The effect of melatonin on in vitro maturation fertilization and early embryo development of mouse oocytes and expression of HMGB1 gene in blastocysts

Abstract

Antioxidants are commonly used for maturation, fertilization and early development of embryos. Melatonin as an antioxidant have been recently proven to be useful for the assisted reproductive technology. In the present study, we evaluated the roles of melatonin in the in vitro maturation, fertilization, development and also the gene expression of high mobility group box-1 (HMGB1) in the blastocysts. The immature oocytes of BDF1 mice were transferred to the media containing different doses of melatonin (10-6, 10-9, 10-12 M). The blastocysts that developed under in vitro fertilization from each group were stained to determine the cell number of embryos and analyzed to determine the expression level of HMGB1 by real-time PCR. The most effective doses of melatonin for maturation of oocytes were 10-6 and 10-12M (P<0.05). Fertilization rate, early development and the cell number of blastocysts were significantly higher in the group that treated with 10-12 M of melatonin comparing to the other groups. The HMGB1 expression decreased in groups that treated with 10-6M and 10-9M of melatonin and increased in the group that treated with 10-12 M of melatonin, but did not show a significant difference (p˃0.05). From the results, it may be concluded that the melatonin could be effective when the embryos undergo maturation, fertilization and early developmental processes. The HMGB1 expression, as a marker of early development in mice embryos, increased in the groups that treated with low doses of melatonin

Keywords:
Blastocyst; Fertilization; HMGB1 gene; In vitro maturation; Melatonin

INTRODUCTION

Obtaining high quality matured oocytes is a critical factor for optimal in vitro fertilization (IVF) and embryo development (^{Xu et al., 2017}). The in vitro maturation (IVM) medium is used for maturation of retrieved immature oocytes and increasing the number of matured oocytes that will be used in IVF or the intracytoplasmic sperm injection technique. In addition, IVM is a technique that can be utilized in the patients with the risk of ovarian hyper stimulation syndrome (^{Jafarabadi et al., 2017}). Nowadays, IVM is one of the most important techniques in assisted reproduction, although its success rate has been controversial (^{Teoh, Maheshwari, 2017}). The reactive oxygen species (ROS) are generated by in vitro manipulation and affects both oocytes maturation and embryos development. These free radicals, can damage DNA, ATP synthesis and mitotic spindle that consequently impair the maturation of immature oocyte (^{Tamura et al., 2013}). Many studies have reported the harmful effects of ROS on the oocyte maturation and pre-implantation embryo development (^{Bahadori, Ramezani, Asghari Nohadani 2011}; ^{Zhou et al., 2016}; Dehghani-Mohammad abadi et al., 2014-These studies revealed that the protection of embryos from oxidative stresses can improve the embryo development in in vitro culture (^{Leon et al., 2004}). Many enzymatic and non-enzymatic antioxidants were suggested to reduce the ROS, during the IVM procedure. Some of these components include taurine, hypotaurine, pyruvate, cysteine and glutathione (Zhou et al., 2016; ^{Moawad, Tan, Taketo, 2017}; ^{Hou et al., 2015}). In addition, the antioxidant effects of melatonin have been recently proven to be useful for the assisted reproductive technology (^{Tian et al., 2014}). Melatonin (N-acetyl-5-methoxytryptamine) is secreted mainly by the pineal gland and has great impact on the reproductive functions of mammals (Tamura et al., 2014; ^{Arnao, Hernández-Ruiz, 2014}; ^{Dragojevic Dikic et al., 2015}). Follicular fluid showed a three-fold higher melatonin than the serum in the pre-ovulatory phase (^{Wang et al., 2017}). After ovulation, this fluid release to the ampulla of oviduct where the fertilization occurs (^{Lyons, Saridogan, Djahanbakhch, 2005}). Therefore, it is assumed that the melatonin plays a critical physiological role in oocyte maturation and fertilization as well as early embryo development (^{Cruz et al., 2014}). In addition, the melatonin represses the expression of CASP3 and BAX genes, which are directly associated with apoptosis. Therefore, the melatonin reduces apoptosis in embryonic cells and increases the development of mice embryo (Dehghani-Mohammad abadi et al., 2014). Today, melatonin is widely used for improving the quality of oocytes that were retrieved from infertile women and matured in in vitro (Tian et al., 2014).

