LH与hCG对昆明小鼠卵母细胞体外成熟的影响

目的研究促黄体素（LH）、人绒毛膜促性腺激素（hCG）对昆明小鼠卵母细胞体外成熟的影响。方法小鼠经注射孕马血清促性腺激素（PMSG）48h后,摘取卵巢获得未成熟卵母细胞,分别在含不同浓度的LH和hCG的成熟液中,或将LH和hCG以不同的浓度组合加入到成熟液,进行体外成熟。结果经15.16h的成熟培养,5个浓度LH组中的极体率均高于对照组,其中200IU/mL组显著高于50IU/mL、400IU/mL、300IU/mL组和对照组（P〈0.05）;5个浓度hCG组的极体率与对照组极体率无显著差异（P〉0.05）;协同组中15IU/mL hCG＋200IU/mL LH组的极体率显著高于对照组和其它各处理组。结论LH对小鼠卵母细胞的体外成熟有一定的促进作用。     

年轻与老龄小鼠未成熟卵母细胞之间的生发泡移植

为研究不同年龄来源的细胞质或细胞核对卵母细胞成熟、钙振荡及核型的影响,在6~8周龄(6W)小鼠与9月龄(9M)和12月龄(12M)小鼠卵母细胞之间进行了生发泡(GV)互换。通过显微操作和电融合获得了5组重组卵母细胞。重组卵母细胞和对照卵母细胞经Sr2 诱导后呈现相似的钙震荡模式。6WGV-6W胞质体组、6WGV-9M胞质体组和6WGV-12M胞质体组成熟卵母细胞染色单体提早分离的比率与6~8周龄对照组比较无差异(P>0.05),但显著低于12月龄对照组(P<0.01)。而9MGV-6W胞质体组和12MGV-6W胞质体组成熟卵母细胞染色单体提早分离的比率则明显增加。这些结果表明由年轻与老龄小鼠之间GV互换所重组的卵母细胞能够正常成熟和产生钙震荡,与衰老相关的减数分裂异常似乎归因于细胞核或染色体而不是细胞质。     

化学联合激活可明显提高小鼠孤雌胚胎发育率

为探讨一种高效的小鼠卵母细胞孤雌激活的方案,进一步提高孤雌囊胚发育率。用不同浓度的氯化锶及不同作用时间的乙醇,并分别联合6-DMAP对不同卵龄小鼠卵母细胞进行活化,统计小鼠卵母细胞卵裂率和体外发育状况。结果显示,15～16h、18～19h和20～21h卵龄组卵母细胞经6mmol/LSrCl2联合6-DMAP处理后,三组的激活率随卵龄增长而升高,其中20～21h卵龄组显著高于15～16h、18～19h组(P<0.05),激活胚胎的发育率以18～19h时最高;6mmol/L和10mmol/L的SrCl2联合6-DMAP均能有效地激活小鼠卵母细胞,激活率分别为76.4%和83.6%,桑葚胚率分别为50.0%和56.3%;70ml/L乙醇联合6-DMAP以处理7min组获得了较好的激活率和囊胚发育率,分别为77.1%和42.4%,囊胚率均显著高于4min和10min处理组(P<0.05)。6-DMAP与SrCl2或乙醇联合应用可以有效抑制第二极体的排出,提高激活胚的二倍体比率;孤雌囊胚的平均细胞数显著低于正常受精囊胚(P<0.05)。不同激活方案对孤雌活化胚的核型和发育能力的作用差异较大,小鼠卵母细胞孤雌激活率与卵龄...     

