Seiji Kusuhara

Estrogen target cells during the early stage of medullary bone osteogenesis: Immunohistochemical detection of estrogen receptors in osteogenic cells of estrogen-treated male Japanese quail

SummaryThe 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.

IMMUNOELECTRON MICROSCOPIC DEMONSTRATION OF ESTROGEN RECEPTORS IN OSTEOGENIC CELLS OF JAPANESE QUAIL

SummaryThe localization of estrogen receptors (ERs) in osteogenic cells was immunoelectron microscopically examined in the femurs of female and estrogen-treated male Japanese quail. An electron dense reaction product showing ER localization was observed in the nuclei of osteoblasts and immature osteocytes in the medullary bone of the female quail. However, reaction product was not seen in the osteoclasts. On the endosteal bone surface of male quail, nuclear reaction product was detected in bone lining cells. After 24 h of estrogen treatment, reaction product was observed in the nuclei of preosteoblasts on the endosteal bone surface. After 48 h, the medullary bone partly appeared along the endosteal surface. Nuclear reaction product was seen in osteoblasts on the medullary bone surface.

Effects of oestrogen and anti‐oestrogen on the cells of the endosteal surface of male Japanese quail

1. The endosteal cellular response occurring in the early stages of medullary bone formation was histologically examined in the femurs of male Japanese quail treated with oestrogen and antioestrogen. 2. The numbers of endosteal cells decreased, and preosteoblasts and osteoblasts increased, on the endosteal surface following oestrogen treatment. 3. After oestrogen and anti-oestrogen treatment the numbers of preosteoblasts and osteoblasts on the endosteal surface did not increase. However, endosteal cells slowly decreased in number and osteoclasts increased. 4. This study demonstrates that differentiation of osteoblasts is induced by oestrogen stimulation and suggests that osteoclasts occur by oestrogen inhibition on the endosteal surface.

Expression and localisation of calbindin D28k in all intestinal segments of the laying hen

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.

Immunoelectron microscopic detection of estrogen target cells in the bone marrow of estrogen-treated male Japanese quail

The localization of estrogen receptors (ERs) in bone marrow cells was studied immunoelectron microscopically in the femurs of estrogen-treated male Japanese quail. The electron-dense reaction product showing ER localization was not detected in the bone marrow cells of the male quail. After 48 h of estrogen treatment, nuclear reaction product was observed in many mononuclear cells in the bone marrow. Most were located in the marrow stroma, and they were divided into undifferentiated and differentiated type cells, characterized by poorly and well-developed cytoplasm, respectively. Some ER-positive cells were situated in the hematopoietic tissue and had moderately developed cytoplasm. Thus, estrogen target cells may be present in the stroma and hematopoietic tissue of bone marrow.

Ultrastructural changes of osteoclasts on hen medullary bone during the egg‐laying cycle

1. The ultrastructure of osteoclasts on hen medullary bone during the egg-laying cycle was observed by electron microscopy. 2. At 0 to 6 h after oviposition, osteoclasts lacking ruffled borders were attached to the bone via the clear zone and appeared to have ceased bone resorption. Small vacuoles were scattered throughout this cytoplasm. 3. At 9 to 21 h after oviposition, most of the osteoclasts had ruffled borders and appeared to be resorbing bone. The ruffled borders at 15 h were well developed, whereas at 9, 18 and 21 h they were poorly developed or showed similarities in structure to the clear zone. The small vacuoles were concentrated under the ruffled borders at 9 h and then decreased. 4. These results demonstrate cyclic changes in osteoclasts during the egg-laying cycle, indicate that ruffled borders form at the beginning of bone resorption and suggest that they are derived from the clear zone and the small vacuoles. The results also indicate that the ruffled borders fuse into the clear zone and disappear at the completion of bone resorption.

