60 specific pathogen-free F1 (ICR) female mice (6-8 weeks of age, 16-20 g in weight) were obtained from the Animal Center of Sun Yat-sen University. All the mice were housed in standard transparent plastic cages (330 × 205 × 180 mm) with five mice in each cage under temperature controlled (25 ± 0.5 ℃ and light controlled conditions (lights on, 06:00-18:00). Mice had free access to food and water [13]. All the protocols and procedures in this study were following the requirements of the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Sun Yat-sen University (Approval no. 201204-008).

Superovulation were achieved by intraperitoneal injection of 7.5 IU (0.15 mL) of pregnant mare serum gonadotropin (PMSG, Sigma, St Louis, MO, USA) into female mice by and after 48 h, the mice were injected with 7.5 IU of human chorionic gonadotropin (hCG, Sigma, CG5). Superovulated females were then mated with single males of the same strain (8 weeks old). At the time of examination, mating was confirmed by the presence of a vaginal plaque in female mice. Mated females were sacrificed by cervical dislocation and the oviducts were flushed 62 h after hCG injection to collect 8-cell embryos [14]. The embryos were rinsed and grown in M16 medium (Sigma, M7292) medium at 37 ℃ with 5% CO₂ before freezing. Control embryos were continuously cultured into blastocysts without freezing. After injection with hCG for 115 h, the development of blastocyst was observed in the control group.

After equilibrating in a DMEM/F12 medium containing 10% glycerol, 10% Dulbecco phosphate-buffered saline (DPBS), IVF-30 and 10% fetal calf serum (FCS) for 15 min, the embryos were pipetted into straws [15] and placed in a programmable freezer (Cryobath CL3300; Cryologic, Australia) for 10 min until they reached -7 ℃ at a cooling speed of 0.7 ℃/min. Forceps were placed in liquid nitrogen and cooled at first. Then, embryos were cooled to -33 ℃ at a cooling speed of 0.3 ℃/min. Finally, embryos were transferred into liquid nitrogen for storage. The straw was removed from liquid nitrogen, held at room temperature for 60 s and then in warm water (25 ℃) for 30 s to thaw the embryos. Cryoprotectant (CPA) was removed from the embryos in four steps: 1) embryos were incubated in medium containing 6% glycerol and 0.3 M sucrose for 5 min, 2) the embryos were incubated in medium containing 3% glycerol and 0.3 M sucrose for 5 min, 3) the embryos were incubated in medium containing 0.3 M sucrose for 5 min, and 4) the embryos were incubated in 20% fetal calf serum (FCS) FCS in phosphate-buffered saline (PBS) for 5 min. The embryos were then cultured at 37 ℃ with 5% CO₂ in air.

Embryos equilibration was initiated in equilibrationsolution (ES) for 15 min at room temperature. The ES contained 7.5% (v/v) ethylene glycol (EG, Sigma, 324558) and 7.5% (v/v) dimethyl sulfoxide (DMSO, Sigma, D2650) was dissolved in human tubal fluid (HTF) medium (Sigma, MR-070) supplemented with 10% human serum albumin (HSA) (Sigma, A9511). After equilibration, the embryos were washed in small drops of vitrification solution containing 15% (v/v) EG for three times and base medium containing of 15% (v/v) DMSO for < 1 min. Then, the embryos were loaded into a modified straw by pulling and cutting a 0.25 mL plastic straw. After the embryos were loaded, the straw was covered with another straw with the end heat-sealed and the straw was plunged into liquid nitrogen. After cryostorage, the embryos were warmed by removing the straws from the liquid nitrogen and keeping them in the air for 10 s followed by placing them in a 37 ℃ water bath for 1 min. The contents of the straws were then expelled into a 0.28 mol/L sucrose solution at 37 ℃ for 1 min. The embryos were placed in a 0.17 mol/L sucrose solution for 3 min and then in base medium for 5 min at RT (room temperature) to further rehydrate and remove of cryoprotectants [16].

The lower back of the anaesthetized 2.5 days pseudo pregnant female mice was cleaned and then shaved. A small cut (1 cm) was made in the body wall just above each ovary. Then, under an operating microscope, frozen-thawed embryos or fresh 8-cell stage embryos were transferred to each uterine horn and were implanted, 10 litters of each embryo treatment group were generated. Finally, the skin incision was sutured.

All offsprings were kept throughout the experiment. Mice were weighed daily from birth until 8 weeks. At 8 weeks, the mice were euthanized and the fat was dissected out of each mouse. The fat was collected and weighed.

