Expression of the progesterone receptor, bcl-2 and bax in clomiphene citrate treated rat uteri
Clomiphene citrate (CC) is a synthetic oestrogen receptor modulator that may act to promote or inhibit oestrogenic responses depending on the type of tissue or organism. CC acts as a superovulator and has been widely prescribed in the treatment of female infertility. However, pregnancy rates after CC treatment are low, possibly due to the CC causing morphological and other unidentified changes to the uterus. This study investigated the changes caused by CC on the progesterone receptor (PR), the anti-apoptotic protein, Bcl-2, and the pro-apoptotic protein, Bax. PR with its ligand, progesterone, is important for the maintenance of a pregnancy. Bcl-2 is a cell survival molecule and acts in opposition to Bax, which promotes apoptosis, which is important in allowing an attaching blastocyst to infiltrate the maternal endometrium. A high physiological dose of CC (1.25 mg) was administered to ovariectomised rats, either as a single treatment, or prior to a hormonal treatment regime characteristic of pre-implantation animals. The single CC treated animals were compared with single 5 mg progesterone (P4) treated animals and vehicle controls. The animals treated with CC in conjunction with the ovarian hormone milieu (CCPPPE treatment) were compared to PPPE treated animals or to the vehicle controls. The effects of CC, P4, PPPE and SPPPE on the morphology of the uteri were investigated by light microscopy. Expression of the PR, Bcl-2 and Bax were investigated using immunohistochemistry and enzyme linked immunosorbent (ELISA) techniques. The present study showed that CC treatment affects the microanatomy of the uterine compartments, particularly the shape of the lumen and the luminal epithelial height. The expressions of PR, Bcl-2 and Bax in the entire uterus were not significantly affected by the various treatments applied to the ovariectomised rats, as measured using ELISA. However, expression of the proteins in the various uterine compartments was altered by CC treatment. The single CC and CCPPPE treatments had an oestrogenic effect with regards to PR expression in the uteri as seen in the immunolocalisation, whereas the single P4 treatment decreased the PR expression, as expected. CC treatment caused patchiness in both Bcl-2 and Bax expression surrounding the endometrial lumen. Moreover, treatment with CC maintained expression of Bcl-2 in the luminal epithelium, whereas the expression of Bax shifted away from the luminal epithelium. This suggests that CC treatment promotes cell survival of the luminal epithelium, which may diminish the ability of the blastocyst to infiltrate the maternal tissue. CC action is both dose sensitive and has a cumulative effect with multiple treatments. Future studies would aim to separate the various uterine compartments in order to quantitatively assess the actions of CC on expression in the PR, Bcl-2 and Bax. In addition, it would be interesting to investigate how the accumulation of subsequent CC doses affects the expression of apoptotic markers, as this would be a more realistic model for human infertility treatment.