Endoplasmic Reticulum Stress Inducer Tunicamycin Reduces Porcine Embryo Development by Disturbing Blastocoel Formation and Expansion

TL;DR

Tunicamycin, an ER stress inducer, harms pig embryo development by disrupting blastocoel formation and cell growth.

Contribution

Demonstrates how ER stress impacts blastocyst formation and gene expression in porcine embryos.

Findings

Tunicamycin increases ER stress markers ATF6, CHOP, and GRP78 in porcine embryos.

Tunicamycin impairs blastocoel formation, expansion, and cell proliferation in blastocysts.

Tunicamycin disrupts the expression of E-cadherin, Oct4, Sox2, and Cdx2 in porcine embryos.

Abstract

This study investigated the impact of endoplasmic reticulum (ER) stress on preimplantation embryos during blastocyst formation and expansion in pigs. Porcine embryos were treated with ER stress inducer tunicamycin (TM). TM increased the mRNA levels of ER stress-related markers ATF6, CHOP, and GRP78. It also impaired porcine blastocyst quality by inhibiting blastocoel formation and expansion and cell proliferation, and by disrupting the expression patterns of E-cadherin, Oct4, Sox2 and Cdx2. These findings provide insights into the molecular events associated with ER stress-related embryonic developmental defects.

The formation and expansion capacity of blastocysts plays a very important role in successful implantation. During mammalian embryo development derived from in vitro production (IVP), early embryos are highly susceptible to various cellular stresses, including endoplasmic reticulum (ER) stress, which has been identified in IVP embryos, suggesting that ER stress modulation is crucial for IVP embryo development. In this study, we aimed to evaluate the influences of ER stress on preimplantation embryos during blastocyst formation and expansion potential in pigs. Tunicamycin (TM), an ER stress inducer, was employed in porcine embryos, significantly increasing the mRNA levels of ER stress-related markers ATF6, CHOP, and GRP78. When one-cell embryos were cultured in the presence of TM, the blastocyst formation and diameter (reflecting the blastocyst expansion capacity) were significantly inhibited in a dose-dependent manner. When morula-stage porcine embryos were cultured in TM, the blastocyst formation rate, blastocyst diameter, total cells and EdU-positive cell numbers were significantly lower than the TM-free control group. TM reduced the potential of blastocoel recovery (ex-expansion) in blastocysts collapsed by cytochalasin D and impeded blastocyst expansion. In addition, TM reduced the mRNA levels of CDH1 and TJP1 and affected the normal expression pattern of E-cadherin, Oct4, Sox2 and Cdx2 in porcine blastocysts. Taken together, these findings suggest that TM treatment during embryo development in vitro interferes with the formation and expansion capacity of the blastocoel in pigs.

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1. Introduction

During the eight-cell into morula stage of mammalian embryo development, blastomeres begin to bind steadily together in a process called compaction, whereafter the morula differentiates into a blastocyst, the final stage in the development of the pre-implanted egg. The blastocyst is generally regarded as a useful model for assessing physiological and biochemical embryology [1], being composed of an inner cell mass and trophectoderm, as well as a fluid-filled cavity (blastocoel) [2]. Blastocoel formation requires coordinated morula compaction and trophectoderm (TE) differentiation [3] and depends on the establishment of functional intercellular junctions and efficient transepithelial ion and fluid transport [2]. Proper blastocoel expansion is essential for creating a suitable signaling microenvironment that supports pluripotency maintenance and lineage specification. In contrast, defective blastocoel formation or insufficient blastocoel expansion are often associated with abnormal expression of lineage-specific transcription factors [4], defects that ultimately lead to reduced blastocyst quality and impaired implantation potential.