The high mobility group box-1 (HMGB1) gene has multiple functions in DNA transcription and repair, differentiation, extracellular signaling and inhibition of apoptosis (^{Lee et al., 2014}; ^{Ugrinova, Pasheva, 2017}). The expression of HMGB1 in mice embryos promotes embryonic development to the blastocyst stage through suppressing the expression of CASP3 and BAX genes and consequently reducing the apoptosis in blastomeres of embryos (Cui, Shen, Kim, 2017). As previously mentioned, melatonin has multiple protective effects including oocyte maturation, IVF and embryo development, reducing apoptosis and enhancing embryonic development. However, the effect of melatonin on the expression of HMGB1 has not been evaluated yet. Regarding the effect of antioxidants on ROS production, the expression level of HMGB1 differs in different tissues. For instance, one study reported that antioxidants reduced the expression of HMGB1 in pancreas (^{Tang et al., 2007}). In contrary, it has been shown that oxidants induced the expression of HMGB1 in lymphatic tissues, especially in macrophages and monocytes (^{Kang et al., 2011}). Also a recent study showed that melatonin enhanced the expression of HMGB1 in in vitro matured oocytes (^{Salimi et al., 2014}). Therefore, this study evaluated the effect of melatonin on the IVM, IVF, cleavage and blastocyst formation rate of immature mice oocytes and also on the expression of HMGB1 in blastocysts.

MATERIAL AND METHODS

All chemicals were purchased from Sigma Chemical Corporation (St. Louis, MO, USA) except where noted otherwise. The experiments on animal were approved by the Institutional Animal Care at the Shahid Beheshti University of Medical Sciences (Tehran, Iran). The B6D2F1 mice were procured from the Pasteur Institute of Iran and maintained in temperature and humidity-controlled rooms under 12 hours dark and 12 hours light cycle.

Oocyte Collection and IVM

Immature oocytes at the germinal vesicle (GV) stage were collected from 6- to 8-week-old BDF1 female mice, 48 hours after injection of 10 IU of pregnant mare serum gonadotropin (PMSG). The mice were scarified by cervical dislocation. The immature oocytes were retrieved 48 hours after PMSG injection and cultured in a tissue cell culture medium (TCM) 199 supplemented with 5% fetal bovine serum (FBS). The GV stage oocytes with complete granulosa cell layer were released by puncturing ovarian antral follicles with a 28G needle. The immature oocytes with GV and complete granolosa cell layer were chosen and transferred to 50 μL droplets of TCM-199 supplemented with 10% of FBS, 0.2 mM of sodium pyruvate, 2 mM of L-Glutamin, 10 μg/mL of luteinizing hormone, 10 μg/mL of follicle stimulating hormone, 1 μg/mL of 17β estradiol and different doses of melatonin (10^-6, 10^-9, 10^-12 M). Afterwards, the oocytes were incubated at 37 ºC in 5% CO₂ for 22-24 hours.

Assessment of Nuclear Maturation

After the IVM period, the oocytes were denuded from cumulus cells using 1 mg/mL hyaluronidase and the pipetting procedure. The nuclear status of oocytes was evaluated by 10 μg/mL Hoechst (33342) under a fluorescent microscope (Olympus, Tokyo, Japan). The oocytes were classified as metaphase I (MI, containing oocytes with a metaphase plate without polar body) and metaphase II (MII, containing a metaphasic plate with polar body) stages.

Measurement of ROS by Chemiluminescence Assay

Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)- based assay is widely used to measure global ROS production in the medium. Briefly, after maturation, the IVM medium of all the groups was collected and centrifuged at 600 g for 7 minutes. Then, 400 μL of the supernatant was removed and mixed with the Luminol (50 mM). Consequently, the mixture was placed in the Luminometer (LKB 953, Wallac Inc., and Gaithersburg, MD, USA) for 15 minutes and the data were expressed as relative light units (RLU/s).

