胚胎植入及其分子机制研究进展

哺乳动物的胚胎植入是生殖过程的一个重要环节,是妊娠的关键。包括胚胎对母体子宫内膜的识别、黏附、侵入等过程。正常情况下,胚胎仅在一个极短的时间内有机会附着到子宫内膜上,即胚泡滋养层达到侵入状态,子宫内膜达到接受状态,且两者同步发生,胚泡才会成功植入。胚胎植入的成功必须具备许多条件,需要足够的激素分泌,合体滋养层细胞形成,以及胚泡与子宫内膜同步发育与相互配合等。这一过程的调控机制十分复杂,至今仍是生殖医学领域中尚未解决的问题。文章就胚胎植入的基本过程及其分子机制做简要综述。     

冢畜胚胎着床和母体妊娠识别的分子调控

着床是指胚胎发育到胚泡阶段 ,与此同时子宫增殖和分化到可接受状态的同步化发育 ,并最后形成胎盘的过程。猪、马、牛、羊等都是表面着床 ,胚泡与子宫内膜只是表面接触 ,并未嵌入子宫内膜中。黄体产生的孕酮对于妊娠维持、子宫内膜接受态的建立以及着床后的妊娠维持都很重要。妊娠早期胚胎诱导母体的内分泌变化以维持卵巢黄体孕酮的持续分泌 ,这是母体对妊娠最早的生理反应 ,以使妊娠与正常的卵巢周期相区别 ,称为母体妊娠识别 ,它是着床起始必不可少的环节。最近证实 ,雌激素启动着床是通过诱导子宫上皮分泌细胞因子、生长因子以及粘蛋白等…     

ephrin A1基因在附植后期猪繁殖组织表达变化及与表观性状的关联分析

本研究旨在明确促红细胞生成素产生肝细胞配体A1(ephrin A1)在猪附植后期的mRNA和蛋白表达规律,分析高胚胎数母猪和低胚胎数母猪之间表达差异,以及这些差异与繁殖性状的关系。选择妊娠第22天和24天(附植后期)母猪,屠宰后采集6种繁殖组织样,利用Real-time定量PCR(qPCR)和免疫印迹方法检测这些组织样品中ephrin A1基因的mRNA和蛋白表达量,并将基因表达量与繁殖性状进行关联分析。妊娠第22天,高胚猪和低胚猪的黄体数和总胚胎数均差异显著(P0.05)。Real-time qPCR结果显示:妊娠第22天,子宫内膜附植点的表达量显著高于尿囊绒毛膜或胚胎(P0.05),高胚猪的胚胎表达量小于低胚猪(P0.05);妊娠第24天,ephrin A1在组织中的mRNA表达量由高到低依次为:子宫内膜附植点、子宫内膜附植点间、子宫颈、卵巢黄体和胚胎;妊娠第22天或24天,妊娠猪ephrin A1的mRNA表达量均高于空怀猪(P0.05)。Western blot结果显示,妊娠第24天,ephrin A1蛋白在子宫内膜附植点的表达量最高,其次为子宫内膜附植点间和卵巢黄体(P0.05)。ephrin A1的表达与胚胎长度呈正相关,相关系数为0.31～0.59;相较于胚胎重、总胚胎数、正常胚胎数和卵巢重性状,ephrin A1表达与胚胎长度之间出现最高相关系数。研究结果表明,ephrin A1表达在猪附植后期的调控过程中可能发挥重要作用,子宫内膜附着位点可能是ephrin A1基因表达的主要靶组织,ephrin A1的表达量增加可能与胚胎生长(长度和重量)和卵巢重量增加有关。     

猪早期胚胎在体内发育及分布的研究

猪是我国重要的家畜之一。然而过去对其胚胎早期发育过程的研究报道甚少。本项研究对猪受精后至１９２ｈ前的早期胚胎发育过程以及在其生殖道内的分布进行了研究。结果表明，猪胚胎胚龄７２ｈ左右时，胚胎均分布在输卵管内，猪胚胎发育期为８细胞前期；７２ｈ至１２０ｈ期间，猪胚胎已进入子宫角，分布比较集中于靠近宫管结合部，胚期已发育至桑椹期：１２０－１９２ｈ胎龄的猪胚已发育形成囊胚；到１９２ｈ时，胚胎已分布在整个子宫     

