Journal of Animal Reproduction and Biotechnology 2020; 35(1): 12-20
Published online March 31, 2020
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Dongjie Zhou, Yingjie Niu and Xiang-Shun Cui*
Department of Animal Science, Chungbuk National University, Cheongju 28644, Korea
Cell adhesion plays an important role in the differentiation of the morphogenesis and the trophectoderm epithelium of the blastocyst. In the porcine embryo, CDH1 mediated adhesion initiates at compaction before blastocyst formation, regulated post-translationally via protein kinase C and other signaling molecules. Here we focus on muscle RAS oncogene homolog (M-RAS), which is the closest relative to the RAS related proteins and shares most regulatory and effector interactions. To characterize the effects of M-RAS on embryo compaction, we used gain- and loss-offunction strategies in porcine embryos, in which M-RAS gene structure and protein sequence are conserved. We showed that knockdown of M-RAS in zygotes reduced embryo development abilities and CDH1 expression. Moreover, the phosphorylation of ERK was also decreased in M-RAS KD embryos. Overexpression of M-RAS allows M-RAS KD embryos to rescue the embryo compaction and blastocyst formation. Collectively, these results highlight novel conserved and multiple effects of M-RAS during porcine embryo development.
Keywords: blastocyst formation, CDH1, embryo compaction, muscle RAS oncogene homolog (M-RAS)
M-Ras shares ∼50% sequence identity with the canonical Ras proteins (Kimmelman et al., 1997), and, although it can interact with some of the well-established Ras effectors, such as Raf, PI3K, and RalGDS. M-Ras also binds to distinct effectors, including certain RapGEFs and the Shoc2 scaffold (Herrmann et al., 1996; Rebhun et al., 2000; Gao et al., 2001; Rodriguez-Viciana et al., 2006). In contrast to the canonical Ras proteins, constitutively active M-Ras has a low transforming activity and is rarely mutated in human cancer (Quilliam et al., 1999); however, gain-of-function mutations in M-RAS have been identified in patients with Noonan syndrome (Higgins et al., 2017). Notably, mutations in one of the distinct effectors of M-RAS, the SHOC2 scaffold, have also been detected in Noonan patients (Cordeddu and Di, 2009; Hannig et al., 2014), and, likewise, Shoc2 has been implicated in cell migratory events (Young et al., 2013; Kaduwal et al., 2015; Jeoung et al., 2016).
Subsequent biochemical studies have shown that the direct binding of Shoc2 to active GTP-bound M-Ras allows the Shoc2 scaffold to nucleate a ternary complex consisting of active M-Ras, Shoc2 and the catalytic subunit of PP1 (PP1c) (Rodriguez-Viciana et al., 2006). In RTK-mediated signaling, the M-Ras/Shoc2/PP1c ternary complex functions to dephosphorylate a negative regulatory 14-3-3 binding site on the Raf kinases, which promotes Raf binding to the canonical Ras proteins and facilitates ERK cascade activation (Rodriguez-Viciana et al., 2006; Matsunaga-Udagawa et al., 2010; Galperin et al., 2012). This complex has been implicated in the dynamic regulation of ERK activity and cell polarity in some cancer cell lines (Young et al., 2013). Moreover, M-Ras/Shoc2 signaling contributes to junction turnover by modulating the Cdh1/p120-catenin interaction and, in turn, the junctional expression of Cdh1. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphor-regulation of p120-catenin and required downstream ERK cascade activation (Kota et al., 2019).
Compaction appears to occur in all mammalian embryos but at different cell cycles. In the pig, compaction and cell polarization occur more gradually than in the mouse and coincide with relocation of CDH1 to contact sites, the process not completing long before blastocyst formation (Reima et al., 1993). In the developing embryo CDH1 is a key player during morula compaction and
To further elucidate the biological functions of the M-RAS on porcine embryonic compaction, we investigated the mechanism by which M-RAS and CDH1 contribute to the regulation of compaction.
Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
All animal studies were performed in strict accordance with the institutional guideline and prior approval from the Institutional Animal Care and Use Committee (IACUC) of the Chungbuk National University, Republic of Korea.