The mice forelimbs were placed flat on the induction bar of the Grip Strength Meter (GPM-100; Melquest, Toyama, Japan). When the mouse grasped the bar, the mouse's tail dragged in parallel, and the peak pulling force (in grams) was recorded on a digital force transducer. The grasping device would automatically record the maximum grasping force. These measurements were taken three times. The maximum value of the mouse's grasp force was measured at 8 weeks.

Whole blood was collected simultaneously from all mice by caudal venipuncture. Serum was collected from plasma via centrifugation and then fstocked at -20 ℃. Serum glucose and insulin concentrations were measured using 15-μL duplicate samples and an ELISA kit (Arbor Assays).

Total RNAs were isolated from 10 brains if offspring were pooled and constituted one biological replicate per group (control, slow frozen, and vitrified) using TRIzol reagent (Invitrogen) by following the manufacturer’s protocols. The total RNAs were dissolved in 10 μL RNAse-free water, and cDNAs were synthesized by reverse transcribing from 2 μL of each sample using reverse transcription kit (Takara). The sequence of primers used to determine the relative transcript abundance of PGC-1α, FTO, GFAP, and MBP are summarized in Table 1.

Total proteins were extracted from10 brains if offspring were pooled and constituted one biological replicate per group. The lysates from brains were prepared in 200 mL of lysis buffer. Proteins were separated using 6-16% gradient polyacrylamide gels and then the proteins were transferred onto polyvinylidene fluoride membranes. After blocked in 5% bovine serum albumin (BSA), the membranes were incubated with FTO (ab94482) Rabbit mAb (1 : 1,000; Abcam), PGC-1α (sc-518025) Mouse mAb (1 : 500; Santa Cruz), MBP (sc-52070) Mouse mAb (1 : 500; Santa Cruz), or GFAP (ab4648) Mouse mAb (1 : 500; Abcam). The blots were then incubated with secondary antibodies. Densitometry of band intensities was performed and intensities were normalized to GAPDH (ab8243) Mouse mAb (1 : 2,000; Abcam) or α-tubulin (ab7291) Mouse mAb (1 : 2,000; Abcam) protein expression. Immunoblots were quantified using Image J.

Cortical neurons grown on coverslips were fixed with 2% paraformaldehyde (PFA) in PBS for 15 min at 37 ℃. After blocked with 5% BSA/PBS for 1 hour at room temperature, the nerons were then incubated with primary antibodies against MBP (1 : 500) and GFAP (1 : 1500) for 2 h at room temperature, and then stained with fluorescent conjugated secondary antibodies (Alexa Fluor 488 and 555, 1 : 500) for 1 h at room temperature. The immunostained coverslips were mounted onto slides with FluorSave (Calbiochem), and a Delta Vision microscope was used for fluorescent analysis. All of the images were taken with a 60× oil immersion lens.

The MWM test was conducted in a homogeneously illuminated room at 8:30 am. Each mouse was tested at postnatal day 60. Each mouse was trained in three blocks per day, and each block contained a 1-min trial. Mouse movement, escape latency, path length, mean speed, percentage of time in the target quadrant, and crossing number were recorded and analyzed as previously described [17].

All the data were statistically analyzed using the Student’s t-test with Excel (Microsoft). All experiments were performed with at least three biological replicates. Error bars in the Figures represent ± SD.

This study aimed to determine the effect of embryo vitrification and slow freezing on number of offspring. The control and slow frozen groups showed a reduced number of offspring per litter compared to the Vitrified group without statistical significance, which indicated that the postnatal birth rates were similar in the vitrified, slow frozen, and control groups in all treatments using t-test.

To test whether vitrification or slow freezing affects postnatal physiology and development, offsprings from the vitrified, slow frozen, and control groups were weighed daily and the mean weekly weights were calculated. Mice in the slow frozen and vitrified groups were significantly heavier than mice in the control group during weeks 4-8 (Figure 1A). However, there was no difference in body weight between mice in the slow frozen and vitrified groups during weeks 0-8. The amount of body fat in mice in the slow frozen and vitrified groups was markedly higher when compared with mice in the control group (Figure 1B). However, no differences were found in muscle distribution (sarcomere length) among the three groups at 8 weeks (Figure 1C). Serum glucose and insulin levels were measured with an ELISA kit, and there was no difference among the three groups (Figure 1D-E). There was also no difference in the forelimb grip test results among the three groups (Figure 1F). These results indicate that mice in the slow frozen and vitrified groups were similar to mice in the control group in muscle distribution, forelimb grip strength, and index parameters of metabolic health. However, the body weight and body fat of mice in the slow frozen and vitrified groups were higher than the mice in the control group.

Figure 1.

