Does endurance running before orchidectomy prevent osteopenia in rats?

This experiment was performed to study the effects on femoral bone of endurance training performed during the 3 months before orchidectomy in rats which were then killed 90 days later. A total of 70 male Wistar rats were used at 8 weeks old. One day 0 of the experiment, 10 rats were killed by cervical dislocation and used as first controls. Among the 60 others, 30 were selected for treadmill running (60% maximal oxygen uptake, 1 h x day(-1), 6 days x week(-1) for 90 days). The 30 other rats remained at rest. On day 90, 10 exercised (IE) and resting (IR) rats were killed and used as intermediary controls. Among the 20 other animals of each group, 10 were surgically castrated (CXE, CXR) or 10 sham-operated (SHE, SHR) and killed on day 180. On day 90 femoral failure load (three-point bending test) was greater in IE than in IR. Simultaneously, the deoxypyridinolinuria was lower in IE than in IR. On day 180, femoral bones were thinner in CXR than in CXE. The lowest values for trabecular bone are in the distal femoral metaphysis were measured in CXE and CXR rats, but the value measured in CXE was no different from that measured in SHR. Simultaneously total femoral bone density was lower in CXR than in SHE, while no difference concerning femoral metaphyseal density was observed between CXE and SHR. These results confirmed that endurance running increased femoral bone growth and modelling and femoral trabecular area, and thereby peak bone mass, in 8-month-old male rats. In resting animals, castrated after the training period, androgen deficiency decreased femoral density, mineral content and trabecular area. This decrease was not observed in castrated but previously exercised rats. Thus, by increasing peak bone mass, it was considered that endurance training may have a preventive effect against orchidectomy-induced bone loss.

Isolation of cells from ovine fetal long bone and characterization of their osteoblastic activities during in vitro mineralization.

Studies about bone formation and regulation are complex due to a close relationship between bone cells. Primary cell cultures allow to understand osteoblastic function. We isolated cells from the cortical metacarpal bone of 85 or 120 day-old ovine fetuses by an enzymatic method. After first passage and cell amplification, the growth medium (DMEM, ascorbic acid and fetal calf serum 10%) was replaced at confluence by a mineralization medium (MM: DMEM, ascorbic acid, beta-glycerophosphate, insulin). Alkaline phosphatase (ALP) activity in cell-matrix layer increased after 4 days of cultures in MM and maximized at day 6. We also measured osteocalcin, ALP and IGF-I secretion simultaneously during mineralization. PTH, PTHrP and 1.25(OH)2D3 decreased ALP activity in cell-matrix layer after 4 days of treatment in MM without fetal calf serum (FCS). Cells from 120 day-old fetuses were cultivated in MM with 10% FCS during 32 days to induce mineralization. Inorganic phosphorus concentration increased in medium between days 5 and 12, Ca concentration decreased in medium after 12 days of culture. Mineralization started at day 12, in the same time ALP activity appeared in medium. Osteocalcin secretion increased between days 6 and 12, decreased at day 14 and increased from day 16 until day 32. Ovine fetal bone cells produced IGF-I until first days of culture in MM. Such ovine osteoblast phenotype cells having the capacity to differentiate and mineralize in vitro would be a model to study the endocrine regulation of osteoblastic function in large mammals.

Calcitonin mRNA expression and plasma calciotropic hormones in fetal lambs.

