Melatonin protects against defects induced by Enniatin B1 during porcine early embryo development

Effect of EB1 on the development of early embryos

To clarify the detrimental effects of EB1 on early embryo development, the dose and time dependent manners of EB1 were investigated. The results displayed that the development competence of early embryos was reduced after EB1 treatment with different concentrations, and 10, 25 or 50 μM EB1 resulted in the significantly lower cleavage and blastocyst rates and blastocyst cell number compared with the untreated group (Figure 1 and Supplementary Table 3, P<0.05). Considering that 25 or 50 μM EB1 resulted in the even more significantly reduced blastocyst rate and was the high dose for early embryos, 10 μM EB1 was chosen for the subsequent treatment. For analysis of the time effects of EB1 on early embryos, when the treatment time was shortened to 12 h, no significant development differences were observed compared with the untreated group, whereas the rates of cleavage and blastocyst were significantly higher than those of embryos treated for 24 h (Supplementary Table 4, P<0.05). Thus, EB1 exerted the detrimental effects on early embryos, and treating embryos with 10 μM EB1 for 24 h significantly reduced the development competence and quality of embryos.

Effect of melatonin on the development of EB1-exposed embryos

To overcome the detrimental effects of EB1 on early embryos, melatonin was also added to treat embryos. As shown in Figure 2 and Supplementary Table 5, EB1 significantly reduced the cleavage and blastocyst rates and blastocyst cell number, while melatonin (MT group) significantly upregulated the cleavage and blastocyst rates and blastocyst cell number compared with the untreated (CON) group (P<0.05). Moreover, when EB1-exposed embryos were treated with melatonin (ME group), the cleavage and blastocyst rates and blastocyst cell number were significantly higher than those in the EB1 group, and no significant differences of blastocyst rate and blastocyst cell number were observed compared with the CON group though the cleavage rate was still significantly lower (P<0.05). Thus, melatonin effectively protected the development competence and quality of embryos from EB1 exposure.

Effect of melatonin on nuclear remodeling in EB1-exposed embryos

After sperm penetrated into oocyte, sperm nucleus performed decondensation and swelling and formed paternal pronucleus, which then fused with maternal pronucleus to form the diploid zygote pronucleus, and then zygote carried out mitosis, meaning that nuclear division and cytokinesis successively occurred (Figure 3A). Based on the morphology changes of sperm nucleus, nuclear remodeling progress was mainly divided into sperm nuclear condensation, sperm nuclear decondensation including swelling, paternal pronucleus including formation and fusion with maternal pronucleus, and nuclear division, and oocyte with no sperm penetration and embryo cytokinesis were also examined during the first cell cycle of early embryos (Figure 3B and Supplementary Table 6). In the EB1 group, the significantly upregulated rates of sperm nuclear condensation at 2 h, 4 h, 6 h, 12 h and 18 h, sperm nuclear decondensation at 6 h, 12 h, 18 h and 24 h, and paternal pronucleus at 24 h, and downregulated rates of paternal pronucleus at 2 h, 4 h, 6 h and 12 h, nuclear division at 18 h and 24 h, and cytokinesis at 18 h and 24 h were observed, while the MT group displayed the significantly lower percentages of sperm nuclear condensation at 2 h, 4 h and 6 h and sperm nuclear decondensation at 6 h, and higher percentages of paternal pronucleus at 2 h, 4 h and 6 h, nuclear division at 12 h, and cytokinesis at 18 h and 24 h compared with the CON group (P<0.05). Importantly, the ME group showed the significantly downregulated rates of sperm nuclear condensation at 2 h, 4 h, 6 h, 12 h and 18 h, sperm nuclear decondensation at 12 h, 18 h and 24 h, and paternal pronucleus at 24 h, and upregulated rates of paternal pronucleus at 2 h, 4 h, 6 h and 12 h, nuclear division at 18 h and 24 h, and cytokinesis at 18 h and 24 h compared with the EB1 group, and only the significantly increased percentage of sperm nuclear decondensation at 6 h, and decreased percentages of paternal pronucleus at 6 h and cytokinesis at 24 h compared with the CON group (P<0.05). All these results demonstrated that EB1 delayed nuclear remodeling progress, while melatonin ameliorated the disrupted nuclear remodeling induced by EB1 during the first cell cycle of early embryos.

