The present study identifies uterine fluid (UF) proteins that display differential abundance during the embryo-permissive phase in nonconception and conception cycles in rats. UF samples were collected from nonpregnant rats in the proestrous (n=17) and metestrous (n=18) phases and also from pregnant (n=17) and pseudopregnant (n=17) rats on day 4 post coitus. UF protein profile in the metestrous phase was compared with that in the proestrous phase. Similarly, UF protein profile of the pregnant rats was compared with that of the pseudopregnant rats. Two-dimensional PAGE, followed by densitometric analysis of the paired protein spots, revealed differential abundance of 44 proteins in the metestrous phase, compared with that in the proestrous phase. Of these, 29 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight or liquid chromatography–tandem mass spectrometry. Functional groups such as proteases, protease inhibitors, and oxidoreductases were enriched in differentially abundant proteins. Total protease activity in UF was found to be significantly (P<0.05; t-test) higher in the proestrous phase, compared with that in the metestrous phase. Furthermore, 41 UF proteins were found to be differentially abundant in pregnant rats, compared with pseudopregnant rats. Of these, 11 proteins could be identified. Immunoblotting analysis confirmed significantly higher (P<0.05; t-test) abundance of β-actin, Rho-specific guanine nucleotide dissociation inhibitor alpha (Rho-GDIα), and peroxiredoxin-2 and -6 in the metestrous phase, compared with that in the proestrous phase. Compared with pseudopregnant rats, pregnant rats had significantly higher (P<0.05; t-test) levels of UF β-actin and Rho-GDIα. Furthermore, these proteins could be detected in the culture supernatants of endometrial epithelial cell lines, thereby providing an evidence of their secretion from endometrial epithelial cells. Data obtained from the study expand our knowledge on the uterine milieu that favours embryo implantation.
Sumit Bhutada, R R Katkam, Tarla Nandedkar, S M Metkari, U K Chaudhari, Sneha Varghese, S D Kholkute, and Geetanjali Sachdeva
Kashmira Bane, Junita Desouza, Asma Rojewale, Rajendra Katkam, Gwendolyn Fernandes, Raj Sawant, Uddhavraj Dudhedia, Neeta Warty, Anahita Chauhan, Uddhav Chaudhari, Rahul Gajbhiye, and Geetanjali Sachdeva
Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of double-stranded DNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Towards this, we focused on the X-Ray Repair Cross-Complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (p<0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the GEO database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in-vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AFX and 8-hydroxy-2’-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (ThESCs) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AFX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.