蛋白质营养对动物卵泡发育的影响及其作用机制

卵泡的发育对于雌性动物繁殖性能具有重要影响.蛋白质是机体酶、激素合成以及组织器官构建的原料,对于卵泡发育必不可少.蛋白质营养状态能通过肝脏雌激素受体(ER)影响胰岛素样生长因子Ⅰ (IGF-Ⅰ)、瘦素(leptin)、胰岛素(insulin)等代谢信号,进而传递至下丘脑-垂体-性腺(HPG)轴,对卵泡发育进行调控.本文综述了蛋白质营养对卵泡发育的影响及其作用机制.     

基于iTRAQ技术的水牛卵泡液蛋白质样品预处理的优化

[目的]优化水牛卵泡液蛋白质差异表达样品制备程序,为相对和绝对定量同位素标签(iTRAQ)技术在蛋白质差异表达分析中的应用奠定基础.[方法]在蛋白质样品溶液酶解过程中,选择两种不同沉淀剂沉淀蛋白质,分别比较蛋白质酶解、iTRAQ标记反应和多肽色谱分离的效果;在纳升液相色谱(nano-LC)分离样品前用快速分离小柱进行处理,鉴定色谱分离效果,以确定蛋白质样品预处理的优化程序.[结果]相对于丙酮,使用含0.1％乙酸的丙酮—乙醇混合液(1∶1)作为沉淀剂可获得较好的蛋白质沉淀效果,蛋白沉淀为白色,蛋白质浓度为5.76μg/μL.相对于蛋白质溶液在还原烷基化后直接使用胰蛋白酶酶解,在酶解前沉淀蛋白质溶液可使蛋白样品的酶解效率更高,获得的肽段峰更丰富、峰值更高;可使多肽和iTRAQ试剂间产生有效的标记反应,母离子具有较高的碎裂效率,获得的二级碎片峰更丰富,iTRAQ试剂的特征报告离子116 m/z较117 m/z的谱图强,辨识度高;获得的多肽nano-LC分离色谱峰更丰富,峰值和分辨率更高.相对于直接进行LC分离,在nano-LC分离前用快速分离小柱对多肽溶液进行杂质处理,也可获得丰富的多肽nano-LC色谱峰,且峰值和分辨率较高.[结论]在iTRAQ试剂标记、蛋白质液相分离和差异分析前,使用适宜的沉淀剂处理蛋白质溶液,并以快速分离小柱除去多肽混合液小分子杂质,可使质谱获得更准确的鉴定结果.     

绵羊FecX1基因对排卵率调控的研究进展

绵羊为基因调控排卵率的研究提供了一个有价值的模型。Inverdale(FecX1)基因是在Romney绵羊中发现的一个多胎性基因,定位于绵羊的X染色体上,这是继FecB之后在绵羊中发现的又一个多胎性基因。本文综述了FecX1基因对绵羊排卵率调控研究的最新进展。     

BFF,rbGH对牛卵泡卵母细胞体外受精后发育的影响

利用屠宰黄牛卵巢，对２～５ｍｍ卵泡卵母细胞的体外成熟（ＩＶＭ）、体外受精（ＩＶＦ）、受精卵的体外培养（ＩＶＣ）进行了系列研究。结果表明，在成熟培养液中单独添加１０％Ｄ０ＥＣＳ（发情当天牛血清）获得了卵泡卵母细胞受精后较高的卵裂率（６４．７％）、桑椹胚发育率（３９．９％）和囊胚发育率（２４．８％），说明在成熟培养液中单独添加Ｄ０ＥＣＳ是可行的；在含１０％Ｄ０ＥＣＳ的成熟培养液中再添加１０％或２０％ＢＦＦ（牛卵泡液），均能提高受精卵的卵裂率以及桑椹胚和囊胚的发育率，以添加２０％ＢＦＦ效果较好，其囊胚发育率（３５．０％）显著高于对照组（２４．８％，Ｐ＜０．０５）；在卵泡卵母细胞成熟培养系统和受精卵共同培养系统中添加１０μｇ／Ｌ重组牛生长因子（ｒｂＧＨ），虽对体外受精卵的卵裂率无显著影响（Ｐ＞０．０５），但能显著提高卵裂胚的囊胚发育率（Ｐ＜０．０５）     