Effect of melatonin on apoptosis in EB1-exposed embryos

To further explore the antagonistic effects of melatonin on EB1 in early embryos, apoptosis related indexes were analyzed. As shown in Figures 4, 5, compared with the CON group, the EB1 group showed the significantly increased number and rate of apoptotic blastocyst cells, downregulated the expression of antioxidant genes (Sod1 and Gpx4) at the 4-cell and blastocyst stages, antiapoptotic factor Bcl2l1 at the 4-cell and blastocyst stages and antioxidant gene Cat during embryo development progress, and upregulated the transcription of proapoptotic genes (Bax and Caspase3) at the 4-cell and blastocyst stages, while the MT group displayed the significantly decreased number and rate of apoptotic blastocyst cells, higher expression levels of Sod1 at the blastocyst stage and Gpx4, Cat and Bcl2l1 at the 4-cell and blastocyst stages, and lower the transcription levels of Bax and Caspase3 at the 4-cell and blastocyst stages, respectively (P<0.05). In the ME group, the significantly decreased number and rate of apoptotic blastocyst cells, upregulated expression of Sod1 at the blastocyst stage, Gpx4 and Bcl2l1 at the 4-cell and blastocyst stages and Cat during embryo development progress, and downregulated transcription of Bax and Caspase3 at the 4-cell and blastocyst stages were observed compared with the EB1 group, and only the significantly increased rate of apoptotic blastocyst cells, lower expression level of Gpx4 at the 4-cell stage, and higher transcription level of Bax at the 4-cell stage were shown compared with the CON group (P<0.05), suggesting that EB1-induced embryo apoptosis could be effectively blocked by melatonin. Thus, EB1 triggered embryo apoptosis, while melatonin reduced EB1-induced embryo apoptosis.

Effect of melatonin on DNA methylation reprogramming and gene expression in EB1-exposed embryos

Here, transcription levels of Dnmts and Tets regulating DNA methylation reprogramming were examined in early embryos (Figure 6). The similar expression patterns of Dnmt1 and Tet3 with the downward trend, Dnmt3a with the first decreased and then increased trend, and Tet1 with the upward trend during embryo development progress were observed among the CON, EB1, MT and ME groups. When compared with the CON group, the EB1 group showed the significantly higher expression levels of Dnmt1 and Dnmt3a at the 4-cell stage and lower transcription levels of Dnmt1 and Dnmt3a at the blastocyst stage, Tet1 at the 4-cell and blastocyst stages and Tet3 during embryo development progress, the MT group displayed the significantly lower expression level of Dnmt3a at the 4-cell stage and higher transcription levels of Dnmt1, Dnmt3a, Tet1 and Tet3 at the blastocyst stage, and the ME group only took the significantly lower expression levels of Dnmt1 at the blastocyst stage and Tet3 at the 1-cell and 4-cell stages (P<0.05). Importantly, the significantly decreased transcription of Dnmt1 and Dnmt3a at the 4-cell stage and increased expression of Dnmt1, Dnmt3a and Tet1 at the blastocyst stage and Tet3 at the 4-cell and blastocyst stages were observed in the ME group compared with the EB1 group (P<0.05). Thus, melatonin effectively ameliorated the disturbed expression levels of DNA methylation reprogramming related genes in EB1-exposed embryos.

Then, DNA methylation reprogramming of genome (CenRep) and pluripotent genes (Oct4, Nanog and Sox2) was investigated. As shown in Figures 7, 8, the CON, EB1, MT and ME groups displayed the similar patterns of DNA demethylation and then remethylation of CenRep and DNA demethylation of Oct4, Nanog and Sox2 during embryo development progress, however, CenRep DNA remethylation did not obviously occur at the blastocyst stage in the EB1 group. When compared with the CON group, the EB1 group displayed the significantly higher DNA methylation levels of CenRep at the 4-cell stage and Oct4, Nanog and Sox2 at the 4-cell and blastocyst stages (P<0.05), while no significant DNA methylation differences of CenRep, Oct4, Nanog and Sox2 were observed in the MT or ME group. Furthermore, DNA methylation levels of CenRep at the 4-cell stage and Sox2 at the 4-cell and blastocyst stages were significantly lower in the ME group than the EB1 group (P<0.05). Thus, melatonin largely rescued the disrupted DNA methylation reprogramming in EB1-treated embryos.

The expression patterns of genes related to early embryo development were further detected (Figure 9). As for zygotic activation related gene Eif1a, compared with the CON group, the EB1 group displayed the significantly decreased expression level, while the MT group showed the significantly increased transcription level (P<0.05), and the ME group took the similar expression level. Moreover, the expression level of Eif1a in the ME group was significantly higher than that in the EB1 group (P<0.05). When the expression patterns of Oct4, Nanog and Sox2 were detected, similar expression trends were observed among the CON, EB1, MT and ME groups. However, compared with the CON group, the EB1 group showed the significantly lower expression levels of Oct4, Nanog and Sox2 at the 4-cell and blastocyst stages, the MT group displayed the significantly higher transcription levels of Oct4 at the blastocyst stage and Nanog and Sox2 at the 4-cell and blastocyst stages, and the ME group only took the significantly lower expression levels of Nanog at the 4-cell and blastocyst stages (P<0.05). Moreover, the significantly upregulated expression levels of Oct4, Nanog and Sox2 at the 4-cell and blastocyst stages were observed in the ME group compared with the EB1 group (P<0.05). These results displayed that melatonin ameliorated EB1-induced disturbed expression of genes in early embryos. Taken together, melatonin protected early embryos against the disrupted DNA methylation reprogramming and gene expression induced by EB1.

