Tremoleda Jordi L, Stout Tom A E, Lagutina Irina, Lazzari Giovanna, Bevers Mart M, Colenbrander Ben, Galli Cesare
Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands.
Biol Reprod. 2003 Dec;69(6):1895-906. doi: 10.1095/biolreprod.103.018515. Epub 2003 Aug 6.
Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.
马胚胎体外生产（IVP）过程中的囊胚形成率令人失望，在培养中形成囊胚的胚胎无法产生具有特征性且至关重要的糖蛋白囊。本研究的目的是评估IVP对马胚胎发育和囊形成的影响。IVP胚胎通过将体外成熟的卵母细胞进行胞浆内精子注射产生，然后在合成输卵管液（SOF）中培养或临时转移至母羊输卵管。对照胚胎在排卵后6 - 9天从母马子宫中冲洗获得。用光学显微镜评估胚胎形态，并用多光子扫描共聚焦显微镜检查微丝（AlexaFluor - 鬼笔环肽染色）的分布和凋亡率（具有碎片化或末端脱氧核苷酸转移酶介导的dUTP缺口末端标记阳性细胞核的细胞）。为了研究培养对囊形成的影响，用针对囊糖蛋白的单克隆抗体（OC - 1）对孕体进行染色。转移至绵羊输卵管的合子的囊胚率（16%）高于在SOF中培养的合子（6.3%）。第7天的IVP胚胎体积小且致密，细胞相对较少，几乎没有或没有囊胚腔，内细胞团不明显。IVP胚胎凋亡细胞百分比高（10%，而体内胚胎为0.3%）且微丝分布不规则。尽管它们分泌囊糖蛋白，但后者并未形成正常的囊，而是渗透到透明带中或留在滋养外胚层表面的斑块中。这些结果表明，囊的初始层由OC - 1反应性糖蛋白组成，并且体外胚胎发育受到阻碍且异常，由于糖蛋白聚集失败导致囊形成失败。