— Effect of embryo vitrification and slow freezing on postnatal physiology. (A) The weights of offspring from the vitrified, slow frozen, and control groups from weeks 0-8. The data are shown as mean ± SEM ** p < 0.001, denote differences between control and slow frozen. ^## p < 0.001, denote differences between control and vitrified. (B) The body fat of offspring from the vitrified, slow frozen, and control groups at 8 weeks. The data are shown as mean ± SEM, n = 30 (10 offspring were pooled and constituted one biological replicate, three separate biological replicates were repeated.). *** p < 0.0001. (C) Skeletal muscle distribution in offspring from the vitrified, slow frozen and control groups at 8 weeks. The data are shown as mean ± SEM, n = 30. (D-E) Serum glucose and insulin concentrations in offspring from the vitrified, slow frozen, and control groups at 8 weeks. The data are shown as mean ± SEM, n = 30. (F) Forelimb grip strength in offspring from the vitrified, slow frozen, and control groups at 8 weeks. The data are shown as mean ± SEM, n = 30.

We next investigated the movement coordination function in fresh-embryo group, slow freezing and vitrified-embryo group to provide further evidence for the effect of vitrification on the development of offspring in mice. The offsprings in the three groups were evaluated by the MWM test to determine whether vitrification and slow freezing influence spatial learning, and no difference was found among the slow frozen, vitrified, and control groups (Figure 2A-C). The escape latency, including times across the platform, percent time in the target quadrant, and escape path length analyses showed no difference among the three groups, indicating that normal spatial learning in offspring from the slow frozen and vitrified groups.

Figure 2.

— MWM analyses of offspring from the vitrified, slow frozen, and control groups. (A-C) No significant difference in spatial learning impairment, which including escape latency, times across the platform, time spent in the target quadrant, and escape path length was found in offspring from the vitrified, slow frozen, and control groups. n = 10 for each subgroup. Three repeated measures analysis of variance. The data are shown as mean ± SEM, p > 0.05.

To determine whether the increased weight and body fat in offspring from the slow frozen and vitrified groups were resulted from altered gene expression, we evaluated the expression of two fat and obesity-related genes, PGC-1α and FTO. The results from RT-qPCR and Western blot showed that mRNA and protein expression of PGC-1α and FTO were higher in the slow frozen and vitrified groups than in the control group (Figure 3A-B), indicating that the increased weight and body fat of offspring in the slow frozen and vitrified groups may result from increased expression of PGC-1α and FTO.

Figure 3.

— Increased expression of the weight-associated genes FTO and PGC-1 in offspring from the vitrified and slow frozen groups. (A) Cerebral cortices were lysed and subjected to RT-qPCR analysis to determine mRNA levels. Each cDNA sample was normalized to the reference gene GAPDH. mRNA levels of FTO and PGC-1 were higher in offspring from the slow frozen and vitrified groups than in offspring from the control group. (B) Cerebral cortices were lysed and subjected to Western blot analysis to determine protein levels. Each protein sample was normalized to the reference protein GAPDH. Protein levels of FTO and PGC-1 were higher in offsprings from the slow frozen and vitrified groups than in offspring from the control group. Data are shown as mean ± SEM, n = 3 (10 offsprings were pooled and constituted one biological replicate, three separate biological replicate). ^** p < 0.01, ^*** p < 0.001.

Since many reports suggested that GFAP and MBP play important roles in brain development, we determined the effect of embryo vitrification and slow freezing on cerebral development by examining the expression of GFAP and MBP. We found similar expression levels of GFAP and MBP in offsprings from all three groups (Figure 4A). We also confirmed the distribution of GFAP and MBP in the cerebral cortex by immunofluorescence and immunohistochemistry (Figure 4B-C). These data indicated that there were no differences in content and distribution of GFAP and MBP in offspring from the slow frozen, vitrified, and control groups.

Figure 4.

— Expression and location of GFAP and MBP were similar in offsprings from the slow frozen, vitrified, and control groups. (A) Cerebral cortices were lysed and subjected to Western blot analysis to determine protein levels. Each protein sample was normalized to the reference protein GAPDH. GFAP and MBP expression were similar in offsprings from the slow frozen, vitrified, and control groups. (B) GFAP and MBP expression in the cortical neurons of offsprings from the slow frozen, vitrified, and control groups were analyzed at 8 weeks by immunofluorescence with anti-GFAP (red, left) and anti-MBP (red, right) antibodies. Scale bar, 100 μm. (C) GFAP and MBP expression in the cortical neurons of offsprings from the slow frozen, vitrified, and control groups were analyzed at 8 weeks by immunohistochemistry with anti-GFAP and anti-MBP antibodies. The arrow shows positive MBP staining. Scale bar, 100 μm. The results are representative of three independent experiments.