After fertilization, the mouse embryo goes through a series of divisions forming multiple blastomeres and forms blastocyst which then invades to the uterus (Tarkowski, 1959; Rossant, 1976). In the case of mice, embryonic genome activation occurs in the 2-cell phase and after the 8-cell phase, it develops into a morula through a compression process (Zeng and Schultz, 2005). This stage is when the fate is determined by the inner cell mass (ICM) and the trophectoderm (TE) as the outer layer of the cell (Ziomek et al., 1980). The trophoblast, also known as the outer cell mass, forms extra-embryonic tissues, which in turn produces the placenta, chorionic membrane, and umbilical cord. Embryonic cells, inner cell mass, develop into embryos (Mihajlović and Bruce, 2017). In the last stage before implantation, blastocyst gradually increases blastocele and develops into expanded late blastocyst. Late blastocyst consists of three cell types: epiblast, primitive endoderm, and trophectoderm (Chazaud et al., 2006). As medical methods for overcoming infertility due to various causes, there are known that assisted reproduction technology (ART) through intrauterine insemination (IUI) and in vitro fertilization (IVF). It is known that the success of embryo transfer depends on the stage and quality of blastocyst development (Lindner and Wright, 1983). In vitro produced embryos are exposed to different culture conditions than in vivo and caused to various effects and damage (Rizos et al., 2008). Therefore in vitro-cultured embryos have reasonably higher lipid accumulation in the cytoplasm, chromosomal abnormality and reduced total number of cells (Mahdavinezhad et al., 2019). The condition of in vitro culture system is important to produce high quality embryos (Hansen and Block, 2004; Rizos et al., 2017). However, despite improvements in the quality of in vitro culture media to enhance the quality of blastocyst, the rate of success related artificial reproductive technique is still low (Marrs et al., 1984; Quinn et al., 1985; Lane and Gardner, 2007; Richter, 2008). In mouse and human embryos, 15-50% of embryos after fertilization die during embryonic development before implantation and only 30-50% of embryos had been reported to grow to blastocyst in vitro (Levy, 2001).

Lipopolysaccharide (LPS) is an endotoxin that forms the Gram-negative bacteria of the structure of cell wall (Jacob et al., 1977). LPS consists of three structural domains: the core oligosaccharide, lipid A, and O antigen (Tobias et al., 1989; Bidne et al., 2018). Toll-like receptor 4 (TLR4) is a cell-surface receptor that recognizes pathogen associated molecular patterns (PAMPs) (Raetz and Whitfield, 2002). LPS introduced in vivo is selectively recognized by the LPS binding protein (LBP) and rapidly transfer to the cluster of differentiation 14 (CD14). The CD14 transfer LPS to complex of TLR4 and myeloid differentiation factor 2 (MD2), the accessory proteins. Afterwards, TLR4/MD2 activates myeloid differentiation factor 88 (MyD88)- dependent or independent intracellular signaling pathways and finally activates nuclear factor-κB (NF-κB) (Barnes, 1997). The NF-kb is a transcription factor for expression of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 (Cavaillon, 2018). When inflammatory stimulated by those cytokines, the production of reactive oxygen species (ROS) such as superoxide (O2.-), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) is promoted, and cell damage is induced (Li et al., 2010). Therefore, LPS has been widely used as an experimental model to study the inflammatory response. Furthermore, LPS has a detrimental effect on female fertility, such as suppression of the secretion of reproductive hormones, impaired control of follicular activation, and decreased primitive follicular pool (Bromfield and Sheldon, 2013; Magata et al., 2014; Bidne et al., 2018;) In female mice exposed to LPS, adverse effects such as fetal preterm labor, fetal intrauterine growth restriction (IUGR), and reduction of trophoblast proliferation are caused (Leazer et al., 2002; Xu et al., 2006).