Prepubertal porcine ovaries were transported from a local abattoir (Farm Story Hannang, Chungwon, Chungbuk, Republic of Korea) within one hour of harvest. Cumulus-oocyte complexes (COCs) were aspirated from 3-6 mm of ovarian follicles using 18-gauge needles attached to a 10-mL syringe. High-density cumulus oocytes were collected and washed three times with HEPE-PVA medium (HEPES medium supplemented with 0.01% polyvinyl alcohol). After washing, 60-80 COCs were cultured in 500 mL of IVM medium consist of M-199 (Invitrogen, Carlsbad, CA) containing 20 ng/mL epidermal growth factor, 1 g/mL insulin, 75 g/mL kanamycin, 0.91 mM Na pyruvate, 0.57 mM L-cysteine, 10% (v/v) porcine follicular fluid, 0.5 μg/mL follicle stimulating hormone (FSH), and 0.5 mg/mL luteinizing hormone (LH) at 38.5oC in an atmosphere containing 5% CO2 at 100% humidity.
For parthenogenetic activation, mature oocytes were denuded by gentle pipetting in 1 mg/mL hyaluronidase until all the cumulus cells were removed from around the oocyte. Oocytes were then washed three times in PBS-BSA (DPBS added in 0.1% BSA) and activated using an Electro Cell Manipulator 2001 (BTX, Inc., San Diego, CA, USA). The electric pulse was stimulated at 110 V/cm twice for 60 μsec in 280 mM mannitol medium supplemented with 0.1 mM CaCl2, 0.05 mM MgCl2, 0.01% polyvinyl alcohol (PVA, w/v), and 0.5 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid. Activated oocytes were treated in PZM-5 medium containing 7.5 μg/mL cytochalasin B in an incubator for 3 h to suppress extrusion of the pseudo second polar body. Embryos were thoroughly washed and cultured in bicarbonate buffered PZM-5 supplemented with 4 mg/mL BSA in 4-well plates for 6 d at 38.5°C and 5% CO2. Two-cell and 4-cell cleavage rate and morula and blastocyst formation rate were examined, respectively, at 24, 48, 96, and 144 h after activation. To determine the total cell number, day-6 blastocysts were randomly collected and stained with 10 mg/mL Hoechst 33342 in PBS for 5 min.
The open reading frames of pig
For knockdown or overexpression experiments,
After washing 3 times with PBS and PVA, embryos were fixed in 3.7% paraformaldehyde for 30 min at room temperature, permeabilized with PBS and PVA containing 0.5% Triton X-100 at room temperature for 30 min and incubated in PBS and PVA containing 1.0% BSA at room temperature for 1 h. These embryos were incubated overnight at 4°C with anti-M-RAS (1:100,14213-1-AP; Proteintech), anti-CDH1 (1:100, 610182, BD Biosciences), or anti-ZO1 (1:100; 33-9100, Invitrogen) diluted in blocking solution. After washing 3 times with PBS and PVA, the embryos were incubated at room temperature for 1 h with goat anti-rabbit IgG, rabbit anti-goat IgG, or anti-mouse IgG. The oocytes and embryos were stained with 10 mg/mL Hoechst 33342 for 5 min, washed 3 times with PBS and PVA, mounted onto slides, and examined using a confocal microscope (Zeiss LSM 710 Meta). Images were processed using Zen software (v.8.0; Zeiss).
Embryos were collected, and mRNA was extracted from a pool of 30 embryos per group using the DynaBeads mRNA Direct Kit (61012; Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was obtained by reverse transcription of mRNA using the Oligo (deoxythymine) 20 primer and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Amplification was conducted as follows: 95°C for 3 min followed by 40 cycles of 95°C for 15 s, 60°C for 25 s, 72°C for 10 s, and final extension at 72°C for 5 min. Target genes was M-RAS. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The primers used to amplify each gene are shown in Table 1. mRNA quantification data were analyzed using the 2-ΔΔCt method (Livak and Schmittgen, 2001).
