Received Feb 11; Accepted Apr Associated Data The datasets supporting the conclusions of this article are included within the article. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate Pc17, Paromatase and CPR expression in vaginal mucosa of Galea spixii Spix cavy by immuno-histochemical and western immunoblot analyses. Methods Stages of estrous cyclicity were monitored by vaginal exfoliative cytology.
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Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate Pc17, Paromatase and CPR expression in vaginal mucosa of Galea spixii Spix cavy by immuno-histochemical and western immunoblot analyses. Methods Stages of estrous cyclicity were monitored by vaginal exfoliative cytology. After euthanasia, vaginal tissues were retrieved, fixed and frozen at diestrus, proestrus, estrus and metestrus.
The ovaries and testis were used as positive control tissues for immunohistochemistry. Results Data from cytological study allowed identification of different estrous cycle phases. Immunohistochemical analysis showed different sites of expression of steroidogenic enzymes along with tissue response throughout different phases of the estrous cycle. However, further studies are needed to characterize the derived hormones synthesized by, and the enzymes activities associated with, vaginal tissues.
Conclusion Current results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression.
Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating androgens and estrogens.
Background Galea spixii Spix cavy are rodents of the Caviidae family and subfamily Caviinae [ 1 ] that live in backland vegetation of the Brazilian northeastern region [ 2 ]. Most animals are bred in captivity for food [ 3 , 4 ], in part to help preserve the species which is on the endangered list of the International Union of Conservation of Nature [ 5 ].
Spix cavy has also been employed as experimental models in research on placental function [ 2 , 6 ]. Based on vaginal exfoliative cytology, the estrous cycle lasts 14—19 days and the vaginal epithelium undergoes variations related to each estrous cycle phases [ 4 ] in concert with ovarian follicular development and ovulation [ 7 ]. Laboratory rodents including mice, rats and guinea pigs are commonly used as experimental models since they have a relatively short estrous cycle and gestation length, and are easy to handle [ 8 , 9 , 10 , 11 ].
Guinea pigs Cavia porcellus , like Spix cavy were employed in some of the earliest studies elucidating morphological and physiological changes in the reproductive tract and vagina during reproductive cyclicity [ 12 , 13 , 14 , 15 ]. Moreover, the biology of the vaginal mucosa has clinical significance. For instance, atrophy of the vaginal epithelium in post-menopausal women can have serious consequences for quality of life [ 16 ] requiring therapies aimed at restoring estrogenic stimulation lost as a result of decreased ovarian synthesis [ 17 ].
Though many treatment strategies employ local application of estrogens themselves [ 18 ], others have found success using dehydroepiandrosterone DHEA [ 19 ]. DHEA has little bioactivity of its own, but seems to provide benefits similar to estrogens [ 19 ], suggesting it may be used as a substrate for estrogen synthesis in the vaginal wall itself. Indeed, data has been reported that supports this possibility, specifically demonstrating the expression of steroidogenic enzymes in the vaginal epithelium [ 16 , 20 , 21 , 22 ].
Exfoliative vaginal cytology Reproductive cycles were monitored using exfoliative vaginal cytology. Two full cycles were monitored for each animal prior to euthanasia and tissue collection, analyzed and photodocumented by light microscopy. The predominant cells in each smear were classified as superficial, large and small intermediate, parabasal and neutrophils throughout the cycle. Then the estrous cycles phases were characterized according the different cell types present in each phase [ 4 , 7 , 12 , 13 ].
After euthanasia, the genital organs were then exposed and samples of tissues from the vaginal tube were retrieved in each phase of the cycle along with ovaries used as positive control tissue.
Alan J. The primary antiserum was omitted for negative controls. Sections were counter-stained with hematoxylin and mounted with coverslips. Western immuno-blotting The specificity of staining for Pc17 was verified by western immuno-blotting.
The proteins were visualized by chemiluminescence. Results Detecting the phases of the estrous cycle Vaginal cytology allowed characterization of cycle stage. Superficial nucleated cells were predominant when sperm were also evident in the smear in those female co-housed with males and were therefore a reliable indication of estrus.