In Vitro Fertilization (IVF)

Mature oocytes were transferred to 100 μL culture droplets of KSOM that supplemented with 15 mg/mL bovine serum albumin (BSA). Next, for sperm preparation, two male BDF1 mice (10-week-old) were sacrificed by cervical dislocation. The vas deferens was cut and immediately transferred to the Ham’s F10 medium that supplemented with 5% BSA. After incubation for 15 minutes, the sperm suspension was centrifuged at 3,000 rpm for 3 minutes. The supernatant was discarded and 300 μL Ham’s F10 containing 5% BSA added to the pellet. The swim-up procedure was performed at 37 ºC in 5% CO₂ for 45 minutes. After collecting the motile spermatozoa, 5×10⁶ sperms were added to the IVF medium containing the mature oocytes and incubated at 37 ºC under 5% CO₂ for 6 hours.

In Vitro Culture

Zygotes were collected and transferred to 30 μL droplets of the culture medium (KSOM supplemented with 4 mg/mL BSA) under mineral oil and incubated at 37 ºC under 5% CO₂ for 5 days. The number of 2, 4, and 8-cell embryos as well as morula and blastocyst were recorded at 24, 48, 72, 96 hours after fertilization.

Differential Staining

Differential staining was performed to determine the number of trophoectoderm cells, inner cell mass (ICM) and total blastocyst cells. Briefly, the zona pellucida of the blastocyst was removed by acid Tyrode’s solution and the blastocyst was transferred to the M2 medium containing rabbit anti-mouse serum at the ratio of 1:2 and incubated for 30 minutes. Afterwards, they were washed with M2 medium and incubated (30 min) in 30% guinea pig complement (in M2) medium that containing 10 mg/mL propidium iodide and 10 mg/mL Hoechst H33342. After fixation with ethanol, the blastocysts were mounted on slides with using of glycerol and finally observed by a fluorescent microscope.

RNA Extraction and cDNA Synthesis

The relative transcript levels of the HMGB1 were determined by real-time PCR. PCR reactions were performed on an applied Bio-Rad thermo cycler. At least two blastocysts were analyzed for each group. The blastocysts were transferred to the bottom of a 0.2 mL Eppendorf tube containing 1.5 μL lysis buffer. Next, 2 μL of poly N and 5 μL water were added to the embryo and placed in a thermal cycler for 5 min at 75 ºC. The tubes were taken on the ice and 9 μL of the reaction mixture consisting of 5x RT buffer, 200u RT enzyme, 10 mM dNTP and 10u RNase inhibitor added to the samples. The amplification program for the reverse transcription step was performed as follows: 10 min at 25 ºC, 15 min at 37 ºC, 45 min at 42 ºC and 10 min at 72 ºC [23]. After the reverse transcriptase reaction, the samples were kept at 4 ºC for 24 h.

HMGB1 Expression by Quantitative PCR

Real-time quantitative PCR was performed to assess the expression of the HMGB1 (Forward: 5’ -AAGTATGAGAAGGA TATTGCTG - 3’ Reverse: 5’ - CCAACTTATTCATCATCATCATC- 3’, Accession number: NM_010439.3) with using of Rotor-Gene Q instrument (QIAGEN). Real-time PCR reactions were carried out in a total volume of 13 µL according to the manuals of the DNA Master SYBR Green I mix (Roche Applied Sciences). The primer concentrations were adjusted to 1 µM for the reaction of each gene. The cycling parameters were 5 sec at 95 ºC as holding time, 40 cycles of 3 min at 95 ºC, 15 sec at 60 ºC and 10 sec at 72 ºC. The amplification reactions was confirmed by melting curve analysis. The Hprt1 (Forward: 5’ - TCCCAGCGTCGTGATTAG - 3’; Reverse: 5’ -CGAGCAAGTCTTTCA GT CC - 3’, Accession number: NM_013556.2) was used as internal house-keeping gene. Three replications were performed and the mRNA level of each sample was normalized to that of Hprt mRNA level. The relative levels of mRNA were analyzed by the REST 2009 Software (QIAGEN).

