Endochondral Bone Growth Research Articles

Raf signaling stimulates and represses the human collagen X promoter through distinguishable elements.

Endochondral bone growth is regulated through the rates of proliferation and differentiation of growth plate chondrocytes. While little is known about the intracellular events controlling these processes, the protein kinase c-Raf, a central component of the cellular signal transduction machinery, has recently been shown to be expressed only by differentiated, hypertrophic chondrocytes. The involvement of c-Raf in the transcriptional regulation of the hypertrophic chondrocyte-specific collagen X gene was investigated using cotransfections of collagen X reporter plasmids and expression vectors for mutant c-Raf proteins. Both activated and dominant-negative forms of c-Raf reduced the activity of the collagen X promoter to approximately 30%. The element mediating the repressing effect of activated c-Raf was located between nucleotides -2864 and -2410 of the promoter, whereas the effect of the dominant-negative form of c-Raf was conferred by the 462 nucleotides immediately upstream of the transcription start site. Inhibition of MEK1/2 and ERK1/2, downstream components of Raf-signaling, also caused repression of basal collagen X promoter activity. These data suggest that c-Raf regulates collagen X promoter activity positively and negatively through different cis-acting elements and represent the first evidence of c-Raf activity described in hypertrophic chondrocytes.

A Lys644Glu substitution in fibroblast growth factor receptor 3 (FGFR3) causes dwarfism in mice by activation of STATs and ink4 cell cycle inhibitors.

Missense mutations of human fibroblast growth factor receptor 3 (FGFR3) result in several skeletal dysplasias, including hypochondroplasia, achondroplasia and thanatophoric dysplasia. To study the function of FGFR3 in bone growth and to create animal models for the FGFR3-related inherited skeletal disorders, we introduced a point mutation (Lys644Glu) into the murine FGFR3 genome using a knock-in approach. We found that the Lys644Glu mutation resulted in retarded endochondral bone growth with severity directly linked to the expression level of the mutated Fgfr3. Mice heterozygous for the mutation ( Fgfr3(TD/+) ) expressed the mutant allele at approximately 20% of the wild-type level and exhibited a mild bone dysplasia. However, when the copy number of the mutant allele increased from one (Fgfr3(TD/+) to two (Fgfr3(TD/TD), the retardation of bone growth became more severe and showed phenotypes resembling those of achondroplasia patients, characterized by a dramatically reduced proliferation of growth plate cartilage, macrocephaly and shortening of the long bones, which was most pronounced in the femur. Molecular analysis revealed that expression of the mutant receptor caused the activation of Stat1, Stat5a and Stat5b, and the up-regulation of p16, p18 and p19 cell cycle inhibitors, leading to dramatic expansion of the resting zone of chondrocytes at the expense of the proliferating chondrocytes. The mutant growth plates consequently were in a less active state and generated fewer maturing and hypertrophic chondrocytes. These data provide direct genetic evidence that the point mutations in FGFR3 cause human skeletal dysplasias and uncover a mechanism through which the FGFR3 signals regulate bone growth by modulating expression of Stats and ink4 cell cycle inhibitors.

Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth and activating mutations in Fgfr3 cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, we targeted the expression of an activated FGFR3 to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. We found that FGFR3 inhibited endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulated the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium.

Regulation of sensory neuron precursor proliferation by cyclic GMP-dependent protein kinase.

Cyclic GMP (cGMP) is a molecular messenger involved in diverse cellular processes. Recently, cGMP-dependent protein kinase (cGK) type II was determined to be a regulator of endochondral ossification and bone growth, identifying a role for cGMP in the regulation of cellular proliferation. Here, we demonstrate the presence of cGK type I (cGKI) in cells of the developing trigeminal ganglia. cGKI occurs in some proliferating precursors as evidenced by double labeling with an antibody to cGKI and 5-bromo-2'-deoxyuridine(BrdU) incorporation. Inhibition of cGKI with KT5823 or Rp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) in chick embryos results in a 30-40% decrease in trigeminal ganglia cell number, and this effect is independent of nitric oxide synthase (NOS). In addition, inhibition of cGKI with Rp-8-pCPT-cGMPS results in a 60% decrease in BrdU incorporation in the trigeminal ganglia of embryonic day 5 chicks. We find that PC12 cells expressing cGKI proliferate more rapidly and incorporate more BrdU than do control cells. The cGKI inhibitor Rp-8-pCPT-cGMPS decreases proliferation and BrdU incorporation in transfected PC12 cells but has no effect on control cells. The PC12 cells do not express NOS, indicating that this effect is also independent of NOS. Thus, cGKI regulates the proliferation of sensory neurons as a result of activation of a NOS-independent pathway, representing a novel pathway by which the number of sensory neurons is regulated.

