C57BL/6J超排小鼠取卵时间对体外受精效果的影响

目的 探讨雌性小鼠注射绒毛膜促性腺激素(HCG)后，取卵时间对体外受精率的影响。方法 选用3～4周龄的野生型C57BL/6J雌鼠，体质量10～13 g,通过腹腔注射血清促性腺激素(PMSG)和HCG联合使用^[1]做超排处理。我们将超排后雌鼠取卵时间分为6个时间段，分别为13、14、15、16、17、18 h后取卵母细胞与新鲜精子做体外受精。取3月龄雄性小鼠附睾里精子，在HTF液里获能1 h后，用于体外受精。实验共分3组，每组12只雌鼠用于超排处理，共得到2-细胞胚胎数分别为258、199和243枚；分别移植到当天见栓的假孕鼠输卵管内，得到出生仔鼠分别为98只、87只、95只；出生率分别为37.98%、43.72%和39.09%。结果 总取卵数和2-细胞发育率，超排后15 h取卵发育受精率最高，这说明小鼠卵母细胞超排后14 h少数成熟，15～16 h完全成熟，排卵17 h后则开始出现退化。结论 3组实验最高受精率比较：第1组15 h后取的卵母细胞团受精率最高为79.17%、第2组15 h后取的卵母细胞团受精率最高为75.68%、第3组16 h后取的卵母细胞团受精率最...     

卵母细胞老化对小鼠体细胞克隆胚发育的影响

就小鼠卵母细胞的卵龄对克隆胚的体外发育能力的影响进行了检测,以确定最佳的取卵时间,以及取卵后进行核移植操作的可耐受的时间。采用PMSG和hCG超排B6D2F1雌鼠。在体内老化实验中,分别于注射hCG后13,15,17,20h取卵用于核移植操作;在体外老化实验中,所有的卵均于注射hCG后13h取出并培养,然后在13,15,17,20h进行核移植操作。每个时间点的核移植操作在1h内完成,重构的胚胎培养1h后进行激活。结果显示:在体内老化实验中,注射hCG后13h取卵,获得的克隆囊胚发育率最高,为56.0%,15h取卵仍维持了其支持胚胎体外发育的能力,但17h及更长时间后取的卵,重构后的囊胚发育率显著下降(18.1%)。注射hCG后15h取的卵孤雌发育率最高(囊胚发育率为90.1%),并在17h仍维持较高的孤雌发育能力,在20h显著下降。在体外老化试验中,于注射hCG后13h取卵,分别于13,15,17h重构,都获得了较高的囊胚发育率(分别为57.7%,52.2%,46.3%),而在注射hCG后20h重构,囊胚发育率显著下降(14.8%)。体外老化的孤雌胚在注射hCG后22h激活时囊胚发育率显著下降。结果表明:卵母细胞在体内和体外老化都影响克隆胚的体外发育,最佳取卵时间为注射hCG后13h,取卵后立即用于重构可获得最佳囊胚发育率;体内、外老化卵支持重构胚较好发育的时间间隔是不同的,体内老化卵支持重构胚发育能力从注射hCG后17h开始下降,体外老化卵则发生在20h。这些结果可以指导操作,有利于在应用核移植进行其他基础研究时降低操作引起的误差。     

羔山羊的超数排卵及体外受精

以ＦＳＨ或ＦＳＨ－ＬＨ四种不同处理方法对２９只６８～１１０日龄羔山羊进行超排处理，结果共采集卵泡卵母细胞７０８枚，平均每只羔羊采卵２４．４枚．卵母细胞经体外培养２４ｈ后有８１．２％的卵发育为成熟卵．分别用经钙离子载体Ａ＿（２３１７８）或肝素进行获能处理的精子对体外培养成熟的卵子进行体外受精，结果精子穿入卵率分别为５４．８％和３４．４％，发育至２～４细胞胚的卵裂率分别为２８．６％和４１．９％．采用ＦＳＨ－ＬＨ（肌注）进行超排处理得到的卵子经体外受精处理后，其卵裂率明显高于采用其它超排方法得到卵子的卵裂率．     