The object of this study was to measure basal levels of calciotropic hormones during gestation in the ovine fetus. Thyroid glands and plasma samples were collected from 49 Ile de France x (Limousine x Romanov) fetuses (range, 80 to 145 d). Total thyroidal RNA was extracted for calcitonin (CT) mRNA analysis. Plasma immunoreactive concentrations of CT, parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP), and 1,25-dihydroxy-vitamin D [1,25-(OH)2D] were assayed. Plasma Ca and P concentrations were measured by atomic absorption spectrophotometry and colorimetry, respectively. The ratio of thyroidal weight/body weight decreased twice between Days 80 and 145 of gestation. Simultaneously, the CT mRNA rate increased but did not significantly vary after 89 d of gestation. Plasma CT concentration increased between 80 and 89 d and then did not significantly vary until Day 145. We could not detect PTH in ovine fetuses plasma; PTHrP concentration did not vary significantly between Days 80 and 145. The concentration of 1,25-(OH)2D decreased in plasma essentially between Days 110 and 145. In conclusion, between Days 80 and 145 of gestation, CT, PTHrP, and 1,25-(OH)2D concentrations can be easily measured in plasma from ovine fetuses, but plasma PTH was undetectable. In basal conditions, no relationship could be demonstrated between any hormonal concentrations in the fetal plasma and that of Ca or P.

Metacarpal growth and systemic markers of bone metabolism in the ovine fetus.

Bone metabolism was assessed in 56 Ile de Francex(RomanovxLimousine) fetal lambs killed between Day 80 and Day 145 of gestation. In each fetus, the length, width and weight, as well as the calcium and phosphorus content of the left diaphyseal metacarpal bone were measured. Plasma alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities, bone ALP (B-ALP), osteocalcin (OC) and insulin-like growth factor-I (IGF-I) were assayed in these fetuses and in six newborn lambs from birth until one month after birth. Fetal growth is characterized by an increase in bodyweight, bone size and bone mineral content from Day 80 to Day 132 of gestation. These parameters did not significantly vary until birth. Plasma concentrations of IGF-1, OC and B-ALP increased from Day 80 to Day 132. Between Day 132 and birth, plasma IGF-I and B-ALP concentrations did not significantly vary, whereas plasma OC concentration decreased, confirming the usefulness of OC as a marker of osteoblastic activity and bone formation in the ovine species. The increase in plasma IGF-I, B-ALP and OC concentrations observed during the first two weeks of postnatal life indicate an intense skeletal growth in these animals, which was confirmed by the bodyweight growth curve.

Steroid hormones and retinoic acid interact in the regulation of calcitonin and calcitonin gene-related peptide secretion and messenger ribonucleic acid levels in CA-77 C cells.

Northern hybridizations were used to evaluate the modulated action of retinoic acid (R.A.) in presence of dexamethasone (Dex) and/or calcitriol (1,25-(OH)2D3) on calcitonin (CT) and calcitonin gene-related peptide (CGRP) mRNA steady state levels in the murine CA-77 C cell line. Dex was found to increase both CT and CGRP mRNAs in a time-and-dose-dependent way without changing the alternative splicing. A slight but significant increase in the steady-state CT mRNA level was found 3 days after addition of 10(-10) M Dex; the same dose slightly decreased the CGRP mRNA level; concentrations of Dex > or = 10(-9) M elevated both mRNAs. Calcium from 1-4 mM in short-term (1 hr. and 4 hrs.) or long-term stimulations (1 day and 4 days), with or without Dex cotreatment was ineffective. Dex alone (10(-6) M) elicited a 2-fold increase in CGRP mRNA and a 9-fold increase in CT mRNA steady state levels after 6 days of treatment whereas addition of 5.10(-5) M R.A. alone for 6 days decreased both the CGRP and the CT mRNA steady state level (12- and 4-fold decreases, respectively). Our results showed that 5.10(-7) M R.A. blunted in part (-30%) the rise of CT and CGRP mRNA induced under Dex; whereas doses > or = 5.10(-6) M maximally decreased both CT and CGRP mRNA (2- and 9-fold decreases, respectively). The fall under R.A. alone was enhanced when CA-77 cells were cotreated during 6 days with 10(-7) M 1,25-(OH)2D3 (-68% versus -37%). Moreover, the fall in CGRP mRNA (18-fold) of CA-77 cells treated simultaneously with 10(-6) M Dex, 5.10(-6) M R.A. and 10(-7) M 1,25-(OH)2D3 was greater than the decrease (9-fold) observed when the same dose of R.A. blunted the Dex induction. The results obtained by RIA for the CT and CGRP C cell content and release in the culture medium strengthened those observed on both CT and CGRP mRNAs, since a good parallelism was observed between the peptide biosynthesis, secretion and the mRNA levels. Our data suggest that R.A. and 1,25-(OH)2D3 exert a stronger inhibition of the CT gene by a likely coupled action of the two compounds probably via the formation of an heterodimer receptor.