雌二醇和孕酮对小鼠卵母细胞体外成熟的影响

目的研究雌二醇（E2）、孕酮（P4）对昆明小鼠卵母细胞体外成熟的影响。方法小鼠经注射孕马血清促性腺激素（PMSG）后48 h,摘取卵巢获得未成熟卵母细胞,分别在单独含不同浓度的E2或P4的成熟液中,或在同时含有不同浓度E2和P4的成熟液中,进行体外成熟,并对经过E2和P4处理成熟的卵母细胞进行体外受精。结果经15-16 h的成熟培养,各E2处理组的卵母细胞第一极体排出率虽然均略高于对照组,但它们之间无显著差异;各组卵母细胞体外受精48 h后,1000 ng/mL E2处理组的卵裂率显著低于其他各处理组和对照组,但各组的囊胚率之间无显著差异。各P4处理组的极体率和卵裂率,与对照组相比无显著差异,但是各处理组的囊胚率均极显著低于与对照组;两种激素协同处理组中[1000 ng/mL E2＋1000 ng/mL P4]组的极体率显著高于其他各处理组;而与对照组的极体率差异不显著。结论P4对小鼠卵母细胞的后期发育能力有一定的抑制作用,而E2却对小鼠卵母细胞的成熟及其发育潜力无明显作用。     

两种甲状腺功能亢进症动物模型的对比研究

目的：复制两种甲状腺功能亢进症动物模型,对比研究它们成型性及可持续性的异同,为研究治疗甲亢的药物提供良好的评价载体。方法优甲乐模型：大鼠灌胃给予优甲乐50μg/100 g鼠重共21 d,于末次给药后第1、7、14天分别于眼底取血和接取尿液,检测相关指标;小肠结肠炎耶尔森氏菌模型：大鼠尾静脉注射小肠结肠炎耶尔森氏菌,于第0、5、10、15、20天共注射5次,并于末次注射后第5、15、25天大鼠眼底取血和接取尿液,检测相关指标。结果优甲乐模型：造模后T3、T4、FT3、FT4显著升高,但造模型后1周即恢复正常,尿液代谢的PCA图中显示模型组整体代谢网络偏离空白组,2周后恢复正常;小肠结肠炎耶尔森氏菌模型：造模后第15天和25天时T3和T4升高,第25天时TSH显著升高,尿液代谢的PCA图中模型组偏离空白组,而第15天和25天时未回到空白组位置。结论优甲乐甲亢模型成型性好但恢复迅速,作为药效评价时适合预防给药;而小肠结肠炎耶尔森氏菌甲亢模型成型性好并且持续性较好,作为药效评价时可采取治疗给药方式。     

胞浆内精子注射技术生产小鼠

以piezo操作系统为技术支撑 ,在掌握小鼠卵母细胞胞浆内精子注射技术 (ICSI)的基础上 ,进行了ICSI技术生产试管小鼠的尝试。来自成年昆明 (KM)小鼠附睾尾的新鲜精子 ,剪切去尾后 ,直接将精子头注射到B6D2F1小鼠卵母细胞质中 ,注射后 1h ,83.3%的卵母细胞存活。6h时 ,84.0 %的成活卵子成功受精 ,形成原核 ,排出PB2 体外培养的ICSI胚胎 ,卵裂率 (98%vs 94.7% )和 4-细胞期胚胎比率 (89.5%vs 92.1% )均与培养的体内受精卵没有差异 (P >0.05 ) ;但是 ,桑椹胚(63.8%vs84.2% )和囊胚发育率 (25.7%vs68.4% )极显著地 (P <0.01)低于对照组。120枚原核期胚胎移植给 7只假孕受体后 ,4只受孕小鼠共产出 28只ICSI小鼠 (23.3% )。健康成年的 25只ICSI小鼠都没有明显的生理和行为异常。随机选择其中的 20只小鼠 ,分别进行ICSI小鼠间、ICSI与KM小鼠间共 12组的交配 ,结果所有雌鼠妊娠产仔。在成功建立小鼠ICSI技术的基础上,成功获得了我国的首例ICSI小鼠,并且证明这些ICSI小鼠都具有正常的繁殖后代的能力。     