不同化学激活方法对小鼠卵母细胞孤雌激活效果的影响

通过比较几种常用的化学激活方法对小鼠卵母细胞的孤雌激活和孤雌生殖胚胎体外发育的影响,从而筛选出小鼠卵母细胞孤雌激活的适宜方法.选取成熟小鼠卵母细胞,随机分为以下9组进行激活处理:①离子霉素(I)+6-二甲氨基嘌呤(D);②I+D+细胞松弛素B(CB);③I+放线菌酮(C);④I+C+CB;⑤φ(乙醇)=7%的乙醇(E)+D;⑥E+D+CB;⑦E+C;⑧E+C+CB;⑨Sr2++CB.比较了这9种化学激活方法以及SrCl2的作用时间和卵龄对小鼠卵母细胞激活效果的影响.实验结果表明:小鼠卵母细胞孤雌激活率与激活措施和卵龄有关;小鼠卵母细胞可以被I+D+CB或SrCl2+CB有效激活,激活率及孵化囊胚发育率均高于其他激活方法(P0.05),且以SrCl2+CB最为有效.hCG注射后20h为小鼠卵母细胞进行SrCl2激活的最适卵龄,其最佳处理时间为4h.对小鼠卵母细胞而言,6-DMAP比放线菌酮的激活效果好.     

牛体外受精技术研究的现状与展望

系统综述了牛体外受精技术研究的现状及存在的问题．并对今后的研究方向和前景提出了展望。牛卵母细胞可在不含血清的成熟培养液中获得受精及胚胎发育能力，促卵泡素、促黄体素及上皮细胞生长因子均对这一过程具促进作用。肝素是迄今最为有效的精子获能处理方法，但在受精过程中对精子获能起决定作用的是卵丘细胞和卵母细胞本身。提高受精质量可明显提高受精卵的胚胎发育能力。体外受精卵可通过改善培养条件或与体细胞进行复合培养发育到囊胚阶段．但后者更为稳定可靠。目前，牛卵母细胞的体外受精分裂率可达８０％，囊胚发育率达４０％左右．两枚鲜胚的移植妊娠率可达５０％～６０％．而两枚冻胚的移植妊娠率仅３０％～５０％。因此，尚需进一步提高体外受精胚胎的活力和改进胚胎的冷冻保存技术。     

皱纹盘鲍精子的激活及超低温保存研究

用不同浓度的氨海水激活解剖获得的皱纹盘鲍精子并进行激活率和受精率的测定.以自然海水作基础溶液,以二甲基亚砜(DMSO)和甘油(Gly)做保护剂,在不同降温程序下,将解剖获得和经诱导自然排放的皱纹盘鲍精子置于液氮罐中保存24 h,解冻后检测精子的成活率.结果表明,加入自然海水后有64.27%的解剖精子被激活,但不能受精,经氨海水处理后的精子在0.02‰浓度条件下激活率和受精率最高,分别为85.60%、67.42%.自然排放和解剖获得的精子在14%DMSO、4℃平衡15 min,-20℃时平衡5min,-80℃时平衡5 min条件下精子存活率最高,分别为48.15%和64.01%.     

卵母细胞的卵龄对猪核移植效率的影响

对核移植中，猪卵母细胞的卵龄对核移植效率的影响进行了研究。结果表明：体外成熟培养４８ｈ的猪卵母细胞最适合作为核受体其激活率达７８．３％，并无裂解：去核率高达９０％；在作为核受体进行核移植时，融合率为７５％，重构胚胎的卵裂率为６４．６％，卵龄进一步增大，激活率略有增加，但去核率，融合率和卵裂率均下降。     

受精滴中精子不同平衡时间对牛卵母细胞体外受精的影响

为寻找精子在受精滴中的最佳平衡时间, 以加入精子时间为０ ｈ （对照组）, 分别在０ｈ, ０．５ ｈ, １ ｈ, ２ ｈ, ４ ｈ 随机加入等数卵母细胞到各受精滴, 结果显示： ０ ｈ, ０．５ ｈ 组卵裂率较高, 分别为８７．７％ 、８４．７％ , 与其他组相比差异显著（ Ｐ＜ ０．０１）; ０．５ｈ 组的囊胚率最高,为６７．５％ ,与对照组的５７．７％ 比较差异显著（ Ｐ＜ ０．０１）。结果表明,受精滴中精子经过０．５ｈ 平衡后, 弱精及死精粘聚沉淀, 余下活力强的健壮精子, 此时加入牛卵母细胞受精, 可获得较高卵裂率及囊胚率     

小鼠附睾精子的显微受精

目的：建立显微受精的方法,并探讨小鼠附睾精子在显微注射进入卵母细胞后的受精能力。方法：用显微注射法把小鼠附睾头和附睾尾精子注入卵母细胞的胞质内或卵周隙进行显微受精。结果：把单个附睾尾精子注入卵细胞质中,培养后１４个存活的卵细胞中,有５个卵裂为２－细胞期胚胎;将单个附睾头精子注入卵母细胞中,有２５个存活,其中９个受精发育为２－细胞期胚胎;将附睾尾精子注入卵周隙进行带下受精,３０个存活的卵细胞中有４个     