STATISTICAL ANALYSIS

All statistical analyses were performed with using SPSS 22 Software (SPSS, Chicago, IL, USA). The means of MI and MII oocytes, 2,4 and 8-cell embryos, morula and blastocyst were compared by the non-parametric analysis test (Kruskal-Wallis). The total cells of blastocysts in experimental groups were compared by using the Tukey’s HSD post-hoc test. The relative levels of mRNA were analyzed by REST 2009 Software (QIAGEN). The obtained data were expressed as means ± SEM. Statistically significant difference was accepted at P≤0.05.

RESULTS

Oocyte Maturation

A number of 1255 of immature oocytes were transferred to IVM media that supplemented with different doses of melatonin. It was observed that the number of matured (MII) oocytes was significantly increased in the media that supplemented with 10^-6 and 10^-12 M of melatonin (85% and 79%, respectively). The highest maturation rate was achieved at the highest concentration of melatonin (i.e., 10^-6 M). Although a positive effect was observed on nuclear maturation in the group that supplemented with 10^-9 M of melatonin, it was not significant compared to the control group (Figures 1 A and B).

FIGURE 1
The maturation rate of the oocytes and ROS concentration in culture media: (A); The fluorescent intensity of Hoechst H33342 stained nuclear mice oocytes after maturation; immature oocytes shown by MI and mature oocytes shown by MII, scale bar 50 µ (B); the chart of oocytes maturation (MII) in IVM media containing different doses of melatonin (10^-6, 10^-9, 10^-12 M) (C); the chart of the ROS concentration in the maturation medium after IVM. The means of MI and MII oocytes were compared by using the non-parametric analysis test (Kruskal-Wallis). ^a,b.c Columns with different lowercase letters differ significantly (P<0.05). Data were presented as mean ± SEM.

Effect of Melatonin on ROS Concentration

According to the obtained results, the ROS concentration in the media after IVM was significantly different in the oocytes that treated with different doses of melatonin (p<0.05). The ROS concentration in the medium that supplemented with 10^-6 M of melatonin was significantly decreased compared to the others groups, except the 10^-9 M treatment (p<0.05). Moreover, the ROS concentration in the medium that supplemented with 10^-12 M of melatonin was significantly decreased compared to the others groups, except the 10^-9 M treatment (p<0.05) (Figure 1C). However, the ROS concentration in the medium that supplemented with 10^-9 M of melatonin was not significantly different with that in the 10^-6 and 10^-12 groups (p˃0.05), but was lower than that in the control and sham groups. Thus, melatonin decreased the ROS concentration proportional to the concentration of melatonin (p<0.05).

IVF and In Vitro Culture

The rate of fertilization in the medium that supplemented with 10^-12 M of melatonin obtained 80.9%, which was significantly different with control and other groups (p<0.05). The cleavage and blastocyst formation rates in the medium that supplemented with 10^-12 M of melatonin were significantly improved compared to the control group (p<0.05) (Table 1).

TABLE I
Development of the IVF/2-cell mouse embryo cultured in the medium supplemented with different concentrations of melatonin. The means of 2-cell, 4-cell, and 8-cell embryos as well as morula and blastocyst were compared by the non-parametric analysis test (Kruskal-Wallis). Data are presented as mean ± SEM. ^{a, b, c,d} Numbers with different superscript letters in the same column differ significantly (P<0.05).

Group	Oocyte number	2-cell stage N(% ±S.D)	4-cell stage N(% ± S.D)	8-cell stage N(% ± S.D)	Morula N(% ± S.D)	Blastocyst N(% ± S.D)
Control	151	92 (60.90±3.9) a	53 (61.75±7.1)a	52 (60.63±7) a	37 (44.76±9.5)a	22 (24.76±4.49) a
DMSO	165	103 (63.15±2) a	83 (76.96±3.2) b,c	70 (67.8±2)a	56 (53.86± 3.41)a,b	37 (35.12±5) a,c
Melatonin 10-6 M	247	163 (63.65±3.55) a	125 (77.4±7.6) c	109 (65.14±6.1) a.	100 (60.1±4.4)b	79 (48.14±5.14) b,c
Melatonin10-9 M	229	113 (60.22±2.57) a	79 (74±4.3)d,b,c	77 (72.88±5.4) b	56(53.05±4.39)a,b	45 (40.96±7.72) c
Melatonin 10-12 M	213	171 (80.91±6.36) b	51 (86.36±5.37)e,c	149 (84.14±9.61)c	129(69.5±18.48)c,b	105 (53.88±21.27)d,b

