Comparison on the effects of three sex hormones on the fetal rat calvarial osteoblasts

Abstract

17β-estradiol(E₂), progesterone (P) and testosterone (T) were investigated for their effects on the proliferation and differentiation of primary rat calvarial osteoblastsin vitro. Rat calvarial osteoblasts were cultured with 10⁻¹⁰ mol/L E₂, 10⁻⁹–10⁻⁶ mol/L P and 10⁻¹⁰ mol/L T for 20 days. Cell proliferation was determined by³H-thymidine incorporation and cell counting. Cell differentiation was examined by measuring alkaline phosphatase (ALP) activity, osteocalcin gene expression and production, bone nodule formation in different periods of culture. Our results showed that no effect of three sex hormones was observed on cell proliferation, but, the responses of cell differentiation to the hormones were more or less different. Among these three hormones used in this study, P appeared to have multi-stimulating effect on cell differentiation. Effect of T seemed not so significant as that of P on cell differentiation. Although ALP activity and osteocalcin production were increased significantly by T, it had slight effect on osteocalcin mRNA and bone nodule formation. Besides, E₂ did not demonstrate any effect on cell differentiation. It is concluded that the proliferation of rat calvarial osteoblasts was independent of the presence of sex hormones. However, the differentiation of these cells were stimulated in different levels and to different extent either by P or T. P appeared to be a multi-stimulator on differentiation of such cells.

Keywords

17β-estradiol / progesterone / testosterone / osteoblast / differentiation

References

[1] BellowsC G, AubinJ E, HeerscheJ N M, et al.. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calci Tissue Int, 1986, 38: 143-143

[2] ChenL, JorgenS, FogedN T. ALP induction by β-glycerophosphate during the non-mineralization phase in vitro. J Tongji Med Univ, 1996, 16: 20-20

[3] GrayT K, FlynnT C, GrayK M, et al.. 17β-estradiol acts directly on the clonal osteoblastic cell line UMR106. Proc Natl Acad Sci USA, 1978, 84: 6267-6267

[4] ChomczynskiP, SacchiN. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 1987, 162: 156-156

[5] EriksenE F, ColvardD S, BergN J, et al.. Evidence of estrogen receptors in normal human osteoblast-like cells. Science, 1988, 241: 84-84

[6] KeetingP E, ScottR E, ColvardD S, et al.. Lack of a direct effect of estrogen on proliferation and differentiation of normal human osteoblast-like cells. J Bon Miner Res, 1991, 6: 297-297

[7] CentrellaM, HorowitzC H, WozneyJ M, et al.. Transforming growth factor-β gene family member and bone. Endoc Rev, 1994, 15: 27-27

[8] HorowitzM C. Cytokines and estrogen in bone:anti-osteoporotic effects. Science, 1993, 260: 626-626

[9] SchevenB A A, DamenC A, HamiltonN J, et al.. Stimulatory effects of estrogen and progesterone on proliferation and differentiation of normal human osteoblast-like cells in vitro. Biochem Biophys Res Commun, 1992, 186: 54-54

[10] TertineggI, HeerscheJ N M. Progesterone stimulates bone nodule formation in rat calvarial cell cultures while estrogen has no effect. J Bone Miner Res, 1992, 7(1): S220-S220

[11] VerhaarH J J, DamenC A, DuursmaSA, et al.. VerhaarH J J, et al.. Progesterone enhances transforming growth factor-β secretion by normal adult human osteoblast-like cells in culture: a comparison with 17β-estradiol. Doctoral thesis: Progestin and bone cell metabolism, 1993The HagueAddix77-77

[12] BellowsC G, AubinJ E. Determination of number of osteoprogenitor present in isolated fetal rat calvarial cells in vitro. Dev Biol, 1989, 133: 8-8

[13] KasperkC, FitzsimmonsR, StrongD, et al.. Studies of the mechanism by which androgens enhance mitogenesis and differentiation in bone cells. J Clin Endocrinol Metab, 1990, 71: 1322-1322