Inhibitor mediated WNT and MEK/ERK signalling affects apoptosis and the expression of quality related genes in bovine in vitro obtained blastocysts

https://doi.org/10.1016/j.bbrc.2019.01.113Get rights and content

Highlights

  • WNT and MEK/ERK signalling pathways affect bovine embryo quality.

  • Activation of pluripotency related signalling reduces apoptosis in preimplantation embryos.

  • Altering of signalling pathways induce SDHA expression, suggesting oxidative stress conditions.

Abstract

Culture conditions determine embryo quality, which may be affected on many levels (timing of development, blastomere count, transcripts, metabolite content, apoptosis). Molecular interactions of signalling pathways like MEK/ERK and WNT/β-catenin are critical for cell-to-cell communication and cellular differentiation. Both pathways are important regulators of apoptosis. We have aimed to verify the prolonged effect of MEK/ERK silencing and WNT activation by chemical inhibitors (2i or 3i systems) on bovine IVP embryos. Apoptotic index, total cell count and transcription of embryo quality markers were evaluated. A higher rate of apoptosis was observed in 2i blastocysts, but was not accompanied by changes in transcript content of genes controlling apoptosis (BAX, BCL2, BAK, BAX/BCL2 ratio). Therefore, alternative pathways of apoptotic activation cannot be ruled out. The expression of genes related to embryo quality (HSPA1A, SLC2A1) was not affected. GJA1 transcripts were significantly higher in 3i blastocysts, what indicates a stimulatory effect of the applied inhibitors on cell-to-cell interactions. The lowest mRNA level of the IFNT2 gene was found in 2i embryos. A variation in the SDHA gene transcript was observed (with the highest content in the 3i blastocysts), what may suggest their reduced quality. It may be concluded that the modifications of culture conditions (activation of the WNT and silencing of the MEK/ERK signalling) might alter pathways crucial for embryo development without causing embryonic death.

Introduction

Studies of physiological mechanisms and interactions between the preimplantation embryo and it's in vivo or in vitro environment are essential to define optimal in vitro culture (IVC) conditions [1].

Proper embryogenesis relies on signalling, which in addition to directing cell fate also controls proliferation and cell survival with apoptosis being at its centre. Although apoptosis is described as beneficial for the developing embryo, there must be a certain, still unknown upper threshold causing growth retardation and embryonic death. The incidence of apoptosis (apoptotic index, AI) in bovine in vitro produced (IVP) embryos varies from 3.1% to 9.4% and for in vivo derived embryos it lies between 4.2 and 6.1% [2,3]. Apoptosis is induced via two main pathways, both regulated by a cascade of molecular events controlled by pro-(BAX, BAK) and anti-(BCL2) apoptotic genes. The proportion between BAX and BCL2 proteins determines the faith (survival or death) of a cell [4].

Other important regulators of early development are the signalling pathways, critical for cell-to-cell communication and the induction of cellular differentiation, which include mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AktWingless (WNT)/β-catenin.

MAPK family plays an important role in many cellular processes including apoptosis. Active WNT pathway is crucial for pluripotency maintenance. It acts as a regulator of embryonic cell patterning, differentiation, cell adhesion, survival and apoptosis. Depending on the specific cellular environment stimuli, the WNT signalling can either foster or restrain apoptosis. It regulates the early and late stages of apoptosis in both development and cell injury [5]. The important mechanisms of regulation include signals acting via the WNT-BMP signalling, β-catenin and glycogen synthase kinase 3 (GSK3). Although β-catenin was proven to be the main signalling molecule of the WNT pathway, adenomatous polyposis coli protein (APC) is a critical component of the GSK3β complex necessary for WNT activity. The role of APC in apoptosis regulation depends on its length – overexpression of full length APC protein induces apoptosis, and the expression of truncated protein maintains an anti-apoptotic environment [6]. In response to continuous WNT signalling (which may be caused by mutations in APC), β-catenin is stabilised and constantly transferred to the nucleus where it binds to the T cell factor/lymphoid enhancer factor (TCF/LEF). As a result, a continuous expression of c-MYC, a transcription factor important for cell cycle progression, apoptosis and terminal differentiation is maintained.