During embryonic development, high levels of adenosine triphosphate (ATP) produced by mitochondria are required (Sharma and Agarwal, 2004). When ATP is produced, reactive oxygen species (ROS) are produced from electron transport system, which is present in the inner mitochondrial membrane. ROS include O2.- (superoxide anion), H2O2 (hydrogen peroxide) and •OH (hydroxyl radical) (Goud et al., 2008). ROS play roles as secondary messenger in signaling, but excessive accumulation of ROS can lead to DNA damage and lipid peroxidation. Lipid peroxidation is known to inhibit cell function and can lead to cell death (Chatterjee and Walker, 2017). Antioxidants such as superoxide dismutase (SOD), peroxisomal catalase (CAT), glutathione peroxidase (GPx) and peroxiredoxin (Prx) exist in vivo and can remove free radicals (Ighodaro and Akinloye, 2018). However, when excessive ROS is produced or dysfunction of antioxidant system, oxidative stress cannot be reduced by endogenous antioxidant enzymes. It has been reported that excessive ROS produced during in vitro-cultured embryo degrade embryo quality and delay embryonic development (Cebral et al., 2007). Melatonin (N-acetyl-5-methoxytryptamine) is a hormone that is primarily synthesized in the mammalian pineal gland and secreted into the cerebrospinal fluid and blood (Lerner et al., 1958; Jang et al., 2017). And many recent studies have shown that melatonin is produced in a variety of tissues, including reproductive tissues such as the ovaries and placenta (Jang et al., 2017). Tryptophan, an essential amino acid, is a precursor to serotonin and then melatonin is derived from serotonin through two enzymes: N-acetylserotonin and hydroxyindole-O-methyltransferase (HIOMT) (Stehle et al., 2011). Among the exogenous antioxidants, melatonin is an antioxidant that is attracting attention in recent studies because it has easy access to cells, organelles, and nuclear membranes due to its amphiphilic properties (He et al., 2016). Melatonin improves endogenous antioxidant capacity by altering the activity of antioxidant enzymes such as SOD and CAT. It is also an antioxidant that directly removes various oxidizing agents including LPS (Mayo et al., 2002). During the process of in vitro fertilization (IVF), the embryos are easily exposed to environments that contain free radicals or permit their generation in comparison to in vivo (Karagenc et al., 2004). Various antioxidant strategies are being studied to prevent oxidative damage, and exogenous antioxidants may be used as a method of preventing oxidative stress (OS) in assisted reproductive technology environments (Truong et al., 2016; Truong and Gardner, 2017). Melatonin has been reported to influence the development and fertilization of early embryos through the role of ROS scavenger (Tamura et al., 2008; Tian et al., 2017). In clinical practice, it was also confirmed that oral administration of melatonin improved embryo fertility and embryo viability in patients who participated in assisted reproductive procedures (Eryilmaz et al., 2011; Batıoğlu et al., 2012). However, the regulatory effect of melatonin on LPS-treated embryo culture remains unclear. Therefore, we aimed to investigate the effect of LPS and melatonin in early embryo development of mouse in this study.

Animal care and experimentation

All animal experiments were used ICR mice (8-10 weeks old male, 7-8 weeks old female) provided by JA Bio (Suwon-si, Korea). Mice were housed in animal facility of Konkuk university kept on 23 ± 1℃ and 50 ± 2% humidity under the light:dark (12 h:12 h) cycle. Animals had access to standard rodent feed ad libitum. All experiments were performed with the approval of Konkuk University’s Institutional Animal Care and Use Committee (IACUC) (approved number KU20199).

Hormone treatment and oocytes preparation

Female mice were intraperitoneally injected with 7.5 IU of pregnant mare serum gonadotropin (PMSG) (Cat.7101, Daesung Micro. Lab, Suwon-si, Korea) diluted at 50 IU/mL in sterile diluent (Cat. CBP3076, DYNE BIO, Korea) to induce superovulation. At 50 hours interval mice were administered with 7.5 IU of human chorionic gonadotropin (hCG) (Cat.CG5-1VL, Sigma-Aldrich, St. Louis, MO, USA) diluted at 50 IU/mL in sterile diluent. After 16 hours, the fallopian tubes were removed and placed in M2 medium containing antibiotics (penicillin/streptomycin 1%). We used two types of in vitro culture medium which were human tubal fluid medium (HTF) (Cat.MR-070-D, Millipore, USA) that used up to the 2-cell stage and KSOM medium (Cat.MR-121-D, Millipore, USA) that used until the blastocyst stage. All culture media were used covered with light mineral oil (Cat.9305, Irvine Scientific, USA) by equilibrating for at least 30 minutes before use at 37℃ in the presence of 5% CO2. Oocytes were introduced from the ampulla of oviduct into HTF medium. All collected oocytes were counted and only the high-quality cumulus-oocyte complexes (COCs) were based on their morphological appearance.

