1. Introduction
In eukaryotic cells, the endoplasmic reticulum (ER) is the major intracellular organelle responsible for protein synthesis [
1,
2]. The accumulation of misfolded or unfolded proteins in the ER lumen can disrupt ER homeostasis and activate ER stress [
3,
4]. The unfolded protein response (UPR) is an adaptive mechanism of the ER; its activation allows cells to respond to ER stress conditions [
5,
6]. Although the precise molecular mechanisms of the UPR remain poorly described in mammalian embryo development [
4], it is generally accepted that the UPR is regulated by three major sensors: Double-stranded RNA-activated protein kinase-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) [
7]. As a pro-survival response, the UPR works to alleviate the accumulation of misfolded proteins and restore ER function [
8]. However, in cases where ER stress becomes prolonged or too severe for UPR-based mitigation, apoptosis is triggered.
New protein synthesis due to the translation of maternal mRNA is extremely important for oocyte maturation and embryo development [
9,
10]. During oocyte maturation and preimplantation embryo development, the ER acts as a major site for the biosynthesis of proteins, lipids and secretory proteins, and thus plays a key role in meeting the oocyte’s increased demand for new proteins. These functional proteins must be folded properly in the ER to maintain appropriate oocyte maturation and embryo development. Thus, regulation of ER stress/homeostasis is likely to be an important mechanism during these processes [
9].
Developing gametes and embryos may encounter various types of exogenous stress in an in vitro culture system [
11,
12]. It is becoming more and more apparent that some of these adverse factors negatively impact ER functions and protein synthesis, resulting in the activation of ER stress and the UPR signaling pathways [
11]. Indeed, ER stress and UPR signaling appear to play critical roles during oocyte meiotic resumption and preimplantation embryo development. Inhibition of UPR signaling by ER stress inhibitors has been shown to improve oocyte maturation and early embryo development in pigs, mice, bovines, etc. [
4,
13,
14,
15,
16], suggesting that ER stress is detrimental to mammalian oocyte maturation and preimplantation embryo development. Thus, understanding the mechanistic relationships between ER stress and in vitro development could help the improvement of the maturation of oocytes and early development of embryos.
This review examines what currently is known regarding the involvement, potential impacts and mechanisms of ER stress and UPR signaling in mammalian (excluding human) oocyte maturation and preimplantation embryonic development.
4. Activation and Induction of Endoplasmic Reticulum Stress in Oocytes and Preimplantation Embryos
Mammalian oocytes and preimplantation embryos are usually sensitive to variations in many exogenous factors, including temperature, osmotic stress, shear stress, chemical exposure, oxidative stress, etc. [
11,
43]. All of these factors are known to induce ER stress [
17], and reduce embryo developmental potential through alterations in gene expression, epigenetic mechanisms and metabolism [
44,
45].
During oocyte maturation and early embryo development, various enzymes and metabolic pathways produce endogenous reactive oxygen species (ROS) [
46]. The accumulation of ROS in cells leads to cell membrane lipid peroxidation, blockade of RNA transcription, damage of DNA and decreases in protein synthesis [
47,
48,
49]. It has been reported that oxidative stress can induce ER stress and UPR signaling by impeding correct protein folding and calcium homeostasis [
50]. Oxidative stress can decrease the formation rate of bovine IVF-derived blastocysts by upregulating the mRNA expression levels of IRE1, ATF4, ATF6 and CHOP [
12]. ER stress and oxidative stress are usually found together [
43,
51]; ER stress can produce ROS [
52], whereas ROS can induce ER stress by disturbing correct protein folding/transport and altering calcium homeostasis [
50].
Tunicamycin (TM) is a chemical reagent that inhibits N-glycosylation, which is often essential for protein folding; TM is generally used to induce ER stress [
53,
54]. TM has been reported to negatively affect oocyte maturation and embryo development by promoting ER protein misfolding and inducing apoptosis in mice, pig and cattle [
4,
13,
14,
16,
37,
40]. TM-induced ER stress usually has a negative influence on embryo development. For example, mouse embryos treated with more than 5 μg/mL TM were completely blocked at the 2-cell stage and failed to develop into blastocysts [
16], while bovine and pig PA- or IVF-derived embryos failed to develop to the blastocyst stage when exposed to 5 μg/mL TM during in vitro culture [
4,
14,
55]. In porcine SCNT-derived embryos, in contrast, as little as 1 μg/mL TM was found to block the ability of embryos to develop to the blastocyst stage [
13]. This suggests that SCNT-derived embryos are more sensitive to TM than IVF-derived embryos, perhaps because cloned embryos exposed to electrofusion-mediated activation are subject to an increased ER stress response [
13].
Electrofusion, which is the most common method employed to create a cloned embryo, typically increases intracellular calcium ions, decreases maturation-promoting factor activity and enhances improper nuclear remodeling [
39,
56]. SCNT-derived embryos derived from electrofusion reportedly undergo activation of ER stress and UPR signaling via upregulation of the mRNAs for XBP1-s, GRP78 and ATF6, leading to decreases in the developmental competence and quality of SCNT embryos [
39]. Similarly, the author of a porcine SCNT study speculated that the nuclear transfer process used during SCNT (e.g., electrofusion) could induce extra ER stress [
57]. Therefore, it may be useful to decrease ER stress during SCNT embryo production by bypassing electrofusion, as a means to improve SCNT embryo development.