Oocyte in vitro maturation

Oocyte in vitro maturation has been reported in our previous study [35]. Briefly, ovaries were collected from a local slaughterhouse and transported to laboratory. Follicles were aspirated, and follicular contents were washed with HEPES-buffered Tyrode’s lactate. Cumulus-oocyte complexes (COCs) were recovered, washed and cultured in maturation medium. After 42 h, COCs were vortexed in hyaluronidase to remove cumulus cells. Only oocytes with the visible polar body, regular morphology and homogenous cytoplasm were used in the subsequent experiments.

In vitro fertilization (IVF) and embryo culture, treatment and collection

The procedures for IVF and embryo culture have been described in our previous reports [13, 21]. Briefly, spermatozoa were diluted with modified Tris-buffered medium, and matured oocytes were transferred into fertilization medium and co-incubated with spermatozoa. Then, embryos were washed and cultured in porcine zygote medium-3 (PZM-3) for the subsequent development, and the cleavage and blastocyst rates were evaluated at 48 h and 156 h, respectively.

For embryo treatment, embryos were cultured in PZM-3 with 0, 5, 10, 25 or 50 μM EB1 for 12 h or 24 h, or co-incubated with 0.1 μM melatonin during the development progress [21].

For embryo collection, 1-cell, 4-cell and blastocyst embryos in the CON (untreated), EB1 (10 μM EB1 for 24 h), MT (0.1 μM melatonin) and ME (10 μM EB1 for 24 h and 0.1 μM melatonin) groups were collected at 12 h, 48 h and 156 h, respectively.

Examination of nuclear remodeling and blastocyst cell number

Embryos at 2 h, 4 h, 6 h, 12 h, 18 h, 24 h and 156 h were treated with acidic Tyrode's solution to remove zona pellucida, fixed in 4% paraformaldehyde, and stained with Hoechst 33342, respectively [21]. Then, nuclear status and blastocyst cell number were examined, and the percentage of every nuclear status and average cell number per blastocyst were calculated.

Assessment of embryo apoptosis

Detection of embryo apoptosis using the TUNEL method with an In Situ Cell Death Detection Kit (Roche) has been described in our previous work [29]. Briefly, blastocysts were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, incubated in the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling reaction medium, and stained with Hoechst 33342. Then, the number of apoptotic cells per blastocyst was counted.

Quantitative real-time PCR

Measurement of gene expression with quantitative real-time PCR has been applied in our previous study [33]. Briefly, total RNA was extracted from 50 pooled embryos at each stage using a RNeasy Micro Kit (Qiagen). Reverse transcription was performed using a PrimeScript^® RT Reagent Kit (TaKaRa). For quantitative real-time PCR, reactions were performed in the 96-well optical reaction plate using a SYBR^® Premix ExTaq^TM II kit (TaKaRa) and a 7500 Real-Time PCR System. For every sample, the cycle threshold (CT) values were obtained from three replicates. The primers were presented in Supplementary Table 1. Relative expression levels of genes were analyzed using the 2^−ΔΔCT method.

Bisulfite sequencing

Bisulfite sequencing has been reported in our previous work [31]. Briefly, pooled embryos were digested and treated with sodium bisulfite to convert all unmethylated cytosine to uracil using an EZ DNA Methylation-Direct^TM Kit (Zymo Research). For samples of 500, 200 and 50 pooled embryos at the 1-cell, 4-cell and blastocyst stages, digestion was performed in the M-Digestion Buffer, cytosine to thymine conversation was carried out at 98° C for 10 min and 64° C for 2.5 h, and samples were desalted and purified. Subsequently, nested PCR was performed to amplify the target regions of genes using the primers described in Supplementary Table 2. The amplified products were verified, cloned into T-Vectors and sequenced. The sequenced results were analyzed to get the methylated (the black circle) and unmethylated (the white circle) CpG sites, and then DNA methylation level for every sequenced result was calculated by dividing the number of methylated CpG sites by the total number of CpG sites not including the mutated or single nucleotide polymorphism (SNP) CpG sites.

Statistical analysis

Differences in data (mean ± SEM) were analyzed with the SPSS statistical software. Statistical analyses of data concerning embryo development, blastocyst cell number, nuclear remodeling, embryo apoptosis, gene expression and DNA methylation were performed with t-test when the comparison was made between two groups or one-way analysis of variance when there were more than two groups. For all analyses, differences were considered to be statistically significant when P<0.05, and ^a-ddifferent superscripts were applied to indicate statistically significant differences.