A Review of Nutritional and Metabolic Factors Involved in Dyschondroplasia in Poultry

Whitehead, C.C. 1998. A review of nutritional and metabolic factors involved in dyschondroplasia in poultry. J. Appl. Anim. Res., 13: 1–16. Dyschondroplasia is caused by a defect in endochondral bone growth which interrupts the process of growth plate chondrocyte differentiation and leads to the build up of a mass of avascular cartilage. It occurs most frequently as tibial dyschondroplasia (TD) in broilers and can result in bone growth abnormality contributing to leg weakness. A number of nutritional factors, including calcium, phosphorus, monovalent ions, protein, amino acids, thiocarbonates, trace minerals and vitamins, have been shown to affect the incidence of TD. Vitamin D metabolites are probably the most important nutritional factors, because dietary supplementation with 1-hydroxylated metabolites, particularly 1,25-dihydroxyvitamin D, can prevent TD completely. Other metabolites, including 25-hydroxyvitamin D, may also be partially effective at higher dietary concentrations. The full aetiology of TD is not understood, but the mechanism of action of vitamin D metabolites may involve stimulation of differentiation by interaction with local or systemic cell regulatory factors.

Deer Antler Does Not Represent a Typical Endochondral Growth System: Immunoidentification of Collagen Type X but Little Collagen Type II in Growing Antler Tissue

The collagen isotypes present at early (6 week) and late (5 month) stages of growing deer antler were isolated and identified. Pepsin-digested collagens were separated by differential salt fractionation, SDS-PAGE and Western blotting and subsequently identified by immunostaining. Cyanogen bromide digestion of antler tissue was used to establish a collagen type-specific pattern of peptides, and these were also identified by immunoblotting. Collagen type I was found to be the major collagen in both early- and late-stage antler. Collagen type II was present in the young antler in small amounts but was not confined to the soft “cartilaginous” tip of the antler. Collagen type XI was found in the pepsin digest of the young antler, but collagen type IX was not present at either stage of antler growth. Collagen type X was found in the young antler in all fractions studied. Microscopic study showed that the deer antler did not possess a discrete growth plate as found in endochondral bone growth. Unequivocal immunolocalization of the different collagen types in the antler were unsuccessful. These results show that, despite the presence in the antler of many cartilage collagens, growth does not occur through a simple endochondral process.

The Apert syndrome hand: pathologic anatomy and clinical manifestations.

The Apert syndrome hand demonstrates many typical clinical features including syndactyly, symbrachyphalangism, and growth disturbances. This is due to the grossly abnormal anatomy of both the skeletal and soft-tissue structures associated with a progressive disease process. This paper presents a clinical, radiologic, and histologic analysis of the Apert syndrome hand anatomy and correlates it with the clinical manifestations. It also links hand and craniofacial dysplasia to other regions of the skeleton as well as the overall disease process. From our analysis, we conclude that there is a genetic anomaly causing variable and uncoordinated differentiation of the mesenchyme at the time of embryologic separation into its various skeletal components, particularly in the distal limb bud and craniofacial skeleton. This disease process continues postnatally in endochondral bone growth center malformation and malfunction as well as ectopic cartilage ossification in soft tissues. We discuss the role of abnormal musculotendinous anatomy and altered biomechanical forces in relation to these processes.

The expression of transforming growth factor-β by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-beta (TGF-beta) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-beta 1 to -beta 3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-beta mRNA and conditioned medium was assayed for TGF-beta activity and isoform composition. Active TGF-beta was only detected in 10(-8) M 1,25(OH)2D3-treated cultures (8.37 ng active TGF-beta/mg protein). There was a significant decrease in total (latent-active) TGF-beta activity in conditioned medium of 10(-12) M (23.4%; P < 0.05) and 10(-10) M (20.7%; P < 0.05) 1,25(OH)2D3-treated cultures but 10(-8) M 1,25(OH)2D3 significantly increased (30.9%; P < 0.01) TGF-beta activity. The amounts of TGF-beta 1, -beta 2 and -beta 3 isoforms produced were similar in control, 10(-10) or 10(-12) M 1,25(OH)2D3-treated cultures but the conditioned medium of 10(-8) M 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-beta mRNA demonstrated differential control of TGF-beta gene expression with TGF-beta 1 and -beta 3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10(-8), 10(-10) and 10(-12) M) whilst TGF-beta 2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-beta secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth.

