细胞成熟和体外受精等影响

探讨了四氯化碳对小鼠生殖作用的影响.采用小鼠卵母细胞体外培养、体外受精的方法研究了四氯化碳对小鼠卵母细胞的成熟和体外受精的影响.结果表明:四氯化碳对小鼠脏器、超排卵的卵母细胞数量和卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率.四氯化碳可以破坏卵母细胞减数分裂进行,降低卵母细胞的受精能力,具有潜在的生殖毒性.     

Sources of cumulus expansion enabling factor (CEEF) in porcine follicles

It was shown that expansion of porcine cumulus did not depend on oocyte-secreted factor(s), and it is therefore presumed that porcine CEEF may not be produced exclusively by the oocyte. In this experiment, we used mouse oocytectomized complexes (OOX), which were incapable of CEEF production, to assess the secretion of CEEF by evacuated zona, oocytes of different quality and somatic cells in the porcine follicles. The results showed that: (ⅰ) Evacuated zonae from both porcine and mouse oocytes did not produce CEEF. (ⅱ) Porcine oocytes of A, B and C types from 3—6 mm follicles were not significantly different in both production and activity of CEEF. (ⅲ) Both porcine OOX from 3—6 mm follicles and granulose cells from < 1 mm follicles secreted CEEF in a large quantity, independent of gonadotropins; mural granulose cells from 3—6 mm follicles, however, produced neglectable amount of CEEF. (ⅳ) The follicular fluid from 3—6 mm porcine follicles contained CEEF activity that was concentration-dependent, and thus it enabled cumulus expansion in 60% mouse OOX when used at 10% of concentration, but the expansion rate of mouse OOX decreased to 9% when the concentration was increased to 50%. (ⅴ) Mouse OOX cultured in porcine CEEF-containing M199 expanded only in the presence of gonadotropins, suggesting that the activity of porcine CEEF is hormone-dependent.     

Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes

The c-mos proto-oncogene is expressed as a maternal mRNA in oocytes and early embryos of Xenopus laevis, but its translation product pp39mos is detectable only during progesterone-induced oocyte maturation. Microinjection of mos-specific antisense oligonucleotides into oocytes not only prevents expression of pp39mos, but also blocks germinal vesicle breakdown, indicating that it functions during reinitiation of meiotic division.     

Roles of protein kinase C in oocyte meiotic maturation and fertilization

Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis, release the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian oocytes, and there is evidence showing that classical PKCs may be the principal mediator of oocyte cortical reaction.     

体外培养时间和表皮生长因子对牦牛卵母细胞成熟及孤雌胚胎体外发育的影响

研究在成熟液中添加表皮生长因子（Epidermal growth factor,EGF）以及成熟时间对牦牛卵母细胞体外成熟及孤雌胚胎体外发育的影响,以确立牦牛卵母细胞体外成熟的最佳体系.结果表明,成熟液中添加EGF组的成熟率明显高于未添加组（P＜0.05）,并且牦牛卵母细胞的成熟率随着EGF添加的浓度增加也不断上升;随着体外培养时间的延长,成熟率逐渐增加,培养24 h后成熟率趋于稳定;胚胎体外培养液中添加EGF能显著提高孤雌胚胎的8-细胞和囊胚形成率（P＜0.05）.由此推测：在体外培养22-24 h,且添加40 μg/mL EGF最有利于牦牛卵母细胞体外成熟;胚胎培养液中添加40μg/mLEGF能显著提高牦牛孤雌胚胎的体外发育能力.     

Meiotic initiation by the mos protein in Xenopus.

When fully grown Xenopus oocytes are stimulated by progesterone, a period of protein synthesis is necessary for maturation. Synthesis of the mos proto-oncogene product, pp39mos, is necessary for the activation of M-phase promoting factor (MPF) in meiosis I. On the basis that mos is translated de novo on hormonal stimulation of Xenopus oocytes and that injecting mos RNA into oocytes induces their maturation, we have proposed that the mos protein is a candidate initiator of oocyte maturation, needed to trigger the conversion of precursor MPF into its active form. To determine whether mos is the only protein required for initiating maturation, we have produced a soluble, active recombinant mos protein and injected it into Xenopus oocytes. We report here that in the absence of protein synthesis that mos protein efficiently induces germinal vesicle breakdown and the activation of MPF. The oocytes, however, do not proceed into meiosis II. Thus, the mos protein fulfills the requirements of an initiator protein, but the synthesis of one or more additional proteins may be necessary to complete oocyte maturation.     

Function and interaction of maturation-promoting factor and mitogen-activated protein kinase during meiotic maturation and fertilization of oocyte

Mitogen-activated protein kinase (MAP kinase) cascade and maturation-promoting factor (MPF) play very important roles during meiotic maturation and fertilization of oocyte. Interaction between MAP kinase and MPF influences meiotic maturation and fertilization of oocyte throughout the animal kingdom, including stimulation of germinal vesicle breakdown (GVBD), suppression of DNA replication, control of meiotic chromosome segregation, maintenance of metaphase II arrest, and resumption and completion of second meiosis. This review focuses on the function and interaction of MAP kinase and MPF during meiotic maturation and fertilization of oocyte.     

中华蟾蜍卵巢性类固醇激素受体的免疫细胞化学研究

为探讨两栖动物卵巢内不同性类固醇激素受体在卵子发育中的调控作用.采用免疫细胞化学方法,对中华蟾蜍不同发育时期卵泡中的雌激素受体、孕激素受体和雄激素受体进行了定位检测.结果发现,三种受体在不同发育时期的卵泡中均有阳性反应:雌激素受体在卵黄合成期和卵黄合成后期的滤泡细胞表达为强阳性,在卵母细胞胞质和核膜中呈弱阳性反应;孕激素受体在卵黄合成早期的滤泡细胞和卵母细胞质、核膜中表达均较弱,在卵黄合成后期表达均增强;雄激素受体在卵黄合成早期的滤泡细胞和卵母细胞胞质、核膜中表达也均较弱,在卵黄合成后期卵母细胞胞质和核膜中表达略有增强.