Interfering Effects of In Vitro Fertilization and Vitrification on Expression of Gtl2 and Dlk1 in Mouse Blastocysts

All chemicals and reagents were obtained from Sigma Chemical Company (St. Louis, USA) unless otherwise noted.

We obtained 6-8 weeks old female and 10-weeks old male NMRI mice from Royan Institute (Tehran, Iran) to use in this study. The mice were accommodated under the controlled conditions of 12 hours light: 12 hours dark photoperiod at room temperature (22 ± 2°C) and 50 ± 10% humidity with ad libitum use of food and water. The animals were killed by cervical dislocation.

Female mice were superovulated by intraperitoneal (IP) injection of 10 IU pregnant mare serum gonadotropin (PMSG; Pregnecol®, Australia), followed 48 hours later by 10 IU human chorionic gonadotropin (hCG; Pregnyl). The experiment was carried out on three treatment groups: control, IVF, and vitrification as shown in Figure 1.

In the control group, after hCG injection, female mice were mated with male mice. Successful mating was verified by the detection of a vaginal plug, the next day morning. Fresh blastocysts were collected from the mice uteri by flushing the uterine horns with FHM flushing media 94 hours posthCG, according to the previous study (13). The blastocysts were used for RNA extraction and reverse transcription.

In the IVF and vitrification groups, we collected the cumulus oocyte complexes containing metaphase II (MII) oocytes from the oviduct ampullae 14-16 hours after hCG injection. The oocytes were released into FHM medium and then transferred to 50 μl droplets of human tubal fluid medium (HTF) supplemented with 4 mg/ml bovine serum albumin (BSA).

IVF was performed as formerly explained (14). Sperms were collected from the male mice. The cauda epididymides and vas deferens were isolated and placed in a petri dish containing previously equilibrated HTF medium (37°C, 5% CO2 in air). The sperms were passively released into the culture by using pointed forceps and a razor blade. The suspended sperms were incubated at 37°C for 45 minutes to allow capacitation. Capacitated motile spermatozoa were added to 50 μl IVF drops to reach 1 × 10⁶ sperm/ml concentrations. Subsequently, they were incubated in a humidified atmosphere of 5% CO₂ in air at 37°C under mineral oil for 5-6 hours. Next, the in vitro-derived zygotes were washed in FHM medium and cultured in potassium simplex optimized medium (KSOM) supplemented with 4% BSA under the same conditions to allow for further development. After 24 hours, we divided the 2-cell embryos into two groups. In the IVF group, the embryos were maintained in KSOM for 72 hours until the blastocyst stage. In the vitrification group, the 2-cell embryos were vitrified/warmed and then cultured under the same conditions as the IVF group, for 72 hours, to reach the blastocyst stage. Finally, the rates of development at the 4-cell, 8-cell, morula and blastocyst stages were assessed in both groups. The blastocysts were used for RNA extraction and reverse transcription.

In the vitrification group, the 2-cell embryos were vitrified by the cryotop method with Kitazato Vitrification Kit (Kitazato Biopharmaceuticals, Japan), as previously described (15). Briefly, embryos were equilibrated in equilibration solution (ES) with 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO). After 3 minutes, the embryos were exposed to the vitrification solution (VS) containing 15% (v/v) EG, 15% (v/v) DMSO and 0.5 mol/l sucrose for less than 1 minute. Next, 3-5 embryos with minimal VS were loaded onto the inner surface of the cryotop and immediately submerged in liquid nitrogen (LN2), followed by capping and storing in LN2 for up to 2 weeks. Vitrification processes were carried out at room temperature. For warming, the embryos were exposed to decrease concentrations of sucrose on a 37°C hot plate, as follows: 0.5 M sucrose for 1 minute, 0.25 M sucrose for 3 minute and 0.125 M sucrose for 5 minute. Finally, the embryos were placed for 3 minute in a 0 M washing solution and they were assessed for survival by observing the intactness of zona pellucida and blastomeres. The surviving 2-cell embryos were cultured in KSOM medium in an incubator at 37°C and 6% CO₂ to allow further development to the blastocyst stage. All media used for warming were incubated at 37°C for 30 minutes before warming.

