Immunolocalization of steroidogenic enzymes in the vaginal mucous of Galea spixii during the estrous cycle

Abstract

Background: The synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome P450 (P450arom) catalyzing estrogen synthesis, both of which must complex with the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) for activity. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate P450c17, P450aromatase 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.

Keywords: Androgens; Cytochrome P450; Estrogens; Estrous cycle; Vaginal epithelium.

Fig. 1

Immunolocalization of the enzyme cytochrome P450 aromatase in the vaginal epithelium and ovary of Spix cavy. a immunoreactivity in cells (arrow) of the connective tissue (ct); (e) epithelium in the vaginal wall at diestrus. b immunoreactivity in basal cells (b) of the epithelium in the vaginal wall (c) cornified cells of the epithelium; (ct) connective tissue in the vaginal wall at proestrus. c immunoreactivity in cells (arrow) of the connective tissue (ct); (e) epithelium in the vaginal wall at metestrus. d immunoreactivity in basal cells (b) of the epithelium; (c) cornified cells; (ct) connective tissue in the vaginal wall at estrus. e no immunoreactivity in the epithelium (e) or connective tissue (ct) in the absence of primary antiserum, negative control. f immunoreactivity in the cells of the granulosa layer (gc) of the ovary tissue; oocyte (o); positive control. Bars = 20 μm

Fig. 2

Immunolocalization of the enzyme cytochrome P450c 17 in the vaginal epithelium, and testes of Spix cavy. a immunoreactivity in basal cells (arrow) of the epithelium (e); (ct) connective tissue at diestrus. b immunoreactivity in surface cells (arrow) of the epithelium (e); (ct) connective tissue at proestrus. c immunoreactivity in epithelium cells (arrow); (ct) connective tissue at metestrus. d immunoreactivity (arrow) in basal cells (b); (c) cornified cells; (ct) connective tissue at estrus. e no immunoreactivity in epithelium cells (e) or connective tissue (ct). Negative control. f immunoreactivity in Leydig cells of the testicle (arrow). Spermatozoa (arrowhead). Positive control. Bars = 20 μm

Fig. 3

Immunolocalization of the enzyme NADPH cytochrome P450 reductase in the vaginal epithelium and testes of Spix cavy. a immunoreactivity in cells (arrow) of the epithelium (e) and connective tissue (ct) at diestrus. Bar = 20 μm. b immunoreactivity in the basal cells of the epithelium (b) and of the connective tissue (ct); (c) cornified surface cells of the epithelium at proestrus. Bar = 10 μm c immunoreactivity in cells (arrows) of the epithelium and connective tissue (ct); (c) cornified cells of the epithelium at metaestrus. d immunoreactivity in cells (arrow) of the connective tissue (ct) and basal cells (b) of the epithelium; cornified cells of the epithelium at estrus e no immunoreactivity in cells of the epithelium (e) or connective tissue (ct). Negative control. f immunoreactivity in Leydig cells of the testicle (arrow). Spermatozoa (arrowhead). Positive control. Bars = 20 μm

Fig. 4

Western immuno-blotting test for enzyme cytochrome P450c17 in the vaginal tissue of Spix cavy in several phases of the estrous cycle. Metaestrus (m), diestrus (d), proestrus (p), estrus (e) and adult testicles (T)