Bone morphogenetic protein (BMP)-3/osteogenin has a diverse spectrum of biological functions ranging from bone induction to developmental organogenesis. To clarify the role of BMP-3 during bone formation and maintenance, we investigated the expression of BMP-3 mRNA by in situ hybridization (ISH) on 4% paraformaldehyde fixed decalcified bone of normal and fractured bone of adult mice and in the whole body of the mouse fetus with digoxigenin-labeled single-stranded DNA probes generated by PCR. Our modified in situ hybridization technique at the electron microscopic level was also applied to identify specific cell types expressing BMP3 during endochondral ossification in the developing fetal bone. Besides its expression in the developing fetal bronchial epithelium and glial cells of the brain, BMP-3 expression was observed mainly on chondrocytic cells during maturation in the normal growth plate and the fractured callus of the adult long bone and the developing fetal woven bone, suggesting that BMP-3 expression was related not to differentiation of mesenchymal cells into the chondrocytic cell lineage but to the differentiation of immature chondrocytic cells into more mature chondrocytes.

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The relationship between neuronal nitric oxide synthase (n-NOS), and heme oxygenase-2 (HO-2), the enzyme synthesizing CO, in the myenteric plexuses of the canine esophagus were examined by means of double staining technique of NADPH-diaphorase (NADPH-d) histochemistry and HO-2 immunohistochemistry. The coexistent ratio was examined regarding three portions of the esophagus; the cervical, intrathoracic and subdiaphragmatic portions. In the myenteric neurons of all portions, n-NOS frequently coexisted with HO-2 but HO-2 only partially coexisted with n-NOS. Thus, NO cooperates with CO but CO has some functions independent of NO. Many NADPH-d reactive nerve fibers were detected but there were very few HO-2 immunoreactive nerve fibers around the blood vessels and exocrine glands in the esophagus. As a neurotransmitter, NO may be more important than CO because many NADPH-d reactive nerve fibers could be detected in the esophagus. Another possibility is that CO may act as a neuromodulator, rather than as a neurotransmitter.

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To see the cellular localization mechanism of membrane proteins in the hepatocyte in situ, we introduced cDNAs of facilitated-diffusion sugar transporters, GLUT1, GLUT3, GLUT4, and GLUT5, and a Na⁺-dependent active sugar transporter SGLT1 into the rat liver by the in vivo electroporation method and the particle gun method, and the localization of their products was analyzed by immunofluorescence microscopy. SGLT1 was strictly restricted to the bile canalicular (apical) domain, whereas GLUT1 was found in the sinusoidal (basolateral) membrane of hepatocytes. GLUT3 and GLUT5 were present along the whole aspects of the plasma membrane with a tendency for the bile canalicular membrane to be enriched with transporters as compared with the sinusoidal membrane. GLUT4 remained in the intracellular compartments. Simultaneous expression of two of the transporters confirmed these results. Compared with membrane localization of sugar transporters in MDCK cells, GLUT1, GLUT4 and SGLT1 exhibited a similar localization pattern. On the other hand, localization of GLUT3 and GLUT5 was different from that in MDCK cells. These observations suggest that hepatocytes in situ may have a different localization mechanism for sugar transporters from that in MDCK cells. In addition, the in vivo electroporation and the particle gun methods seem to be useful tools for the introduction and analysis of foreign genes in the liver in situ.

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We investigated the expression of reactive oxygen species (ROS)-related enzyme mRNA to clarify the role of ROS in reproduction. In the present study, we investigated the expression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in rat testes by in situ hybridization. To prepare digoxigenin-labeled probes, we obtained 5'-phosphorylated oligonucleotides containing sense and antisense sequences (93-mer) with EcoR I or Hind III restriction sites as protruding cohesive ends (total 99-mer). Then, both oligonucleotides were annealed and cloned into a pGEM4Z vector. The resultant plasmid DNA was linearized with EcoR I or Hind III and used as a template for the synthesis of labeled sense or antisense riboprobes. PHGPx mRNA was expressed stage-specifically during spermatogenesis in adult rats. It was not expressed during spermatogonia, but its expression first appeared in stage VII pachytene spermatocytes, became evident in stage VIII pachytene spermatocytes and increased gradually until becoming diplotene spermatocytes. It decreased slightly during the metaphase of meiosis, and then gradually increased as the spermatids differentiated. The expression was marked in spermatids between steps 7 and 12, and the maximum expression was observed in step 9-10 spermatids. Expression was diminished completely in step 18 spermatids. These results suggest that PHGPx plays an essential role in spermatogenesis.

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Axons containing substance P (SP) serve bone tissue, however, the role of SP in bone metabolism, particularly on osteoclastic bone resorption, is unknown. Therefore, we examined the distribution of neurokinin 1-receptors (NK₁-R), which have a high affinity to SP, in rat osteoclasts in vivo, and investigated the effects of SP on osteoclast formation and osteoclastic bone resorption in vitro. Using electron microscopy, immunoreactive products of NK₁-R were seen in the plasma membrane and cytoplasm of osteoclasts, and were also observed in preosteoclast-like cells. Cell suspensions containing osteoclasts were prepared from neonatal rats and cultured on ivory slices. The addition of 10^-10-10^-6 M SP caused the number of mono- and multi-nucleated tartrate-resistant acid phosphatase positive (TRAP(+)) cells to increase. The increase in TRAP(+) cells with the addition of 10^-8 M SP was inhibited by treatment with the SP receptor antagonist. In cultures on glass coverslips, timelapse studies show that SP induced cell spreading within 5 min and maintained the spreading. The number of resorption pits excavated by the osteoclasts and the resorption area per osteoclast increased in a 48-hour incubation with 10^-8 M SP. These results suggest that SP stimulates osteoclast formation and activates osteoclastic bone resorption through NK₁-R.

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