Camaioni A, Hascall V C, Yanagishita M, Salustri A
Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Sep 25;268(27):20473-81.
Compact cumulus cell-oocyte complexes (COCs) isolated from preovulatory mouse follicles undergo expansion in vitro when high levels of hyaluronic acid (HA) are synthesized and organized into an extracellular matrix. We studied the effects of fetal bovine serum (FBS) and of exogenous HA and HA-oligomers on the expansion process. Maximum retention of HA in the COC matrix, and hence complete COC expansion, occurs when 1% FBS is continuously present during the first 18 h of culture. Irrespective of the culture time, HA synthesized when serum is absent is primarily in the medium, whereas HA synthesized when serum is present is primarily in the cell matrix. These findings support the hypothesis that the serum factor, identified as an inter-alpha-trypsin inhibitor by Chen et al. (Chen, L., Mao, S. J., and Larsen, W. J. (1992) J. Biol. Chem. 267, 12380-12386), is a structural component of the matrix. Addition of exogenous HA or of HA oligomers of decasaccharide size (GlcUA-GlcNAc)5 or larger effectively displaces endogenously synthesized HA from the matrix into the medium, thereby preventing COC expansion. Addition of exogenous chondroitin sulfate affects neither matrix organization nor COC expansion, thus indicating specificity of the binding of some structural component(s) to HA. Fully expanded COCs disassemble when cultured longer than 18 h, a process which occurs also in vivo and which correlates with loss of oocyte fertilizability both in vivo and in vitro. This process involves release of macromolecular HA from the matrix into the medium, with loss of 50% of the HA in the first 8 h of incubation after full expansion. The release is not facilitated when HA oligomers, long enough to prevent matrix formation, are added to the culture medium after the COCs are fully expanded. This suggests that cooperative binding to HA of either the serum factor, an endogenously synthesized factor(s), or both is required to stabilize the fully expanded COC matrix.
从排卵前小鼠卵泡中分离出的致密卵丘细胞 - 卵母细胞复合体（COCs），当合成高水平的透明质酸（HA）并将其组织成细胞外基质时，会在体外发生扩张。我们研究了胎牛血清（FBS）以及外源性HA和HA - 寡聚体对扩张过程的影响。当在培养的前18小时持续存在1% FBS时，HA在COC基质中的保留量最大，从而实现COC的完全扩张。无论培养时间如何，无血清时合成的HA主要存在于培养基中，而有血清时合成的HA主要存在于细胞基质中。这些发现支持了这样一种假设，即被Chen等人（Chen, L., Mao, S. J., and Larsen, W. J. (1992) J. Biol. Chem. 267, 12380 - 12386）鉴定为α - 胰蛋白酶抑制剂的血清因子是基质的一种结构成分。添加外源性HA或十糖大小（GlcUA - GlcNAc）5或更大的HA寡聚体可有效地将内源性合成的HA从基质中置换到培养基中，从而阻止COC扩张。添加外源性硫酸软骨素既不影响基质组织也不影响COC扩张，因此表明某些结构成分与HA结合具有特异性。当培养超过18小时时，完全扩张的COCs会解体，这一过程在体内也会发生，并且与体内和体外卵母细胞受精能力的丧失相关。这个过程涉及大分子HA从基质释放到培养基中，在完全扩张后的孵育的前8小时内，HA损失50%。当在COCs完全扩张后向培养基中添加足够长以防止基质形成的HA寡聚体时，这种释放不会加速。这表明血清因子、内源性合成因子或两者与HA的协同结合是稳定完全扩张的COC基质所必需的。
