We examined whether estrogen receptor (ER)alpha is required for estrogen to stimulate cancellous bone formation in long bones of male mice. 17 beta-Estradiol (E(2)) was administered to ER alpha(-/-) male mice or wild-type (WT) littermate controls at 40, 400, or 4000 microg/kg by daily sc injection for 28 d and histomorphometric analysis performed at the distal femoral metaphysis. In WT mice, treatment with E(2) (40 microg/kg per d) increased the proportion of cancellous bone surfaces undergoing mineralization and stimulated mineral apposition rate. In addition, higher doses of E(2) induced the formation of new cancellous bone formation surfaces in WT mice. In contrast, E(2) had little effect on any of these parameters in ER alpha(-/-) mice. Immunohistochemistry was subsequently performed using an ER alpha-specific C-terminal polyclonal antibody. In WT mice, ER alpha was expressed both by cancellous osteoblasts and a significant proportion of mononuclear bone marrow cells. Immunoreactivity was also observed in cancellous osteoblasts of ER alpha(-/-) mice, resulting from expression of the activation function-1-deficient 46-kDa ER alpha isoform previously reported to be expressed in normal osteoblasts and bones of ER alpha(-/-) mice. Taken together, our results suggest that estrogen stimulates bone formation in mouse long bones via a mechanism that requires the presence of full-length ER alpha possessing activation function-1.

We had long-term comparative investigation on the reproduction and hormones in fish (protandrous black porgy, Acanthopagrus schlegelii) and scleractinian coral (Euphyllia ancora). In fish, the hypothalamic-pituitary-gonadal axis releases GnRH, LH/FSH and sex steroids (estradiol, testosterone, 11-ketotestosterone, 17,20ß-dihydroxy-pregnen-3one, 17,20ß,21-trihydroxy-4-pregnen-3-one) for the regulation of reproduction, and gamete growth and maturation. We hypothesize that the endocrine system or hormones play an important role as an endogenous factor both in the synchronous mass spawning of corals and fish seasonal reproduction. We comparatively identified the vitellogenesis in coral and fish. Immunoreactive testosterone and estradiol in the free and glucuronided forms were identified and consistently detected in the coral polys throughout the year. Peak levels of free estradiol and glucuronided estradiol were obtained in the coral tissue just prior to spawning. The presence of specific aromatase activity was demonstrated in coral tissue according to the tritiated-androstenedione as a substrate and tritiated H2O formation, with a significantly high aromatase activity detected during the spawning period. Higher concentrations of free estradiol than glucuronided estradiol were detected in the coral tissue throughout the year. In contrast, higher levels of glucuronided estradiol than free estradiol and glucuronided testosterone were found in seawater during coral mass spawning. Furthermore, immunoreactivity and biological activity of immunoreactive gonadotropin-releasing hormone (irGnRH) were detected and peak levels of irGnRH were observed in coral tissue during spawning. Coral extracts and mammalian (m)GnRH agonist had a similar dose dependent effect on the release of LH in the black porgy fish pituitary cells; while mGnRH receptor antagonist blocked the stimulatory effects of coral extracts. Our recent studies demonstrated that a steroidogenic enzyme 17ß-hydroxysteroid dehydrogenase type 14 transcript and protein were detected in the coral tissues. Some hormones are possibly conserved in the regulation of seasonable reproduction of fish and coral.

We recently found that estrogen receptor (ER) antagonists prevent high-dose estrogen from inducing the formation of new cancellous bone within the medullary cavity of mouse long bones. In the present investigation, we studied the role of specific ER subtypes in this response by examining whether this is impaired in female ERβ−/− mice previously generated by targeted gene deletion. Vehicle or 17β-estradiol (E2) (range 4–4,000 μg · kg−1 · day−1) was administered to intact female ERβ−/− mice and wild-type littermates by subcutaneous injection for 28 days. The osteogenic response was subsequently assessed by histomorphometry performed on longitudinal and cross sections of the tibia. E2 was found to cause an equivalent increase in cancellous bone formation in ERβ−/− mice and littermate controls, as assessed at the proximal and distal regions of the proximal tibial metaphysis. E2 also resulted in a similar increase in endosteal mineral apposition rate in these two genotypes, as assessed at the tibial diaphysis. In contrast, cortical area in ERβ−/− mice was found to be greater than that in wild types irrespective of E2 treatment, as was tibial bone mineral density as measured by dual-energy X-ray absorptiometry, consistent with previous reports of increased cortical bone mass in these animals. We conclude that, although ERβ acts as a negative modulator of cortical modeling, this isoform does not appear to contribute to high-dose estrogen's ability to induce new cancellous bone formation in mouse long bones.

It is well recognized that high-dose estrogen induces a marked osteogenic response in long bones of female mice. In light of evidence which suggests that nitric oxide synthase (NOS) plays a role in regulation of osteoblast activity, we analyzed whether NOS is involved in mediating this response. Intact female mice were administered 17β-estradiol (E2) either alone or in combination with NG-nitro-l-arginine methylester (L-NAME) or aminoguanidine (AG), over 24 days. The former inhibits both constitutive and inducible isoforms of NOS, whereas the latter is a selective inhibitor of inducible NOS. Bone mineral density (BMD) of the femur was subsequently measured by dual-energy X-ray absorptiometry (DXA), and histomorphometry performed at the proximal metaphysis on longitudinal tibial sections. As expected, E2 given alone led to a marked accumulation of cancellous bone at the proximal tibial metaphysis, associated with a significant gain in femoral BMD, and an increase in cancellous mineralizing surfaces as assessed by histomorphometry. Neither L-NAME nor AG affected cancellous histomorphometric indices when given alone. However, when administered in combination with L-NAME, the magnitude of the skeletal response to E2 was significantly reduced. The tendency for L-NAME to reduce estrogen-induced bone formation within the proximal tibial metaphysis was more marked distally compared with proximally. In contrast, AG showed no tendency to suppress the osteogenic response to E2. Subsequently, we examined the effect of E2 administration on expression within mouse femoral bone marrow of endothelial NOS (eNOS), which is the predominant constitutive isoform of NOS within bone. No change in eNOS mRNA levels was observed following E2 administration, as assessed by reverse transcription-polymerase chain reaction (RT-PCR). Taken together, our results suggest that eNOS plays a role in mediating estrogen-induced bone formation in intact female mice, possibly as a consequence of posttranscriptional regulation of eNOS activity by estrogen.