These data suggest that the diabetic state produces dramatic changes in vaginal tissue structure and function, characterized by decreased blood flow, atrophy of the muscularis and attenuation of epithelial proliferation. These changes were also accompanied by alterations in sex steroid hormone receptors and key enzymes that regulate blood flow. Previous studies in the rat demonstrated that preservation of normal vaginal tissue structure and blood flow are dependent upon estrogen signaling [13, 26]. On a gross level, the histological and physiological changes in the present study resemble those observed in vaginal tissue from ovariectomized animals in previous studies [13, 15, 26]. Similar to ovariectomized animals, mean plasma estradiol concentration and mean uterine wet weight was decreased in the diabetic group. However, unlike ovariectomized animals, plasma testosterone levels were significantly elevated in the diabetic group. While the reasons for this increase remain unclear, previous studies have demonstrated that ovarian aromatase activity is insulin-dependent [27–29]. In the STZ-induced diabetic rat, the lack of insulin production may inhibit aromatase activity, causing an increase in testosterone due to decreased estrogen biosynthesis. In addition, female rats repeatedly subjected to physical and psychological stress protocols are reported to have significantly elevated plasma testosterone , suggesting that increased testosterone may be part of a systemic stress response.
It remains to be established whether the lower levels of circulating plasma estradiol are responsible for the changes in the diabetic rats. Yet, given that vaginal tissue from diabetic animals exhibited decreased ERα protein in the nucleus, it is likely that additional mechanisms that interfere with estrogen receptor signaling are triggered. This perspective is further supported by the observation that in vaginal tissue of ovariectomized animals, ERα increases in the nuclear compartment but decreases in the cytosolic compartment . If decreased plasma estradiol alone were responsible for the alterations in vaginal tissue structure and function, one would expect similar intracellular distributions of ERα in both diabetic and ovariectomized animals. Since the severity and duration of estrogen deficiency is different in the ovariectomized rat, additional studies are necessary to define the time course of changes in the diabetic rat and the dose-response relationship between estrogen and vaginal structure and function. The decreased levels of nuclear AR in diabetic rat vaginal tissue suggests that testosterone action may also be disrupted in the diabetic state. While testosterone has been shown to affect adrenergic nerve density in the rat vagina , the role of androgens in regulating vaginal structure and function in diabetes remains to be examined.
Further parallels between the diabetic and ovariectomized states are highlighted by the reductions in arginase I and eNOS in vaginal tissue. Previous studies have shown that arginase in rabbit vagina  and eNOS in rat vagina  are decreased in ovariectomized animals and up-regulated after estrogen administration. However, some of these changes appear to be tissue and species specific, since estradiol can have the opposite effect on eNOS expression or activity in rabbits [33, 34]. While arginase has been shown to modulate vascular responses in female genital tissue, it has also been shown in other tissues to be critical for regulating cell growth [35, 36]. Lower arginase levels may be partially responsible for the atrophic appearance of the vaginal muscularis and epithelium in diabetic animals. In addition, arginase activity can decrease NO synthesis through substrate competition (i.e. by decreasing intracellular arginine pools) and the lower levels of arginase in diabetic rat vagina may also be reflective of a compensatory down-regulation in the local vasculature to maintain basal perfusion.
This compensatory response may also involve the observed changes in PKG. Since PKG mediates the intracellular response to NO, decreased NO production and/or increased NO scavenging in diabetic animals could lead to a compensatory up-regulation in PKG. Alternatively, the protein level and enzyme activity of PKG have been reported to increase in the lung following chronic hypoxia . It is possible that decreased blood flow to the vagina in diabetic animals may have produced hypoxic conditions that triggered the up-regulation of PKG. Interestingly, PKG was significantly decreased in penile tissue of diabetic male rabbits . This contrasting finding may be attributed to any number of differences, including tissue, species, sex, duration of diabetes or mechanism of function.
Thus, data from the current study, together with our previous findings, suggest that diabetes may adversely affect estrogen production and/or ER signaling. This hypothesis is derived from several lines of evidence that include parallel changes with regard to blood flow, tissue structure, and protein expression of arginase and eNOS that are observed in both diabetic and ovariectomized (estrogen deficient) animals. The dissimilar changes of nuclear ERα levels in diabetic and ovariectomized animals, and the observation that many diabetic rats are in diestrus (phase of estrous cycle with lowest estrogen level), further support a pathophysiological mechanism that includes disruptions in estrogen action.
Previous laboratory studies have indicated that diabetes disrupts estrogen signaling in a variety of tissues. In the pituitary of diabetic animals, ER binding was reduced and ERα levels decreased, potentially affecting sexual receptivity and reproduction [39, 40]. Nuclear retention of estradiol-bound ER has been shown to be of shorter duration in both the pituitary and uteri of diabetic animals [41, 42]. Further, pituitary responsiveness to LH-RH was reduced, inhibiting ovulation in diabetic animals . In STZ-induced diabetic mice, estradiol restored cell proliferation in the dentate gyrus and subventricular zone  and high glucose has been shown to block the effects of estradiol in human vascular cell growth . Diabetes is also known to adversely affect renal function and estradiol treatment counteracts this pathophysiological effect in diabetic animals . More specific investigation into disease mechanisms affecting ER regulation indicate that oxidative stress, a well-known consequence of diabetes, differentially regulates the expression of ERα and ERβ in different cell types . Thus, the diabetic state may cause dysregulation of estrogen action within both central and peripheral estrogen-sensitive tissues.
It should be noted that our study is a preliminary report and has several important limitations. Histological changes in the vagina were only assessed on a gross level and not analyzed by objective morphometric methods. More detailed studies examining diabetes-induced structural changes in the vagina are currently planned. These studies would quantitatively determine structural changes in the various layers of the vagina, blood vessel density, innervation, etc. Further, since the Western blot assays were performed with whole tissue extracts, the cellular localization of the biochemical markers that were examined in this study remains unclear. Immunohistochemical staining of these markers would provide additional information pertinent to the effects of diabetic impairment of vaginal function. Future histological studies should also examine changes in structure and protein expression in different regions of the vagina. Only tissue sections from the mid-vagina were used in the current study since this is the region where blood flow was measured. Also, tissues from multiple animals in each treatment group were pooled for Western blot analyses. Values reported as percent change (relative to control) were intended to be indicative of general trends and should not be taken as exhaustive quantitative determinations. Finally, vaginal smears were not performed daily to assess potential disruptions in the estrous cycle and the increased number of diabetic rats in diestrus may have been purely coincidental. However, the decrease in plasma estradiol and uterine weight, the general atrophic appearance of the vagina, and the reduced levels of nuclear ERα in vaginal tissue of diabetic animals are consistent observations that are suggestive of disruptions in steroid hormone synthesis and signaling. We did not determine ERβ levels in our study since ERα has been shown to be the predominant subtype expressed in the rat vagina .
While the data from this study are limited in their interpretation with respect to the clinical state of diabetes, this model provides data supporting clinical observations of decreased genital arousal in women with diabetes mellitus. If indeed STZ-induced diabetes in the rat disrupts estrogen signaling, then it would be logical to investigate in future studies the role of estrogen therapy in diabetes to ameliorate the complications of diabetes.