Humane care and use of animals

Here, 8-week-old female and 10-week-old male BDF1 mice were used for the experiments. All mice were bred and maintained under controlled temperature (∼22°C) with a 14-h light and 10-h dark cycle at the Animal Center, Guangdong Second Provincial General Hospital, Guangzhou, Guangdong. All animal procedures were approved by the Animal Center. All animal procedures in this study were performed in compliance with the Guide for Care and Use of Laboratory Animals of Guangdong Second Provincial General Hospital (No. 2014-KYLLM-065).

Collection of metaphase II (MII) oocytes in mice

Mice were administered intraperitoneal injections of 7.5 IU pregnant mare serum gonadotrophin (PMSG; Ningbo Sansheng Hormone Factory, Ninbo, China) followed by intraperitoneal injection of 7.5 IU human chorionic gonadotrophin (hCG; Ningbo Sansheng) 48 h later. The mice were euthanized by cervical dislocation 13–14 h after the hCG injection, and the oviducts were excised and placed in 1 ml of M2 medium (Sigma-Aldrich Chemical Co. St. Louis, MO, USA). Cumulus–oocyte complexes (COCs) were released by tearing the ampullae of the oviducts. The cumulus cells were enzymatically removed by treatment with 80 IU/ml hyaluronidase (Sigma-Aldrich) and mechanically dissociated with a glass pipette. Only morphologically normal mature MII oocytes, as determined by the presence of a first polar body, were used.

Routine droplet merge equilibration and vitrification of oocytes

In total, 2, 4, 6, 8 or 10 MII oocytes were classified to DM2, DM4. DM6, DM8 or DM10 groups, respectively, were placed into the holding medium (H) and left undisturbed for 1 min. The holding medium was mixed with equilibration solution 1 (ES1) using the tip of a transfer pipette, and two droplets were spontaneously mixed for 2 min. Then ES2 was merged with the holding medium + ES1, and the two droplets were spontaneously mixed for 2 min. The oocytes from the merged drop were transferred to ES3 and exposed undisturbed for 6–10 min. Then, oocyte(s) from ES3 were transferred to a vitrification solution (VS) for 90 s before they were loaded. The oocytes were gently but thoroughly pipetted in a VS drop to ensure a complete rinse. Different numbers of oocytes were loaded onto the tips of a Cryotop (Kuwayama et al., Reference Kuwayama, Vajta, Ieda and Kato2005a, Reference Kuwayama, Vajta, Kato and Leibo2005b; Kitazato Co., Minato-ku, Japan) within 90 s, taking care not to exceed 110 s following the initial exposure to the VS. Finally, once the vitrification was complete, the Cryotop was immediately capped and dropped into liquid nitrogen for storage (Figure 1A). The vitrification and thawing kits were purchased from Irvine Scientific Inc. (Santa Ana, CA). Procedures were performed strictly according to the manufacturer’s instruction manual.

Figure 1.

Schematic diagram of droplet merge and multi-gradient equilibration vitrification protocols. Mature mouse oocytes were transferred into holding medium (H) for a duration of 1 min and equilibrated through two different methods in equilibration solution (ES) or ES/H mixture, and treated with vitrification solution (VS) for 90 s. After the oocytes were loaded onto a Cryotop, they were immersed in liquid nitrogen. (A) Routine droplet merge and vitrification. (B) Modified multi-gradient equilibration and vitrification.

Modified multi-gradient equilibration and vitrification of oocytes

In total, 2, 4, 6, 8 or 10 oocytes were classified into MG2, MG4. MG6, MG8 or MG10 groups, respectively, and were suspended in holding medium (H), 4H1ES [80% H + 20% equilibration solution (ES)], 3H2ES (60% H + 40% ES), 2H3ES (40% H + 60% ES), and 1H4ES (20% H + 80% ES), respectively, for 1 min each, then transferred to ES and incubated for 6–10 min. The oocytes were then transferred to VS and incubated for 90 s (<110 s) at room temperature. The oocytes were loaded onto the tip of a Cryotop (Kitazato, Japan), capped, and immediately dropped into liquid nitrogen for storage (Figure 1B). All the oocytes were cryopreserved until they were thawed for analysis. Each vitrification via this protocol was repeated 25 times, similar to the droplet merge vitrification method.

Thawing of oocytes

Oocytes were thawed by direct immersion of the Cryotop in pre-warmed TS1 solution at 37°C for 1 min. The warmed oocytes were sequentially transferred to TS2 for 3 min each, washed twice with TS3 medium, and finally transferred to human tubal fluid (HTF) medium (Millipore Co., Billerica, MA, USA). They were incubated at 37°C in 5% CO₂ in humidified air for 2 h. The survival of oocytes was assessed by morphological analysis, which involved examining the integrity of the plasma membrane and the discoloration of ooplasm after the thawed oocytes were recovered. The oocytes that survived were inseminated by intracytoplasmic injection of spermatozoa.