Nupr1 mRNA在小鼠早期妊娠子宫中的表达

试验旨在研究核蛋白1(nuclear protein 1,Nupr1)mRNA在小鼠早期妊娠子宫中的表达,探讨Nupr1与小鼠胚胎着床的相关性。通过建立小鼠早期妊娠模型、假孕妊娠模型、延迟着床及激活模型、人工蜕膜化模型和激素处理模型,采用原位杂交的方法检测Nupr1mRNA在小鼠各种模型子宫组织中的定位表达情况,并应用实时荧光定量PCR法检测早期妊娠模型和假孕妊娠模型中Nupr1mRNA的相对表达量。结果显示,Nupr1mRNA在小鼠早期妊娠第1~4天子宫的腔上皮和腺上皮表达,第5~8天表达于蜕膜区域;假孕妊娠第1~5天,Nupr1mRNA主要表达于小鼠子宫腔上皮和腺上皮;延迟着床模型中信号表达于在小鼠子宫的腔上皮和腺上皮,与正常妊娠第4天结果相似;延迟激活模型中信号表达于蜕膜区,与早期妊娠第5天表达结果相似;人工蜕膜化模型中信号表达于蜕膜区,而蜕膜对照组中信号表达于腔上皮和腺上皮;17β-雌二醇(oestrogen,E2)处理组信号表达于腔上皮和腺上皮,信号增强,孕酮(progesterone,P4)和E2共同处理表达无明显变化;实时荧光定量PCR结果显示,正常妊娠第2天Nupr1mRNA相对表达量较高,假孕妊娠第2天Nupr1mRNA相对表达量也较高。本研究结果表明,Nupr1mRNA在小鼠子宫中的表达与小鼠早期妊娠过程相关,Nupr1mRNA在腔上皮和腺上皮的表达可能受激素调节,在子宫基质中的表达与蜕膜化及活化胚泡相关。     

NO在小鼠早期胚胎吸收过程中的作用

给妊娠 7d小鼠尾静脉注射细菌脂多糖 (L PS)诱导早期胚胎吸收。注射 L PS后 12、2 4、36 h,以 EL ISA、比色法检测血清和子宫匀浆中 Th1型细胞因子 IFN- γ、IL- 12与 NO的含量变化 ;免疫组织化学法观察 3种一氧化氮合酶(n NOS,e NOS,i NOS)和 Th2型细胞在小鼠子宫表达的变化。此外 ,还观察了 NO供体硝普钠 (SNP)诱导孕鼠流产效果及氨基胍 (AG)对抗 L PS诱导孕鼠流产的效果。结果显示 ,相对于正常妊娠组 ,L PS处理组孕鼠子宫匀浆 NO含量及 IFN- γ、IL- 12水平极显著升高 (P<0 .0 1) ,血清 NO含量也极显著升高 (P<0 .0 1) ;n NOS、i NOS在 L PS处理组小鼠子宫可见阳性标记 ,而 e NOS未见阳性标记 ;大量 Th2型阳性细胞标记仅在正常妊娠组小鼠子宫内膜基质可见 ,L PS处理组未见阳性细胞。腹腔注射 SNP致使孕鼠早期胚胎吸收 ,然而妊娠 6～ 9d腹腔注射 AG却未能降低 L PS诱导的孕鼠早期胚胎吸收。上述结果提示 ,L PS处理后 ,Th1型免疫反应增强 ;源自升高表达的 i NOS的子宫局部高浓度 NO可能作为一种效应分子介导小鼠早期胚胎吸收。     