Osmotic stress, cryopreservation and shear stress during embryo handling can also induce the ER stress response. Culture media of an appropriate osmolality has been shown to improve oocyte maturation and embryo development [
58,
59], whereas hyperosmolarity of the culture media has been shown to decrease embryo developmental capacity by inducing ER stress and apoptosis [
16]. For example, the addition of 50 mM sorbitol to the culture medium completely arrested mouse embryos at the 2-cell stage, while the addition of 25 mM sorbitol significantly decreased the blastocyst formation rate by increasing XBP1 protein expression [
16]. Cryopreservation can also induce ER stress and UPR pathway activation. Zhao et al. reported that the protein level of XBP1 was significantly higher in vitrified-warmed mouse oocytes than in fresh mouse oocytes, suggesting that the freeze-thawing procedure triggers ER stress in mouse oocytes [
40]. Similarly, sheep COCs subjected to vitrification showed increased expression levels of ER stress markers, such as the mRNAs for ATF4, ATF6, GRP78 and CHOP, when compared to controls, suggesting that sheep oocyte cryopreservation is associated with ER stress and activation of the UPR signaling pathways [
42]. Embryo pipetting often produces shear stress, which can damage oocytes and negatively impact embryo development [
60]. Although transient shear stress may not negatively influence embryos, prolonged or repeated handling can have such effects [
61]. The IRE1 arm of the UPR pathway is reportedly activated in response to embryo collection techniques [
26]. Overall, the studies described above indicate that ER stress and the UPR signaling pathways are inducible and activated in mammalian oocytes and preimplantation embryos. The impacts of common ER stress activators on mammalian oocyte maturation and embryo development are summarized in
Table 2.
Table 2.The impact of endoplasmic reticulum stress inducers on mammalian oocyte maturation and embryo development.
6. Relief of Endoplasmic Reticulum Stress Reduces Apoptosis, Improves Oocyte Maturation and Enhances the Developmental Potential of Embryos
The ER stress inhibitor-induced decrease of ER stress-induced UPR signaling not only improves oocyte maturation and preimplantation embryo development potential, it also prevents ER stress-mediated apoptosis (
Table 3,
Figure 4). Tauroursodeoxycholic acid (TUDCA), which is a bile acid that acts as a potent chemical chaperone to inhibit ER stress in vitro [
71], has been widely used to alleviate ER stress during in vitro oocyte maturation and/or embryo development. The beneficial role of TUDCA is typically attributed to suppression of the UPR [
72,
73].
Figure 4.The influences of endoplasmic reticulum (ER) stress on oocyte maturation and preimplantation embryo development. Oocyte maturation and/or embryo culture environments associated with prolonged or severe ER stress (e.g., treatment with the ER stress inducer, TM) can significantly reduce oocyte maturation, decrease embryo developmental potential and increase apoptosis (top box with light pink color). When ER stress is inhibited by an ER stress inhibitor (e.g., TUDCA), maturation and development improve and the apoptotic index decreases (bottom box with light green color). Oocyte maturation is shown to the left of the broken black line, while embryo development is presented on the right. COCs, cumulus oocyte-complexes; GV, germinal vesicle; M II, metaphase II.
Table 3.The positive influence of Endoplasmic Reticulum stress inhibitor supplementation during IVM/IVC on mammalian oocyte maturation and/or embryo development.
TUDCA was reported to significantly improve porcine oocyte maturation by triggering the MAPK pathway and enhance the developmental capacity of early PA-derived embryos by preventing ER stress-induced apoptosis [
4]. Inhibition of ER stress by supplementation with TUDCA reportedly reduces the incidence of DNA double-strand breaks (DSBs) and improves preimplantation embryo development [
38]. Incubation of porcine IVF-derived embryos with TUDCA was shown to improve the blastocyst formation rate and total cell and inner cell mass (ICM) cell numbers, while decreasing the apoptosis rate and the mRNA level of pro-apoptotic BAX [
55]. Similarly, in cattle, TUDCA supplementation of the culture medium can enhance the blastocyst formation rate, trophectoderm proportion and cell survival [
12]. TUDCA (50 μM) improved the cleavage rate of buffalo IVF-derived embryos and attenuated TM-induced apoptosis by decreasing the expression levels of BAX and ER chaperones [
14]. In mice, the addition of TUDCA to the culture medium improved the rate at which 2-cell embryos developed to blastocysts, reduced apoptosis [
16] and improved the implantation and live birth rates of transferred embryos [
57]. TUDCA was also shown to improve the viability and subsequent embryo developmental potential of vitrified-warmed mice oocytes by reducing cryopreservation-induced ER stress [
40].
