Melatonin Improves The Developmental Competence of Goat Oocytes

Abattoir ovaries were obtained from goat and transferred in physiological saline at 35°C within 4 hours after collection. Cumulus oocyte complexes (COCs) were aspirated from follicles with 2-6 mm diameters. The procedure of in vitro oocyte maturation was performed as described previously (22). Briefly, selected COCs with more than two layers of cumulus were washed three times in HEPES-buffered tissue culture medium-199 (HTCM- 199) containing 10% fetal bovine serum (FBS). After washing, COCs (n=10) were transferred into 50 µL micro- drops of HTCM-199 supplemented with 10% FBS, 1µg/mL 17ß-estradiol, 5.0 µg/mL luteinizing hormone (LH), 0.5 µg/mL follicle-stimulating hormone (FSH), 100 IU/mL penicillin, 100 µg/mL streptomycin and melatonin under mineral oil. All cultures were incubated in maximum humidity with 5% CO₂ at 38.5°C for 24 hours.

After 24 hours of culturing the cells, treatment groups with 10^-6 M melatonin (M-10^-6), 10^-9 M melatonin (M-10^-9) and 10^-12 M melatonin (M-10^-12), and the control group (without melatonin) were used in the designed experiments. The following were analyzed for each treatment and control group: nuclear maturation, GSH content, ROS levels, global DNA methylation, gene expression and developmental competence after parthenogenetic activation (PA). For each condition three to five replicates were used.

For evaluating the transition from germinal vesicle (GV) to metaphase II (M-II) stage, COCs were striped from the cumulus cells mechanically in the presence of hyaloronidase and were fixed in 4% paraformaldehyde. Then, oocytes were washed in phosphate buffered saline (PBS) and stained with 5 µg/mL bisbenzimide (Hoechst 33342, excitation: 346 emission: 460) for 5 minutes. Stained oocytes were evaluated using epifluorescence microscope (Nikon Eclipse-600) for first polar body extrusion (23).

Glutathione content of oocytes was measured as described previously (24). Briefly, denuded oocytes were incubated in tyrodes medium (TLH) containing 5 mg/mL polyvinylalcohol (PVA) and 10µM CellTracker Blue (excitation: 371 emission: 464) for 30 minutes at 38.5°C. After incubation, oocytes were washed in PBS and observed using epi-fluorescence microscope (Nikon clips-300). Digital images were captured and analyzed by Image J software.

The ROS production in centrifuged culture medium following IVM was measured by the chemiluminescence. One microliter of luminal (50 mM) dissolved in dimethyl sulfoxide was added to 400 µl of the supernatant. The global ROS levels were evaluated by measuring chemiluminescence with a luminometer (LKB 953, Wallac, Gaithersburg, MD) for 15 minutes, and the results were expressed in relative light units (RLU)/s (25, 26).

Parthenogenetic activation method was described earlier (22). Briefly, after the maturation period, oocytes were stripped from cumulus cells by vortexing. Denuded oocytes were exposed to 5 mM inomycine for 5 minutes in HTCM and then washed three times in Charles Rosenkrans 1 with amino acid (CR1aa) medium. Afterward, oocytes were incubated for 2 hours in CR1aa medium contain 2 mM 6-dimethylaminopurine. Finally, activated oocytes (n=6) were transferred to 20 µl droplets of CR1aa plus 3 mg/mL bovine serum albumin (BSA) under mineral oil at 38.5°C, 5% O₂, 5% CO₂ and maximum humidify for 3 days, and then the medium was refreshed with 10% FBS. The cleavage and blastocyst rate were determined on day 3 and 8 post activation, respectively.

After fixation in 4% paraformaldehyde, the oocytes were permeabilised with 1% Triton X-100 in PBS for 1 hour, then washed in Tween-20 in 1% PBS/BSA and treated with 2 N HCl for 1 hour at room temperature. After washing in Tween-20 in PBS, the samples were blocked in 0.5% Triton X-100 in 1% PBS/BSA for 1 hour. After blocking, the oocytes were incubated with primary anti-5-methyl cytosine antibody (mouse monoclonal, Abcam, Cambridge, UK) at 1:200 in the blocking buffer for 1 hour at room temprature. After incubation with the primary antibody, the samples were washed in PBS/BSA and incubated with phycoerythrin- conjugated secondary antibody (Molecular Probes, Invitrogen, Carlsbad, CA, USA). After the final wash in PBS/BSA, the DNA of oocytes was stained with 1 µg/mL Hoechst 33342 for 15 minutes. Oocytes were mounted on slides and observed with a Nikon (Eclips-300) fluorescence microscope and the fluorescence intensity of the oocytes was analyzed by Image J software (27).

