东北细毛羊胎羊卵巢卵泡超微结构研究

为给胚胎工程研究提供基础材料,应用电子显微镜对东北细毛羊不同胎龄胎羊卵巢卵泡的超微结构进行了观察研究,结果表明:1)7周龄胎儿卵巢形成了原始卵泡雏形,10周龄才形成结构较完整的原始卵泡。2)卵母细胞呈圆形或椭圆形。卵母细胞膜双层不规则;核仁呈网状,单核仁或双核仁;核大多位于卵母细胞的边缘;并发现由双核构成的卵母细胞和胞质中形成不含细胞器的“透明区”;在10周胎龄卵巢,发现有1条线正将1个卵母细胞一分为二。早期线粒体多数圆形,但发现了1个线状的线粒体。随着卵母细胞发育,除圆形以外,还出现了类似菱形、不规则型、幼稚型的线粒体。单个粗面内质网可见,但高尔基体却很少见到。12周龄原始卵泡形成类似“透明带”物质。3)卵泡细胞核多数不规则,个别呈扁长形或纺锤形;卵泡细胞环绕于卵母细胞周围,早期数量少,随着卵泡发育逐渐增多。4)东北细毛羊胚胎卵巢卵母细胞发育的各阶段都有一定数量卵母细胞发生退行性变化———闭锁。     

授粉方式对板栗胚胎发育的影响

为研究受精对板栗胚胎发育的影响,以“遵玉”品种10年生树为试验材料,通过控制授粉试验,观察不同授粉处理板栗胚珠、总苞和子房的形态发育及其氮素和碳水化合物含量的变化趋势。结果表明,受精不良导致空苞的产生,人工授粉板栗空苞率为0%,自然授粉板栗空苞率为12.37%,不授粉板栗空苞率为100%。在板栗果实发育早期,不授粉板栗与人工授粉、自然授粉板栗的胚珠大小、总苞和子房外观形态及碳水化合物含量变化差异不显著,但其氮素含量显著小于其他2个授粉处理板栗;授粉后16 d,正常发育胚珠大小显著大于发育异常胚珠,此时人工授粉、自然授粉板栗总苞和子房体积显著大于不授粉的板栗子房,碳水化合物含量差异不显著;幼胚发育期,人工授粉、自然授粉板栗子房可溶性糖、蔗糖和淀粉含量显著高于不授粉板栗子房的含量。     

卵巢颗粒细胞自噬与PI3K/AKT/FOXO3a信号通路的相关性

细胞自噬是哺乳动物细胞物质代谢的一个重要机制,与细胞凋亡共同参与卵巢卵泡的发育和闭锁,并发挥重要的作用。近年研究发现,磷脂酰肌醇3-激酶/蛋白激酶B（phosphatidylinositol 3-kinase/protein kinase B,PI3K/AKT）信号通路参与卵巢疾病的发生。PI3K和AKT的过度激活可使原始卵泡过早发育以及卵泡过快凋亡,卵巢颗粒细胞作为卵泡发育重要的支持细胞,其功能的减退或凋亡很可能引发一系列女性内分泌方面的疾病。FOXO3a转录因子是PI3K/AKT信号通路下游的重要靶蛋白之一,参与抗增殖和凋亡。本文就关于卵巢颗粒细胞自噬与PI3K/AKT/FOXO3a信号通路的相关进展加以综述。     

磷酸二酯酶在猪卵巢的表达与相关中药对其活性的影响

为了测定猪卵巢组织磷酸二酯酶（PDE）同工酶基因表达类型与活性规律，研究相关中药的作用机理，为繁殖障碍性疾病治疗积累资料。作者提取中国实验用小型猪卵巢组织总RNA，应用反转录聚合酶链反应（RT-PCR）测定PDE同工酶在其中的表达。选取催情散等复方及单味药，运用高效液相色谱法（HPLC）根据作用前后cAMP/cGMP含量的变化，分析对PDE活性的影响。结果检测的18个PDE亚型中除PDE4C外，PDE 1A、1B、1C、2A、3A、3B、4A、4B、4D、5A、7A、7B、8A、8B、9A、10A、11A mRNA在卵巢组织中均有表达。卵巢组织PDE活性较强，其中cGMP-PDE活性大于cAMP-PDE活性。催情散对卵巢组织cGMP-PDE活性均有较强的抑制作用，其中单味药淫阳藿作用最强。研究结果表明卵巢组织含有丰富的PDE同工酶，在通过环核苷酸水平卵巢机能调节中发挥着重要作用，中药催情散的作用与显著抑制cGMP-PDE活性有关。     

高频饮用牛奶和葡萄糖对雌性大鼠生殖机能的影响

选用22d断奶SD大鼠高频饮用牛奶或葡萄糖40d,分别记录各组动物的阴道开张时间和阴道黏液、每周日增重、卵巢及子宫所占体重百分比、血糖水平,收集卵巢进行组织学处理以观察卵泡发育情况,探讨高频饮用牛奶和葡萄糖对雌性大鼠初情期的影响.结果显示,奶组和糖组大鼠较对照组阴道开张时间来得早,预示初情期可能提前,而且,奶组和糖组大鼠的卵巢占体重百分比较对照组高(P＜0.05).牛奶组血糖水平比对照组高,但糖组与对照组血糖水平差异不显著.通过对卵巢进行石蜡切片、HE染色及卵泡计数,结果显示:奶组的闭锁卵泡、每张切片卵泡总平均数都比对照组多(P＜0.05),而葡萄糖组的黄体数比对照组少(P＜0.05).试验表明高频饮用牛奶和葡萄糖可对性成熟前雌性大鼠的生殖机能产生一定影响.     