The blastocyst formation rate, total cell number, ICM cell number and apoptotic rate were significantly lower and higher in electrofusion-mediated SCNT embryos compared to Sendai virus-mediated SCNT embryos or IVF-derived embryos, respectively; this reflects the increased activation of ER stress by the electrofusion process, suggesting that ER stress plays a negative role in early SCNT-derived embryos [
39]. However, TUDCA treatment significantly enhanced the formation rate and quality of electrofusion-mediated SCNT blastocysts by reducing apoptosis and increasing cell numbers [
39]. The presence of TUDCA in porcine in vitro maturation (IVM) medium did not improve the cleavage or blastocyst formation rates of SCNT embryos, but inclusion of TUDCA in the in vitro culture (IVC) medium appeared to enhance the blastocyst formation rate and quality of porcine SCNT-derived embryos by alleviating ER stress, reducing the ROS level, increasing the GSH level and decreasing apoptosis [
13]. A recent study in bovine SCNT-derived embryos showed that TUDCA treatment of the donor cell can also significantly enhance the fusion rate, cleavage rate, blastocyst formation rate and total cell number while decreasing the apoptotic index [
41]. These studies collectively suggest that ER stress is a common event in mammalian oocyte IVM and/or embryo IVC systems, and that relieving ER stress by TUDCA treatment can improve oocyte maturation, enhance embryo developmental potential and reduce apoptosis.
Melatonin (N-acetyl-5methoxytryptamine), which is an indole that is mainly synthesized from tryptophan by the pineal gland in animals, contributes to many important physiological functions, such as sleep, temperature regulation, metabolism, the circadian rhythm and seasonal reproduction [
74,
75,
76,
77,
78]. It is also a free radical scavenger, anti-oxidant and anti-apoptotic factor [
79]. Unlike other free radical scavengers, melatonin is multifunctional and universal: It can directly obliterate toxic oxygen derivatives, prevent ROS generation, and induce anti-oxidative enzymes [
80,
81,
82]. Melatonin reportedly has beneficial influences when added to the culture medium during the oocyte maturation and embryo development of pigs, cattle and mice [
80,
83,
84]. As mentioned above, oxidative stress (e.g., ROS) contributes to UPR activation; thus, melatonin can exert its functions on cell viability by regulating the UPR signaling pathways or reducing ER stress via its anti-oxidant and anti-apoptotic properties. Although melatonin has repeatedly been used to reduce ER stress in cells [
85,
86], relatively few studies have focused on the mechanisms through which melatonin decreases ER stress during mammalian oocyte maturation and/or preimplantation embryo development. One study in a porcine oocyte maturation system revealed that adding melatonin to the maturation medium improved meiotic maturation by reducing ER stress, suggesting that melatonin critically modulates UPR signaling and reduces ER stress during oocyte IVM in pig [
3]. Numerous factors involved in in vitro culture can induce ER stress, which damages oocyte maturation and embryo development. The use of melatonin as an anti-oxidant and anti-apoptotic factor could relieve ER stress by modulating UPR signaling pathways, which could yield improved oocyte maturation and/or embryo developmental potential. The mechanisms through which melatonin acts on ER stress and the UPR signaling pathways during mammalian oocyte maturation and preimplantation embryo development await further clarification.
In addition to TUDCA and melatonin, the HDAC (histone deacetylase) inhibitor, valproic acid, or glutathione (GSH) can also reduce ER stress by regulating the UPR pathways. In bovine SCNT embryos, valproic acid was reported to significantly reduce the mRNA levels of XBP1-s (an ER stress marker), CHOP and BAX (a pro-apoptotic gene) while increasing those of the ER molecular chaperone, BiP, and the anti-apoptotic gene, BCL-xl; these findings suggested that valproic acid could improve the developmental potential of bovine SCNT-derived embryos by alleviating ER stress and reducing apoptosis [
15]. GSH reportedly inhibits ER stress during mouse embryo development [
37]. It was also found to significantly decrease the mRNA expression levels of ATF6, ATF4, BiP, CHOP and PERK, and increase the cell numbers and apoptosis index in blastocysts; thus, GSH appears to improve the developmental capacity and quality of mouse embryos by alleviating ER stress and apoptosis [
37]. Salubrinal, a selective eIF2α dephosphorylation inhibitor, has been reported to protect cells from ER stress [
87]. In mouse COCs, palmitic acid-mediated ER stress reportedly impairs mitochondrial membrane potential, pentraxin-3 secretion and embryonic development; however, salubrinal treatment was shown to significantly improve these deficiencies by reducing ATF4, ATF6 and XBP1 gene expression levels, suggesting that salubrinal could reverse the cellular dysfunctions induced by ER stress and improve oocyte development [
29].
The studies above show that supplementation of the in vitro culture medium with chemical inhibitors of ER stress could serve as a beneficial approach to preventing ER stress-induced oocyte damage and alterations of embryo development. The effects of various ER stress inhibitors on mammalian oocyte maturation and embryo development are summarized in
Table 3. ER stress often occurs alongside other stress responses, particularly oxidative stress [
11]. ER stress itself induces ROS generation, whereas ROS contributes to the activation of ER stress and UPR signaling, so it is possible that some anti-oxidants may hold promise as potential inhibitors of ER stress.