Effects of dexamethasone, calcium and 1,25-dihydroxycholecalciferol on calcitonin and calcitonin gene-related peptide mRNA levels from the CA-77 C cell line.

We used the CA-77 cell, a murine C-cell line derived from a medullary thyroid carcinoma, to study the effects of glucocorticoids, calcium, and vitamin D metabolites on calcitonin (CT) gene expression. Total RNA was isolated, and CT and CT gene-related peptide (CGRP) mRNAs were measured by Northern hybridizations using specific probes. A control mRNA probe (cyclophilin) was used to quantitate the specificity of the changes in CT and CGRP mRNAs. The CA-77 C cell line cultured in basal conditions with a medium deprived of fetal calf serum, but was supplemented by insulin, expressed mainly the CGRP mRNA. Dexamethasone was found to increase both CT and CGRP mRNAs in a time- and dose-dependent way without changing the alternative splicing. A slight but significant increase in the steady-state CT mRNA level was found 3 days after addition of 10(-10) M dexamethasone; the same dose slightly decreased the CGRP mRNA level; concentrations of dexamethasone > or = 10(-9) M elevated both mRNAs. A twelve-fold increase for CT mRNA, and an eightfold increase in CGRP mRNA occurred 3 days after administration of 10(-6) M dexamethasone. Kinetic data revealed inductions of both mRNAs 24 hours after exposure to 10(-7) M dexamethasone, and highest CT and CGRP mRNAs levels were observed after 72 hours of treatment. Calcium from 1-4 mM in short-term (1 hour and 4 hour) or long-term stimulations (1 day and 4 days), with or without dexamethasone cotreatment was ineffective. CT and CGRP mRNAs levels were both half-reduced 48 hours after addition of 10(-7) M 1,25-dihydroxycholecalciferol; this effect was transient, as it disappeared 2 days later.

Effects of 1,25-dihydroxycholecalciferol and calcium on calcitonin mRNA levels in suckling rats.

The chronic administration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) to 9-day-old suckling rats induced no change on day 13 in the calcitonin (CT) mRNA steady-state level of thyroid glands measured by Northern hybridization. Thyroidal CT contents were decreased in relation to increased plasma calcium levels in animals treated with 0.1 or 1 microgram 1,25-(OH)2D3/kg. Using a lower dose (0.01 microgram/kg), neither plasma calcium, nor thyroidal CT contents were changed. No correlation was found between CT mRNA levels and thyroidal CT contents as well as for plasma CT levels and thyroidal CT contents since hormone in blood remained unchanged after treatment by the active vitamin D3 metabolite. Intraperitoneal calcium administration in fasted 13-day-old rats was associated with a 5-fold increase in plasma CT 30 min after injection, but CT mRNA levels were unchanged within 240 min. By contrast, stomach gavage with calcium in fasted 13-day-old rats induced a sustained increase in plasma CT (X2), and a 4-fold increase in the steady-state level of CT mRNA. Calcium per se is a potent stimulator of CT release in suckling rats, but did not change the amount of CT mRNA. However, gastrointestinal factors may be implied directly or indirectly in the increased CT mRNA level after calcium gavage. In conclusion, 1,25-(OH)2D3 which is known to affect CT gene expression in adult rats is ineffective in 13-day-old suckling rats. This observation may be related to developmental changes in the amount of 1,25-(OH)2D3 receptors of C cells.