伊犁鹅产蛋前后各期卵巢转录谱的构建及卵泡发育相关基因的分析

【目的】构建产蛋前后各期伊犁鹅卵巢转录组文库,结合生物信息学分析揭示不同产蛋期伊犁鹅卵巢组织差异表达基因,并鉴定出影响鹅卵巢发育的关键基因,为伊犁鹅的繁殖调控提供理论参考。【方法】选择处于开产期（KL组）、产蛋期（CL组）及休产期（XL组）的伊犁鹅各4只,屠宰后取其卵巢组织,用于构建卵巢转录组文库。通过新基因挖掘、基因差异表达、基因注释以及蛋白互作网络分析筛选出与鹅卵泡发育相关的候选基因;随机挑选8个差异表达基因,应用实时荧光定量PCR验证其表达情况。【结果】伊犁鹅卵巢组织学结果表明,在开产期时,伊犁鹅卵巢表面有大量初级卵泡,而产蛋期卵巢则显示出卵泡的层级性,在休产期卵巢中可观察到卵泡出现向内凹陷、闭锁的现象。通过转录组测序（RNA-Seq）,从构建的12个伊犁鹅卵巢cDNA文库中获得有效读数57 811 186~85 328 377条,Q30值均>93.38%,每个样品所产的测序读数于鹅参考基因组上的比对率在82.79%~89.24%。在卵巢中注释的新基因共有1 112个,单核苷酸多态性（SNP）位点总数为1 642 273~2 425 069个;SNP和插入缺失（InDel）均主要注释于内含子区域;各时期的可变剪切类型主要集中为最后1个外显子可变剪切（TSS）及第1个外显子可变剪切（TTS）。在KL vs CL、XL vs CL及XL vs KL组中分别有337、1 136和525个差异表达基因,共有差异表达基因为α2A肾上腺素能受体（ADRA2A）、表皮蛋白（CP）、非转移性黑色素瘤糖蛋白B （GPNMB）及α-1-抗胰蛋白酶样（LOC106033756）。GO功能富集分析发现,差异表达基因主要富集在肽基酪氨酸磷酸化的正调控、细胞黏附及质膜外侧等过程。KEGG通路富集分析发现,差异表达基因主要显著富集于神经活性配体-受体相互作用、ECM-受体相互作用及类固醇生物合成等。结合蛋白互作网络分析,筛选到与鹅卵巢发育相关的潜在调控因子Bruton’s酪氨酸激酶（BTK）、血小板源性生长因子受体α（PDGFRA）、整合素β3（ITGB3）等。实时荧光定量PCR结果显示,RNA-Seq结果准确可靠。【结论】本研究揭示了产蛋前后各期伊犁鹅卵巢组织中的基因表达差异,筛选到影响伊犁鹅卵巢发育的神经活性配体-受体相互作用、ECM-受体相互作用、类固醇生物合成等重要通路与BTK、PDGFRA和ITGB3等关键候选基因,为了解鹅卵巢组织调控产蛋性能的分子机制提供了理论支撑。     

Evidence that 17α,20β,21-trihydroxy-4-pregnen-3-one is a maturation-inducing steroid in spotted seatrout

Ovarian steroidogenesis during final oocyte maturation (FOM) in the spotted seatrout (Cynoscion nebulosus) was investigated by incubating ovarian fragments with tritiated pregnenolone, followed by chromatographic separation of the radioactive products. The major tritiated steroid produced during FOM comigrated with 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-dihydro-11-deoxycortisol, 20β-S) on HPLC and TLC. Only minor amounts of radioactive material coeluted with 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P), 11-deoxycorticosterone (DOC), estradiol-17β and testosterone standards in the HPLC system. Additional chromatography by TLC confirmed the presence of radioactive estradiol-17β and testosterone but not 17α,20β-P and DOC. All the ovarian steroids producedin vitro during FOM were assayed for their ability to induce germinal vesicle breakdown (GVBD) of spotted seatrout oocytes. Twenty grams of ovarian tissue were incubated with human chorionic gonadotropin and exogenous pregnenolone. The steroidal products were purified by HPLC and TLC. Most of the maturation-inducing activity was confined to steroidal material which comigrated in these systems with 20β-S. This material was active at a concentration of 1 ng steroid/ml medium in the GVBD assay. Smaller amounts of material which coeluted with 11-deoxycortisol, DOC, 17α,20β-P and several minor unidentified fractions induced GVBD at concentrations of 10 ng steroid(s)/ml. The structure-activity relationships of authentic steroids in inducing GVBD of spotted seatrout oocytes was investigated. Hydroxylation at the 17α, 20β or 21 positions increased potency to induce GVBD. Steroids with multiple hydroxyl groups at the 17α and 20β positions (17α, 20β-P) and at the 17α, 20β, and 21 positions (20β-S) had maximum biological activity in the GVBD bioassay. The results suggest that 20β-S is a major maturation-inducing steroid in spotted seatrout.     

In vitro steroidogenesis by gonads of the Russian sturgeon, Acipenser gueldenstaedti Brandt

Ovaries and testes from the Russian sturgeon at different stages of sexual maturity were incubated with ;sup3;H-androstenedione or ³H-pregnenolone. The major metabolites of androstenedione in both sexes were testosterone and 5- and 5ß-androstanediols, but no evidence was found for the gonadal production of 11-oxygenated androgens. Pregnenolone was converted to 17-hydroxyprogesterone, androstenedione and testosterone together with reduced metabolites. 11-Oxygenated androgens were found in female serum by gas chromatography-mass spectrometry (GC-MS), in serum of both sexes by radioimmunoassay (RIA), and was detectable by RIA in interrenal but not gonadal tissue. The results suggest that sturgeon may differ from teleosts in that 11-oxygenation may take place in extragonadal tissues.