Mammalian embryos are highly susceptible to various forms of stress during in vitro culture [5], of which endoplasmic reticulum (ER) stress is an important response that plays a crucial role in maintaining cellular homeostasis [6]. Under ER stress, unfolded protein response pathways are activated, enabling cells to adapt to transient stress; however, excessive or prolonged ER stress can disrupt normal cellular functions and induce apoptosis [7,8]. Tunicamycin (TM) is a naturally occurring antibiotic that induces ER stress by inhibiting the first step in the biosynthesis of N-linked glycans in proteins, resulting in the accumulation of unfolded or misfolded proteins within the ER lumen [9]. TM is widely used in research to study the role of N-glycosylation in cell proliferation [10], apoptosis [11], and tumor cell sensitivity to anticancer drugs [12]. It is also commonly used to induce ER stress in mammalian embryos [13,14,15,16] for in vitro production experimental purposes.

ER stress or TM-induced ER stress impair oocyte maturation and subsequent embryo development by promoting ROS generation; mitochondrial dysfunction and apoptosis have been reported widely in pigs [13,17,18,19], mice [20,21,22] and cattle [14,23,24].

Although accumulating evidence indicates that ER stress negatively affects blastocyst formation and embryo quality, most studies focus on overall developmental competence or blastocyst formation rates. The specific effects of ER stress on blastocoel formation and expansion, as well as the underlying cellular and molecular mechanisms, remain poorly understood. Therefore, in this study, we used porcine embryos as a model to investigate the molecular events through which ER stress affects embryo development. In particular, we focused on its impact on blastocoel formation and expansion capacity.

2. Materials and Methods

2.1. Chemicals

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), unless otherwise indicated.

2.2. Tunicamycin (TM) Treatment

TM (Sigma-Aldrich, T7765) was prepared in DMSO (Sigma-Aldrich, D2650) as a 5 mg/mL stock solution at 4 °C, which was used to make fresh TM dosages in final concentrations of 0, 1, 2, 5 and 10 μg/mL in porcine zygote medium with 3 mg/mL BSA, PZM-3.

2.3. Oocyte Recovery and In Vitro Maturation

Porcine ovaries were collected from pre-pubertal gilts at a local abattoir and transported to the laboratory in PBS solution, with 75 μg/mL potassium penicillin G and 50 mg/mL streptomycin sulfate added within 2 h. Porcine COCs (cumulus oocyte complexes) were obtained from antral follicles (3 to 8 mm in diameter) visible on the ovarian surface using an 18-gauge needle attached to a 10 mL disposable syringe. Oocytes with a uniform ooplasm and more than two layers of cumulus cells were considered for in vitro maturation (IVM). For IVM, groups of about 50–60 COCs were incubated in 500 μL of maturation medium placed in each well of a four-well multidish at 38.5 °C for 22 h in humidity-saturated air containing 5% CO_2_. After 22 h maturation, COCs were cultured in the same medium without hormone for an additional 22 h.

The medium used for oocyte maturation was TCM-199 (Sigma Aldrich, M4530) supplemented with 3.5 mM D-glucose (Sigma Aldrich, G6152), 0.57 mM L-cysteine (Sigma Aldrich, C6852), 0.91 mM sodium pyruvate (Sigma Aldrich, P4562), 75 μg/mL penicillin (Sigma Aldrich, P4687), 50 μg/mL streptomycin (Sigma Aldrich, S1277), 10 ng/mL epidermal growth factor (EGF, Sigma Aldrich, E4127), 10 IU/mL pregnant mare serum gonadotropin (PMSG, ProSpec, Rehovot, Israel, cat#: hor-272-b), 10 IU/mL human chorionic gonadotropin (hCG, Intervet International B. V., Boxmeer, The Netherlands, A002A01) and 10% porcine follicular fluid.