Fibroblast Growth Factor Receptor 3 Is a Negative Regulator of Bone Growth

Endochondral ossification is a major mode of bone formation that occurs as chondrocytes undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. We have identified a role for fibroblast growth factor receptor 3 (FGFR-3) in this process by disrupting the murine Fgfr-3 gene to produce severe and progressive bone dysplasia with enhanced and prolonged endochondral bone growth. This growth is accompanied by expansion of proliferating and hypertrophic chondrocytes within the cartilaginous growth plate. Thus, FGFR-3 appears to regulate endochondral ossification by an essentially negative mechanism, limiting rather than promoting osteogenesis. In light of these mouse results, certain human disorders, such as achondroplasia, can be interpreted as gain-of-function mutations that activate the fundamentally negative growth control exerted by the FGFR-3 kinase.

Regulators of chondrocyte differentiation in tibial dyschondroplasia: an in vivo and in vitro study.

Tibial dyschondroplasia (TD) is a disorder of endochondral bone growth and results in the retention of a mass of unmineralized, avascular cartilage extending into the metaphysis. We have studied various parameters of chondrocyte differentiation, both in isolated chick chondrocytes and growth plate sections, in an attempt to determine whether the inhibition in chondrocyte differentiation seen in TD is a consequence of an inherent incapability of chondrocytes to differentiate terminally and mineralize. Results from in vitro experiments indicated that both normal and lesion chondrocytes synthesized a matrix that stained with antibodies to types II and X collagen and displayed foci of mineralization. Alkaline phosphatase activity in lesion chondrocytes was significantly increased in comparison to that in normal hypertrophic chondrocytes. In addition, normal and lesion chondrocytes in culture synthesized transforming growth factor-beta and 24,25(OH)2D3 but not 1,25(OH)2D3. There was no significant difference in the production rate of these growth regulators between normal and lesion chondrocytes. In contrast, in growth plate sections, alkaline phosphatase activity was markedly reduced in the lesion chondrocytes and sites of mineralization were not evident. Type II collagen was located throughout the growth plate and lesion, but type X collagen was not present within the lesion except at sites of vascularization. These results indicate that, in culture, lesion chondrocytes have the ability to differentiate terminally and mineralize, and suggest that the primary abnormality in TD is related to a developmental fault which is only operative in vivo. This may include a defect in cartilage vascularization and/or impairment of chondrocyte differentiation by mechanisms that have not yet been elucidated but may involve the abnormal production of regulatory factors.

Insulin-like growth factors.

The insulin-like growth factors I and II are single chain polypeptides homologous to proinsulin. IGF I and IGF II contribute to cell regulation and stimulate protein synthesis via signaling through type I receptors which are homologous to insulin receptors and activate phosphorylation cascades. IGFs enhance the proliferation of chondocytes and the proliferation of their collagen and proteoglycan matrix; IGFs stimulate longitudinal (endochondral) bone growth. Throughout life, IGFs are constitutvely expressed ubiquitous factors which help to maintain the survival of differentiated cells, Increased expression is found during growth and tissue repair, Six specific binding proteins, IGFBP 1-6, allow additional tissue compartment specific control of IGF activity; IGFBP production favours storage and IGFBP cleavage leads to activation.

Systemic administration of rhIGF-I or rhIGF-I/IGFBP-3 increases cortical bone and lean body mass in ovariectomized rats

The purpose of this study was to compare dose-related effects on cortical bone and lean body mass following subcutaneous administration of rhIGF-I alone, or bound to an equimolar amount of rhIGFBP-3 to adult Ovx rats. At the age of 16 weeks, rats were ovariectomized or sham-operated and were allowed 8 weeks to develop osteopenia. After being divided into control (saline treated) or treatment groups, rats were injected daily during an 8-week period with 0.9 and 2.6 mg/kg of rhIGF-I, or with 0.9, 2.6, and 7.5 mg/kg of rhIGF-I bound to rhIGFBP-3. Fluorescent bone markers were given 9 and 2 days prior to necropsy. Body weights and lean body mass were monitored throughout the experiment. Cortical bone histomorphometry was performed on tibial cross-sections at the tibiofibular junction, and endochondral bone growth was measured at the distal femoral metaphysis. All rats treated with rhIGF-I or the rhIGF-I/IGFBP-3 complex had increased body weights, corresponding to a dose-dependent increase in lean body mass. Endochondral growth was slightly increased in all experimental groups, but was not dose-dependent. A dramatic increase in periosteal, modeling-dependent formation, coupled with decreased or unchanged resorption on the endocortical envelope resulted in a dose-dependent increase in cortical thickness and cross-sectional area in groups treated with the complex of rhIGF-I/IGFBP-3. This complex appeared to be more effective in promoting positive musculoskeletal changes than rhIGF-I alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic Administration of rhIGF-I or rhIGF-I/IGFBP-3 Increases Cortical Bone and Lean Body Mass in Ovariectomized Rats

