Estrogen deposits extra mineral into bones of female rats in puberty, but simultaneously seems to suppress the responsiveness of female skeleton to mechanical loading

Abstract

To first test the possible effect of gender on the responsiveness of growing rat skeleton to mechanical loading, 5-week-old littermates of 25 male and 25 female rats were subjected to either free-cage activity or treadmill training for a period of 14 weeks (experiment 1). Using peripheral quantitative computed tomography (pQCT) and mechanical testing of the femoral neck, we observed female rats exhibiting a clearly lower responsiveness to external loading than male rats (+3.0% vs +25% in cross-sectional area (CSA), +4.2% vs +27% in the bone mineral content (BMC), −0.6% vs +10% in volumetric bone mineral density (BMD), and +4.7% vs +28% in fracture strength (F_max) of the femoral neck). Also, relative to the mechanical demands placed on the skeleton, the bones of the young female rats were considerably denser (>50%) than those of the males. In the subsequent experiment 2, we repeated the above-noted first experiment with 33-week-old rats and observed virtually identical exercise-induced benefits (+2.1% vs +10% in CSA, +3.4% vs +18% in BMC, +2.5% vs +23% in BMD, and −1.1% vs +27% in F_max in females vs males, respectively) and the growth/puberty-related condensation of mineral into female bones. Finally, in experiment 3, 60 littermates of 3-week-old female rats were first subjected to sham operation or ovariectomy and then further randomized to exercise or control groups, respectively, to study whether the condensation of mineral into female bones and their lower responsiveness to loading were attributable to the effects of estrogen. At the end of the 16-week intervention, our pQCT and mechanical testing analysis showed not only the anticipated effect of reduced bone density in the ovariectomized rats (∼ −20%) but also the hypothesized better responsiveness to mechanical loading in these estrogen-depleted rats (−3.5% vs +9.1% in CSA, −0.4% vs +12% in BMC, +4.4% vs +9.6% in BMD, and −4.2% vs +16% in F_max in SHAM vs OVX, respectively). In conclusion, the results of our series of three experiments suggest that as such estrogen seems to have very little primary effect on the sensitivity of female bone to respond to external loading, but rather deposits extra stock of mineral into female bones in puberty. This estrogen-driven extra condensation of the female skeleton seems to persist into adulthood, simultaneously damping the responsiveness of the female skeleton to mechanical loading.

Introduction

The primary function of the bones is to bear the muscle contraction- and gravity-induced mechanical forces exerted on them without breaking, consequently enabling efficient locomotion of the body [1], [2]. To successfully carry out this locomotive function, bones are believed to have a loading-driven feedback system which, homologous to a thermostat controlling the temperature inside a house, senses the incident mechanical strain within the loaded bones and subsequently removes bone tissue from sites where the loading is marginal and forms new bone tissue at sites subjected to increased loading in order to provide each bone with the mechanically appropriate size, shape, and architecture [3], [4], [5], [6], [7].

Estrogen, the predominant female sex hormone, has commonly been considered the most important nonmechanical (endocrine) regulator of bone metabolism, taking part in both the accretion of bone mass in adolescence and the loss of bone in senescence [8], [9]. The actions of estrogen have also been linked to the mechanical control of bone homeostasis [10], [11], [12], [13], [14]. Somewhat contradictory to the above-summarized fundamental principle of the skeleton’s adaptation to mechanical loading, the bone loss due to estrogen deficiency continues over a prolonged interval without anticipated compensatory response to increasing mechanical strain experienced by the surviving bone. Thus, it has been postulated that estrogen deficiency actually increases the threshold at which bone cells respond to mechanical strain [3], [11], [12]. In fact, it was recently proposed that postmenopausal osteoporosis per se would be a failure of the bone’s adaptation to functional loading [10].

Although estrogen is the predominant female sex hormone, some believe that estrogen actually is the major biologically active bone steroid in males, too [15], [16], [17]. Recent data also suggest that in both sexes the strain-related proliferation of osteoblasts, the bone-forming cells of the skeleton, is mediated at least in part through a common pathway, the estrogen receptor [18]. Notwithstanding this biological finding, there is some indirect evidence, based on the side-to-side differences of male and female tennis players, that the male skeleton appears clearly more responsive to similar mechanical loading than the female skeleton [19], [20]. The credibility of the cross-sectional data is, however, subject to differences in intensity and frequency of incident loading, to genetic variability, and to many other confounding factors between the given groups of male and female tennis players.

In the light of the above, the initial objective of this study was to evaluate whether there is any sex-related difference in skeletal responsiveness to mechanical loading (treadmill running) between both growing (experiment 1) and mature (experiment 2) female and male rats. As an extension to the findings of the first two experiments, a third experiment (experiment 3) was carried out to assess the notion that the skeletal effects of estrogen would be coupled with the mechanical loading-induced regulation of bone integrity [10], [11], [12] by comparing the skeletal responsiveness of sham-operated and ovariectomized female rats.