2.4. Embryo Production by Parthenogenetic Activation

After IVM, the cumulus cells were removed from oocytes by vortexing in maturation medium supplemented with 0.1% hyaluronidase. For parthenogenetic activation (PA), denuded oocytes with the first polar body were washed and equilibrated in an activation solution (0.3 M D-mannitol supplemented with 0.1 mM MgSO_4_, 0.05 mM CaCl_2_ and 0.01% PVA) for 5 min. Electric stimulation was induced with a direct current pulse of 1.1 kV/cm for 100 μs using an Electro Cell Manipulator 2001 (BTX, San Diego, CA, USA). The activated oocytes were then incubated in embryo culture medium (PZM-3) supplemented with 7.5 μg/mL cytochalasin B (CB) for 4 h. Thereafter, the presumptive zygotes were washed and transferred into a four-well multidish containing 500 μL of pre-equilibrated CB-free culture medium at 38.5 °C in a humid 5% CO_2_ atmosphere.

2.5. Evaluating Influences of TM-Induced ER Stress on Blastocyst Formation and Expansion

First, we investigated the developmental potential of porcine embryos that were continuously treated from the one-cell stage with different concentrations (0, 1, 2, 5 and 10 μg/mL) of TM in PZM-3 culture medium. Next, morula-stage embryos, at which point blastocoel formation begins, were collected on day 5 of culture and treated for two days in PZM-3 with or without TM. Blastocyst formation was morphologically evaluated using a stereomicroscope with an ocular scale to capture pictures. Blastocyst expansion capacity was evaluated by estimating their diameters using Image J software (version 1.47). To determine the total cell number, blastocysts were washed in PBS-PVA and fixed in 4% (v/v) paraformaldehyde for 30 min. The samples were then treated with DAPI and observed and imaged under an epifluorescence microscope (CKX53, Olympus, Tokyo, Japan) using a 40 X objective lens (NA 0.65) and a digital camera (Olympus C270).

2.6. Evaluating Influence of TM-Induced ER Stress on Blastocoel Recovery (Re-Expansion)

Cavitary blastocysts on day 6 of culture were collected and treated with 0.5 μg/mL cytochalasin D (Sigma Aldrich, C8273) for 1 h to completely collapse the porcine blastocoel. The collapsed embryos were randomly divided into two groups and then cultured in PZM-3 with or without TM for additional 24 h to allow blastocoel recovery, after which the recovery capacity was morphologically evaluated using a stereomicroscope. Blastocyst expansion capacity was evaluated by checking their diameters using Image J software (version 1.47).

2.7. Evaluating Influence of TM-Induced ER Stress on Cell Proliferation in the Blastocyst

Blastocysts derived from morulae treated with or without TM for 2 days were used to check cell proliferation by first being immunostained with EdU (5-ethynyl-2′-deoxyuridine) staining using Click-iT EdU Imaging kits (C10337, Invitrogen, Eugene, OR, USA). EdU is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis or S-phase synthesis in proliferating cells [25]. Briefly, porcine blastocysts were incubated in 10 μΜ EdU medium for 3 h and then treated with 4% paraformaldehyde for 15 min. Samples were washed in 3% BSA then permeabilized with 0.5% Triton X-100 for 20 min. After brief flushing in 3% BSA, the samples were incubated for 30 min in the dark in a Click-iT reaction cocktail including Alexa Fluor 488 azide (green) according to the instructions provided by the manufacturer. After EdU labeling, samples were mounted on glass slides using VECTASHIELD mounting medium containing DAPI (blue) and pictures were captured using a Zeiss laser-scanning confocal microscope (LSM5 Live, Carl Zeiss, Oberkochen, Germany) equipped with X 40 objectives (NA 1.0) and running Zeiss LSM Image Browser software (ver. 4.2. SP1 Image Browser software). Finally, the total cell and EdU-positive cell numbers were counted by checking nuclei with different colors (blue and green represent total and EdU-positive cells in blastocysts, respectively).