RNA extraction, complementary DNA (cDNA) synthesis, and quantitative reverse-transcription PCR (qRTPCR) analysis were carried out according to the previous study protocols (16). Briefly, two blastocysts in each replicate of each experiment were pipetted into microtubes containing 1.5 μl lysis buffer. We added 5 μl nucleasefree water and 2 μl random hexamer to each sample and then placed the samples in a BioRad thermocycler for 5 minutes at 75°C. Immediately afterwards, the microtubes that contained the reaction product were placed on ice, followed by the addition of 5x RT buffer, 200 u RT enzyme, 10 mM dNTP, and 10 U RNase inhibitor to each reaction for cDNA synthesis. Reverse transcription (RT) reaction was performed in the thermocycler with the following amplification program: 25°C for 10 minutes, 37°C for 15 minutes, 42°C for 45 minutes and 72°C for 10 minutes. The samples were left at 4°C overnight. PCR mixture, consisted of 5 μl Master Mix (Taq DNA Polymerase Mix Red-MgCl; Amplicon, Denmark), 3 μl nuclease-free water, 1 μl cDNA, and 1 μl specific primer (Table 1) was added to each PCR microtube to amplify cDNA product. The endogenous control (β2m) and the investigated genes were amplified according to the following PCR cycle: 94°C for 3 minues (denaturation), 94°C for 30 seconds (denaturation), 60°Cfor 45 seconds (annealing) and 72°C for 45 seconds (extension), followed by 40 cycles. A final elongation step was carried out at 72°C for 10 minutes. The amplification products were loaded and run alongside a DNA ladder on a 2% agarose gel in TAE and, after 25 minutes, they were observed under short-wave UV.

qRT-PCR was executed to evaluate the amount of Dlk1 and Gtl2 expressions by using a Rotor Gene Q instrument (Qiagen, USA). Table 1 lists the primer sequences applied for qRT-PCR. qRT-PCR reaction were conducted in a total volume of 13 μl reaction containing 1 μM of each primer for the indicated genes and 1 μM of the synthesized cDNA based on the manual for the DNA Master SYBR Green 1 mix (Roche Applied Sciences, Germany). Cycling program for the RT-PCR was as follows: 2 minutes at 95°C, and 40 cycles of 5 seconds at 95°C, 30 seconds at 60°C, 10 seconds at 72°C. Melting curve examination for all amplification reactions confirmed the particular amplification peaks and lack of primer-dimer formation. β2m was the endogenous internal house-keeping gene for RT-PCR data normalization. We used the Relative Expression Software Tool (REST, version 2009) for qRTPCR data analysis.

Statistical analyses were performed by applying the Statistical Package for the Social Science software, version 16 (SPSS, USA). Cleavage and developmental ratio to blastocysts stage between IVF and vitrification groups were compared by the non-parametric Mann- Whitney test. The relative gene expression levels of Gtl2 and Dlk1 were analyzed by REST software (Qiagen). P<0.05 was regarded as statistically significant.

We assessed the effect of vitrification on developmental competence of preimplantation embryos. The 2-cell embryos obtained from IVF in three runs were divided into two groups. Totally, for the IVF group, there were 170 cultured 2-cell embryos. In the vitrification group, 166 embryos (2-cell) were vitrified/ thawed. The vitrification group had a survival rate of 96.72% ± 2.93, after vitrification and warming. We compared the percentage rates of the 4-cell, 8-cell and morula stages between the IVF and vitrification groups. There was no significant difference between these two groups, in terms of cleavage rate. The blastocyst (64.04% ± 10.16) and hatching (48.51% ± 10.92) rates in the vitrification group were significantly lower than the blastocyst (82.63% ± 2.56; P<0.037) and hatching (69.22% ± 5.20; P<0.041) rates in the IVF group (Table 2).

qRT-PCR was implemented to appraise the expression levels of the lncRNA Gtl2 and Dlk1 gene in blastocysts. Gtl2 expression was down-regulated in the IVF and vitrification groups compared to the control group. Gtl2 was less expressed in the vitrification group compared to the IVF group (P<0.05, Fig . 2A). Dlk1 was up-regulated in the IVF and vitrification groups compared to the control group (P<0.05). There was no difference between the IVF and vitrification groups, in terms of Dlk1 expression (P<0.05, Fig .2B).