A total of 100 porcine embryos per group were placed in 1-time SDS sample buffer and heated at 98°C for 10 min. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Next, the membranes were blocked in Tris buffered saline containing 0.1% Tween 20 and 5% nonfat milk for 1 h and then incubated at 4°C overnight with anti-M-RAS, ERK, pERK, CHD1 or b-tubulin, wash three time by TBST. Then incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:20000; Santa Cruz Biotechnology). Membranes were developed by SuperSignal West Femto Trial Kit (34094, Thermo Scientifc, Waltham, MA, USA). Blots were visualized by a charge-coupled device camera and UviSoft software (Uvitec, Cambridge, United Kingdom).
Each experiment was repeated at least 3 times, and representative images are shown in figures. All data were subjected to Student’s t test. All percentage data were subjected to arcsine transformation prior to statistical analysis and presented as the means 6 SEM. Significance was set at a value of
We evaluated the expression and subcellular location of M-RAS at different stages during porcine embryo development (Fig. 1). M-RAS was expressed in the cytoplasm at zygote stage and highly expressed on the membrane from 2-cell stage until morula stage. The signal was detected on the nucleus from blastocyst (Fig. 1A). Moreover, the protein expression was decreased during preimplantation development (Fig. 1B). The mRNA expression level of M-RAS was detected by qPCR.
To investigate the role of M-Ras in compaction during porcine embryo development, M-RAS double-stranded RNA (
To more directly assess the role of M-RAS in regulating intercellular adhesion, we examined whether the presence or absence of M-RAS had any effects on E-cadherin localization in different stages of embryos (Fig. 3). In M-RAS KD embryos, the decrease in cell-cell adhesion coincided with a lower expression of E-cadherin shown by immune fluorescence and western blotting (Fig. 3A, 3B). However, no detectable change in ZO-1 (also known as tight junction protein-1) was present in
To confirm that the role of the M-RAS on embryonic compaction, we first produced the cRNA of activated M-RASQ71L and injected together with dsRNA of
M-RAS has variable expression throughout the body, with high expression in the heart, brain, breast, and ovaries. M-RAS encodes a 208-amino acid, membrane-associated protein which participates in Ras/MAPK pathway signaling.
M-RAS is a member of the Ras super family of proteins, which includes HRAS, KRAS, NRAS, Rit1, and RRAS (Aoki et al., 2005; Schubbert et al., 2006; Cirstea and Kdvorsky, 2010; Aoki et al., 2013; Flex et al., 2014). All Ras proteins belong to a class of small GTPases and cycle between an active GTP-bound state and an inactive, GDP-bound state. In the active, GTP-bound state, Ras proteins undergo a conformational change allowing binding to other effector proteins and participation in signaling cascades. The intrinsic Ras GTPase hydrolyzes Ras-GTP back to Ras-GDP, with the aid of GTPase-activating proteins (GAPs), to terminate activity and signaling.
M-RAS specifically functions within the Ras/MAPK pathway by forming a ternary complex with SHOC2 and PP1. The formation of this complex localizes SHOC2 and PP1 to the cell membrane where SHOC2 acts as a regulatory subunit of PP1c, targeting it to specifically dephosphorylate the P-S259 inhibitory site of RAF1 kinase. The removal of this inhibitory phosphate stimulates Raf1 activity and, thereby, activity of downstream MAPK pathway signaling (Rodriguez-Viciana et al., 2006). M-RAS, therefore, makes a critical contribution to ERK activation by receptor tyrosine kinases and downstream MAPK signaling (Young et al., 2013). Our results suggest that M-RAS regulate the cell adhesion proteins through the activity of this M-RAS/SHOC2/PP1C complex.
Elevated expression of E-cadherin at adhesion junctions is known to increase cell-cell adhesiveness, and binding of p120-catenin to the juxta membrane domain of E-cadherin promotes the surface stability of E-cadherin by preventing its endocytosis and degradation. In particular, we found that E-CADHEREN protein level of
This work was financially supported by the National Research Foundation (NRF) of Korea grant funded by the Korea government (ministry of education science and technology) (No. 2018R1A2B6001173) and Research Year of Chungbuk National University in 2019, Republic of Korea.
Xiang-Shun Cui, Dongjie Zhou designed the experiment. Dongjie Zhou conducted the experiments, analyzed the results, and wrote the article. Yingjie Niu helped with the analyses of the results and figures. Xiang-Shun Cui revised the manuscript.
D Zhou, PhD Student,
Y Niu, Post-Doc.,
XS Cui, Professor,