For each group, three pools of biological replicates containing (n=10) mature oocytes and their surrounding cumulus cells were used for total RNA isolation. RNA pellets were dissolved in sterile water and cDNA was synthesized using M-MULVE Reverse transcriptase. Briefly, 2 µg total RNA was mixed with 5 mM Random Hexamer. Five µL water was added to 2 µL of oocytes and incubated at 75ºC for 5 minutes for the reaction to take occur. Then 10 µL RT buffer, 10 mM dNTPs, 10 µL RNase inhibitor and 200 U reverse transcriptase were added to reach a total volume of 20 µL. Reverse transcriptase-polymerase chain reaction (RT-PCR) was done in an applied Bio Rad thermocycler. After the reverse transcriptase reaction was finished, the samples were maintained at 4ºC overnight. PCR reaction was performed in total volumes of 26 µL that included 2 µL cDNA, 2 µL of each primer and 1.25 µL tag polymerase, 20.75 µL Master Mix (Takara, Japan). The PCR primers for each gene are listed in Table 1. The endogenous control (YWHAZ) and the three investigated genes were amplified with PCR cycle program at 94ºC for 3 minutes followed by 40 cycles of 94ºC for 30 seconds and 72ºC for 45 seconds. The number of cycles varied between 30 and 40, depending on the abundance of a particular mRNA. Ten microliters of PCR product were mixed with 1 mL loading buffer and electrophoreses was carried out on a 2% agarose gel in TAE for 25 minutes. The ovary was used as a positive control for melatonin receptors (28).

Real-time quantitative RT-PCR was performed to assess the expression of the investigated genes by using Rotor Gene Q instrument (QIAGEN, Germany). Real time PCR reactions were carried out in a total volume of 13 µL according to the manufacturer’s manuals for DNA Master SYBR Green I Mix (Takara, Japan). The primer concentrations were adjusted to 1 µM for each gene. The cycling parameters were 5 seconds at 95ºC, 3 minutes at 95ºC for denaturation, 15 seconds at 60ºC, 10 seconds at 72ºC for amplification and 40 cycles of extension. Expression of YWHAZ transcript was used as the internal housekeeping gene. Three replications were performed and the mRNA level of each sample was normalized to that of YWHAZ mRNA level. The relative levels of mRNA were analyzed by the REST software (Qiagen, Germany) (6).

The nuclear maturation of oocytes, cleavage and blastocyst rates were compared by x² analysis. The intracellular GSH content and ROS levels were analyzed by one-way ANOVA followed by Tukey’s test via SPSS 22 for windows (SPSS, Chicago, IL, USA). Relative gene expression levels of different genes were evaluated by REST software. A P<0.05 was considered statistically significant. The data are expressed as mean ± SD.

The effect of melatonin on nuclear maturation of goat oocytes was examined. Supplementing the IVM medium with melatonin significantly increased the rate of M-II oocytes at M-10-12 group (88%) when compared to the control group (76.1%) (Table 2). No significant difference (P>0.05) was observed between the other groups.

The obtained results from fluorescence intensity experiment (Fig .1A) indicate that GSH content was significantly higher in melatonin-treated groups compared to the control (P<0.05).

Our findings demonstrated that different concentrations of melatonin (i.e. M-10-6, M-10-9 and M-10-12) had no effect on cleavage rate after PA when compared to the control group. However, the blastocyst formation were higher (P<0.05) in M-10^-6 (55.4%), M-10^-9 (49.2%) and M-10^-12 (51%) groups as compared to control group (34.7%). Moreover, in terms of blastocyst formation, no difference (P>0.05) was observed among melatonin-treated groups (Table 3).

The present data indicate that M-10-12 group had a significant effect on ROS levels in comparison to the control groups (Fig .1B).

Representative images of labeling for 5-methyl cytosine in goat oocytes are shown in Figure 2A-D. Results from quantitative analysis of these images by Image J software showed significantly different methylation levels between the control and M-1012 groups (Fig .2E).

The expression of DNMTs genes were analyzed by quantitative real-time PCR in mature oocytes (Fig .3A). The expression of DNMT1 in M-10^-12 group was significantly lower (P<0.05) in comparison to the control group. Our observations indicated that the expression of DNMT3a was lower significantly in all melatonin-treated groups compared to the control group. The expression of DNMT3b was significantly lowered in the oocytes with melatonin 10^-6 treatment compared to the control groups (P<0.05).

The expression of MTNR1A gene was detected via RT- PCR in both oocytes and cumulus cells, in response to melatonin addition to the IVM medium (Fig .3B, C). This result shows that MTNR1A exists in both oocytes and cumulus cells independent from the presence of melatonin in the maturation medium.