乙醇及6-DMAP对小鼠卵母细胞孤雌激活的研究

实验研究了乙醇、6-DMAP以及二者联合使用时对注射hCG后18小时采集的小鼠卵母细胞孤雌激活的效果。结果证明:(1)用5％的乙醇分别作用5和10分钟及10％的乙醇分别作用5和10分钟,小鼠卵母细胞的孤雌激活率分别为41.3％、63.7％、57.9％和85.6％。说明在一定范围内,随着乙醇浓度和作用时间的增加,小鼠卵母细胞孤雌激活率有上升的趋势。(2)用2mM 6-DMAP作用2、4和6小时,小鼠卵母细胞的孤雌激活率分别为 12.0％、25.0％和40.0％。说明随着6-DMAP作用时间的增加,小鼠卵母细胞的孤雌激活率有所升高。(3)用5％乙醇作用5分钟,再用含有2mmol/L 6-DMAP的培养液培养6小时,小鼠卵母细胞的孤雌激活率可达65.5％,明显高于单独使用5％乙醇作用5分钟或单独使用2mmol/L 6-DMAP作用6小时卵母细胞的孤雌激活率。(4)用10％的乙醇作用5分钟,再用含有2mmol/L 6-DMAP的培养液培养6小时,小鼠卵母细胞的孤雌激活率达到100％,远远高于单独使用10％乙醇作用5分钟或单独使用2mmol/L 6-DMAP作用6小时卵母细胞的孤雌激活率。(5)在单独使用乙醇刺激时,激活卵母细胞中直接卵裂(2-细胞)的比率随乙醇作用强度的增加而增加,最高达62.5％;但6-DMAP则抑制激活卵母细胞的直接卵裂,增加二原核卵的比例。     

小鼠精子注入兔卵母细胞受精研究

利用显微操作仪将小鼠精子注入家兔卵母细胞的胞质内和透明带下,对鼠兔异种精卵互作和异种受精胚胎的发育进行了研究,并对注射精子的数量及卵的体外成熟时间等影响鼠兔异种显微受精的因素进行了探讨,结果如下:(1)将小鼠精子分别注入兔卵胞质内和透明带下,均能激活兔卵母细胞,导致精核解聚和原核形成;(2)小鼠精子注入兔卵胞质内和透明带下受精,杂种胚胎体外培养能发育到8-细胞期;(3)鼠兔异种受精4-细胞胚胎染色体标本制备观察结果表明,它们为正常二倍体;(4)鼠兔异种受精4-细胞胚胎的超微结构观察结果表明,它们极近似兔正常4-细胞胚胎的超微结构;(5)将小鼠精子注入兔卵透明带下,注射5—10个精子组卵的受精率(32.4%)和卵裂率(16.2%)均高于注射单个精子组的,但二组间差异不显著(P>0.05);DM 15%NCS液中体外成熟培养11—12h兔卵透明带下注入1—2个小鼠精子后的受精率(42.3%)和卵裂率(30.8%)均高于体外成熟培养24—25h组的,但二组间差异未达到显著水平(P>0.05)。     

蜂蜜对洛哌丁胺诱导的 便秘小鼠肠道微生态的影响

目的观察蜂蜜对洛哌丁胺诱导的便秘小鼠肠道微生态的影响。方法雄性Balb/c小鼠30只,随机分成两组:正常对照组(10只)、模型组(20只),同时模型组再分为自然恢复组(10只)、蜂蜜组(10只)。应用洛哌丁胺制备便秘小鼠模型,分别于造模后、给蜂蜜后第5天对每只小鼠进行称重、采便;之后于第12天称重、采便,处死小鼠,进行小肠推进率、结肠组织血管活性肠肽(VIP)、P物质(SP)、5-羟色胺(5-HT)测定和肠道菌群检测。结果洛哌丁胺造模后的第5天,模型组小鼠粪便含水量及体质量下降;蜂蜜组小鼠体质量下降,但粪便含水量无明显变化。蜂蜜治疗至第12天时,蜂蜜组小鼠与模型组比SP增加,粪便含水量及小肠推进率增加,蜂蜜组小鼠与模型组比VIP、5-HT变化不明显。此外蜂蜜干预后便秘小鼠肠道中拟杆菌属和厚壁菌属的细菌丰度有所降低,Alistipes obesi的含量增加。结论蜂蜜对小鼠5-HT分泌水平无明显影响;可以提高便秘小鼠结肠组织中SP含量、增加粪便含水量、加快小肠推进率;调节肠道微生态失衡,对便秘发挥一定的治疗作用。     

