Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation in vitro.

To study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse-chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCl extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCl extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time-dependent loss in sulphate from the BSP was evident. Using a 24 h pulse-labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12-day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation.     

Studies on bone enzymes. The assay of acid hydrolases and other enzymes in bone tissue.

1. Nine acid hydrolases, cytochrome oxidase, alkaline phenylphosphatase and catalase were demonstrated in 0.25m-sucrose homogenates of newborn-rat calvaria. The acid hydrolases were: acid phenylphosphatase, acid beta-glycerophosphatase, beta-glucuronidase, beta-N-acetylglucosaminidase (beta-N-acetylaminodeoxyglucosidase), acid ribonuclease and acid deoxyribonuclease, showing optimum activity at about pH5; cathepsin, beta-galactosidase and hyaluronidase, with optimum activity at about pH3.6. 2. The main kinetic characters of these enzymes have been studied and methods for their quantitative assay have been worked out. The activities present in bone are given and compared with those found in liver. 3. Acid-phosphatase activity was assayed with phenyl phosphate and beta-glycerophosphate as substrates: activities with these two substrates appeared to be due to two different enzymes. Acid phenylphosphatase is particularly labile and is readily inactivated by various physical or chemical agents.     

Radionecrosis of normal tissue: studies on mouse tails.

Studies on the radionecrosis of mouse tails demonstrate the following modifications to the dose necessary for necrosis in 50 per cent of tails (the ND50): (a) There is very little reduction in ND50 values for irradiated lengths of tail from 2 cm to almost the whole tail, but there is a sharp increase in dose for lengths less than 1.5 cm. (b) The ND50 is high for unanesthetized mice irradiated in air, due to tissue hypoxia. (c) The hypoxia can be reduced by varying amounts by applying heat to the tail, or by flowing oxygen over the tail surface, or by anesthetizing the animal. (d) The ingress of oxygen through the surface can be reduced by placing a clamp round the proximal tail. These features are discussed with reference to the state and possible position of the target cells, and to the use of this assay technique in comparative studies.     

Phosphoproteins of chicken bone matrix. Proof of synthesis in bone tissue

Twelve-day-old embryonic chick mandibles were cultured in vitro for 6 days. Measurements of the weights of the explants, their mineral and protein components, and the EDTA-extractable proteins established that bone tissue synthesizes O-phosphoserine- and O-phosphothreonine-containing phosphoproteins which are similar to those present in embryonic and postnatal chicken bone matrix. The synthesis of the phosphoproteins was further confirmed by the demonstration that radioactively labeled O-phosphoserine and O-phosphothreonine were identified in bone and in the EDTA-extractable phosphoproteins after pulse-labeling chick mandibles in vitro with radioactively labeled serine and threonine, respectively.     

Morphometric studies on mink testicular tissue

The mink, a seasonal breeder of great economic importance, shows a high incidence of male infertility. This problem has forced investigators to find methods of assaying male mink infertility. In this study, morphometric studies have been performed on testicular tissue of a total of 31 males eliminated from breeding after testicular palpation, sperm test, and estimation of serum testosterone concentrations. Males having low sperm quality or disturbed testicular development (n=24) had significantly (p<0.01) lower numbers of spermatocytes, spermatids, and freefloating luminal spermatozoa. compared with males with good sperm quality (n=7). No differences were found in the numbers of spermatogonia, Sertoli, and Leydig cells. Other morphometric parameters such as mean diameter, mean area, mean volume, percentage of area, and surface area per volume of nuclei are also presented for each cell type in the testis. It may be concluded that the sperm test is best suited for assessing fertility in mink. Severe disturbances in testicular development can be detected by testicular palpation and serum testosterone measurements.