ICM, Trophectoderm (TE) and Total Cell Number of Blastocysts

Differential staining was performed to determine the cell number of ICM and TE as well as the total cell number of blastocysts (Figure 2, A). The group that supplemented with 10^-12 M of melatonin showed a significant increase in cell number of ICM and total cell number (p<0.05) compared to the control group. However, there was no significantly difference between the groups in terms of trophectoderm cells (Figures 2 A and B).

FIGURE 2
The fluorescence micrograph of differentially labeled mouse blastocysts and chart of the cell number of embryos: (A); the fluorescence micrograph of a differentially labeled mouse blastocyst showing trophectoderm nuclei stains red with propidium iodide and inner cell mass nuclei stains blue with Hoechst H33342 in different doses of melatonin (10^-6, 10^-9, 10^-12 M), scale bar 50 µ (B); the chart of cell number, ICM and TE in different groups. Total cells of blastocyst in experimental groups were compared by using the Turkey’s HSD post-hoc test. ^a,b.c Columns with different lowercase letters differ significantly (P<0.05). Data were presented as mean ± SEM.

HMGB1 Gene Expression

The expression level of the HMGB1 was analyzed by real-time PCR in blastocysts of all the groups. The HMGB1 expression decreased in the groups that supplemented with 10^-6M and 10^-9M of melatonin and DMSO compared to the control group (p≤0.05). The HMGB1 expression in the group that supplemented with 10^-12 M of melatonin increased compared to the control group, but did not show a significant difference (p˃0.05) (Figure 3).

FIGURE 3
Relative expression levels of the HMGB1 gene expression in mice blastocysts Relative expression levels of the HMGB1 gene in blastocysts cultured with different doses of melatonin and without melatonin. The mRNA levels of HMGB1 were analyzed with real-time PCR and mRNA levels were normalized to Hprt mRNA level. The relative levels of mRNA were analyzed by REST 2009 Software (QIAGEN). ^{a, b} Numbers with different lowercase letters differ significantly (P<0.05). Data were presented as mean ± SD.

DISCUSSION

The components of the culture medium, including proteins, hormones and antioxidants play important roles in the maturation of oocytes and development of the early embryos (^{Rizos et al., 2002}). Previously, melatonin was used as an antioxidant for the improvement of oocyte maturation, fertilization and cleavage. However, the most effective dose of melatonin for optimal oocyte maturation in an IVM medium has not been determined so far. (^{Sirard, 2001}). Therefore, this study was designed to evaluate the effects of melatonin on nuclear and cytoplasm maturation of the oocytes.

In this study, it was revealed that melatonin decreased the ROS concentration, proportional to the concentration of melatonin. The ROS concentration in the media with 10^-6 and 10^-12 M of melatonin was lowest and highest in all the groups, respectively. Furthermore, our results revealed that all the three doses of melatonin enhanced nuclear maturation by optimization of the ROS concentration. Interestingly, in the groups that were treated with 10^-6 and 10^-12 M of melatonin, the nuclear maturation was significantly increased comparing to the control group. These results are in agree with the previous studies showing that melatonin significantly increased the maturation and fertilization rate of mouse immature oocytes in micro molar concentrations (^{Farahavar, Shahne, 2010}; Seo, So, Hyun, 2016; ^{Mokhber et al., 2016}). The rate of fertilization depends on the quality of oocytes maturation. It has been shown that the fertilization rate of in vitro matured oocytes is lower than that of in vivo matured oocytes (Bahadori, Ramezani, Asghari Nohadani, 2016). The cleavage rate and embryonic development in the blastocyst stage were significantly higher in the group supplemented with 10^-12M of melatonin comparing to the control and other groups. These results are in contrast to previous report by Bahadori, Ramezani, Asghari Nohadani, 2016 who showing the 10^-9 M dose of melatonin was more suitable for oocytes maturation, fertilization and cleavage (Bahadori, Ramezani, Asghari Nohadani, 2016). These results suggested that high doses of melatonin increased the maturity of oocytes while higher fertilization and cleavage rate detected in low doses of melatonin. Therefore, it should be considered that low levels of ROS (produced by high dose of melatonin) promote the meiosis of oocytes and release the first polar body while high levels of ROS (produced by low dose of melatonin) give rise to fertilization and cleavage rates. Consequently, medium supplemented with 10^-12M of melatonin have optimum ROS levels to induce the oocytes maturation, fertilization and development of embryos.

