Estrogen-responsive organs are affected by estrogen imprinting well into adulthood [27
]. These effects can depend on the duration of exposure during gestation as well as on the levels of estrogen [28
]. Bone and articular cartilage have already been characterized as estrogen-responsive tissues [30
]. More recent evidence suggests that the IVD contains estrogen receptors as well [32
Developmental exposure to DES between 9 and 16 days of gestation was shown to increase both trabecular and cortical femoral bone mass in female offspring [10
]. No similar studies evaluated the effect on either male offspring or the lumbar vertebrae. Although sexual dimorphism does exist, when it comes to the bone response to estrogen, estrogen-deficient males still have low bone mass [33
]. Estrogen-receptor alpha (ERα) and beta (ERβ) are mainly responsible for the effects of estrogen on bone in both genders [34
]. The involvement of estrogen receptor-related receptor (ERR) was also recently identified in the effects of estrogen on bone [35
]. Diethylstilbestrol is known to have an agonistic effect on both ERα and ERR [35
]. In light of these data, it would be expected that an increased BMD would be noted in both males and females, which would most likely be due to an increase in BMC.
The current study demonstrated that the BMC and TBA increased in adult females but decreased the overall bone size in males. This corresponds to more-fragile bone in females and a feminized phenotype in males because of the decreased bone size. The epigenetic changes induced by DES can thus be responsible for altering how bone responds to estrogen exposure [37
]. Hypermethylation of histone 2 occurred in cell cultures exposed to DES and may play a part in the effect on bone [39
]. The effects were similar in both lumbar and femoral bone, although lumbar bone was more sensitive to lower doses of DES. Clinically, this might imply vertebral fractures earlier in life, which can be associated with significant morbidity [40
]. If other exercises that involve impact or high rates of load, such as running or jumping, as opposed to swimming, were evaluated, it is possible that lumbar and vertebral bone would have similar results. DES cohorts can thus potentially benefit from earlier screening with bone scans to detect those vulnerable to vertebral or hip fracture.
In this work, because the mothers were all epigenetic siblings and were treated exactly the same way during gestation (except for DES dose given), no reason exists to believe that pups born to different mothers have any reason to be different, except for the dose of DES to which they were exposed. All pups that received the same dose of DES during gestation were treated as being part of the same pool and randomized to swim or sedentary groups accordingly. The only difference between the sedentary and exercise groups was the exercise regimen. According to our results, it seems that exercise does potentiate the effects of DES. It could also be that DES affects how the musculoskeletal system responds to swimming. The fact that these differences were observed without weight-bearing exercise makes the results more striking. One of the limitations of the study was the small sample size. The sample size used in this study was determined based on the expense of data collection and expedience.
Humans were typically exposed to DES throughout gestation until birth [8
]. The brief exposure in this study was chosen at a time of organogenesis; however, the overall dose of DES in this study is much less than the comparable dose used on pregnant women. Therefore, the present results are more likely to underestimate the effects of DES because the exposure was brief and at a lower cumulative dose. In this study, no dose-response relation was found. At first glance, this is not what one would expect. However, the lack of a dose response is actually typical for steroid hormones [38
]. This is because estrogen receptors can have different downstream actions, depending on whether they were exposed to low or high doses of estrogen [41
] This is why it is quite common to observe an effect at low and high doses of estrogen (or estrogen agonists) but not at intermediate doses.
DES is traditionally thought of as a pure estrogen agonist, but can actually have an antagonistic effect in some circumstances. It has an agonistic effect on ERα but an antagonistic effect on ERR-γ [42
]. DES was most studied in terms of its effect on the reproductive system, and it was found to have a net agonist effect on these tissues (endometrium, breast, prostate) [33
]. How a tissue responds to DES would largely be determined by the receptor distribution in that tissue. A prevalence of ERα in articular cartilage, for example, would lead us to suspect an agonistic effect. Furthermore, it should be noted that the effects observed in this study are due to "priming" with DES at a critical stage, which differs from exposing an adult animal to DES. This priming can change the receptor distribution in a particular tissue or in the downstream pathways that a particular receptor stimulates. These exact effects are not known at present and must be further elucidated.
Contradictory evidence exists about the effects of estrogen on articular cartilage [43
]. Most studies, however, have concentrated on the effects of adult estrogen exposure on articular cartilage, which contains ERα [44
]. Little is known about how estrogen imprinting with DES can affect articular cartilage. The present study found that even a brief 4-day fetal exposure to DES can increase markers of articular cartilage degeneration in adult mice. Safranin O staining demonstrated consistent decrease in proteoglycans for both males and females at all doses of DES. This suggests that in utero
DES exposure can impair the ability of adult articular cartilage to maintain its proteoglycan content. Estrogen was shown to help maintain the integrity of articular cartilage in sheep and rats [45
]. The detrimental effects seen here could be due to DES inhibiting the response of articular cartilage to estrogen in both adult males and females.
The intervertebral disc [IVD) was shown to express estrogen receptor β (ERβ) in the annulus fibrosus [32
]. Diethylstilbestrol has an agonistic effect on ERβ, although it has a higher affinity for ERα [47
]. These findings suggest that the IVD is an estrogen-responsive tissue, but no work has been done to study the effect of in utero
exposure to DES on the IVD. The present study suggests that IVD in both male and female mice fetally exposed to DES are more susceptible to disc degeneration. Swimming ameliorated some of these effects but exaggerated others, suggesting different mechanisms of response to in utero
exposure to DES. Swimming was studied as an alternative to weight-bearing exercise because it can have beneficial effects on bone turnover, strength, and structure [49
]. More animals must be tested to corroborate that these degenerative changes are consistent.
Other than ERβ, no other estrogen receptors were detected in the IVD [51
]. The altered proteoglycan content in DES mice can thus be a secondary effect. Sexual dimorphism was also noted in the IVD response to in utero
DES exposure, replicating the pattern noted in bone and articular cartilage.