山羊早期胚胎发育的初步研究

本实验以黑龙江地方山羊为材料,经FSH超数排卵后,在不同时间屠宰母山羊,并冲洗输卵管及子宫,获取新鲜卵及各发育时间的胚胎。实验中发现山羊的排卵时间为发情开始后约30小时。受精卵的第一次卵裂的发生在排卵24小时以后。2细胞、4细胞、8细胞、16细胞、桑椹及胚泡期胚胎所处的时间分别为排卵后约32～42、48～52、62、72、96小时以及7～8天。16细胞期以前的胚胎移行于输卵管中,桑椹胚及胚泡则移行于子宫中。在每一时间从每只羊中所收集的胚胎基本上处于几个相邻的发育时期。在桑椹胚的动物极有明显的突起,这可能是内细胞群已开始形成的标志。到胚泡期时,胚胎体积增大,透明带变薄。正在孵化或已经孵化的胚泡中,内细胞群外端无滋养层细胞包围。     

白血病抑制因子及其受体与子宫内膜的关系

白血病抑制因子(LIF)是一种多生物活性的细胞因子,研究表明,LIF在人及动物的子宫内膜的腺上皮细胞、妊娠期蜕膜及胎盘上均有表达;胚泡是动物生殖过程中的关键,一些细胞因子起重要作用,它们能够充当子宫内膜微环境的"局部调节者",LIF对哺乳动物胚胎发育到胚泡阶段具有一定的作用,LIF在子宫内膜中特异性表达,促进胚胎生长发育和启动胚泡植入;LIF在生殖周期中影响子宫的功能及调节子宫内膜生长,这可能与LIF作用于子宫使其能发生蜕膜反应有关;LIF通过与LIF受体及gp130蛋白作用而发挥重要的生物学功能。LIF在哺乳动物早期妊娠中的作用机制具有重要意义。文章对LIF及其受体与子宫内膜的关系做了综述。     

家禽胚胎的早期发育影响其死亡率的因素

一、引言 家禽胚胎死亡并不是在整个孵化过程中随机地死亡，一般而言，胚胎在孵化的早期及后期阶段的死亡率要高于中间阶段。根据观察统计发现，鸡胚在孵化过程中的第2至第4天死亡机率较高，而火鸡胚胎在第3天至第6天死亡机率较高。家禽胚胎在早期发育过程中在生理及遗传方面要发生许多变化。     

山羊胚胎分割及同卵双生试验

选择山羊晚期桑椹胚、囊胚、孵出囊胚和孵出增大胚泡,用简化分割法二分。将19对裸半胚移植于18只受体羊,结果有12只妊娠,其中两只胚胎消失,两只流产,其余8只足月分娩,共产半胚羔11只。晚期桑椹胚、囊胚、孵出囊胚和孵出增大胚泡各组的半胚发育为羔羊的发育率分别为12.5％(1/8)、20％(2/10)、25％(3/12)和62.5％(5/8)。前三组均未获得同卵双生羔羊。在第四组,将4对裸半胚移植于4只受体,有3只妊娠,足月分娩半胚羔5只,其中两对为同卵双生。本研究证明,对称分割山羊孵出增大胚泡,不仅其半胚在体内仍可继续发育形成正常胎儿,而且不装透明带移植其裸半胚,仍能获得较高的同卵双生率。山羊孵出增大胚泡更适宜用简化分割法分割。     

Expression and Hormonal Regulation of E‐Cadherin in Canine Uterus During Early Pregnancy

E‐cadherin, a Ca^{2 +} ‐dependent cell adhesion molecule, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of E‐cadherin in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. E‐cadherin mRNA expression was at a low level in the glandular epithelium on days 6, 12 and 17 of pregnancy. On days 20 and 23 of pregnancy, E‐cadherin mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium and declined in villi and placenta on day 28 of pregnancy. During oestrous cycle, a moderate level of E‐cadherin mRNA expression was found in the luminal and glandular epithelium of canine uteri at oestrus stage. The same expression was also found at anoestrus stage. Progesterone slightly induced the expression of E‐cadherin mRNA in the luminal and glandular epithelium of ovariectomized canine uterus. These results suggest that E‐cadherin expression is closely related to canine implantation and can be up‐regulated by progesterone.     