Sperm preparation

Sperm collection was performed from the cauda epididymis. Sperm was flowed into HTF drop from the cauda epididymis with use of a fine scissors. Before insemination, spermatozoa were incubated in 200 µL of HTF media under light mineral oil at 37℃ with 5% CO2 for 30 min. After preparation, the spermatozoa concentration was determined using a hemocytometer and added in the fertilization dish drop (final concentration 1 × 106 cell /mL).

Culture and observation of embryos

After 4 hours of in vitro fertilization, only morphologically normal COCs were selected and used. Embryos were washed 3 times with HTF medium and cultured into each concentration groups culture medium of the LPS or/and melatonin. Embryos were cultured in each medium under light mineral oil in a humidified incubator at 37℃ with 5% CO2. Developmental rates at 2-cell (cleavage; 24 h), morula (compaction; 72 h), blastocyst (96 h), and hatched blastocyst (after 96 h) stages were investigated. The optimal concentration of each substrate was selected by evaluating the embryonic developmental rate.

Treatment of LPS and melatonin

Lipopolysaccharides from Escherichia coli O111:B4 (Cat.L4391, Sigma-Aldrich, St. Louis, MO, USA) were diluted at 1 mg/mL with culture medium and stored at -20℃. To confirm the effect of LPS on early embryonic development, oocytes were maintained at 12.5, 25, 50 and 100 µg/mL LPS or the same amount of HTF medium. Melatonin (Cat.M5250, Sigma-Aldrich, St. Louis, MO, USA) dissolved in dimethyl sulfoxide (DMSO) (Cat.D2650, Sigma-Aldrich, St. Louis, MO, USA) and then further diluted to 100 µM/mL adding culture medium. To prevent DMSO toxicity, all culture media contained less than 0,1% DMSO. Embryos were cultured in different concentration of 0 (control), 0.1, 1, or 10 µM/mL melatonin. In one experiment, at least 20-50 oocytes were used, and the experiment was repeated at least three times.

Statistical analysis

The experimental data were expressed as the standard error of the mean (SEM), and the graphs were produced in Microsoft Excel. One-way analysis of variance test (ANOVA) was performed by Vassar-Stats (http://vassarstats.net) through Tukey’s post-test. All analyzes were considered statistically significant at p-value (p < 0.05). All experiments were repeated in at least three independent experiments.

Effects of LPS on early mouse embryo development

To evaluate the effect of LPS on preimplantation embryonic development, embryos were cultured in the KSOM media at 0 (control) or differential concentration LPS (12.5, 25, 50 or 100 µg/mL) until the blastocyst stage (Fig. 1A). There was no change in the overall 2-cell cleavage rate (Fig. 1B), but there was a significant decrease in the morula compaction rate at an LPS concentration of 100 µg/mL. And the rate of blastocyst and hatching showed a significant reduction from the LPS concentration of 12.5 µg/mL. At LPS concentrations above 50 µg/mL, it was difficult to detect blastocysts and hatching embryos (Fig. 1B and 1C). Based on these results, 12.5 µg/mL LPS was used in subsequent experiments.

Figure 1.The effect of LPS on mouse embryonic developmental competence in vitro. (A) In vitro cultured mouse embryos treated LPS were observed by inverted fluorescence microscopy until ED 4.5 (EVOS^® FL, AMG) (Scale bar = 200 µm). (B) A graph of each development stage. The numbers of embryos tested in each group are at least 20 and experiments were performed repeatedly at least three times. Data are expressed as mean ± standard error mean (SEM) expressed in one-way ANOVA. A significant difference between treatments indicates as *(p < 0.05) and **(p < 0.01). (C) In vitro developmental rates of mouse embryos cultured with LPS.

Effect of melatonin on early mouse embryo developmental rate

Next, the effect of melatonin alone treatment on embryonic development rate was investigated. We cultured fertilized embryos in the presence of 0 (control), 0.1, 1 or 10 µM/mL melatonin until the blastocyst stage (Fig. 2A). In all melatonin experimental groups, there was no significant difference from the control group in division, compression, and blastocyst stage (Fig. 2B and 2C).

Figure 2.The effects of melatonin on the development of mouse embryos in vitro. (A) In vitro cultured mouse embryos were observed by inverted fluorescence microscopy until ED 4.5 (EVOS^® FL, AMG) (Scale bar = 200 µm). (B) A graph of each development stage. The numbers of embryos tested in each group are at least 20 and experiments were performed repeatedly at least three times. Data are expressed as mean ± standard error mean (SEM) expressed in one-way ANOVA. A significant difference between treatments indicates as *(p < 0.05) and **(p < 0.01). (C) In vitro developmental rates of mouse embryos cultured with LPS.

