Porter T E, Couger G S, Dean C E, Hargis B M
Department of Poultry Science, Texas A & M University, College Station 77843, USA.
Endocrinology. 1995 May;136(5):1850-6. doi: 10.1210/endo.136.5.7720629.
In the present study, a reverse hemolytic plaque assay (RHPA) for chicken GH was established and used to study the ontogeny of somatotroph differentiation and functional responsiveness to GH-releasing hormone (GHRH) during chicken embryonic development. Anterior pituitaries from embryos on days 10, 12, 14, and 16 of incubation were isolated and dissociated into single cells with trypsin. The resulting cells were then subjected to the GH plaque assay under basal and GHRH-stimulated conditions. No GH-releasing cells were detected on day 10 or 12 of embryonic development. In contrast, a few somatotrophs (< 2% of all cells) were consistently found on day 14, and a statistically significant population existed on day 16, when 6.3 +/- 1.4% of all anterior pituitary cells secreted GH. Thus, GH-secreting cells differentiated by embryonic day 16. Treatment of pituitary cells from day 16 embryos with GHRH was found to increase the proportion of GH plaque-forming cells during a shortened assay interval from 1.8 +/- 0.3% under basal conditions to 6.7 +/- 1.2% in the presence of GHRH. This nearly 4-fold increase in the proportion of plaque-forming cells indicates that at least 70% of the initial somatotrophs present on day 16 were responsive to the stimulatory effects of GHRH. To test whether the absence of GH cells on day 12 of embryonic development was due to the presence of cells that produced but did not release GH, pituitary cells from day 12 and day 16 embryos were subjected to immunocytochemistry for GH and to the GH RHPA in parallel. No significant differences were found in the percentage of cells that either contained or released GH on the two embryonic ages tested. On day 12, 1.1 +/- 0.8% of all cells contained GH, as determined by immunocytochemistry, whereas 0.5 +/- 0.5% released GH as determined by RHPA. By day 16, the proportions of cells that contained and released GH had increased to 9.5 +/- 0.6 and 11.2 +/- 2.5%, respectively. Taken together, these results indicate that GH-secreting cells differentiate by day 16 of chicken embryonic development and that these initial somatotrophs are responsive to GHRH. Given that growth and metabolism are regulated in part by GH in chick embryos, these findings suggest that these processes may be under hypothalamic control during late embryonic development in the chicken.
在本研究中，建立了一种用于检测鸡生长激素（GH）的反向溶血空斑试验（RHPA），并用于研究鸡胚胎发育过程中生长激素分泌细胞的分化个体发生以及对生长激素释放激素（GHRH）的功能反应性。分离孵化第10、12、14和16天胚胎的垂体前叶，并用胰蛋白酶将其解离为单细胞。然后将所得细胞在基础条件和GHRH刺激条件下进行GH空斑试验。在胚胎发育的第10天或第12天未检测到GH释放细胞。相比之下，在第14天始终能发现少量生长激素分泌细胞（占所有细胞的<2%），在第16天存在统计学上显著的细胞群体，此时所有垂体前叶细胞中有6.3±1.4%分泌GH。因此，分泌GH的细胞在胚胎第16天分化。发现用GHRH处理第16天胚胎的垂体细胞，在缩短的试验间隔期间可增加GH空斑形成细胞的比例，从基础条件下的1.8±0.3%增加到存在GHRH时的6.7±1.2%。空斑形成细胞比例近4倍的增加表明，第16天存在的初始生长激素分泌细胞中至少70%对GHRH的刺激作用有反应。为了测试胚胎发育第12天GH细胞的缺失是否是由于存在产生但不释放GH的细胞，对第12天和第16天胚胎的垂体细胞同时进行GH免疫细胞化学和GH RHPA检测。在所测试的两个胚胎时期，含有或释放GH的细胞百分比未发现显著差异。通过免疫细胞化学测定，在第12天，所有细胞中有1.1±0.8%含有GH，而通过RHPA测定，有0.5±0.5%释放GH。到第16天，含有和释放GH的细胞比例分别增加到9.5±0.6%和11.2±2.5%。综上所述，这些结果表明，分泌GH的细胞在鸡胚胎发育第16天分化，并且这些初始生长激素分泌细胞对GHRH有反应。鉴于鸡胚胎中的生长和代谢部分受GH调节，这些发现表明这些过程在鸡胚胎发育后期可能受下丘脑控制。