Distribution of immunoreactive von Willebrand factor in the microvascular network of bovine cystic follicles

The present study was undertaken to examine whether the von Willebrand factor (vWF) expression in the follicular microvasculature in the cystic follicles differs from that in the atretic follicles. Paraffin sections of healthy, atretic and cystic follicles were immunostained with rabbit monoclonal antibody to vWF. The vWF-positive cells were counted in four different regions of a follicle from the apical to the basal side. In all types of follicles, immunoreactions for vWF were observed in the endothelial cells of capillaries as well as veins and arteries in the theca interna and externa. In the theca interna, vWF-positive areas were significantly lower in the Type A and B cystic follicles compared to advanced and late atretic follicles. In the theca externa, the vWF-positive blood vessels and vWF-positive area were significantly smaller in all types of cystic follicles than in the healthy or atretic follicles. From these results, it is suggested that in the cystic follicles the induction of vWF in the follicular microvasculature system is reduced, which may suppress the degeneration of vascular system. Continuation of stability in vasculature may be one of the factors that delays the tissue regression in the cystic follicles, and also contributes to the accumulation of follicular fluid that originates from the serum.     

共培养系统的体细胞类型和状态对猪胚胎早期发育的影响

经体外成熟、受精和培养获得猪胚胎 ,采用体细胞共培养研究了体细胞类型和状态对胚胎早期发育的影响。取体外成熟的不同直径卵泡 (>5 m m,2～ 5 mm)卵母细胞的卵丘团 (颗粒细胞团 ) ,培养铺层后与猪受精卵共培养 ,组间受精卵的卵裂率和发育能力无显著差异 ;根据卵巢的状况对猪输卵管上皮细胞 (POECs)的状态进行分组 ,受精卵和卵巢表面布满卵泡的 POECs共培养 ,卵裂率显著低于卵巢表面有黄体和 /或红体的 POECs共培养组 (P<0 .0 5 ) ,虽然各组间 3～ 4-细胞的发育率无显著差异 ,但卵巢表面有红体和卵泡的 POECs共培养组的 >4-细胞的发育率显著高于卵巢表面布满卵泡的 POECs共培养组 (P<0 .0 5 ) ;受精卵在共培养系统和非共培养系统中的卵裂率无显著差异 ,受精卵非共培养系统中 3～ 4-细胞的发育能力显著低于颗粒细胞共培养组 (P<0 .0 5 ) ,极显著低于 POECs共培养组 (P<0 .0 1) ,无能力突破 4-细胞继续发育 ,与颗粒细胞单层、POECs单层共培养的受精卵 >4-细胞的发育率分别为 2 4.0 %、5 3.8% ,差异显著 (P<0 .0 5 )。结果表明 ,共培养系统对胚胎体外发育的作用 ,一方面与体细胞类型有关 ,另一方面也受输卵管上皮细胞状态的影响     

Zur Wirkung von Follikelflüssigkeit auf die Befruchtung in vitro gereifter Oozyten des Rindes

