Evaluation and manipulation of the normal, gradual transition to ovarian failure in rats, an estrogen-replete period that mimics the hormonal changes of perimenopause, provides a unique tool for exploring the association of nonestrogenic reproductive hormones with BMD loss during the menopausal transition. Our finding that longitudinal changes in FSH were statistically associated with BMD loss at trabecular-enriched sites during the transition to ovarian failure recapitulates in rats the clinical findings of the Study of Women's Health Across the Nation, a large, longitudinal, community-based study demonstrating a correlation between FSH and decreased BMD of the spine and hip across the perimenopausal transition [3
]. The correlation between FSH and decreased BMD documented here is also consistent with previous reports of a positive correlation between FSH and bone resorption in perimenopausal women [1
]. Thus, our finding of a positive association between FSH and BMD loss in an experimental model of perimenopause reproduces epidemiologic data in perimenopausal women, including the documentation of significant losses in BMD only when FSH is increased five- to sixfold or more over normal cycling levels [7
Decreased ovarian production of inhibins during the transition to ovarian failure is thought to be a primary driver of increased pituitary secretion of FSH in women [9
]. Our data would suggest that supraphysiologic FSH increases in VCD-treated rats are similarly driven by decreases in ovarian production of inhibins A and B in these follicle-depleted rats, with inhibin A being most strongly correlated. Having thus reproduced a second important hormonal feature of perimenopause, these studies further documented a significant correlation of decreased inhibin A with BMD loss in animals transitioning to ovarian failure, an association that was stronger than that of FSH. As inhibin A (vs. inhibin B) has been reported to correlate most strongly with bone resorption in perimenopausal women [13
], these findings in the rat model would thus appear to reproduce the association of decreased inhibin A with bone loss in women during perimenopause.
In contrast to the significant correlations of FSH and inhibin A with bone loss demonstrated in these studies but consistent with previous findings in perimenopausal women [1
], serum levels of estradiol either did not correlate with (in skeletally mature rats) or only weakly correlated with (during persistent estrus) BMD. Because the periods of decreased BMD in VCD-treated animals generally coincided with persistent estrus, one could postulate that loss of cyclic variations in E2
levels during this period could be detrimental to bone. However, the well-documented normalization of BMD in OVX rats in response to continuous E2
replacement would seem to run counter to this theory [40
VCD effects on ovarian inhibin production have not previously been reported, nor to our knowledge have dynamic changes in serum inhibin A versus B levels been characterized over time in normal aging rats. It is thus intriguing to note a divergence between inhibin dynamics in premenopausal women versus normal aging Sprague-Dawley rats, as reported here. Inhibins A and B are both decreased during perimenopause in women [9
], while in normal aging rats inhibin A levels actually increased with age while serum inhibin B levels decreased. It is notable, therefore, that inhibin A levels were uniquely suppressed in VCD-treated animals coincident with the onset of persistent estrus, the period when bone loss was most pronounced. While the design of the studies reported here does not allow for analysis of the ovarian sources of inhibins A and B, in normal rats inhibin beta B-subunits are reported to be preferentially expressed in less mature follicles (secondary and early antral), while inhibin beta A is preferentially expressed in more mature antral follicles [46
]. Additionally, and unique to persistent estrus, inhibin beta A is localized to cystic follicles present during persistent estrus, a period during which the inhibin alpha-subunit is produced by ovarian follicles and the ovarian stroma [47
]. Therefore, it is possible that inhibin B serves as a bio-marker of maturing ovarian follicles in cycling animals and, thus, decreased in all treatment groups with aging, albeit at different rates. In contrast, increasing inhibin A levels with aging in normal rats may reflect an increasing pool of cystic follicles associated with persistent estrus. Future studies will be required to determine whether decreases in inhibin A during persistent estrus in VCD-treated animals occur secondary to changes in follicular number vs. follicular (or stromal) expression of inhibin alpha and beta A.
This study has several limitations. First and foremost is the relative insensitivity of dual-energy X-ray absorptiometry (DXA), the method available to us for tracking skeletal changes in these animals, for assessing trabecular bone loss. The distal femur, while enriched for metabolically active trabecular bone, is comprised of only 20% cancellous bone [37
]. Thus, BMD loss in OVX animals at this site is <20%, as reported here and by others [37
], while methods that can specifically assess trabecular (vs. cortical) bone, such as histomorphometry or microCT, can detect losses of >50% in the trabecular compartment of the distal femur in response to OVX [37
]. Also, while FSH and inhibin A levels significantly correlated with BMD in our studies, accounting for up to 33% of the variation in BMD during persistent estrus, extreme changes in these hormones occurred only with VCD treatment. Thus, the possibility that direct adverse skeletal effects of VCD, unrelated to hormonal changes, mediated and/or contributed to BMD changes cannot be ruled out. However, it should be noted that the delay in BMD loss relative to the time of VCD exposure, particularly in low-dose VCD rats where these events were separated by a 10-month gap, would argue against this possibility.
In summary, these studies provide novel evidence demonstrating that depletion of ovarian follicular reserves in Sprague-Dawley rats by VCD administration can be used as an experimental tool to reproduce and manipulate key hormonal changes of human perimenopause in animals that retain an intact HPO axis. In this model, dose-dependent decreases in ovarian production of inhibins A and B likely result in supraphysiologic increases in FSH, while serum estradiol levels, as in human perimenopause [8
], remain normal to high despite a loss of regular estrus cyclicity. Using this model as a tool, the association of perimenopausal changes in FSH and inhibin A with BMD loss at cancellous-enriched bone sites has been reproduced for the first time in an animal model. Because inhibin A was most strongly correlated with BMD loss in these studies and normalization of FSH in postmenopausal women has previously been demonstrated to have no effect on bone turnover [18
], inhibin A remains a likely candidate in the search for hormonal mediators of perimenopausal bone loss. As all known biological effects of the inhibins can be attributed to their interference with receptor binding of activin [49
], a hormone reported to be selectively increased in women during perimenopause [50
], further examination of inhibin/activin signaling pathways in bone may prove fruitful in future studies probing the mechanistic basis of perimenopausal bone loss.