体外培养小鼠卵母细胞及其生长分化因子-9基因表达

体外培养小鼠的窦前卵泡以得到第二次减数分裂中期(MⅡ)卵母细胞,比较体外发育卵母细胞与体内生长的卵母细胞生长分化因子-9(GDF-9)的基因表达量,探讨GDF-9的表达对卵母细胞体外发育成熟的影响。选择体外培养第2天(D2)、D4、D6、D8、D10、D12卵母细胞作为体外发育组;同窝雌性小鼠出生后D12、D14、D16、D18、D20、D22卵母细胞作为体内发育组;半定量逆转录多聚酶链反应技术分别检测两组MⅠ卵母细胞GDF-9基因表达量。结果体外培养小鼠窦前卵泡可以得到MⅡ期卵母细胞,卵泡成活率、窦腔形成率、卵母细胞成熟率分别达到89·5%、51·8%和56·6%。小鼠卵母细胞GDF-9基因表达量随发育时间的改变而发生变化,而体外发育D8—12卵母细胞GDF-9表达量显著低于同期体内发育卵母细胞(P<0·05)。体外发育D8—12卵母细胞GDF-9基因表达量低于同期体内发育的卵母细胞的原因之一可能是其发育潜能较低。     

碱基切除修复基因Lig3对小鼠胚胎神经发育的影响

摘要 目的：DNA连接酶III（DNA ligase III, Lig3）基因是碱基切除修复通路中的关键基因,在胚胎发育过程中发挥重要作用,通过研究Lig3基因在叶酸代谢障碍状态下的表达情况,探讨其对小鼠胚胎神经发育的影响。方法：采用无特定病原体（specific pathogen free, SPF）级C57BL/6J成年小鼠（8-9周,18-20 g）,雌雄1:1合笼,孕鼠随机分为实验组和对照组,孕7.5天实验组腹腔注射4.5 mg/kg体重甲氨蝶呤（Methotrexate, MTX,二氢叶酸还原酶抑制剂）诱导产生叶酸代谢障碍的小鼠神经管畸形（neural tube defects, NTDs）模型,对照组腹腔注射等体积的生理盐水。孕10.5天体视显微镜下观察胎鼠的发育情况。同时利用200 nM的MTX建立叶酸代谢障碍的小鼠神经干细胞模型。在模型建立成功的基础上,应用实时荧光定量聚合酶链反应(Real time quantitative PCR,RT-qPCR)及免疫印迹（Western blot）等方法研究碱基切除修复通路相关基因Lig3的表达水平。结果：4.5 mg/kg 体重MTX处理孕鼠后胎鼠NTDs的发生率为31.1%（19/61）,而正常对照组未见胎鼠NTDs的发生。在体视显微镜下可见NTDs胎鼠神经管未闭合,而正常胎鼠发育完好。RT-qPCR检测发现叶酸代谢障碍小鼠NTDs 胚胎神经组织中Lig3 mRNA的表达水平明显低于对照组（P<0.05）。Western blot检测发现,与对照组相比,叶酸代谢障碍NTDs胎鼠神经组织中Lig3蛋白水平明显降低（P<0.05）。同时,在MTX处理的神经干细胞中,Lig3的表达水平明显低于对照组（P<0.05）。对凋亡相关蛋白Cleaved caspase-3进行检测发现MTX处理后的NTDs胎鼠神经组织及细胞模型中其表达均明显增加,表明细胞凋亡增加。结论：在叶酸代谢障碍前提下,Lig3表达降低,DNA修复功能减弱,细胞凋亡增加,导致NTDs的发生,为NTDs及出生缺陷的防控提供新思路。     