体外受精条件对牦牛与黄牛异种受精的影响

本实验通过改变体外受精精子浓度(0.5 × 106、2 × 106、8 × 106 个/mL), 精卵共培养时间(12 h、18 h、24 h), 体外受精96 h后是否更换培养液(IVC)以及氧气的含量(5% CO2 和5% CO2、5% O2、90% N2)等培养条件, 观察牦牛精子对黄牛卵母细胞受精后卵裂和早期胚胎发育状况. 结果显示精子浓度为2 × 106个/mL、精卵共培养24～26 h、培养96h后要更换培养液, 在5% CO2、5% O2、90% N2的条件下培养, 受精和早期胚胎的发育.     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育的比较

目的对MII期水牛卵母细胞进行人工诱导激活,可以间接判断体外成熟卵母细胞质量的优劣,并且,卵母细胞的充分激活也是提高核移植效率的关键因素之一。比较了水牛卵母细胞体外成熟时间对孤雌激活胚发育能力的影响以及不同化学激活方法对水牛卵母细胞孤雌激活效果的影响,并在相同条件下,对孤雌激活胚与体外受精胚的发育能力进行了比较。结果表明,水牛卵母细胞体外成熟27 h或30 h的囊胚发育率(19.0%,17.7%)明显高于体外成熟21 h或24 h的囊胚发育率(12.3%,13.8%);Ion联合6_DMAP激活水牛卵母细胞的效果优于其他几组激活方法;在相同培养条件下,孤雌激活胚与体外受精胚的发育能力存在着差异,其中卵裂率差异不显著,但孤雌激活胚的囊胚发育率显著高于体外受精胚(21.7%,13.0%)。     

MAPK regulates cell cycle progression in pig oocytes and fertilized eggs

Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogenactivated protein kinase (MAPK) was detected with SDS-PAGE and Western blotting, and translocation of ERK₂ was observed with immunofluorescent cytochemistry. We found that the quantity of MAPK kept unchanged during oocyte maturation. There was no phosphorylated MAPK in porcine oocytes at the germinal vesicle (GV) stage; a little MAPK was phosphorylated at 20 h of IVM; a high level phosphorylation was detected at 30 h, while MAPK phosphorylation decreased at 36 h; and then MAPK phosphorylation increased again to the peak level from 40 to 60 h. ERK2 translocated from the peripheral cytoplasm to inner cytoplasm and nuclear area during oocyte maturation. There was nearly no phosphorylated MAPK at 18 and 20 h of electrically activated oocytes, but phosphorylation increased at 22 h. There was no phosphorylated MAPK at 12 h of IVF, while phosphorylation resumed at 16 h. These results suggest that MAPK may play an important regulatory role in MI-MII transition, pronucleus formation and the initiation of the first mitosis in pig eggs.     

光裸方格星虫(Sipunculus nudus)人工繁殖及生物学的初步研究

对经济海产动物光裸方格星虫(SipunculusnudusLinnaeus)若干繁殖生物学作初步研究.主要报导,干露5～8h,成熟星虫亲体可排放精、卵并达成受精和正常胚胎发育,它是行之有效的人工催产方法.产卵(精子团)于体腔与排卵(活动精子)于体外是两个必经的先后生殖过程.体腔液中的精、卵必需通过肾管(兼有排泄和生殖的功能器官)的再成熟,排放才能受精及发育.取自厦门及其附近海域的生殖种群雌雄性比为1:1.2.中细沙质泥的底质为亲体暂养的适宜底质.耐饥饿实验结果表明,43h内亲体仍有50%存活,并且仍可作亲体使用.测定5批体壁干湿重关系,其直线回归为y=0.1810x-0.0078(R2=0.9366,),回归系数与个体大小、季节不呈相关.     

Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse acti-vation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.     

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.     

人输卵管液和输卵管上皮细胞培养液诱发精子的顶体反应

目的：探讨人输卵管液和上皮细胞培养液对人精子顶体反应的诱发作用。方法：用Ｈａｍ′ｓＦ１０作对照组，输卵管液和上皮细胞培养液为实验组，分别与生育力组（ｎ＝２０）和不育组（ｎ＝２０）的精子进行共孵育，用精子顶体三色染色法对顶体状态进行评价。结果：共孵育３ｈ时与Ｈａｍ′ｓＦ１０对照组相比较，两种输卵管液均可显著提高生育力组和不育组的精子顶反应率（Ｐ〈０．０５），但这两种输卵管液处理的生育力组与不育组之间     

Reconstruction of human embryos derived from somatic cells

Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into M Ⅱ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyteactivation and 2PN formation, we removed the female PN.By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation,and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into the blastocyst stage in vitro.