The Expression of GPR30 in Iron-Overloaded Atypical Ovarian Epithelium and Ectopic Endometrium Is Closely Correlated with Transferrin Receptor and Pi3K

Abstract

Background: The mechanism of atypical hyperplasia of the ovarian epithelium and ectopic endometrium caused by iron overload remains unclear. Accordingly, we investigated possible effects on the human ovarian ectopic endometrium and ovarian epithelium by producing a high-iron environment with rat ovaries.

Objective: Human ovarian ectopic endometrium with atypical hyperplasia was collected, and the correlation between transferrin receptor GPR30 and Pi3K protein expression was studied by immunohistochemistry staining. Twenty SPF Sprague-Dawley female rats were microinjected with iron into one side of the ovary once a month, and the other ovary was used as the control. After 10 months of microinjection, the iron histological analysis was examined by Prussian blue staining, and ovarian endometrium morphology was assessed by HE staining. Abnormal lesion changes were measured by Pi3K staining. Evaluation of GPR30 was performed using reverse transcription PCR (RT-PCR) and western blotting, and the interrelationship between GPR30 and Pi3K was also assayed.

Results: GPR30 was significantly increased and correlated with the transferrin receptor and Pi3K in atypical human ovarian ectopic endometrium. Iron overload was confirmed in the 20 microinjected ovary cortexes, epithelial hyperplasia was observed in 12 ovaries, and papillary atypical hyperplasia was noted in eight ovaries. The RNA and protein levels of GPR30 were significantly increased in atypical hyperplasia compared to hyperplasia tissue samples. A positive relationship between GPR30 and Pi3K was found (P = 0.001).

Conclusion: The results suggest that persistent iron exposure may be a potential stimulus for ovarian endometriosis with atypical changes, and the abnormal increase in the new estrogen receptor GPR30 is closely related to this process.

Figure 1

HE Staining of Atypical Hyperplasia Human Ovarian Endometrium. (a): Ovarian ectopic endometrium. (b): Atypical ovarian ectopic endometrium.

Figure 2

Rats were subjected to a small incision at the right back body surface (a) of the ovary (b). Iron dextran for injection was microinjected into the subenvelope of the ovary (c).

Figure 3

GPR30, iron metabolites TfR and Pi3K protein were more positively expressed in the atypical human ovarian endometrium (b, d, f) than in the normal control group (a, c, e).

Figure 4

Ferric iron (blue) deposits of ovaries with Prussian blue staining in the endometriosis model group (b) by repeated injections of iron, with most of the iron (Fe⁺²) accumulated in macrophages. There were no ferric iron deposits identified in control ovary tissue (a). Repeated microinjections of iron led to different morphologic changes in the ovarian epithelium: multinucleated giant cells (d), epithelial hyperplasia (e), and papillary hyperplasia with atypical cells (f) in iron-dextrin-injected ovaries. Image (c) is control ovary tissue injected with glucose water. No cellular morphology changes were observed in the control ovary tissues, which clearly indicate that the tissue reaction observed was not induced by surgical trauma or dextrin as an iron carrier substance.

Figure 5

GPR30 mRNA (346 bp) and protein were significantly more expressed in the atypical group (c) than in the epithelial hyperplasia (b) and control groups (a). These data suggest that repeated microinjection of iron may have an effect on the expression of GPR30, with increased expression of GPR30 most obvious in atypical alterations of the ovarian epithelium.

Figure 6

Pi3K and GPR30 proteins (brown granules located in the nucleus and cytoplasm) were significantly increased in expression in the ovarian epithelial cells of the atypical group (b, d) than in the control group (a, c). This finding suggests that the occurrence of atypical ovarian epithelia by repeated microinjection of iron can lead to the upregulation of GPR30 and Pi3K. Images are shown at 400× magnification.