Enzymohistochemical Studies on the Formation and Resorption of Bones in Laying Hens

白色レグホーン種の産卵鶏を用いて,大腿骨のMedullary bone (MB)とCortical bone (CB)における破骨細胞,骨芽細胞および骨細胞のAlkaline phosphatase (ALP), Acid phosphatase (ACP), Succinate dehydrogenase (SDH)およびLactate dehydrogenase (LDH)の活性を卵管内の卵の位置と関連づけて組織化学的に調べた.卵が卵白分泌部または峡部に存在する時期において,MBにおける破骨細胞のACPとSDHは中等度の活性,LDHは強い活性を示した.骨芽細胞のALPは強い活性,SDHとLDHは中等度の活性をあらわした.骨細胞のSDHとLDHはともに弱い活性を示した.CBにおける破骨細胞のACPとSDHは強い活性,LDHは中等度の活性を示した.骨芽細胞のALP, SDHおよびLDHはともに弱い活性,骨細胞のACPは中等度の活性,LDHは弱い活性をあらわした.これらの所見はMBの形成促進とCBの吸収促進を示唆している.卵が子宮部(カルシウム分泌部)あるいは腟部に存在している時期および産卵直後において,MBにおける破骨細胞のACPとSDHの活性は強く,LDHの活性は中等度であった,骨芽細胞のALP, SDHおよびLDHの活性は弱かった.骨細胞のACP,SDHおよびLDHは弱い活性を示した.CBにおける破骨細胞のACPとSDHは中等度の活性,LDHは強い活性をあらわした.骨芽細胞の酵素はいずれも弱い活性を示した.骨細胞のACPとLDHの活性は弱かった.これらのことはMBの吸収が活発であるが,CBの形成と吸収は不活発であることを示している.

Histochemical observations on the enzymes of chicken yolk sac membrane.

Synopsis In the endodermal cells of the yolk sac membrane of chicken embryos incubated for 12 to 18 d, the activity of glucose‐6‐phosphatase (G‐6‐Pase) was high, the activity of acid phosphatase (Acid Pase) was moderate, the activities of lactate dehydrogenase (LDH) and of glyceraldehyde‐3‐phosphate dehydrogenase (G‐3‐PDH) were low, and succinate dehydrogenase (SDH) and alkaline phosphatase (Alk Pase) activities were not detected. On the day before hatching, however, SDH became evident with low activity, and the activities of Acid Pase, LDH and G‐3‐PDH increased and remained higher until the day after. Five days after hatching, there were marked decreases in the activities of all the enzymes except SDH. Alk Pase was not detected at any time. The function of the yolk sac endo‐derm in the absorption of yolk is discussed in the light of these observations.

Effects of 25‐hydroxy‐cholecalciferol on the development of osteochondrosis in swine

The principal objective of this experiment was to evaluate the effect of 25-hydroxy-cholecalciferol (25-OH-D3 ) on the development of osteochondrosis in 6- to 110-kg castrated male pigs. The growth rate and serum calcium and inorganic phosphate levels neither increased nor decreased in response to supplementation of 25-OH-D3 . However, supplemental 25-OH-D3 significantly increased serum levels of 25-OH-D3 and 1α,25-hydroxy-cholecalciferol without any influence on bone mineral density. The 25-OH-D3 -treated group had significant (P < 0.05) reduced incidence of osteochondrotic lesions compared to the control group as evidenced by macroscopically examining the articular cartilage of the distal humerus (32.4% vs. 59.3%) and distal femur (47.1% vs. 87.5%). Likewise, supplemental 25-OH-D3 significantly reduced osteochondrotic lesions over the control when histologically examining humerus (20.6% vs. 43.8%) and femur (52.9% vs. 87.5%). The results of this experiment suggested that 25-OH-D3 supplementation in pig diets had a tendency to promote normal endochondral ossification, inhibit osteochondrosis progression and possibly regenerate destroyed cartilage tissue.

Histochemical identification of oestrogen target cells in the medullary bone of laying hens

1. Oestrogen target cells in the medullary bone of laying hens were examined using a histochemical method with fluorescein isothiocyanate labelled oestradiol. 2. Specific fluorescence was found to occur strongly in osteoblasts and weakly in osteoclasts on the medullary bone surface. 3. This study suggests that osteoblasts on the medullary bone surface are oestrogen target cells.