Elsevier

Journal of Proteomics

Volume 125, 1 July 2015, Pages 76-88
Journal of Proteomics

Proteomic analysis of porcine endometrial tissue during peri-implantation period reveals altered protein abundance

https://doi.org/10.1016/j.jprot.2015.05.003Get rights and content

Highlights

  • 2D-DIGE revealed alterations in porcine endometrial proteome.

  • Identified proteins associated with generating endometrial receptivity

  • Identified proteins that altered in abundance in the presence of conceptuses

  • Epithelial remodeling and acute-phase response are significantly altered pathways.

Abstract

In mammals, successful pregnancy depends upon the readiness of uterus for implantation, followed by correct communication between the endometrium and the developing conceptus. The objective of this study was to elucidate changes in protein abundance associated with progression of estrous cycle and pregnancy from Day 9 to Day 12. We analyzed porcine endometrial tissue lysates by 2D-DIGE. Abundance of several proteins was altered depending upon the pregnancy status of animals. MALDI-TOF/TOF was used to identify a number of these proteins. Endometrial proteins that increased from Day 9 to Day 12 of cycle included annexin A4, beta-actin, apolipoprotein, ceruloplasmin and afamin. Changes in protein abundances associated with conceptus secreted factors, including haptoglobin, prolyl-4-hydroxylase, aldose-reductase and transthyretin, were also observed. Functional analysis revealed that endometrial proteins with altered abundance on Day 12 irrespective of the reproductive status were related to growth and remodeling, acute phase response and free radical scavenging, whereas transport and small molecule biochemistry were the functions activated in the pregnant endometrium as compared to the cyclic endometrium. These data provide information on dynamic physiological processes associated with uterine endometrial function of the cyclic and pregnant endometrium during period of maternal recognition of pregnancy in pigs and may potentially demonstrate a protein profile associated with successful pregnancy.

Biological significance

In pigs, the fertility rates are generally very high but the early embryonic loss that occurs during the second and third weeks of gestation critically affects the potential litter size. Temporal changes that take place in the uterine environment during the period of early pregnancy in pigs and a cross-talk between the uterus and the embryo play an important role in embryonic survival and successful pregnancy. A better understanding of the molecular changes associated with these processes will pave way for understanding of endometrial functions and help towards increasing embryo survival. In this study, we present a 2D-DIGE based analysis of changes in porcine endometrial proteome that are associated with progression of cycle and progression of pregnancy. The network analysis of the results clearly revealed the pathways that are involved in rendering the endometrium receptive to the presence of embryo and also the changes that are result of molecular communication between the endometrium and the conceptuses. This comprehensive identification of proteomic changes in the porcine endometrium could be a foundation for targeted studies of proteins and pathways potentially involved in abnormal endometrial receptivity, placentation and embryo loss.

Introduction

The establishment of pregnancy requires the participation of a receptive endometrium and the development of the embryo to the implantation competent stage [1], [2]. In mammalian species, including pigs, the endometrium undergoes a transformation in response to the physiological changes triggered by ovarian hormones in different stages of the cycle to prepare for embryo attachment and implantation [3]. These transformations involve changes to the endometrial structure and spatiotemporal alterations in molecular profiles. The endometrium also secretes a wide array of growth factors, proteins and cytokines, which constitute the histotroph, an important source of energy and nutrition to a growing embryo [4], [5]. In both cycling and pregnant animals, similar molecular changes occur in the endometrium up to the initiation of conceptus elongation [6], suggesting that the changes responsible for the formation of a receptive endometrium are initially under maternal control. The endometrial receptivity triggered by ovarian steroids is later modified by the molecular signaling between the embryo and the endometrium. In pigs, this signaling between the embryo and endometrium starts around Days 11 to 12 of pregnancy when there is a surge of estrogens secreted by the conceptuses in the uterine lumen [7]. This period is also known as the maternal recognition of pregnancy in pigs. It has been hypothesized that an increase in estrogen concentration stimulates the secretion of endometrial proteins, promoting trophoblast growth, which are critical for implantation [8].

Using the candidate gene approach, many studies have evaluated gene and protein expressions in the porcine endometrium to elucidate the molecular mechanisms responsible for the establishment of pregnancy in pigs [3], [9], [10]. These efforts, along with global gene expression studies, have tremendously advanced our understanding of porcine pregnancy and endometrial biology. In recent years, while many global gene expression studies in the porcine endometrium have been conducted on differential gene expression during pregnancy [11], [12], [13], [14], there are very few reports regarding global changes in porcine endometrial proteome during pregnancy [6], [15]. Kayser et al. [6] reported changes in uterine fluid proteome during early porcine pregnancy and some proteins reported by them are common to this study. However, as uterine fluid does not contain high abundance cellular proteins, it is expected to have a less complex proteome than that of endometrial tissue. Given the complexity of the endometrium, the uterine fluid and endometrial proteome would not be expected to be quantitatively or qualitatively identical, though common proteins could be identified.

In our study, 2D-DIGE was used to construct a 2D map of the cycling and pregnant porcine endometrium to identify the proteins that are differentially expressed with the progression of the estrous cycle and pregnancy. We observed both estrous cycle and pregnancy-dependent changes and also identified a number of proteins that have not previously been identified in the porcine endometrium.

Section snippets

Animals

All procedures involving the use of animals were approved by the Animal Ethics Committee, University of Warmia and Mazury in Olsztyn, Poland, and were conducted in accordance with the national guidelines for agricultural animal care. Estrous induction and synchronization were achieved in 16 crossbred gilts (Sus scrofa domesticus) weighing ~ 100 kg by hormonal treatment with administration of an intramuscular injection of 750 IU of equine chorionic gonadotropin followed by 500 IU of human chorionic

DIGE-based profiling of endometrial proteins on Days 9 and 12 of the estrous cycle

A DIGE-based proteomic approach was used to identify proteins that were differentially expressed with the progression of the estrous cycle from Day 9 to Day 12. These factors might be responsible for endometrial preparation for the possible presence of an embryo in the uterus. Proteins from Day 9 and 12 endometrial lysates were separated using IEF. As the protein components of endometrial lysate were better resolved in the pH range of 4–7 as compared to 3–11, we used IPG strips in the pH ranges

Discussion

In mammals, the endometrium has a critical role in the development and survival of the conceptus. Consequently, there has been extensive study of the molecular processes associated with the progression of the estrous cycle, endometrial receptivity and embryonic-maternal cross-talk [17], [18], [19], [20], [21]. However, a qualitative proteomic study of the protein profile of the porcine endometrium defining endometrial function is lacking in pigs. In our current study, we have presented 1)

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgment

This work was supported by a Project 2011/01/B/NZ4/03542 from the National Science Centre, funds appropriated to the Institute of Animal Reproduction and Food Research, Olsztyn, Poland.

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