Developmental and molecular biological studies of in vitro produced cat embryos in different culture systems

EXP. 1 aimed to investigate the effect of roscovitine (ROS) on the developmental competence of cat oocytes matured in vitro. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 µM ROS for 24 h and were either fixed to assess the stages of nuclear maturation or additionally matured in vitro for 24 h before fixation. Cumulus cells from the COCs treated with ROS were examined for late apoptosis. The developmental competence of cat oocytes after ROS treatment and in vitro fertilization was determined. ROS reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. ROS at 12.5 and 25 µM demonstrated less efficiency to arrest the oocytes compared with other doses. However, higher doses of ROS induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation (P<0.05). ROS at 12.5 and 25 µM, which gave rise to the highest rate of mature stage (MII) (P<0.05), were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 µM ROS prior to in vitro maturation decreased the developmental competence of cat oocytes compared with non–ROS-treated controls (P<0.05). In conclusion, ROS reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence. EXP. 2 aimed to define the effects of culture media and culture volume in cat embryos. Groups of 8 to 10 embryos were cultured in SOF, modified Tyrode’s solution and MK-1 medium in different volumes (20-, 50- and 100-µl drops). SOF supplemented with different concentrations of glucose (1.5, 3.0 and 6.0 mM) was used to examine the effect of glucose in culture medium on embryo development. Quantitative polymerase chain reaction was used to determine the relative transcripts of BAX, BCL-2 and GLUT-1 genes in blastocysts derived from various concentrations of glucose. SOF and MK-1 supported feline embryo development better than modified Tyrode’s solution (P<0.05). Embryos cultured in 20-µl droplets showed decreased development in all three media (P<0.05). Increasing the glucose concentration in SOF to 6.0 mM adversely affected embryo development and tended to increase the BCL-2 transcript in blastocysts (P<0.05). In conclusion, type of culture medium, culture volume and glucose concentration affected the development of domestic cat embryos. Decreased culture volume (20 µl) and high glucose concentration (6 mM) negatively affected embryo development. The increase of anti-apoptotic BCL-2 expression found in blastocysts cultured in 6.0 mM glucose may prevent an increase of apoptosis. In the present study, it was clearly demonstrated that differential gene expression occurred in embryos with similar morphology. EXP. 3 aimed to determine the effects of embryo density and number on feline embryo development. Embryos were randomly cultured in group (n=10 and 5) or singly in different medium volume (12.5, 25, 50, 100 and 200 µl). They were examined for their developmental competence and fragmentation of blastocyst cell nuclei using DNA labeling. Only expanded blastocysts acquired from different density and numbers were collected to examine their mRNA transcripts of BAX, BCL-2 and HSP70 genes using quantitative polymerase chain reaction. Embryos cultured in groups tended to develope better than those cultured singly. For group cultured embryos (n=10), embryos acquired from low culture density (1:5, 1:10 and 1:20) could develop better than those acquired from high density (1:1.25 and 1:2.5) (P<0.05). Moreover, fragmentation of the blastocyst cell nuclei tended to increase in high culture density. On the other hand, there was no significant difference of developmental competence among embryos cultured singly in varied densities. Blastocysts derived from high culture density (1:1.25) also significantly up-regulated BAX and HSP70 transcripts comparing with those of low culture densities (1:5 and 1:20) (P<0.05). However, there was no significant difference in relative transcripts among varied embryo numbers (n=10, 5, 1) cultured in fixed 200 µl culture medium. In conclusion, high density negatively affected the developmental competence and up-regulated pro-apoptotic (BAX) and stress response (HSP70) transcripts in embryos. This highlighted the mechanisms used to protect the embryo against suboptimal culture condition.