There are two methods for evaluation of blastocyst quality, first, the total cell number of blastocysts and ICM. In a previous study, it was shown that adding 10^-9 M of melatonin to the culture medium caused an increase in total cell number of mice blastocysts (Tian et al., 2010). The present study showed that supplementation of the medium with 10^-12 M of melatonin increased the fertilization and cleavage rate, as well as ICM and total cell number of blastocysts. The second method to evaluate the quality of in vitro embryos is gene expression analysis. The HMGB1 gene is a transcription factor that repairs DNA and suppresses apoptotic genes expression (TRIL, BAX and Casp8) (^{Liu et al., 2007}).

Furthermore, the HMGB1 inhibits the signaling pathway of the P53. According to the above criteria, the expression of the HMGB1 can decrease the mortality of embryos and, consequently, improve the development of embryos and blastocyst formation (Cui, Shen, Kim. 2001). According this report, there is a variable expression of the HMGB1 during embryo development, so that a high and low level of expression in the zygote and 2-cell stage was seen respectively. In the final step, the HMGB1 increased again in the morula and blastocyst stage. They concluded that when the expression of the HMGB1 increased in embryos, the proliferation and development of embryos were significantly improved. On the other hand, the increased expression of the HMGB1 in in vitro matured oocytes occurred in the presence of 10 ^-12 M melatonin. (^{Salimi et al., 2014}). Their results are in disagree with previous report which shows the antioxidant, increased the HMGB1 expression (^{Tang et al., 2007}). However, this paper was the first study to evaluate the expression level of HMGB1 in blastocysts (obtained from in vitro matured oocytes) that treated with different doses of melatonin. According to the results of this study, the expression level of the HMGB1 significantly decreased in the blastocysts that treated with 10^-6 and 10^-9 M of melatonin. These results are in agree with the previous studies on non-embryonic tissues which revealed a decrease in the HMGB1 expression in response to high-dose antioxidants. The optimal antioxidant activities are induced by low-dose of melatonin as it leads to increased expression level of the HMGB1 as well as efficient fertilization, cleavage and good-quality embryo. In this study, the expression level of the HMGB1 increased in the group that treated with 10^-12 M of melatonin comparing to the other groups, but did not show a significant difference with the control group. The increasing of the HMGB1 in blastocysts that treated with 10¹² M of melatonin correlated with, high fertilization, cleavage and blastocyst formation rate as well as good embryo quality. Therefore, it can be found that low-dose of melatonin decrease the ROS production and increases the expression of HMGB1 that positively affects the development of embryos. However, high dose of melatonin highly decreases the ROS level that is not suitable for the HMGB1 expression. An optimal amount of ROS is required for development, fertilization and expression of the HMGB1, which is provided at the concentration of 10-¹² M of melatonin. Oxidative stress caused by ROS does not always have a negative impact on embryo quality. An efficient concentration of ROS is required for the induction of anti-apoptotic genes and embryo development. The optimal antioxidant effect of melatonin can be induced in low-dose and led to increase the HMGB1 expression as well as to fertilization, cleavage and good quality embryo. As mentioned before, there is a direct relationship between the cleavage and development of embryos with the expression level of the HMGB1. Therefore, it can be assumed that low-dose of melatonin provide the optimal ROS level, which is required for proper expression of the HMGB1 and high embryo development.