Expression of Nupr1 mRNA in Mouse Uterus During Early Pregnancy

The test was aimed to study the expression of nuclear protein 1 (Nupr1) mRNA in mouse uterus during early pregnancy.The method of in situ hybridization was used to investigate Nupr1 mRNA expression in animal models that included early pregnancy,pseudopregnancy,delayed implantation and activation,artificial decidualization and hormonal treatments.The relative expression level of Nupr1 mRNA was detected in early pregnancy and pseudopregnancy using Real-time PCR.During mouse early pregnancy,the signal of Nupr1 mRNA was detected in luminal epithelium and glandular epithelium during the 1st to 4th day and in the decidua area during the 5th to 8th day.Nupr1 mRNA was mainly expressed in the luminal epithelium and glandular epithelium of mose uterus on the 1st to 5th day of pseudopregnancy.The signal was detected in luminal epithelium and glandular epithelium of the mouse uterus in the delayed implantation,which was similar to the results of early pregnancy on the 4th day.The signal was detected in decidua in the model of delayed activation,which was similar to the results of early pregnancy on the 5th day.The expression of Nupr1 mRNA in the model of artificial decidualization was detected in decidua area.In the control of artificial decidualization the slight signal appeared in luminal epithelium and glandular epithelium of the mouse uterus.After treated with oestrogen (E₂) the signal appeared in luminal epithelium and glandular epithelium of the mouse uterus,and the signal was enhanced.After treated with both of E₂ and progesterone (P₄), the expression of the signal was not changed significantly.Real-time PCR result showed that the relative expression on the 2nd day was higher than other days in early pregnancy and pseudopregnancy.The results indicated that the expression of Nupr1 mRNA in mouse uterus was related to the process of mouse early pregnancy.The expression of signal in luminal epithelium and glandular epithelium of the mouse uterus might be regulated by hormones.Nupr1 mRNA expression in uterine stroma was associated with decidualization and active blastocysts.     

猪胚胎早期卵裂时间与其发育潜能关系的初步研究

旨在探讨初次卵裂时间对猪孤雌胚胎发育潜能及其基因相对表达水平的影响。本试验从健康母猪卵巢上抽取卵母细胞进行体外成熟培养，将猪孤雌激活胚胎分为早期卵裂组（16~22 h）与晚期卵裂组（26~32 h）统计比较卵裂率和囊胚率，并对囊胚的多能性相关基因Oct4、Sox2、Klf4等和凋亡相关基因Bcl-xl、Bax、Caspase-3的相对表达水平进行分析检测。结果表明，猪孤雌激活胚胎在16~22 h发生卵裂的为50%~60%，而26 h之后发生卵裂的不到20%，在18 h前完成第一次卵裂的胚胎囊胚发育率为79%，42 h后发生初次卵裂的胚胎无法发育至囊胚期。猪孤雌激活胚胎早期卵裂组的囊胚发育率显著高于晚期卵裂组（P<0.05）。早期卵裂组囊胚的Oct4、Nanog、Sox2、Klf4基因的表达量显著高于晚期卵裂组（P<0.05），Oct4、Sox2、Klf4基因的相对表达量极显著高于晚期卵裂组（P<0.01），Bax和Caspase-3基因的相对表达水平极显著低于晚期卵裂组（P<0.01），而Bcl-xl作为保护因子其表达量相对于晚期卵裂胚胎（26~32 h）显著上调（P<0.05）。结果显示，初次卵裂时间较早的猪孤雌激活胚胎发育潜能显著高于较晚卵裂胚胎，其囊胚多能性相关基因表达上调，凋亡相关基因表达下调，卵裂时间可作为鉴定猪孤雌激活胚胎发育潜力的重要参数。     

