ALTERATIONS OF MATERNAL ESTROGEN-LEVELS DURING GESTATION AFFECT THE SKELETON OF FEMALE OFFSPRING

Estrogens have important effects on bone turnover in both humans and e xperimental animal models. Moreover, the decreased level of estrogens after menopause appears to be one of the key factors in determining po stmenopausal osteoporosis. The presence of estrogen receptor in both o steoblasts and osteoclasts has suggested a direct role of these steroi d hormones on bone tissue. Thus, this tissue is now regarded as a spec ific estrogen target tissue. Exposure to estrogens during various stag es of development has been shown to irreversibly influence responsive target organs. We have recently shown that transient developmental neo natal exposure (days 1-5 of life) of female mice to estrogen resulted in an augmented bone density in the adult animals. The aim of the pres ent study was to evaluate whether shortterm modification of maternal e strogen levels during pregnancy would induce changes in the skeleton o f the developing fetuses and to identify any long-term alterations tha t may occur. Pregnant mice were injected with varying doses (0.1-100 m u g/kg maternal BW) of the synthetic estrogen diethylstilbestrol (DES) from day 9-16 of pregnancy. Offspring were weaned at 21 days of age, and effects on bone tissue of the female mice were evaluated in adulth ood (6-9 months of age). Prenatal DES treatment(s) did not significant ly affect BW. However, a dose-dependent increase in bone mass, both in the trabecular and cortical compartments, was observed in the prenata l DES-exposed female offspring. Furthermore, long bones of DES-exposed females were shorter than controls. Normal skeletal mineralization ac companied these changes in the bone tissue, as shown by a parallel inc rease in skeletal calcium content. Double tetracycline labeling perfor med in 6-month-old DES-exposed animals showed increase in mineral appo sition rate in adult DES-exposed mice as compared with untreated contr ol animals, although no significant difference in the circulating estr ogen levels was found in animals of this age. Experiments were then pe rformed to evaluate whether perturbation of the estrogen surge at pube rty in these diethylstilbestrol (DES)-exposed mice could reverse the o bserved changes. Femur length was chosen as a marker of potential estr ogenic effect. Prepubertal ovariectomy of the prenatally DES-treated a nimals could only partially reverse the effects observed in the skelet on of the DES-treated animals. Further experiments were performed to e valuate whether these changes could have occurred in utero. CD-1 pregn ant female mice were injected with DES (100 mu g/kg maternal BW) from days 9-15 of gestation. On day 16 of gestation, fetuses were examined and stained by a standard Alizarin Red S and Alcian Blue procedure to visualize calcified and uncalcified skeletal tissue. Estrogen treatmen t induced an increase in the amount of calcified skeleton as compared with untreated controls and also a decrease in the length of long bone s, strongly suggesting a change in both endochondral ossification and endosteal and periosteal bone formation. In summary, these data show, for the first time, that alterations in the maternal estrogenic levels during pregnancy can influence early phases of fetal bone tissue deve lopment and subsequently result in permanent changes in the skeleton. Finally, the effect of this short-term estrogen treatment can be seen in the fetal skeleton, suggesting an estrogen-imprinting effect on bon e cell-programming in fetal life because treatment effects on bone cel l turnover can be observed later in adult life.