CONCLUSION

The oxidative stress (ROS) in the medium does not always have a negative effect on embryos development. An efficient concentration of ROS is required for the induction of anti-apoptotic genes and embryos development. Low-dose of melatonin leads to optimal antioxidant effect, increasing the HMGB1 expression and also the improvement of fertilization, cleavage and good quality embryo.

Acknowledgement

The authors would like to thank the Shahid Beheshti University of Medical Sciences for their contribution to this study and their technical support and helpful assistance in the process of conducting this research.

REFERENCES

• ¹
  Arnao MB, Hernández-Ruiz J. Melatonin: plant growth regulator and/or biostimulator during stress? Trends Plant Sci. 2014;19(12):789-797.
• ²
  Bahadori M, Ramezani M, Asghari Nohadani Z. Melatonin dose-dependent effects on oocyte maturation capacity, in vitro fertilization and blastocyst development in mouse. Kowsar Med J. 2011;16:67-71.
• ³
  Cruz MHC, Leal CLV, da Cruz JF, Tan D-X, Reiter RJ. Role of melatonin on production and preservation of gametes and embryos: a brief review. Anim Reprod Sci. 2014;145(3/4):150-160.
• ⁴
  Cui XS, Shen XH, Kim NH. High mobility group box 1 (HMGB1) is implicated in preimplantation embryo development in the mouse. Mol Reprod Dev. 2008;75(8):1290-1299
• ⁵
  Dehghani-Mohammadabadi M, Salehi M, Farifteh F, Nematollahi S, Arefian E, Hajjarizadeh A, et al. Melatonin modulates the expression of BCL-xl and improve the development of vitrified embryos obtained by IVF in mice. J Assist Reprod Genet. 2014;31(4):453-461.
• ⁶
  Dragojevic Dikic S, Jovanovic AM, Dikic S, Jovanovic T, Jurisic A, Dobrosavljevic A. Melatonin: a "Higgs boson" in human reproduction. Gynecol Endocrinol. 2015;31(2):92-101.
• ⁷
  Farahavar A, Shahne AZ. Effect of melatonin on in vitro maturation of bovine oocytes. Afr J Biotechnol. 2010;9(17):2579-2583.
• ⁸
  Hou X, Zhang L, Han L, Ge J, Ma R, Zhang X, et al. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation. J Cell Sci. 2015;128(13):2319-2329.
• ⁹
  Jafarabadi M, Ramezanzadeh F, Salimi S, Forooghifar T. Comparison of In-Vitro Maturation and In-Vitro Fertilization in Infertile Females with Polycystic Ovary Syndrome. Int J Gynaecol Obstet. 2017;2 (1): e9649
• ¹⁰
  Kang R, Livesey KM, Zeh I, Herbert J, Lotze MT, Tang D. HMGB1 as an autophagy sensor in oxidative stress. Autophagy. 2011;7(8):904-906.
• ¹¹
  Lee S, Kwak MS, Kim S, Shin J-S. The role of high mobility group box 1 in innate immunity. Yonsei Med J. 2014;55(5):1165-1176.
• ¹²
  Leon J, Acuña-Castroviejo D, Sainz RM, Mayo JC, Tan D-X, Reiter RJ. Melatonin and mitochondrial function. Life Sci. 2004;75(7):765-790.
• ¹³
  Liu H, Yao Y-M, Yu Y, Dong N, Yin H-N, Sheng Z-Y. Role of Janus kinase/signal transducer and activator of transcription pathway in regulation of expression and inflammation-promoting activity of high mobility group box protein 1 in rat peritoneal macrophages. Shock. 2007;27(1):55-60.
• ¹⁴
  Lyons R, Saridogan E, Djahanbakhch O. The effect of ovarian follicular fluid and peritoneal fluid on Fallopian tube ciliary beat frequency. Hum Reprod. 2005;21(1):52-56.
• ¹⁵
  Moawad AR, Tan SL, Taketo T. Beneficial effects of glutathione supplementation during vitrification of mouse oocytes at the germinal vesicle stage on their preimplantation development following maturation and fertilization in vitro. Cryobiology 2017;76:98-103.
