Reproduction, Genes and Development Group, Department of Comparative Biomedical Sciences, Royal Veterinary College, Hatfield AL9 7TA, UK.
Mol Hum Reprod. 2013 Sep;19(9):590-9. doi: 10.1093/molehr/gat032. Epub 2013 Apr 25.
Biological functions of hyaluronan (HA) depend on its molecular size. Small size HAs are known to regulate cell proliferation; however, the expression of HA synthases (HASs) and hyaluronidase-2 (HYAL2) and their role during early embryo development have not been previously identified. In this paper, we have shown by immunostaining that HA is produced by bovine in vitro-produced embryos at all stages of early development to blastocyst. Addition of HA-synthesis inhibitor (4-methylumbelliferone; 4MU) to in vitro embryo culture inhibited blastocyst formation. HASs HAS2 and HAS3 mRNA were expressed at all stages of embryo development; however, relative mRNA expression of HAS2 was significantly reduced as the embryos develop to the blastocyst stage. HAS1 was detected during 2- and 4-cell stages but was barely detectable in subsequent stages. HYAL2 mRNA expression was detected in oviducts at the early luteal phase but was only detected in the embryos at morula and blastocyst stages (Day 6 and 7 post-fertilization). Addition of HYAL2 to embryo culture media at Day 2 post-fertilization increased phosphorylated mitogen-activated protein kinases (MAPK1 and 3) in the embryos and improved development to the blastocyst stage and increased embryo cell numbers. Addition of an anti-CD44 antibody or an MAPK inhibitor (U0126) abrogated the positive effects of HYAL2 on blastocyst rates. In conclusion, we demonstrate that the expression of different HAS genes and HYAL2 in bovine embryos varies according to the stage of development and that the supplementation of HYAL2 in vitro mimics oviductal conditions and is shown to improve the blastocyst rate and embryo quality, an effect which requires CD44 activity and MAPK signalling.
透明质酸（HA）的生物学功能取决于其分子大小。已知小分子量的 HA 可调节细胞增殖；然而，HA 合酶（HASs）和透明质酸酶-2（HYAL2）的表达及其在早期胚胎发育中的作用以前尚未确定。在本文中，我们通过免疫染色表明，HA 由牛体外生产的胚胎在早期发育的所有阶段（包括囊胚阶段）产生。在体外胚胎培养中添加 HA 合成抑制剂（4-甲基伞形酮；4MU）会抑制囊胚形成。HASs HAS2 和 HAS3 的 mRNA 在胚胎发育的所有阶段都有表达；然而，随着胚胎发育到囊胚阶段，HAS2 的相对 mRNA 表达显著降低。HAS1 在 2-和 4-细胞阶段被检测到，但在随后的阶段几乎检测不到。HYAL2 mRNA 表达在早期黄体期的输卵管中被检测到，但仅在桑椹胚和囊胚阶段（受精后第 6 天和第 7 天）的胚胎中被检测到。在受精后第 2 天向胚胎培养物中添加 HYAL2 会增加胚胎中磷酸化丝裂原活化蛋白激酶（MAPK1 和 3）的含量，并改善胚胎发育至囊胚阶段的效果，增加胚胎细胞数量。添加抗 CD44 抗体或 MAPK 抑制剂（U0126）会消除 HYAL2 对囊胚率的正向影响。总之，我们证明了不同 HAS 基因和 HYAL2 在牛胚胎中的表达根据发育阶段而变化，并且体外补充 HYAL2 可模拟输卵管条件，并显示出可提高囊胚率和胚胎质量的效果，该效果需要 CD44 活性和 MAPK 信号传导。
