Elsevier

Theriogenology

Volume 92, 1 April 2017, Pages 111-120
Theriogenology

The effect of lysophosphatidic acid together with interferon tau on the global transcriptomic profile in bovine endometrial cells

https://doi.org/10.1016/j.theriogenology.2017.01.021Get rights and content

Highlights

  • The transcriptional profiling studies identified a number of genes that are regulated by LPA.

  • LPA alters the expression of genes that are probably important during the peri-implantation period of pregnancy.

  • The DEGs increased in the LPA + IFNτ-treated endometrial cells are largely in response to IFNτ actions.

Abstract

In cows, lysophosphatidic acid (LPA), which acts in an auto/paracrine manner, serves as a luteotropic factor during early pregnancy by stimulating progesterone and prostaglandin E2 secretion, thus protecting the bovine corpus luteum and early embryo development. Our hypothesis was that LPA exerted some local effects on the bovine endometrium prior to early embryo-maternal interactions and that interferon tau (IFNτ), the pregnancy recognition signal, modulated this action. In the present study, we applied an in vitro model involving whole-transcriptomic profiling to examine the effects of LPA on gene expression in bovine endometrial cells. Microarray analyses revealed 36, 269 and 284 differentially expressed transcripts in bovine endometrial cells in the control vs. LPA, control vs. LPA + IFNτ and LPA vs. LPA + IFNτ groups, respectively. The expression of matrix metalloproteinase 13 (MMP13) and radical S-adenosyl methionine domain containing 2 (RSAD2) was increased in the LPA-treated endometrial cells. Among the transcripts differentially regulated by LPA together with IFNτ, many of the genes were classical- or novel-type I IFN-stimulated genes (ISGs). The results indicated that 10 of the 16 analyzed genes showed a positive correlation with their corresponding microarray data upon real-time PCR validation, indicating a considerable consistency between both techniques. In summary, these transcriptional profiling studies identified a number of genes that were regulated by LPA alone and LPA together with IFNτ in endometrial cells from the bovine uterus. Available studies support the idea that LPA, which acts in an auto/paracrine manner on the endometrium, alters the expression of genes that are probably important for uterine receptivity, maternal immune tolerance to the embryo and conceptus growth and development during early pregnancy. Moreover, the differentially expressed genes (DEGs) that increased in the LPA + IFNτ-treated endometrial cells are largely in response to IFNτ actions and are possibly associated with crucial biological processes during the peri-implantation period of pregnancy.

Introduction

Lysophosphatidic acid (LPA), which exerts a variety of physiological and pathological actions in various animal species [1], [2], [3], [4], [5], [6], has been documented to regulate reproductive performance in cows [7], [8]. We have recently found that LPA can be locally produced and released from the bovine endometrium [9]. We have also documented that LPA concentrations and gene expression for its active receptor LPAR1 in the bovine endometrium were significantly higher during early pregnancy than during the estrous cycle [9]. Moreover, LPA stimulated progesterone (P4) and prostaglandin (PG) E2 secretion in vivo, and mRNA expression of LPAR1 was positively correlated with the expression of the enzyme responsible for luteotropic PGE2 production (PGES) in the bovine endometrium during the estrous cycle and early pregnancy [9], [10]. Bovine embryos at different stages of development are also able to synthesize and secrete LPA; additionally, LPA-mediated cell signaling during early embryonic development may be relevant in early embryo-maternal interactions that lead to the embryonic survival [11]. These data indicate that LPA may play autocrine and/or paracrine roles in the uterus and serve as an important factor in the maintenance of early pregnancy not only in mouse [3], [12], pig [5] and sheep [6] but also in cow [10].

There is much evidence showing that after fertilization, between days 10 and 21 to 25 in ruminants, the conceptus starts to produce interferon tau (IFNτ), which is the pregnancy recognition signal that prevents development of the endometrial luteolytic mechanism [13], [14]. The maximal production of IFNτ falls on days 14–18 [15]. However, Kimura et al. [16] reported that IFNτ production during culture of in vivo-derived embryos increased dramatically (150,000-fold) from day 9–14. The signals from the conceptus occurring after fertilization are not only local, but spread throughout the entire female as measured in the systemic blood a few days before implantation [17], [18]. This peri-implantation period during early pregnancy represents the time when the blastocyst is hatching; from this time, it becomes a free-floating embryo within the lumen of the uterus that is totally dependent on the uterine environment for survival [15]. At this time, early embryo survival mostly depends on the appropriate function of corpus luteum (CL)-adequate P4 synthesis, as well as on IFNτ-dependent inhibition of the development of the endometrial luteolytic mechanism [13], [14]. In contrast, LPA also serves as a luteotropic factor during early pregnancy by stimulating P4 and PGE2 secretion, thus protecting the bovine CL and early embryo development [9].

Studies to date on the endometrial response to various early pregnancy modulating factors, including LPA, have been based on a candidate gene approach. Therefore, a better understanding is needed of all the internal signaling pathways involved in the potential modulation of LPA action by IFNτ. This study will provide a deeper understanding of uterine functions at the time of early pregnancy recognition in cow and can directly influence further investigations.

Section snippets

Bovine endometrial cell culture

All of the animal procedures were approved by the Local Animal Care and Use Committee in Olsztyn, Poland (Agreement No. 79/2008/N). For all experiments, normally cycling Holstein/Polish Black and White (75/25%, respectively) cows (n = 9) were chosen. Bovine uteri were obtained at a local slaughterhouse within 20 min of exsanguinations and were transported on ice to the laboratory within 40 min. The animals were slaughtered on days 2–5 of the estrous cycle to isolate the endometrial cells as

Microarray quality control

The large-scale analysis of the endometrial transcriptome was conducted using a microarray, which is comprised of over 23 000 bovine transcripts. Hierarchical clustering and principal component analysis revealed sufficient differences in the gene expression profiles from endometrial cells stimulated with PBS, LPA and LPA + IFNτ that allowed the genes to cluster into three separate groups based on the treatment conditions (Fig. 1). The total principal component (PC) accounts for 86.8% of the

Discussion

In this study, we used microarray technology to investigate the effects of LPA on the gene expression profile in bovine endometrial cells. Previous reports showed that LPA stimulated PGE2 synthesis via the enhanced mRNA expression of PGE2 synthase in stromal cells on days 8–10 and 16 to 18 of the estrous cycle and pregnancy in the bovine endometrium [10], [24]. Moreover, LPA inhibited PGF synthesis by reducing PGF synthase mRNA expression in epithelial cells on days 8–10 and 16 to 18 of

Conclusions

In summary, the transcriptional profiling studies identified a number of genes that are regulated by LPA and LPA together with IFNτ in endometrial cells from the bovine uterus. The results revealed that 10 of the 16 analyzed genes showed the same expression patterns between the microarray and real-time PCR analysis, indicating considerable consistency between both techniques. Lysophosphatidic acid differentially regulates the expression of genes that are probably important for uterine

Funding

This research was supported by Grants-in-Aid for Scientific Research from the Polish Ministry of Sciences and Higher Education (MNiSW-DPN/DWM/MZ/5751/08/09).

Acknowledgements

Not applicable.

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