Intracytoplasmic spermatozoa injection (ICSI) of oocytes

Epididymal spermatozoa were collected from the cauda epididymis of 8-week-old to 10-week-old BDF1 mice and incubated in HTF medium for 30 min at 37°C in air containing 5% CO₂. Then, 1 µl of spermatozoa suspension was mixed with 10 µl of 10% polyvinyl pyrrolidone (PVP)–HEPES-buffered CZB medium in a ICSI manipulation chamber. ICSI was performed as described previously (Jiang et al., Reference Jiang, Zhu, Zhu, Sun and Chen2005) with a minor modification. Briefly, the sperm head was separated from the tail by piezo pulses at the neck region, and the head was immediately injected into a thawed oocyte. After 10 min of recovery at room temperature, the oocytes were washed at least three times and then placed into KSOMaa medium (Millipore Co.). Fertilization was evaluated by observing the formation of two pronuclei (2PN) 5 h after ICSI. The rates of embryos developed to the blastocyst were assessed on day 3.5 after insemination.

Oct4 staining and cell counting of blastocysts

The thawed blastocysts from both vitrification protocols were fixed and treated as previously described (Kong et al., Reference Kong, Zhu, Wang, Li, Chen and Jiang2013; Zhu et al., Reference Zhu, Jiang, He, Zhang, Sun, Jiang and Wang2015). Briefly, blastocysts were fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 40 min at room temperature. Fixed samples were stored in 0.3% (w/v) bovine serum albumin (BSA)/PBS for 1 week at 4°C for further analysis. For immunofluorescence, samples were permeabilized in 0.1% Triton X-100 and 0.3% BSA in PBS (w/v) at 37°C for 30 min. The samples were washed at least three times with 0.01% Triton X-100/ PBS, and then were incubated in blocking solution (150 mM glycine and 0.3% BSA in PBS) for ∼30 min at 37°C. Then, the samples were treated with primary rabbit anti-Oct4 antibodies (H-134, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and FITC-labelled chicken anti-rabbit antibodies (1:100; Invitrogen, Grand Island, NY, USA), and diluted in blocking solution for 30–40 min each at 37°C or overnight at 4°C. After three washes (each time for 5 min), the nucleus was stained with 10 μg/ml 4′-6-diamidino-2-phenylindole (DAPI). The samples were then mounted on slides with anti-fade mounting medium, and examined under a laser-scanning confocal microscope (Zeiss LSM 510 Meta; Carl Zeiss AG, Oberkochen, Germany). Blastomeres expressing positive Oct4 showed green fluorescence and intact nuclei were blue. The ratio of blastomeres expressing green fluorescence to the total blastomeres with blue nuclei was considered the Oct4-positive rate (Figure 4A). The images shown in the Results section are representative of at least 30 samples from more than 25 experimental replicates.

Based on the results of the number of Oct4-positive blastomeres, blastocysts were graded as Grade A, B, or C. Blastocysts with more than 15 Oct4-positive blastomeres were classified as Grade A blastocysts; those with 8–15 Oct-positive blastomeres were classified as Grade B blastocysts; and those with 0–8 blastomeres were classified as Grade C blastocysts. Blastocysts of grades A and B were considered to be good-quality embryos.

Meiotic spindle and chromosome evaluation of oocytes

Microtubules of oocytes were analyzed as described previously with minor modifications (Zhu et al., Reference Zhu, Chen, Li, Lian, Lei, Han and Sun2003, Reference Zhu, Jiang, He, Zhang, Sun, Jiang and Wang2015) in two vitrification protocols. In brief, after vitrification and thawing, the oocytes were incubated in HTF medium at 37°C for 2 h in an atmosphere containing 5.0% CO₂ and then fixed in 4% paraformaldehyde for 10 min. Then the oocytes were washed in PBS and transferred to 0.25% Triton X-100/ PBS (w/v) for 10 min at room temperature. The oocytes were washed twice for 5 min each and blocked for 1 h at room temperature with 2% BSA in PBS. After washing twice with PBS, the oocytes were incubated with fluorescein isothiocyanate (FITC)-labelled mouse monoclonal antibodies against α-tubulin (Sigma-Aldrich Co.), and diluted with blocking solution (1:100) for 30–40 min at 37°C or overnight at 4°C. After washing three times for 5 min each time, the nuclei were stained with 10 µg/ml DAPI (Sigma-Aldrich Co.). Finally, the samples were mounted in an antifluorescence-fade medium (DABCO, Sigma-Aldrich Co.) on slides. They were then observed under a laser-scanning confocal microscope (Zeiss LSM 510 Meta). Typical barrel-shaped microtubules traversing the two poles and centrally aligned chromosomes were considered normal. Images shown in the Results are representatives of at least 20 samples from each group.

Statistical analysis

Data were analyzed using Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). The survival rates and fertilization rates were compared using one-way analysis of variance (ANOVA) from Prism 6.0. The proportions of oocytes with normal spindle, blastocyst rates and proportions of good-quality blastocyst were compared using chi-squared test. A P-value of <0.05 was considered statistically significant.