Effect of melatonin on LPS-treated early mouse embryos

We investigated whether melatonin treatment could enhance the reduced developmental competence of embryos treated with effective LPS concentration (12.5 µg/mL LPS). Embryos were cultured to the blastocyst stage in culture medium treated with 12.5 µg/mL LPS or/and melatonin (1 µM or 10 µM/mL melatonin) (Fig. 3A and 3B). In the group treated with LPS only, the rate of blastocyst development decreased by approximately 50% compared to the control group (Fig. 3C). However, the treatment of melatonin improved the rates of compaction and blastocyst development of LPS-treated embryos (Fig. 3C and 3D).

Figure 3.Effect of melatonin on developmental rate of LPS treated early mouse embryos. (A) The time schedule of the treatment designs of LPS and melatonin. (B) In vitro cultured mouse embryos of blastocyst stage were observed by inverted fluorescence microscopy (EVOS^® FL, AMG) (Scale bar = 200 µm). (C) A graph of each development stage. The numbers of embryos tested in each group are at least 20 and experiments were performed repeatedly at least three times. Data are expressed as mean ± standard error mean (SEM) expressed in one-way ANOVA. A significant difference between treatments indicates as p-value; **p < 0.01. LPS, in vitro culture medium with 12.5 µg/mL LPS; MT1, melatonin 1 µM/mL; MT10, melatonin 10 µM/mL. (D) In vitro developmental rates of mouse embryos cultured with LPS and melatonin. LPS, in vitro culture medium with 12.5 µg/mL LPS; MT 1, melatonin 1 µM/mL; MT 10, melatonin 10 µM/mL.

Effect of LPS or/and melatonin on levels of ROS in mouse embryo

Then we looked at ROS levels of LPS treated embryos with melatonin. The ROS levels were assessed in embryos of blastocyst developmental stages. Interestingly, the ROS level of LPS treated group significantly increased compared to the control group (Fig. 4). On the other hand, the treatment of melatonin decreased ROS levels compared to the LPS only group (Fig. 4A and 4B).

Figure 4.ROS level with or without melatonin treatment in LPS-treated mouse embryo. (A) Fluorescence microscopy images of intracellular ROS expression in blastocyst stained with CellROX (green) and Hoechst 33342 (blue). (B) A graph of relative ROS expression fluorescence intensity in embryo at stage of blastocyst development. Data are expressed as mean ± standard error mean (SEM) expressed in one-way ANOVA. A significant difference between treatments indicates as p-value; **p < 0.01. LPS, in vitro culture medium with 12.5 µg/mL LPS; MT, melatonin 10 µM/mL.

Lipopolysaccharide (LPS), a component of gram-negative bacteria, is an inflammatory substance and is involved in causing serious toxicity in vivo (Tough et al., 1997). LPS induces the production of inflammatory cytokines such as IL1β, IL6 and TNF-α through TLR 4 in various cell lineages and animals (Barnes, 1997). In addition to the inflammatory response, LPS increases the oxidation of lipids, proteins, and DNA by inducing oxidative stress (Forrester et al., 2018). Embryos collected from female animal injected with LPS intraperitoneally have a high level of DNA damage at the blastocyst stage (Jaiswal et al., 2009). In addition, LPS treatment reduces mitochondrial membrane potential and mass leading to mitochondrial dysfunction in embryos (Kuwabara and Imajoh-Ohmi, 2004; Mokhtari et al., 2021). In addition, the level of ROS is increased and glutathione (GSH) content are significantly reduced in embryos exposed to LPS in vitro (Chen et al., 2006). Moreover, LPS reduced in vitro produced-blastocyst quality by significantly increasing the DNA fragmentation index (Moghadam et al., 2021).

