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.

We reported previously that GH-secreting cells differentiate by day 16 of chicken embryonic development. In the present study, primary cultures of anterior pituitary cells from 10-, 12-, 14-, and 16-day-old chicken embryos were incubated for 2 or 6 days in serum-free medium or medium supplemented with serum from day 12 or day 16 embryos (1% serum by volume). After this culture period, the pituitary cells were recovered and subjected to reverse hemolytic plaque assays for chicken GH. GH-secreting cells did not differentiate spontaneously in serum-free cultures derived from any of the embryonic ages tested, indicating that differentiation of somatotrophs does not occur based only on the relative age of the presumptive GH cell. However, we found that treatment for as little as 2 days with serum from day 16 (but not day 12) embryos stimulated somatotroph differentiation in cultures derived from day 12, 14, and 16 embryos. These results indicate that an activity capable of stimulating GH cell differentiation in vitro is present in day 16 embryonic serum and that the presumptive GH cells were responsive to the somatotroph-differentiating effects of day 16 embryonic serum as early as day 12 of development. Next, serum from day 12, 14, and 16 embryos was evaluated over a 25-fold-range in concentration (0.2-5.0% by volume) using pituitary cells from day 12 embryos as an in vitro bioassay. Treatment with day 12 serum at any of the concentrations tested had no significant effect (P > 0.05), relative to that in serum-free cultures, in which 0.8 +/- 0.4% of all pituitary cells released GH. In contrast, treatment with day 14 and day 16 serum increased the percentage of cells that released GH to 8.0 +/- 1.3% and 12.0 +/- 0.8%, respectively (1% serum by volume). Thus, the level of somatotroph-differentiating activity in embryonic serum was undetectable in day 12 embryos, intermediate in day 14 embryos, and increased to high levels by day 16 of development concomitant with the appearance of GH-secreting cells in vivo reported previously. Next, the specificity of this response to day 16 serum was tested further by treating day 12 cells with peptides known to stimulate GH release in adult animals, GH-releasing hormone and TRH, and a GH-releasing hormone-related peptide (vasoactive intestinal peptide). None of these peptides was found to stimulate somatotroph differentiation at the doses tested (10-9 and 10-7 M).(ABSTRACT TRUNCATED AT 250 WORDS)