The purpose of this study was to compare dose-related effects on cortical bone and lean body mass following subcutaneous administration of rhIGF-I alone, or bound to an equimolar amount of rhIGFBP-3 to adult Ovx rats. At the age of 16 weeks, rats were ovariectomized or sham-operated and were allowed 8 weeks to develop osteopenia. After being divided into control (saline treated) or treatment groups, rats were injected daily during an 8-week period with 0.9 and 2.6 mg/kg of rhIGF-I, or with 0.9, 2.6, and 7.5 mg/kg of rhIGF-I bound to rhIGFBP-3. Fluorescent bone markers were given 9 and 2 days prior to necropsy. Body weights and lean body mass were monitored throughout the experiment. Cortical bone histomorphometry was performed on tibial cross-sections at the tibiofibular junction, and endochondral bone growth was measured at the distal femoral metaphysis. All rats treated with rhIGF-I or the rhIGF-I/IGFBP-3 complex had increased body weights, corresponding to a dose-dependent increase in lean body mass. Endochondral growth was slightly increased in all experimental groups, but was not dose-dependent. A dramatic increase in periosteal, modeling-dependent formation, coupled with decreased or unchanged resorption on the endocortical envelope resulted in a dose-dependent increase in cortical thickness and cross-sectional area in groups treated with the complex of rhIGF-I/IGFBP-3. This complex appeared to be more effective in promoting positive musculoskeletal changes than rhIGF-I alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic administration of recombinant transforming growth factor beta 2 (rTGF-β2) stimulates parameters of cancellous bone formation in juvenile and adult rats

Transforming growth factor β is a multifunctional protein with known actions on bone and bone cells. The ability of TGF-β to stimulate osteogenic parameters in vitro and osteogenesis adjacent to injection sites in vivo is well established. The purpose of this study was to determine if systemic administration of recombinant TGF-β2 (rTGF-β2) could stimulate bone formation in rats of different ages. Juvenile (25-day-old) and adult (160-day-old) rats were treated daily for 5 days and 14 days, respectively, with rTGF-β2 given by subcutaneous injection. Bone formation was measured in cancellous bone of the lumbar vertebrae in juvenile rats and the femoral epiphysis in adult rats. Endochondral bone growth rates were measured in the distal femurs from both juvenile and adult rats using histomorphometric methods. Systemic administration of rTGF-β2 resulted in substantial increases in bone formation rates (both surface and volume referent) in both juvenile and adult rats. In the juvenile rats, rTGF-β2 increased the percent double labeled surface and the mineral appositional rate. In the adult rats, TGF-β2 treatment increased the double labeled surface and also endochondral (longitudinal) growth parameters without changing the number of osteoclasts or the number of osteoclast nuclei per cell. These results demonstrate that short-term systemic administration of rTGF-β2 substantially increases cancellous bone formation rate in rats.

Genital and nongenital teratogenesis of prenatal progestogen therapy: The effects of 17α-hydroxyprogesterone caproate on embryonic and fetal development and endochondral ossification in the C57B1/6J mouse