实验1旨在探究罗科司伐他汀（roscovitine, ROS）对猫卵母细胞体外成熟发育潜能的影响。将卵丘-卵母细胞复合体（cumulus-oocyte complexes, COCs）分为多组，分别置于含0、12.5、25、50、100及200 µM ROS的培养液中培养24 h，一部分固定后用于检测核成熟阶段，另一部分则先继续体外成熟培养24 h后再固定。对经ROS处理的COCs的卵丘细胞进行晚期凋亡检测。同时评估ROS处理后猫卵母细胞经体外受精的发育潜能。

结果显示，ROS可呈剂量依赖性可逆性地将猫卵母细胞阻滞于未成熟阶段。与其他剂量组相比，12.5和25 µM ROS的卵母细胞阻滞效率较低。但高剂量ROS可诱导卵丘细胞凋亡，且在体外成熟后会导致大量卵母细胞退化（P<0.05）。12.5和25 µM ROS组的卵母细胞MII期成熟率最高（P<0.05），因此被用于后续胚胎发育效应的评估。与未经过ROS处理的对照组相比，体外成熟前用12.5和25 µM ROS预处理的猫卵母细胞发育潜能显著降低（P<0.05）。

综上，ROS可可逆性地将猫卵母细胞维持在生发泡阶段，且不会对核成熟产生不利影响，但会损害卵丘细胞活力并降低卵母细胞的发育潜能。

实验2旨在明确培养液类型及培养体积对猫胚胎发育的影响。将8~10枚胚胎分为多组，分别在SOF培养液、改良Tyrode液及MK-1培养液的不同体积液滴（20、50、100 µl）中培养。同时设置添加不同浓度葡萄糖（1.5、3.0及6.0 mM）的SOF培养液，以探究葡萄糖浓度对胚胎发育的影响。采用定量聚合酶链式反应（quantitative polymerase chain reaction, qPCR）检测不同葡萄糖浓度下获得的囊胚中BAX、BCL-2及GLUT-1基因的相对转录水平。

结果显示，SOF与MK-1培养液对猫胚胎发育的支持效果优于改良Tyrode液（P<0.05）。三种培养液在20 µl液滴中培养时，胚胎发育率均显著降低（P<0.05）。将SOF培养液中的葡萄糖浓度提升至6.0 mM会对胚胎发育产生不利影响，且会使囊胚的BCL-2转录水平呈上升趋势（P<0.05）。

综上，培养液类型、培养体积及葡萄糖浓度均会影响家猫胚胎的发育。降低培养体积（20 µl）及提高葡萄糖浓度（6 mM）会对胚胎发育产生负面影响。在6.0 mM葡萄糖中培养的囊胚中检测到的抗凋亡基因BCL-2表达上调，可能会抑制细胞凋亡的发生。本研究明确证实，形态学相似的胚胎可存在差异化的基因表达模式。

实验3旨在确定胚胎密度与数量对猫胚胎发育的影响。将胚胎随机分为群组培养组（n=10和n=5）以及单独培养组，分别置于不同体积的培养液（12.5、25、50、100及200 µl）中培养。通过DNA标记技术检测胚胎的发育潜能及囊胚细胞核碎裂情况。仅收集不同密度与数量组获得的扩张囊胚，采用定量聚合酶链式反应检测其中BAX、BCL-2及HSP70基因的mRNA转录水平。

结果显示，群组培养的胚胎发育效果整体优于单独培养组。对于群组培养的胚胎（n=10），低培养密度组（1:5、1:10及1:20）的胚胎发育效果优于高培养密度组（1:1.25及1:2.5）（P<0.05）。此外，高培养密度组的囊胚细胞核碎裂率呈上升趋势。而单独培养的胚胎在不同密度下的发育潜能无显著差异。与低培养密度组（1:5及1:20）相比，高培养密度组（1:1.25）的囊胚中BAX与HSP70的转录水平显著上调（P<0.05）。但在固定体积200 µl的培养液中，不同胚胎数量组（n=10、5、1）的基因转录水平无显著差异。

综上，高培养密度会降低胚胎的发育潜能，并上调促凋亡基因BAX及应激反应基因HSP70的转录水平。这一结果揭示了胚胎应对亚最优培养环境的保护机制。