Identification of stathmin 1 during peri-implantation period in mouse endometrium by a proteomics-based analysis

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Highlights

  • The protein profiles during murine embryo implantation were clarified.

  • Stathmin 1 might be a potential regulator of embryo implantation.

  • Some other potential regulators were also suggested.

Abstract

In this work we aimed to identify the differentially expressed proteins and their potential roles during peri-implantation period through proteomics-based approach. Adult healthy female mice were mated naturally with fertile males to produce pregnancy. The models of pseudopregnancy, delayed implantation, and artificial decidualization were established. The protein profile between pre-implantation (D1) and implantation (D5) period was compared by two-dimensional electrophoresis (2-DE) and identified by mass spectrometry (MS). 2-DE yielded comparative images presenting over 500 protein spots in D1 and D5 mouse endometrium. 15 proteins were identified, of which stathmin 1, Apo-A1, hnRNP H3, transgelin 2 and arginase 1 were validated by western blotting. Stathmin 1 expression did not change in pseudopregnancy, but activation of implantation, or induction of decidualization increased it dramatically. Under non-pregnant status, progesterone alone or in combination with17β-estradiol increased it dramatically. Our results clarified the protein profile in mouse endometrium during implantation. The specific expression profile of stathmin 1 suggested that it should be involved in implantation and serve as a potential regulator of this process. These findings may contribute to the better understanding of the molecules events during embryo implantation, and subsequently improve the ability to treat infertility.

Introduction

Embryo implantation is established and maintained by a network of complex molecules that are involved in physiological changes of the endometrium, including hormones, cytokines, growth factors, and their interactions [1]. It can only occur over a restricted period after ovulation called “implantation window” lasting about 24 h in mouse spanning early on day 4 to middle of the day 5 of pregnancy. Comparing to the commonly used one-by-one research strategy, high throughput techniques theoretically have the advantages in discovering the molecular mechanism in a global extent.

As well as the significant alterations in transcriptional and post-transcriptional levels, for the events relating to translation, post-translational modification, and subcellular localization, proteomics techniques should be more informative because these events are difficult to detect either by studying nucleotide sequence variation or by measuring the quantity of RNA or miRNA. In previous studies, we identified some proteins in endometrial carcinoma by two-dimensional electrophoresis (2-DE)-based proteomic strategies, some of which might serve as potential genetherapy target or prognosis biomarker of endometrial carcinoma [2], [3]. Considering the fact that the embryo implantation process shares similar biological characteristics with cancer development and progression [4], [5], we presume a significant change of the protein profile during peri-implantation period. Therefore, herein we clarified the proteins profile in embryo implantation process, using 2-DE and mass spectrometry (MS)-based approaches. In addition, a series of animal models were established for strict validation of the unbiased candidates. As a result, we successfully identified 15 differentially expressed proteins, and the specific expression profile of stathmin 1 was further validated in a series of animal models, including pseudopregnancy, delayed implantation, artificial decidualization and ovariectomization. As clinicians, we think these findings can contribute to the better understanding of the molecules events during embryo implantation, and subsequently improve the ability to treat infertility, to prevent early pregnancy loss and to increase success rate of IVF.

Section snippets

Preparation for animal specimens

The Institutional Animal Care and Treatment Committee of Sichuan University reviewed and approved all animal experiments herein (04/21/2013, shown in supplemental file). The adult healthy female C57BL6/J mice (6–8 weeks of age, nonfertile and 18–20 g each) were mated naturally with fertile males to produce pregnancy. The morning of finding a vaginal plug was designated as day 1 (D1) of pregnancy. Implantation was assumed to be taking place on the day 5 (D5), which was confirmed by the

Comparison of the protein expression profiles of mouse endometrium between pre-implantation and implantation period

To obtain a comprehensive comparison of the protein expression profile of mouse endometrium between pre-implantation and implantation period, a comparative 2-DE analysis was conducted with 5 pairs of mouse endometrium on D1 and D5. Image analysis was performed using PDQuest 7.1 software and yielded well resolved and reproducible protein profiles for both D1 and D5 endometrium (Fig. 1). Coomassie staining showed a matching rate of 94.5% and an average of 512 ± 45 and 565 ± 58 spots in D1 and D5

Discussion

In this study, we found 18 spots between pre-implantation and implantation endometrium, and 15 spots were successfully identified by MS/MS. Even protein expression profiles with perfect stability and repeatability were obtained, but the limited number of differentially expressed spots seemed not proportionate to the complexity of molecular crosstalk in embryo implantation. We think it was partially due to the strict selection criteria relating to not less than 2.5-fold change and 40% recurrent

Conflict of interest

There is no conflict of interest.

Acknowledgments

This work was supported by the Natural Science Foundation of China (Grant number: 81170592), Special Fund from National Excellent Doctoral Dissertation (Grant number: 201079), and Program for New Century Excellent Talents in University (Grant number: NCET-10-0594). We thank Prof. Canhua Huang, State Key Laboratory of Biotherapy, West China University Hospital, Sichuan University, for the kind and valuable instruction and assistant in proteomics techniques. We thank Bioms Sci-tech Co., Ltd.

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