The localization of estrogen receptors (ER) in osteogenic cells during the early stage of medullary bone osteogenesis was studied immunohistochemically in the femurs of estrogen-treated male Japanese quail. Alkaline phosphatase (ALP) activity was used as a marker for osteogenic cells. ER immunostaining was observed in the nuclei of weak ALP-positive bone lining cells on the endosteal bone surface of nontreated birds. After 24 hours of estrogen treatment, nuclear immunostaining was detected in ALP-positive preosteoblasts on the endosteal bone surface. After 48 hours, the medullary bone appeared to some degree along the endosteal surface. ER immunostaining was observed in the nuclei of ALP-positive osteoblasts on the medullary bone surface. This study demonstrates that ER are present in osteogenic cells, and suggests that estrogen directly acts on medullary bone osteogenesis.

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The endosteal reaction, the initial step in the formation of medullary bone, was investigated in femurs of estrogen-treated male Japanese quail. Morphologically, the endosteal cells were in an undifferentiated state until 30 h after estrogen treatment and showed characteristics resembling those of resting cells. Many preosteoblasts were seen on the endosteum at 33 h, whereas mitotic figures and fully differentiated osteoblasts were recognized at 36 h after estrogen. The mitotic figures were observed among the preosteoblasts on the endosteum. Autoradiographs showed that the number of endosteal cells labeled by [3H]thymidine injected 1 h before sacrifice was maximal 27 h after the estrogen administration and decreased markedly by 30 h. When a single injection of [3H]thymidine was given at 26 h after estrogen, the highest percent of labeled endosteal cells was observed 1 h later (27 h after estrogen). Labeled preosteoblasts and osteoblasts were observed at 7 h (33 h after estrogen) and 10 h (36 h after estrogen), respectively. Our results show that under the influence of estrogen, endosteal cells are induced to maximally synthesize DNA about 27 h after estrogen. These cells appear to modulate into preosteoblasts in about 6 h and then divide via mitosis to become osteoblasts within an additional 3 h. The development of medullary bone induced by estrogen occurs in a sequential and predictable manner, which makes it a useful system for studying basic problems on bone cell differentiation.

1. The aim of the present study was to investigate expression and localisation of a 28-kDa calcium-binding protein (CaBP-D28k) related to active calcium (Ca) absorption, in the entire intestine of egg-laying hens. 2. Western blotting analysis showed that the entire intestine expressed CaBP-D28k to the following degree: duodenum > jejunum > caecum > ileum > colon. Immunohistochemistry showed strong CaBP-D28k localisation in enterocytes along the villus tip-crypt axis in the duodenum and in villus tips in the caecum and colon. The jejunum and ileum had moderate localisation with respect to the number of immunoreactive cells and staining intensity. 3. These results suggest that laying hens actively absorb Ca in both the large and small intestines.