2.8. Evaluating Influence of TM-Induced ER Stress on the Expression Patterns of E-Cadherin, Oct4, Sox2 and Cdx2 in Blastocysts

Blastocysts were washed in phosphate-buffered saline (PBS, Gibco, Grand Island, NY, USA, 20012-027) containing 0.1% polyvinyl alcohol (PBS-PVA) for 10 min and fixed in 4% paraformaldehyde for 15 min. The samples were then washed with PBS-PVA for 10 min, permeabilized with 0.5% Triton X 100 for 30 min, blocked with 3% BSA (Sigma Aldrich, A3311) for 1 h, washed in PBG for 20 min and incubated overnight at 4 °C with primary antibodies (1:100), after which they were washed in PBS containing 0.5% BSA and 0.1% gelatin (PBG) for 20 min and then treated with secondary antibodies (1:200) in the dark for 1 h. Negative controls [26] were performed by replacing the primary antibodies with normal mouse IgG (A7028; Beyotime, Shanghai, China) and rabbit IgG (A7016; Beyotime). After washing with PBG for 20 min, the samples were mounted on glass slides using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Inc. Burlingame, CA 94010, USA) and images were acquired by a Zeiss laser-scanning confocal microscope. The primary antibodies used included E-cadherin (ab15148; Abcam, Cambridge, UK), Cdx2 (ab76541; Abcam), Oct-3/4 (sc-5279; Santa Cruz Biotechnology, Dallas, TX, USA) and Sox-2 antibody (sc-365823; Santa Cruz Biotechnology). The secondary antibodies used included Donkey anti-Rabbit IgG-FITC (ab6798; Abcam); Goat anti-Mouse IgG H&L (Texas Red^®^) (ab6787; Abcam) and Goat anti-Mouse IgG-FITC (sc-2010; Santa Cruz Biotechnology).

2.9. The mRNA Levels of ER Stress-, Cell-Adhesion- and Tight-Junction-Related Genes Were Measured by Real-Time Quantitative PCR (RT-qPCR)

The details were described in a previously published study [27]. Briefly, blastocysts were collected and stored at −80 °C until RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) to extract total RNA from the blastocysts, and TOPscript™ RT DryMIX kit (Enzynomics, Daejeon, Republic of Korea) was used to synthesize complementary DNA according to the manufacturer’s instructions. Real-time PCR was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in a final reaction volume of 20 μL containing the SYBR^®^ Premix Ex Taq™ kit (Takara Bio Inc., Shiga, Japan). Relative gene expression was analyzed by the 2^−△△^Ct method and GAPDH was used for normalization. The primers used in the present study are listed in Table 1.

2.10. Statistical Analysis

Each experiment was performed with at least three replicates, and embryos were randomly allocated to each experimental group. Statistical analysis was conducted using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and percentage data were subjected to arcsine transformation prior to analysis. Experimental data were analyzed using one-way ANOVA by Fisher’s protected least significant difference (LSD) test or Student’s t-test, with values expressed as the mean ± standard error of the mean (SEM). p < 0.05 was considered significantly different.

3. Results

3.1. Influences of TM-Induced ER Stress on Porcine Blastocyst Formation and ER Stress-Related mRNA Levels

As shown in Figure 1, supplementation with 1 and 2 μg/mL TM did not significantly influence the rates of cleavage and four- to eight-cell-stage embryos, but it significantly reduced the morula and blastocyst formation rates (relative to the number of cultured one-cell stage embryos). The rates of cleavage-, four- to eight-cell- and morula-stage embryos significantly decreased when the TM concentration was greater than or equal to 5 μg/mL, with no blastocyst formation observed. The diameter of blastocysts was significantly smaller in the TM treatment group than controls (Figure 1C). In addition, TM treatment significantly increased the mRNA levels of ER stress-related genes (ATF6, CHOP and GRP78) compared to the TM-free group (Figure 1D).

