Label-Free Proteomics Uncovers Energy Metabolism and Focal Adhesion Regulations Responsive for Endometrium Receptivity

Abstract

The menstrual cycle of the female uterus leads to periodic changes of the endometrium. These changes are important for developing the endometrial receptivity and for achieving competency of embryo implantation. However, the molecular events underlying the endometrial receptivity process remain poorly understood. Here we applied an LC-MS-based label-free quantitative proteomic approach to compare the endometrial tissues in the midsecretory (receptive) phase with the endometrial tissues in the proliferative phase from age-matched woman (n = 6/group). The proteomes of endometrial tissues were extracted using an SDS-based detergent, digested by the filter-aided sample preparation procedures, and subsequently analyzed by nano-LC-MS/MS (Orbitrap XL) with a 4 h gradient. Reliable protein expression profiles were reproducibly obtained from the endometrial tissues in the receptive and proliferative phases. A total of 2138 protein groups were quantified under highly stringent criteria with a false discovery rate of <1% for peptide and protein groups. Among these proteins, 317 proteins had differences in expression that were statistically significant between the receptive and proliferative phases. Direct protein-protein interaction network analyses of these significantly changed proteins showed that the up-regulation of creatine kinase B-type (CKB) in the receptive phase may be related to endometrium receptivity. The interaction network also showed that proteins related to cell-cell adhesion were down-regulated. Moreover, the results from KEGG pathway analyses are consistent with the protein-protein interaction results. The proteins, including alpha-actinin (ACTN), extracellular matrix proteins, integrin alpha-V, and so on, that are involved in the focal adhesion pathway were down-regulated in the receptive phase compared with the proliferative phase, which may facilitate the implantation of the fertilized ovum. Selected proteins were validated by Western blot analysis and indirect immunofluorescence, including the up-regulation of CKB and down-regulation ACTN in the receptive phase. In summary, our proteomic analysis study shows potential for predicting the endometrial remodeling from the proliferative to the receptivity phase in women, and these results also reveal the key biological mechanisms (such as energy metabolism and focal adhesion) underlying human endometrial receptivity.

Proteomes of the Female Genital Tract During the Oestrous Cycle

“…The comparison of previously published transcriptomes (14,20,21,33,34) and tissue proteomes (23,25) in other models with the sheep luminal proteomes from this study provided some information about the possible secretory origin of many proteins, suggesting that the regulation of the protein abundance along the cycle may be associated to a significant extent with endocrine control of the expression of several genes.…”

“…We searched for these genes in seven studies of the transcriptome or proteome of the female tract during the cycle in other species. These include the transcriptome of the bovine cervix (14), the transcriptomes of the horse (21) and bovine (20) endometrium, the proteome of human endometrium (23,25), the transcriptome of bovine oviduct epithelial cells (34), and the proteome of porcine oviduct epithelial cells (33). A total of 59 proteins were found to be regulated similarly, i.e.…”

“…The expression of the transcriptome of the endometrium along the oestrous cycle was investigated in bovine (20), equine (21) and mouse (22). In human, the proteome of the uterus along the menstrual cycle was studied either on endometrium epithelium (23)(24)(25)(26) or on uterine fluid (27)(28)(29). However, the variation of the proteome of the uterine fluid during the oestrous cycle is not known.…”

Iron‐induced energy supply deficiency and mitochondrial fragmentation in neurons

“…The consistency and reproducibility of this proteomic platform have already been demonstrated in our previous studies (Chen et al . ). In the present study, the peptide and protein data filtration were based on stringent criteria of < 1% false discovery rate for both peptide and protein group levels, providing highly confident protein identification.…”

“…The LC‐MS/MS was performed as previously described with some modifications (Chen et al . ). In brief, samples were acidified with formic acid to a final concentration of 0.1% (v/v) and loaded on a 75 μm × 150 mm fused silica column packed in‐house with 3‐μm ReproSil‐Pur C18 beads (120 Å; Dr. Maisch GmbH, Ammerbuch, Germany) using an Easy nano‐UPLC 1000 (Thermo Electron, Waltham, MA, USA).…”