Inhalt Follikelflüssigkeit aus Follikeln des Rindes am Tag des Östrus gewonnen, wurde als Serumanalogon zum Kultivierungsmedium TCM 199 zugesetzt. Die Supplementierung des TCM mit 20%östrischer Follikelflüssigkeit (ÖFF) in der Reifungsphase und während der Zygotenkultivierung erbrachte 6–7 Tage post inseminationem einen Anteil an Morulae und Blastozysten von 41,7% (30,8–47,8%). Eine Reduzierung des Anteils ÖFF auf 10% zog einen Abfall transfertauglicher Rinderembryonen auf 9,2% (2,6–16,9%) nach sich. In einer Kurzzeitkultivierung von bis zu 90 h post inseminationem wurden außerdem verschiedene Serumanaloga, im Anteil von 10%, wie fetales Kälberserum (FKS), ÖFF und Follikelflüssigkeit (NÖFF) aus nicht östrischen Follikeln (≥ 10mm Durchmesser) auf ihre Wirksamkeit während verschiedener In-vitro-Phasen verglichen. Von den drei getesteten Seren erwies sich die ÖFF als der wirksamste Zusatz mit 19,9% morphologisch intakten 8- bis 16-Zeller. Das Ergebnis scheint unabhängig davon, ob die Applikation kontinuierlich während aller In-vitro-Phasen oder nur in der Reifungsphase erfolgte. Eine Schlüsselposition der Oozy tenreifung für die In-vitro-Fertilisation wird besonders deutlich. Contents: Influence of follicular fluid on fertilization of in vitro matured bovine oocytes Follicular fluid obtained from bovine follicles on the day of estrus was added to TCM 199 culture medium as a serum analogue. TCM supplementation with 20% estric follicular fluid (EFF) during maturation and zygote cultivation produced a morulae and blastozyst yield of 41,7% (30,8–47,8%) 6–7 days after insemination. Reduction of the EFF fraction to 10% reduced the yield of transferable bovine embryos to 9,2% (2,6–16,9%). In a short term cultivation experiment lasting up to 90 h after insemination, the effects of 10% supplementation with different serum analogues fetal calf serum (FCS), EFF and follicular fluid (NEFF) fiom non-estric follicles (≥10mm diameter) during various in vitro phases were compared. EFF with a yield of 19,9% morphologically intact 8 to 16 cell stages proved to be the most effective of the three sera. This yield is not apparently affected by whether the serum is applied continuously during all phases of in vitro development or only during the maturation phase. The oocyte maturation phase is obviously the key phase for successful in vitro fertilization.     

卵泡液中总蛋白含量与卵泡直径的关系

通过检测不同直径卵泡的卵泡液中总蛋白含量的变化,从而进一步阐明卵泡液中蛋白成分在卵泡发育过程中的作用。将直径为3~5、5~8、8~10mm的卵泡和囊肿卵泡(>21mm)分为4组(n=10),分别抽取卵泡液。通过TCA/丙酮方法进行样品处理;根据Bradford法检测原理,利用分光光度计对其进行蛋白定量研究。结果表明:3~5mm为4440μg/mL,5~8mm为976μg/mL,8~10mm为686μg/mL,囊肿卵泡(>21mm)为537.2μg/mL。经SPSS13.0软件分析,3~5mm卵泡液中蛋白平均浓度高于其他组(P<0.01);其他3组间差异均不显著(P>0.05);囊肿卵泡中蛋白含量最低。由此可得出,卵泡液中总蛋白浓度随着卵泡直径的增加而降低,表明卵泡液中的蛋白含量与卵泡发育的成熟度呈负相关。而囊肿卵泡的发生很可能和蛋白含量降低有关,为卵泡囊肿发病机制的研究提供新的理论参考。     

Effects of body condition and supplementation on ovarian function,growth factors and response to estrus synchronization in postpartum beef cows in Tamaulipas,Mexico

ABSTRACT

Eighteen beef cows with their calves were assigned to 3 groups considering body conditions scores at calving (BCSC): T1 (high BCSC, maintained throughout the study); T2 (low BCSC, increased during the study); and T3 (low BCSC, maintained throughout). An estrus synchronization protocol was implemented, with fixed time artificial insemination on day 70. Follicular or luteal growth, and pregnancy rate were similar between groups. Of all cows, five showed multiple ovulations (2–6); increasing number of ovulations reduced preovulatory follicle diameter and individual luteal volume (P?<?0.05), but total luteal volume was greater in cows with 2 or 3 ovulations on day 14 after insemination (P?<?0.05). Estradiol and progesterone levels were associated to follicular and luteal growth, respectively. No difference was found in IGF1 or GDF9 levels between groups or associated to fertility. No association was found between BCS, IGF1 or GDF9 levels and the reproductive process.     

绵羊卵泡颗粒细胞体外培养条件的优化

为了探讨绵羊卵巢卵泡颗粒细胞体外培养时FSH(Invitrogen)与胰岛素的最佳质量浓度,试验在无血清条件下培养绵羊卵泡颗粒细胞,并向其中加入不同质量浓度的FSH与胰岛素,体外培养7 d后测定细胞数目以及培养液内雌二醇浓度。结果表明,当培养液内FSH质量浓度为25 ng/mL、胰岛素质量浓度为100ng/mL时,颗粒细胞的数目最多;当FSH质量浓度为1 ng/mL、胰岛素质量浓度为10 ng/mL时,培养液内雌激素质量浓度最高。