The influences of treating morula-stage porcine embryos with (1 μg/mL) or without TM on blastocyst formation and development are shown in Figure 2. Figure 2A shows the morphologies of blastocysts after two-day TM treatment, where more than 88% of the morulae in the control group became blastocysts; however, in the presence of TM, only 69% of morulae developed into blastocysts (Figure 2B). The blastocyst diameter (reflecting the expansion capacity of the blastocoel) was significantly decreased in the TM treatment group when compared to the control group (Figure 2C).

3.2. Influences of TM-Induced ER Stress on Blastocoel Recovery

Cavitary blastocysts were treated with 0.5 μg/mL cytochalasin D for 1 h, resulting in the complete collapse of blastocysts (Figure 3A). Next, these collapsed blastocysts were cultured in medium without or with 1 μg/mL TM for 24 h to allow blastocoel recovery (re-expansion) (Figure 3A). The recovery rate [number of re-expanded blastocysts/total number of collapsed blastocysts) × 100%] was significantly lower in the TM treatment group than in the control group (Figure 3B), and blastocyst diameter significantly decreased in the presence of TM compared with the control (Figure 3C). However, no significant differences in total cell number were observed between the TM-treated and control groups (Figure 3D).

3.3. Influences of TM-Induced ER Stress on Cell Proliferation in the Blastocyst

Cell proliferation potential in the blastocyst was assessed by evaluating the total and EdU-positive cell numbers after EdU staining (Figure 4A). The total (Figure 4B) and EdU-positive (Figure 4C) cell numbers and the ratio of the two (Figure 4D) were significantly lower in the TM treatment groups than the control.

3.4. Influences of TM-Induced ER Stress on E-Cadherin Expression Patterns and Levels of CDH1 and TJP1 mRNA

Figure 5A shows images of blastocysts after E-cadherin protein immunostaining. The E-cadherin protein expression profile proportion appears abnormally and significantly higher in the TM-treated group than the control (Figure 5B). The mRNA levels of cell-adhesion-related gene CDH1 (Cadherin 1) and tight-junction-related gene TJP1 (Tight Junction Protein 1), which is essential for tight junction assembly and blastocoel formation, were significantly lower in the TM-treated group than the control (Figure 5C).

3.5. Influences of TM-Induced ER Stress on the Expression Patterns of Oct4, Sox2 and Cdx2 in the Blastocysts

Figure 6A,C,E show images of blastocysts after Oct4, Sox2 and Cdx2 protein immunostaining, with the expression pattern proportions of these proteins appearing abnormally and significantly higher in the TM-treated vs. control group (Figure 6B,D,F).

4. Discussion

The formation and subsequent expansion of blastocyst cavities serve as key indicators of blastocyst quality, reflecting their capacity for successful implantation and further developmental progression. We investigated the influence of TM treatment on blastocoel formation and expansion and the underlying mechanisms in a pig model. Our results showed that TM treatment reduces porcine blastocyst quality by inhibiting formation and expansion of blastocyst cavities.

To investigate the mechanisms by which ER stress affects cell development, artificial induction of ER stress has been widely employed in experimental studies. As a classical activator of ER stress, tunicamycin (TM) has been extensively used to establish ER stress models [5,14]. In this study, we observed that TM treatment increased the mRNA levels of key ER stress-related genes, such as ATF6, CHOP, and GRP78. These genes are well-known markers of ER stress and play important roles in the cellular response to stress conditions [5].

In mice, exposure to TM at concentrations exceeding 5 µg/mL resulted in developmental arrest at the two-cell stage, completely preventing blastocyst formation [20]. Similarly, bovine [14] and porcine embryos generated by parthenogenetic activation [28] or in vitro fertilization [15] failed to reach the blastocyst stage when cultured in the presence of 5 µg/mL TM. Consistent with previous studies, here we found that 5 µg/mL TM completely blocked porcine blastocyst formation; however, we noticed that the blastocysts derived from TM-treated group not only were fewer in number but also had smaller diameters and blastocoeles than the TM-free control group. We speculate that TM treatment may inhibit blastocyst formation and expansion. To verify this hypothesis, embryos in the morula stage at which blastocoel formation begins were collected and cultured with or without TM for an additional 2 days to assess blastocoel formation and expansion. The results indicated that pre-treatment of porcine morula-stage embryos with TM resulted in inhibited blastocoel formation and expansion, therefore causing a reduction in their quality.