Parabasal cells predominated during metestrus, small intermediate cells during diestrus which transitioned into large, superficial intermediate cells during proestrus. These changes were consistent from cycle to cycle and made it possible to predict the onset of estrus. Immunohistochemistry The vaginal epithelium was typically stratified squamous with cell types that correlated with exfoliative cytological findings, varying among stages: a simple squamous a few cell layers in thickness during metestrus, a slight acceleration of proliferation and increased thickness during the diestrus, then, reaching several cell layers in thickness with cornification of the superficial layers and some loss of nuclei during proestrus.
At estrus, the epithelium showed only one or a few layers of cornified cells and a low proliferation of intermediate and parabasal cells.
Expression of Parom was immunolocalized in cells of the connective tissue of the lamina propria during the diestrous phase Fig. No immunoreactivity was observed in the negative controls for the vaginal epithelium Fig.
Immunoreactivity in the vaginal epithelial cells was weak during metestrus Fig. No immunoreactivity was observed in the absence of the primary antisera Fig. Immunoreactivity for Pc17 was restricted only to Leydig cells of testes Fig. Negative control.
Spermatozoa arrowhead. Positive control. Immunoreactivity was not detected in the absence of the primary antisera Fig. Immunoreactivity was observed primarily in the Leydig cells of testes used as positive control Fig. Testicles of adult males used for positive control also were detected. Metaestrus m , diestrus d , proestrus p , estrus e and adult testicles T Full size image Discussion Current results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression.
Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating estrogens. Lephart et al. Testosterone is known to increase in serum and ovarian tissues in mice during proestrus [ 28 ], and vaginal mucosa expresses androgen receptors [ 16 , 21 ] at levels that vary with age and menopausal status of women.
Berman et al. It is possible that cycle stage-specific, vaginal expression of steroidogenic enzymes is an androgen-stimulated response. Furthermore, Iguchi et al. Therefore, it is likely that vaginal epithelial proliferation is stimulated directly by androgens. Given the stimulatory response of vaginal epithelium to androgens, the possibility that androgen synthesis might occur locally within the vaginal epithelium itself assumes additional relevance.
Confirmation that CPR, the redox partner protein for both Pc17 and Parom, is also expressed in this tissue provides further support for local sex steroid synthesis. Although Pc17 expression in the vaginal wall of Spix cavy was less evident than aromatase and cytochrome P NADPH reductase, immuno-staining in positive controls tissues Leydig cells , together with the apparent molecular size as expected by western immunoblot, supports its presence.
Since levels were detectable only during the proestrous and estrous phases, local synthesis might coincide with vaginal proliferation and augment the effects of ovarian steroids. Equally, increased expression during proestrus might reflect regulation of Pc17 in vaginal epithelium by ovarian androgens.
Further studies must be undertaken to quantify the levels of enzyme induced and the steroids synthesized. With regard to the local production of androgens in the vaginal tissue, Berman et al. The authors also observed that the levels of androgen receptors varied according to age and menopausal status of women. Proliferation of the vaginal epithelium is clearly stimulated by estrogens, and probably androgens, but their effects are modified by progestins [ 30 ] in a complex dose-dependent fashion, highly stimulatory at low levels but inhibitory at higher doses [ 31 ].
Further studies, perhaps using local application of inhibitors of sex steroid synthesis may reveal the physiological importance of local steroid production in vaginal epithelium of Spix cavy, building on results of past studies in guinea pigs [ 30 ] and rats [ 32 ].
Effects of vaginal dehydroepiandrostenedione application, vaginal testosterone, and tissue selective estrogen complexes have been promoted as promising new therapies in post-menopausal women; however, further studies are necessary to confirm their efficacy and safety [ 19 ]. Based on present results, the local synthesis of dehydroepiandrostenedione by Pc17 may be considered in further studies in other species including women.
Notwithstanding potential species differences, Spix cavy may prove to be an excellent experimental model of relevance in studies on vaginal biology and health of post-menstrual women, women treated with aromatase inhibitors [ 17 , 18 ] or other diseases of the female reproduction system [ 16 , 25 , 32 ,
Cobaye à dents jaunes de Spix (Galea spixii)
PREÁ (Cavia aperea e Galea spixii spixii)