The current study assesses the effects of 17 alpha-hydroxyprogesterone caproate on embryo-fetal development, with a particular focus placed on intrauterine endochondral bone development and growth. Primigravid C57 BI/6J mice were administered 17 alpha-hydroxyprogesterone caproate by means of subdermal pellets designed to deliver doses of 0.5, 5.0, and 50.0 mg/kg/day on gestational days 7 through 19. Assuming a dosage regimen of 250 mg per 50 kg per week in humans for the treatment of threatened spontaneous abortion, the doses used were 0.7, 7.0, and 70.0 times the human dose equivalent. The time course of exposure encompassed the period of organogenesis through the late fetal period. No maternal toxicity was noted throughout the course of 17 alpha-hydroxyprogesterone caproate administration. Fetal weight at term and the percentage of resorptions, dead fetuses, male fetuses, and malformations at term were not significantly affected at any evaluated dose when compared with control group values. The steroid did not exert a significant influence on any assessed parameter of endochondral bone development or growth, even at a dosage concentration that far exceeded the human therapeutic dose equivalent. As would be expected, fetal weight was directly correlated with diaphyseal length and also influenced the relationship of cartilage to osseous tissue in both the humeri and femora. There were no 17 alpha-hydroxyprogesterone caproate dose and fetal sexual phenotype interactive effects noted with regard to any developmental or bone growth parameter measured, thus discounting any sexual phenotype-related toxic or teratogenic sensitivities. The data indicate that 17 alpha-hydroxyprogesterone caproate administered at doses of up to 70 times the human dose equivalent did not induce increases in the frequency of genital and, importantly, nongenital teratogenesis in spite of exposure during the critical period of organogenesis. Additionally, the steroid did not affect limb morphogenesis or endochondral ossification.

The control of chondrocyte differentiation during endochondral bone growth in vivo: changes in TGF-beta and the proto-oncogene c-myc.

The expression of transforming growth factor-beta and the c-myc proto-oncogene was studied in situ in the chondrocytes of the tibial growth plate of normal chicks and those with avian tibial dyschondroplasia in which the chondrocytes are developmentally arrested in the transitional phase between proliferation and differentiation. This results in an accumulation of unmineralised and avascular cartilage. Dyschondroplastic chicks showed reduced c-myc expression in the transitional chondrocytes but unaltered levels in the proliferating chondrocytes. Transforming growth factor-beta expression was reduced in the transitional chondrocytes of dyschondroplastic chicks. In areas where the lesion was being repaired there was evidence of increased expression of both c-myc protein and transforming growth factor-beta. Addition of 1,25-dihydroxyvitamin D to the diet, which is known to reduce the incidence of dyschondroplasia, resulted in an increase in c-myc production. These results suggest that both transforming growth factor-beta and the proto-oncogene c-myc may be important elements of the cascade of events that lead to chondrocyte differentiation, hypertrophy and mineralisation.

Characterization of the Mouse Type X Collagen Gene

Type X collagen, a homotrimer of α1(X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously characterized a genomic clone containing the major part of the mouse type X collagen gene (col10a1) and assigned the locus for co110al to mouse chromosome 10 (Apte et al., Eur. J. Biochem. 224: 217–224, 1992). In this paper, through additional characterization of cDNA and genomic clones, we describe the complete organization of the col10a1 gene. The col10a1 gene is 7.2 kb in size and, as in the chick, col10a1 gene transcripts are generated from only three exons. Exon 1 encodes only the 5′ untranslated (5′ UT) region of the mRNA and is separated from exon 2 by an intron 562 by in length. Exon 2 (169 bp) encodes 15 by of 5′ UT message, the translation start plus 17-amino acid residues of the putative signal peptide, and 33 1/3 codons of the putative N-terminal non-collagenous (NC2) domain of type X collagen. Exon 3 is 2854 by in size and encodes 4 2/3 codons of the NC2 domain, the entire collagenous domain of 463 residues (COL), the entire C-terminal non-collagenous (NC1) domain (161 amino acid residues) and the entire 965 by 3′ untranslated (3′ UT) sequence of the mRNA. Two TATA boxes are present in tandem in the col10a1 promotor. Both TATA boxes are active in transcription, generating two populations of transcripts with different 5′-termini; the longer transcript is of low abundance and is detectable only by PCR in newborn mice. The col10a1 promotor contains a CCAAT box as well as other consensus sequence elements required for binding of potential transcription factors. Characterization of the col10a1 gene provides data essential for studies of the regulation of type X collagen expression during mammalian endochondral bone growth and development. Knowledge of the complete structure of the mouse typeX collagen gene will also be useful for the investigation of type X collagen gene abnormalities in murine chondrodysplasias and for the generation of transgenic mice.

Morphometric changes in growth of the rat pelvis after papain administration.