Cytochalasin D could completely collapse the blastocoel, but the blastocoel was able to recover after cytochalasin D was withdrawn from the culture medium [29]. To examine the influence of TM-induced ER stress on blastocoel formation, porcine blastocysts were treated with cytochalasin D to induce blastocoel collapse and subsequently cultured in the presence of TM to allow for blastocoel re-expansion. TM dramatically reduced the blastocoel recovery potential and blastocyst diameter. Our results suggest that TM treatment may inhibit blastocoel formation and expansion, although the molecular basis for the inhibitory effect of ER stress on blastocoel formation and expansion is not known.

During early preimplantation development, mammalian embryos undergo a series of critical events, including compaction, differentiation of trophectoderm (TE) and inner cell mass (ICM) as well as blastocoel formation [30]. The transition from the eight-cell to the morula stage is marked by compaction, during which blastomeres lose distinct outlines and establish close cell–cell contacts largely regulated by E-cadherin [31]. A previous study showed that E-cadherin was clearly expressed at the cell boundaries of blastomeres in high-quality embryos [32]. Our results showed that TM treatment damaged the E-cadherin protein profile in porcine blastocysts, a result consistent with the reduced CDH1 (Cadherin 1) gene in the TM treatment group compared to the control.

Cell junctions are critically involved in lineage segregation between the trophectoderm (TE) and inner cell mass (ICM), as well as in blastocoel formation during preimplantation development [33]. In particular, tight junctions regulate the microenvironment of the inner cell mass by trophectoderm-mediated transport of small molecules such as ions, amino acids, growth factors and so on [4,34]. In the present study, TM treatment inhibited blastocoel formation and reduced the expression of tight junction genes such as TJP1.

The blastocoel is surrounded by the trophectoderm, a single layer of polarized epithelial cells [29]. We observed that the expression of abnormal Caudal-related homeobox protein 2 (Cdx2), a marker of trophectoderm cells, was significantly higher in the TM treatment group compared to the TM-free group, suggesting that TM may affect Cdx2 expression in trophectoderm cells.

Blastocoel formation failure induces ICM death [35]. In ICM cells, Oct4 (Octamer-binding transcription factor 4) and Sox2 (SRY-box transcription factor 2) are key regulators of the pluripotency network [36]. In pigs, Oct4 is expressed in all blastocyst cells and is not restricted to the ICM [37,38]; however, Sox2 is a faithful marker for pluripotency in porcine blastocysts [39]. In the present study, we observed abnormal expression patterns of Oct4 and Sox2 in the TM-treated group, suggesting an association between TM treatment and altered Oct4 and Sox2 expression.

Cell proliferation in the blastocyst is commonly used as a criterion for evaluating blastocyst quality [40,41]. In this study, TM treatment significantly reduced the total and EdU-positive cell numbers in blastocysts, suggesting that TM-induced ER stress reduces embryo quality by inhibiting cell proliferation potential. Interestingly, we observed that during the blastocoel recovery (re-expansion) assay, TM supplementation significantly reduced the recovery and expansion capacity but did not influence total cell numbers in blastocysts. A possible cause of this event is that, during this period, blastocoel recovery was more prominent than cell proliferation.

5. Conclusions

Our findings suggest that TM treatment negatively affects porcine blastocyst quality by inhibiting blastocoel formation and expansion, as well as cell proliferation, and by disrupting the expression patterns of E-cadherin, Oct4, Sox2 and Cdx2. These findings provide insights into the molecular embryonic development deficiency events induced by ER stress and offer valuable guidance for improving in vitro embryo developmental potential.