Since the discovery of mouse embryonic stem cells (ESC), the advances in stem cell research in species for which the classical derivation system fails, have been attributed to the specific signalling pathway inhibitors (i). The 2i or 3i inhibitor systems made it possible to maintain pluripotency by blocking differentiation inducing signalling, obtained by interfering with signalling pathways important for cell differentiation and lineage commitment [7]. Both these systems operate within the WNT and the MEK/ERK signalling, but use a different set of inhibitors. The 3i system consists of MEK/ERKi (PD184352), FGF receptor inhibitor SU5402 and GSK3i (CHIR99021). The first two inhibitors are involved in the suppression of the MAPK/ERK pathway, whereas the inhibition of GSK3 supports the WNT activity. The 2i system includes CHIR99021 and MEKi (PD0325901) which emerges as more potent inhibitor than PD184352. The 2i system was shown to be more efficient for mouse and rat ESC derivation than the 3i system [8]. Studies by our group indicated that the addition of CHIR99021 to bovine IVP medium resulted in an upregulation of pluripotency related factors (OCT4, NANOG, c-MYC, REX1) and a decrease in the trophectoderm specific factor CDX2 (both at the protein and the mRNA level) [9]. Ozawa et al. [10] showed that the exposure of bovine embryos to the 2i system at the time of morula to blastocyst transition facilitated the formation of self-renewing pluripotent cell lines from blastocysts and continuously maintained high NANOG and SOX2 expression. The positive effect of the 3i system on putative bovine ESC survival and maintenance after thawing was also confirmed [11].

Studies demonstrated that all of the key developmental pathways described for mouse and human embryos are actively involved in the regulation of bovine embryo development [9,12]. Numerous genes of the WNT and the MAPK pathways are expressed in bovine oocytes and blastocysts [13,14]. Choosing the right inhibitor is crucial for the outcome of the experiment. For example, AMBMi (amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl) pyrimidine) induces β-catenin and TCF dependant transcriptional activity. In contrast to the effect of CHIR9921, its admission decreased bovine blastocyst formation rate. This may be explained by the fact that CHIR9921 acts on different elements of the WNT pathway [15]. It inhibits GSK3 (crucial for the formation of the destructive complex), thus prevents β-catenin from entering the nucleus and activating the downstream effectors.

The unique culture environment exerts distinct effects on embryo quality. Specific signalling pathways may influence some particular elements of embryo quality determination (such as apoptosis), which will consequently affect embryo development and survival. Knowing the important role of signalling pathways in controlling early development, their involvement in apoptosis regulation, and the importance of apoptosis for proper preimplantation development, we have aimed to verify the effect of the 2i and the 3i culture systems on bovine embryo quality. Our goal was to describe the additional factors which may be detrimental for the outcome of IVP procedures and provide new data for bovine ESC derivation protocols. Our attention was focused on parameters such as the apoptotic index (AI), the total cell count, transcription level of five genes recognized as classical markers of bovine embryos quality (SLC2A1, HSPA1A, GJA1, IFNT2, SDHA) and genes controlling the process of apoptosis (BAX, BCL2, BAK).

Section snippets

Ethics statement

Not needed – the biological material was either collected upon commercial animal slaughter, or purchased from commercial Artificial Insemination Station (bull semen). In vitro obtained bovine embryos were of preimplantation stages (under 1/3 of development) which do not require ethics approval (Animal Protection Act; Art 2.1).

In vitro production of bovine embryos

Unless stated otherwise, all reagents used for IVP media were supplied by Sigma-Aldrich (Poland) and the inhibitors were purchased from Axon Medchem (The Netherlands).

Results

The possible effect of the inhibitor vehicle (DMSO) on embryo development (Suppl. Results) and the effectiveness of the inhibitors was validated by gene expression analysis and showed a significant (P ≤ 0.01) increase in the OCT4 gene expression in both 2i and 3i blastocysts, 9dpi (Suppl Fig.1).

Discussion

Maintaining of a delicate balance between cell survival and apoptosis is needed to differentiate between pathology and normal development. Our results demonstrate that the applied inhibitors reduced the percentage of apoptotic cells in bovine blastocysts, what may be explained by the fact that pluripotency maintenance reduces differentiation signalling and to a certain extent supresses pro-apoptotic signalling. The embryo may compensate the external stimuli, and despite altered signalling still

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was funded by National Science Centre, Poland (NCN) Grant no.: OPUS3 2012/05/B/NZ9/03349.

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    • WNT signalling supported by MEK/ERK inhibition is essential to maintain pluripotency in bovine preimplantation embryo

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    1

    These authors equally contributed to this work.

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