The proteolytic enzyme, papain, was given systematically to evaluate the short- and long-term effects of inhibition of endochondral bone formation on pelvic growth, with emphasis on the innominate bone. Ninety-eight Lewis-strain male rats, used concurrently for craniofacial growth studies, were divided into two groups. Thirty rats from Group I (n = 48) received 2% crude papain i.p. daily from 25-40 days-of-age and were euthanized at 40, 54, and 70 days-of-age. Thirty-five rats from Group II (n = 50) were given papain at the same dose from 25-70 days-of-age and were euthanized at 26, 40, 54, 70, and 120 days. The remaining animals in both groups were the controls. Standardized dorsoventral pelvic radiographs were taken of all 98 animals. Ten linear dimensions were measured on each and the data evaluated statistically. A reduction both in size and rate of growth of the bony pelvis was found. All the anteroposterior and most of the transverse pelvic dimensions were significantly shorter, to a greater extent in the prolonged papain group. Bi-ischial width was increased, perhaps to compensate for pelvic shortening and to accommodate the pelvic contents. The findings may contribute to our better understanding of abnormal endochondral bone growth in the pelvis.

Specific β Estradiol Binding in Cartilage and Serum from Young Mice and Rats is Age Dependent

Various studies have shown a direct effect of beta estradiol on cartilage and bone. Such effects point to the possibility that specific receptors to estradiol exist in the growth plate cartilage as well as in bone. 3H-estradiol specific binding (EB) was therefore investigated in the supernatant of cartilage homogenates from the epiphyses and ribs of young growing mice and rats. High levels of EB were observed in the cytosol fraction of cartilage homogenates in the late fetal stage and in young rats and mice. The EB levels decreased gradually from late fetal stage up to 14 days of age in both groups of animals independent of their sex. Nuclear binding of 3H estradiol was also demonstrated by autoradiography in the chondrocytes of proliferating and hypertrophic zones. Estradiol binding was inhibited by high doses of unlabelled beta-estradiol, but not by alpha estradiol. Binding was also inhibited by tamoxifen and DES but not by testosterone. High levels of estradiol binding (EBS) were also observed in serum from young animals, but not in animals 2 months of age or older. Study of estradiol binding in cartilage and in serum of rats of the same age showed a significant difference in estradiol binding between these two systems. The difference in estradiol binding between serum and cartilage was seen in the response to inhibitors, Scatchard analysis, and temperature dependence. The results of our study imply that there are specific receptors for 17 beta estradiol in growth plate cartilage; they originate from chondrocytes, and their amount decreases with age. The effects of estradiol on endochondral bone growth seems therefore to be receptor mediated.

Effect of gestational sex steroid exposure on limb development and endochondral ossification in the pregnant C57BI/6J mouse: I. Medroxyprogesterone acetate

Although data supporting the teratogenic potential of intrauterine progestin exposure is lacking, concern persists among some individuals within the scientific community that these drugs have the potential for nongenital teratogenesis, especially with regard to limb reduction defects. Our laboratory has been interested in the ontogeny of steroid receptors in the developing embryo and in the role of steroid-receptor interactions in limb development, particularly the process of endochondral ossification. Since limb reduction defects can be produced from abnormal processes that are operative during organogenesis or during midgestation (vascular disruption) we have designed an animal study whereby embryos were exposed to sex steroids throughout organogenesis and fetal development. The present study assesses the effects of medroxyprogesterone acetate (MPA) on intrauterine endochrondral bone development specifically, as well as overall embryo-fetal development. Primagravid C57Bl/6J mice were treated via subdermal pellets which deliver MPA at dosages of 5.0, 50.0, and 500.0 mg/kg/day on gestational days 7 through 19. These doses were 25-, 250-, and 2,500-fold higher on a mg/kg basis than the human dose equivalent (HDE). No increases in nongenital malformations were noted at any evaluated MPA dosage level. At 25 X the HDE, MPA did not influence endochondral bone development as evidenced by a lack of significant effects on assessed bone growth parameters. In the 250- and 2,500-fold HDE dosage groups, MPA was shown to exert an embryotoxic effect inducing 48 and 100% resorptions respectively. Mean embryo weights/litter were significantly reduced by MPA exposure at 250 X the HDE. Intrauterine exposure to 250 X the MPA HDE induced reductions in humeral and femoral diaphyseal length in proportion to a reduction in overall growth. The data demonstrate that MPA, administered at dosages of up to several orders of magnitude in excess of the HDE and which permitted embryo survival, did not induce increases in the frequency of nongenital teratogenesis at any dose or gestational stage. Importantly, limb reduction defects were not noted even in instances where the dosage of MPA induced an inhibition of endochondral bone growth.