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.

No abstract

The effect of 14 or 19 days of dietary capsaicin (18 ppm) on Salmonella enteritidis infection and histological, morphometric, and pH changes of the ceca was investigated. At day 13 or day 18, chicks were challenged with 10(8) colony-forming units of S. enteritidis. Chicks were killed and cultured 24 hours later. The total number of S. enteritidis-organ-culture-positive chicks was significantly lower among chicks fed capsaicin for either 14 or 19 days than among controls (P < 0.05). Subjective histological examination revealed a mild to moderate infiltration of mononuclear cells and heterophils in lamina propria of ceca, as well as epithelial cell proliferation in chicks following either 14 or 19 days of capsaicin administration. Using morphometric analysis, the mean lamina propria thickness and mean epithelial cell thickness in chickens fed capsaicin for 14 or 19 days were significantly greater than in controls (P < 0.05). Capsaicin significantly decreased luminal pH in both trials (P < 0.05). These data indicate that the observed capsaicin-induced resistance to S. enteritidis organ invasion is associated with measurable pH and morphological changes of the cecal mucosa.

The effect of 14 or 19 days of 10% dietary lactose administration on Salmonella enteritidis (SE) colonization and histological, morphometric, and organic acid changes of the ceca were investigated. At Day 13 or 18, chicks were challenged with 10(8) cfu of SE. Chicks were killed and cultured 24 h later. A reduction in the total number of positive SE organ invasions was observed following 14 days (P < .001) or 19 days (P < .005) of treatment in chicks fed with lactose. Histological examination revealed a marked reduction in lamina propria thickness of ceca, as well as subjective epithelial cell proliferation from chicks following either 14 or 19 days of lactose administration. Using morphometric analysis, a reduction in the mean lamina propria thickness in chickens fed with lactose during 14 or 19 days was observed as compared with controls (P < .05). Yet, an increase (P < .05) in the mean epithelial cell length in both lactose-treated groups was observed as compared with controls. Lactose decreased luminal pH (P < .05) and increased the concentration of acetic, propionic, butyric, and lactic acid (P < .05). These data indicate that lactose-induced resistance to SE organ invasion is associated not only with an increase in organic acid concentration but also with measurable morphological changes of the cecal mucosa.

Lamprey gonadotropin-releasing hormone-III (I-GnRH-III), the putative follicle-stimulating hormone (FSH)-releasing factor (FSHRF), exerts a preferential FSH-releasing activity in rats both in vitro and in vivo. To test the hypothesis that I-GnRH-III acts on its own receptors to stimulate gonadotropin release, the functional activity of this peptide at mammalian (m) leutinizing hormone (LH)RH receptors transfected to COS cells was tested. I-GnRH-III activated m-LHRH receptors only at a minimal effective concentration (MEC) of 10–6 M, whereas m-LHRH was active at a MEC of 10–9 M, at least 1,000 times less than that required for I-GnRH-III. In 4-day monolayer cultured cells, I-GnRH-III was similarly extremely weak in releasing either LH or FSH, and, in fact, it released LH at a lower concentration (10–7 M) than that required for FSH release (10–6 M). In this assay, m-LHRH released both FSH and LH significantly at the lowest concentration tested (10–10 M). On the other hand, I-GnRH-III had a high potency to selectively release FSH and not LH from hemipituitaries of male rats. The results suggest that the cultured cells were devoid of FSHRF receptors, thereby resulting in a pattern of FSH and LH release caused by the LHRH receptor. On the other hand, the putative FSH-releasing factor receptor accounts for the selective FSH release by I-GnRH-III when tested on hemipituitaries. Removal of calcium from the medium plus the addition of EGTA, a calcium chelator, suppressed the release of gonadotropins induced by either I-GnRH-III or LHRH, indicating that calcium is required for the action of either peptide. Previous results showed that sodium nitroprusside, a releaser of nitric oxide (NO), causes the release of both FSH and LH from hemipituitaries incubated in vitro. In the present experiments, a competitive inhibitor of NO synthase, L-NG-monomethyl-l-arginine (300 μM) blocked the action of I-GnRH-III or partially purified FSHRF. The results indicate that I-GnRH-III and FSHRF act on putative FSHRF receptors by a calcium-dependent NO pathway.