• ¹⁶
  Mokhber ME, Eimani H, Bigdeli MR, Golgar NA, Abedi R. Effects of crocin supplementation during in vitro maturation of mouse oocytes on glutathione synthesis and cytoplasmic maturation. Int J Fertil Steril. 2016;10(1):53-61.
• ¹⁷
  Rizos D, Ward F, Duffy P, Boland MP, Lonergan P. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev. 2002;61(2):234-248.
• ¹⁸
  Salimi M, Salehi M, Farahani RM, Dehghani M, Abadi M, Novin MG, et al. The effect of melatonin on maturation, glutathione level and expression of HMGB1 gene in brilliant cresyl blue (BCB) stained immature oocyte. J Cell Sci. 2014;15(4):294-301.
• ¹⁹
  Seo M-S, So K-H, Hyun S-H. Effects of (-)-Epicatechin Gallate on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization. J Anim Reprod Biotechnol. 2016;31(3):153-159.
• ²⁰
  Sirard M. Resumption of meiosis: mechanism involved in meiotic progression and its relation with developmental competence. Theriogenology. 2001;55(6):1241-1254.
• ²¹
  Tamura H, Takasaki A, Taketani T, Tanabe M, Kizuka F, Lee L, et al. Melatonin as a free radical scavenger in the ovarian follicle. J Clin Endocrinol Metab. 2013;60(1):1-13.
• ²²
  Tamura H, Takasaki A, Taketani T, Tanabe M, Lee L, Tamura I, et al. Melatonin and female reproduction. J Obstet Gynaecol Res. 2014;40(1):1-11.
• ²³
  Tang D, Shi Y, Kang R, Li T, Xiao W, Wang H, et al. Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1. J Leukoc Biol. 2007; 81(3):741-747.
• ²⁴
  Teoh JP, Maheshwari A. Ongoing Developments in ART and Pregnancy Outcome. Clinical Management of Pregnancies following ART. J Clin Pathol. 2017. p.229-242.
• ²⁵
  Tian X, Wang F, He C, Zhang L, Tan D, Reiter RJ, et al. Beneficial effects of melatonin on bovine oocytes maturation: a mechanistic approach. J Pineal Res. 2014; 57(3):239-247.
• ²⁶
  Tian X-Z, Wen Q, Shi J-M, Liang-Wang, Zeng S-M, Tian J-H, et al. Effects of melatonin on in vitro development of mouse two-cell embryos cultured in HTF medium. J Clin Endocrinol Metab. 2010;35(1):17-23.
• ²⁷
  Ugrinova I, Pasheva E. Chapter Two-HMGB1 Protein: A Therapeutic Target Inside and Outside the Cell. Adv Protein Chem Struct Biol. 2017;107:37-76.
• ²⁸
  Wang S, Liu B, Liu W, Xiao Y, Zhang H, Yang L. The effects of melatonin on bovine uniparental embryos development in vitro and the hormone secretion of COCs. PeerJ. 2017;5:e3485.
• ²⁹
  Xu Y, Zhou T, Shao L, Bei Z, Liu K, Gao C, et al. Gene expression profiles in mouse cumulus cells derived from in vitro matured oocytes with and without blastocyst formation. Gene Expr Patterns. 2017;25/26:46-58.
• ³⁰
  Zhou Z, Jia RX, Zhang G, Wan Y, Zhang Y, Fan Y, et al. Using cysteine/cystine to overcome oxidative stress in goat oocytes and embryos cultured in vitro. Mol Med Rep. 2016;14(2):1219-126.

• Conflict of interest

  The authors declare no competing interests.

• Notes on contributors

  MS designed the experiments; MM and MS performed the experiments; SHP analyzed the data; NS, AJ, MS, and MM contributed reagents/materials/analytical tools; and MS, MN wrote the manuscript. All the authors approved all the revisions and the final paper.

Publication Dates

• Publication in this collection
  11 Oct 2021
• Date of issue
  2021

History

• Received
  20 Oct 2018
• Accepted
  01 Feb 2019