水牛卵母细胞和体外受精早期胚胎端粒酶活性测定

为了解水牛卵母细胞和体外受精（IVF）胚胎早期发育过程中端粒酶的活性变化,本研究利用端粒重复序列扩增法（TRAP）进行了水牛未成熟卵母细胞,成熟卵母细胞和2～4细胞,8～16细胞,桑椹胚以及囊胚各阶段的早期胚胎端粒酶活性的测定。依据电泳条带在成像系统下的光密度值,计算端粒酶的相对活性（RTA）。结果发现,未成熟卵母细胞端粒酶活性比成熟卵母细胞高（P〈0.05）,受精后2～4和8～16细胞胚胎端粒酶活性相对较低,桑椹胚端粒酶活性明显升高（P〈0.05）,囊胚阶段达到最高水平。通过对水牛不同发育阶段胚胎细胞数计数及单细胞相对端粒酶活性的分析比较结果显示,卵母细胞的单细胞端粒酶活性最高,囊胚阶段的最低。单细胞端粒酶活性从未成熟卵母细胞到IVF囊胚阶段呈逐渐降低的趋势。这些结果表明,水牛卵母细胞及早期胚胎的端粒酶活性变化与其成熟、发育阻断及全能性的逐步降低有关。     

妊娠早期蒙古绵羊子宫内膜enJSRV及IFN-τ基因表达水平的分析

本研究旨在检测内源性绵羊肺腺瘤反转录病毒(enJSRV)与干扰素-τ(IFN-τ)在妊娠早期蒙古绵羊子宫内膜组织的表达以及enJSRV的表达与外周血孕酮水平变化的关系。运用TaqMan实时荧光定量PCR技术和电化学发光法对enJSRV和IFN-τ在妊娠早期蒙古绵羊子宫内膜组织相对表达及孕酮水平进行了测定。实时荧光定量PCR结果显示,enJSRV和IFN-τmRNA在妊娠早期绵羊子宫内膜组织有不同程度的表达。SAS统计学软件分析得出,子宫内膜组织中enJSRV mRNA在妊娠12～14d(交配日为0d)表达较高,2～10、16～30d的表达均低于前者。IFN-τmRNA仅在妊娠12～25d表达,14d达其峰值,30d就不能检出,且差异都极显著(P<0.01)。电化学发光法结果显示,孕酮水平在妊娠2d为0.4ng·mL-1,以后升高,妊娠8～16d维持在8.3ng·mL-1左右,在妊娠19～30d孕酮水平有所下降,30d为2.5ng·mL-1。以上结果提示,子宫内膜组织中enJSRV mRNA的表达与外周血孕酮含量及IFN-τ的变化高度相关,且enJSRV在胎盘的形态发生及生殖生物学方面发挥重要作用。     

小鼠子宫内膜凝集素结合特性的生殖周期改变

用４种酶标植物凝集素（ＣｏｎＡ,ＷＧＡ,ＳＢＡ,ＲＣＡ）作探针,研究了小鼠发情周期、妊娠期和产后子宫内膜上皮、固有膜细胞和腺上皮凝集素结合特性的变化。子宫内膜与这４种凝集素的结合,随发情周期而波动。与间情期相比,妊娠期子宫内膜上皮与ＣｏｎＡ的结合,由阴性转为阳性,并随妊娠的持续逐渐加强,产后又突然转为阴性。子宫内膜上皮与ＷＧＡ的结合,在间情期和妊娠１ｄ均为阳性,妊娠７ｄ减为弱阳性,继之又加强,产后达最强。无论妊娠期还是产后,子宫内膜上皮与ＲＣＡ的结合均为强阳性,与ＳＢＡ的结合均为阴性。结果提示,生殖周期子宫内膜与凝集素结合特性的规律性变化,可能与激素调节、胚泡的着床和分娩有关     

Japanese Society for Animal Reproduction: award for outstanding research 2002. The development and prevalence of the transfer technique for frozen-thawed embryos of Japanese black beef cattle in Tochigi Prefecture

The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus), were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of > or =2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17beta (E2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.