牛磺酸抗小鼠动脉粥样硬化的研究

为了探讨牛磺酸抗小鼠动脉粥样硬化的影响及可能机制,将C57BL/6J小鼠分成五组,正常对照组给予基础饲料喂养,高脂组给予高脂饲料,牛磺酸组分别给予含1.0%、3.0%和5.0%牛磺酸的高脂饲料,实验时间为90d。结果表明,高脂组小鼠主动脉内膜和肝脏发生了粥样硬化病变和脂肪变性,而牛磺酸组病变程度随剂量增大而减轻。高脂组较正常对照组血清及肝脏甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-c)、动脉硬化指数(AI)显著升高,高密度脂蛋白胆固醇(HDL-c)显著降低;牛磺酸组较高脂组显著改善。可见牛磺酸可通过改善脂质代谢紊乱发挥抗动脉粥样硬化的作用。     

饥饿小鼠卵母细胞中自噬和凋亡的电镜研究

小鼠（Mus musculus domesticus）原始卵泡形成在出生后3 d内进行得最剧烈,此时有大量卵母细胞丢失。出生后不久原始卵泡库就建立起来,新生鼠都会经历一段时间饥饿再摄入母乳营养,对出生后的子鼠饥饿处理时,出现了自噬和凋亡的动态变化,自噬和凋亡都可以影响细胞的存活,这很可能与卵母细胞的大量丢失有关。在本项研究中,将对照组子鼠正常母乳喂养,处理组子鼠与母鼠分开,完全不给予母乳。分别收取饥饿1.5 d与2 d子鼠的卵巢制作电镜切片,每组3只子鼠,每只子鼠3张电镜切片,每组共统计9张切片。在电镜下观察其形态变化。通过观察发现,饥饿1.5 d的子鼠卵巢与正常1.5 d的子鼠卵巢相比,卵母细胞中的自噬小体数量显著增加。这表明,饥饿处理1.5 d促进了卵母细胞的自噬,这可能有助于维持卵母细胞的形态及存活。饥饿处理2 d的子鼠卵巢显示出不同的结果。饥饿2 d的子鼠处于生命的临界阶段,已出现小部分个体死亡。存活子鼠卵巢的电镜形态学观察发现,与正常哺乳2 d的子鼠卵母细胞相比,饥饿2 d子鼠卵母细胞中自噬小体的数量显著减少,并出现了多数卵母细胞凋亡的现象,出现许多凋亡小体。本实验研究结果显示,饥饿处理影响了原始卵泡形成过程中自噬和凋亡动态变化的过程。     

室温下保存的小鼠屠体卵巢GV卵的发育能力

将ICR系雌性小鼠处死并在10℃、15℃、20℃和25℃下依次保存8、14、24和48 h后,采集其体内的卵巢GV期卵,采用常规方法进行体外成熟和体外受精,获得的2细胞期胚经体外培养或胚胎移植观察其发育能力.其结果,在10℃下保存24 h、15℃下保存14 h、20℃下保存8h和25℃下保存4 h后,其体内附有卵丘细胞的GV卵的体外成熟-体外受精后的2细胞率分别为14%、9%、10%和10%,随着保存温度的提高和保存时间的延长,带有颗粒细胞GV期卵的比率明显降低,同时其GV期卵经体外成熟及体外受精后的2细胞率明显降低.在20℃下保存24 h和25℃下保存14 h时,难以获得形态正常的GV期卵;体外受精获得的2细胞期胚经体外培养,总体上有64%的胚胎发育至扩张囊胚,未见有保存温度和保存时间的显著影响,且利用在15℃保存8 h后的GV卵获得2细胞期胚的移植获得正常新生小鼠.上述结果表明,雌性动物室温条件下死亡后,若能短时间及时采集其体内GV期卵并体外成熟、体外受精,体外培养及胚胎移植技术,就有可能获得新生后代.     

Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.     

In vivo effects of arsenite on meiosis, preimplantation development, and apoptosis in the mouse

Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including metabolic abnormalities accompanied by growth inhibition and carcinogenesis. Much of the toxicity of arsenic is known to stem from its uncoupling effects on mitochondria. Because previously we had shown that mitochondrial dysfunction can disrupt oocyte and embryo development, we investigated effects of arsenite on meiotic progression and early embryo development in mice. Six-week-old CD-1 mice were treated with 0 (solvent as control), 8 mg/kg (a dose previously established in mice as the maternal no-observed-adverse-effect level), and 16 mg/kg doses of sodium arsenite every 2 days for a total of seven i.p. injections ver a period of 14 days. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment, was significantly increased in arsenite-treated groups (25% after 8 mg/kg and 62.5% after 16 mg/kg), compared to normal metaphase II in control oocytes. Further, arsenite treatment significantly decreased cleavage rates of zygotes at 24 h, morula formation at 72 h, and development to blastocysts at 96 h in a dose-dependent manner. The total cell number in developed blastocysts did not differ significantly between the 8 mg/kg arsenite treatment and control groups, but was significantly reduced in the 16 mg/kg arsenite treatment group. Moreover, the percentage of apoptotic nuclei was significantly increased in blastocysts following 16 mg/kg arsenite treatment. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis.     

Spaceflight Effects on Cytochrome P450 Content in Mouse Liver

Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine.     

Development of C3 receptors on B lymphocytes derived from normal and memory marrow cells.

Differentiation of B lymphocytes was studied by adoptive transfer of bone marrow into irradiated mice which were then assayed for generation of splenic lymphocytes with complement receptors. These cells developed to normal numbers between 20 and 25 days following marrow cell transplant. Quantitative enhancement of CRL generation occurred when recipient mice were antigen stimulated prior to assay. CRL levels returned to those of normal adults sooner than those found in mice which were not antigen stimulated. A third form of CRL development occurred in mice that were reconstituted with bone marrow cells from antigen-primed donor mice. Here complement receptor-bearing cells appeared much earlier but increased in numbers at a slower, more constant rate to a maximum level on Day 25. Attempts to T-cell deplete recipient mice did not alter receptor development.     

Short-term preservation of porcine oocytes in ambient temperature: novel approaches

The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.     

The effect of beta glucan on MTX induced testicular damage in rats

We investigated the histopathological effects of methotrexate (MTX), a chemotherapeutic agent, and beta glucan (BG), an antioxidant, on rat testis. We used four groups of Sprague-Dawley male rats: MTX, MTX + BG, BG, and control. The MTX group was exposed to a single dose of MTX on the first day of experiment. The MTX + BG group was exposed to a single dose of MTX and BG on the first day of experiment followed by BG for 4 additional days. The BG group was exposed to BG for 5 days. The control group was given saline for 5 days. On day five, all animals were sacrificed and testicular tissue was evaluated for histopathology and the terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick-end labeling assay (TUNEL) was used to detect apoptosis. The apoptotic index (AI) and testicular damage increased in the MTX group compared to the other three groups. Histopathology was reduced in the MTX + BG group compared to the MTX group. Seminiferous tubule diameter was reduced in the MTX group compared to the BG group; we found no difference between control and BG groups. The thickness of th e germinal epithelium was reduced in the MTX group compared to the other groups. We found no difference in testicular weight among the groups. We compared body weight before and after the experiment; weights in the MTX and MTX + BG groups were significantly reduced compared to controls. In the control groups, we found a statistically significant increase in body weight, whereas there was no change in the BG group. We found that MTX causes deleterious effects on testicular tissue and that beta glucan may be protective.