Page K C, Killian G J, Nyquist S E
Department of Biology, Bucknell University, Lewisburg, Pennsylvania 17837.
Biol Reprod. 1990 Oct;43(4):659-64. doi: 10.1095/biolreprod43.4.659.
This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.
本研究评估了体外培养的支持细胞糖基化对培养时间变化以及对重组基底膜（基质胶）或IV型胶原/层粘连蛋白基质存在情况的反应性。从20日龄大鼠制备原代支持细胞培养物，并用[3H]甘露糖（一种对天冬酰胺连接的寡糖具有特异性的单糖）进行孵育。在培养期的第4天、第6天或第10天收获细胞。分别评估富含细胞表面糖肽的上清液（胰蛋白酶消化产物）和去除表面糖缀合物的细胞沉淀。使用伴刀豆球蛋白A凝集素亲和色谱法将来自这两个样品的蛋白酶消化产物衍生的糖肽分离为三大类：多天线复合型、双天线复合型和高甘露糖型寡糖结构。在培养的第4天和第6天之间，出现在这三类中的每一类中的放射性标记糖肽的比例没有差异。相比之下，在从10日龄培养物中收获的细胞中，观察到含有多天线复合型寡糖的放射性标记糖肽的百分比显著增加。在其他实验中，支持细胞在各种基质上生长：塑料；IV型胶原/层粘连蛋白；或基质胶，一种由层粘连蛋白、IV型胶原、硫酸蛋白聚糖、巢蛋白和内联蛋白组成的重组基底膜（RBM）。与在塑料上生长相比，在RBM上生长显著增加了多天线复合型寡糖的形成，而在IV型胶原/层粘连蛋白上生长的细胞中高甘露糖型糖肽增加。这些研究表明，诸如培养时间和细胞外基质的存在等环境和生理条件显著影响支持细胞培养物中的糖基化模式。