Melatonin (N-acetyl-5-methoxytryptamine) is involved in various physiological processes such as chronobiotic actions, innate immunity, abiotic stress resistance, anti-cancer, antioxidant process and female reproduction (Yu and Tan, 2019). In addition, melatonin effectively removes reactive oxygen species (ROS) and reactive nitrogen species (RNS) and contributes to anti-inflammatory effects by reducing cell damage caused by free radical toxicity (Choi et al., 2011). Therefore, melatonin is used to reduce ROS, which is inevitably produced during in vitro culture compared to in vivo. Melatonin has been reported to increase the rate of oocyte maturation and embryonic development in porcine, bovine, and mice (Yang et al., 2017). In human follicular fluid before ovulation, melatonin levels three times higher than serum levels are identified, protecting the oocytes from oxygen stress and cellular damage (Rönnberg et al., 1990). In patients receiving melatonin treatment, melatonin levels in follicular fluid increased and inversely the concentration of 8-OHdG, a DNA damage marker, decreased significantly (Tamura et al., 2020). In addition, melatonin has been reported to inhibit the release of inflammatory cytokines, chemokines, and the expression of nitric oxide produced by LPS (Choi et al., 2011). Another study found that melatonin protects against LPS-induced fetal death, growth restriction, and premature labor in mice (Chen et al., 2006). Recent studies have reported that co-treatment of LPS and Alpha-lipoic acid, the antioxidant, lowers ROS levels in embryos and recovers the number of ICM and TE cells (Mokhtari et al., 2020). In addition, vitamin D3 reduces the induction of early embryonic loss through anti-inflammatory activity (Zhou et al., 2017). Melatonin is more powerful in scavenging free radicals than vitamin E and vitamin C (Pieri et al., 1994). However, so far, the effect of melatonin on the harmfulness of early embryos exposed to LPS has not been confirmed. The purpose of this study was to investigate the effect of co-administration of LPS and melatonin on embryonic development during in vitro culture. In this study, stage of early embryonic development was identified as four; cleavage, compaction, blastocyst, and hatching rate.

To select the optimal LPS concentration, embryos were incubated in LPS-treated cultures at differential concentrations until the blastocyst stage (at embryonic day 4.5) (Fig. 1). Except for the highest concentration of 100 µg/mL LPS group, compaction developmental rate was not inhibited, but the rate of blastocyst development decreased compared to the control group at all LPS group including 12.5 µg/mL LPS concentration (Fig. 1B). These results are consistent with previous studies showing that LPS treatment inhibits embryonic development of morula and blastocyst (Moghadam et al., 2021; Zhao et al., 2017). Melatonin concentrations in this study were treated with reference to the previously reported optimal melatonin concentrations (0.1, 1, 10 µM) in ICR mice. Studies from other institutes showed that the treatment of melatonin was significantly increased in the embryonic developmental rate compared than control group (Shi et al., 2009; Wang et al., 2013), but in this study, there was a tendency that the developmental rate was improved but there was no statistical significance (Fig. 2). Low concentrations of melatonin were not known to have any toxic effects on mouse embryos (Ishizuka et al., 2000), and no toxicity was observed at the concentration used in this study.

As a result of co-treatment with melatonin (1 or 10 µM), the embryonic developmental rate, which was reduced in the LPS only group, significantly increased (Fig. 3). Embryos are very sensitive to high levels of ROS during development due to their limited antioxidant capacity. ROS can alter most cellular molecule types, as well as cause blockages and delays in early embryonic development (Yang et al., 1998). In mouse embryonic development, ROS production is directly related to increased hydrogen peroxide, apoptosis, and fragmentation of the embryo (Yang et al., 1998). In this study, quantitative analysis of ROS relative fluorescence intensity has been investigated in embryos during the development stages of blastocyst (Fig. 4). Our data showed that the level of ROS was significantly increased in the LPS group. On the other hand, when the melatonin group was administered in combination, the ROS level was significantly decreased (Fig. 4). Arrest of development of embryos cultured in vitro has been reported to be due to the abundance of ROS (Pabon Jr et al., 1989; Goto et al., 1993). The results in this study suggest that ROS excessively accumulated in embryos by LPS is the cause of developmental inhibition.

In conclusion, this study confirmed that LPS adversely affected embryonic development and that the antioxidant melatonin effectively scavenged ROS and increased the rate of early embryonic development in mice. Therefore, these results suggest a way to use the potent antioxidant melatonin to ameliorate the loss of reproductive function that may be caused by LPS exposure and bacterial infection.

I would like to thank Dr. Ok-Hee Lee for valuable discussion and comments for the manuscript.

Conceptualization, Y.C.; data curation, H.P.; formal analysis, H.P.; funding acquisition, H.J. and Y.C. writing and editing, S.M., H.J. and Y.C.

This research was supported by grants from National Research Foundation of The Ministry of Science, ICT & Future Planning (2021R1A2C1011916).

This study was approved by Konkuk University Institutional Animal Care and Use Committee (KU19216, 20199).

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No potential conflict of interest relevant to this article was reported.