Chemical
All chemicals were purchased from Sigma-Aldrich Chemical Co. unless otherwise specified.
Oocytes recovery
Ovaries from adult Sardinia sheep (Ovis Aries, 3-6 years old) were recovered from local abattoirs and transported within 3 h to the laboratory in Phosphate Buffered Saline (PBS) with penicillin (100 mg/mL) and streptomycin (100 mg/mL) at 27–30 °C.
Upon arrival at the laboratory, ovaries were cleaned of any tissue residues, washed twice in PBS and placed in Holding Medium (HM) at pH 7.22 ± 0.1 at 37 °C consisting of TCM-199 with Hepes (N-2-Hidroxyethylpiperazine-N-2-ethansulfonic acid) 25 mmol/L, 50 IU/mL of antibiotics (streptomycin and penicillin), sodium bicarbonate 0.005 mol/L and 0.1% (w/v) of polyvinyl alcohol (PVA). The ovaries were divided sagittally with the aid of a sterile microblade and cumulus oocyte complexes (COCs) were released from the follicles by slicing technique.
In vitro maturation (IVM), in vitro fertilization (IVF) and in vitro embryo culture (IVC)
For in vitro maturation (IVM), COCs with several intact cumulus cells layers and homogenous cytoplasm were selected. COCs were matured in 650 μL of in vitro maturation medium consisting of TCM-199 containing 10% heat-treated estrous sheep serum (ESS), 0.36 mmol/L pyruvate, 100 mmol/L cysteamine, FSH 1 IU/mL and LH 1 IU/mL (Pluset; Bio98, Milan, Italy) under mineral oil, in 4-well dishes (Nunc Cell Culture, Thermo Fisher Scientific, Waltham, Massachusetts, USA) in a humidified atmosphere of 5% CO2, at 38.5 °C.
After 24 h from IVM, COCs were partially stripped of cumulus cells as described by Bogliolo and co-authors [21] and co-incubated with frozen-thawed spermatozoa, selected by the swim-up technique, (1 × 106 spermatozoa/mL) in IVF medium consisting of Synthetic Oviductal Fluid (SOF) [22] supplemented with 2% ESS, 1 g/mL heparin, 1 g/mL hypotaurine for 22 h at 38.5 °C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2 in four well Petri dishes.
The semen was preliminarily evaluated with the aid of the stereomicroscope (mass motility) and of the CASA system (Ivos, Hamilton Thorne, Biosciences).
At the end of IVF (approximately 24 h), presumptive zygotes (n = 15-20) were transferred to 650 μL IVC medium (SOF supplemented with BSA (4 mg/mL), essential amino acids (EAA) and non-essential amino acids (NEAA) at oviductal concentration [23] in 4-well culture dishes and incubated at 38.5 °C under 5% O2, 5% CO2, 90% N2 and maximum humidity. At 30 h post fertilization cleavage rate was recorded and on day 6 and 7 (day 0 = day of IVF) embryos were evaluated for development.
Embryo vitrification
For the experiments of vitrification, blastocysts were classified according to degree of expansion and hatching status as: early blastocyst (EB: blastocyst with a blastocoel less or equal to the half of the embryo volume), fully expanded blastocyst (FEB: a large blastocyst with a blastocoel greater than half of the embryo volume) (Fig. 1).
The “E.Vit” device (FertileSafe, Ness Ziona, Israel) used for vitrification consisted of a 0.25-mL straw with a polycarbonate grid (capsule) with pore diameters of 50 μm inserted at free end of the straw (Fig. 2). At day 6 or 7 of IVC either EB or FEB were placed in embryo handling medium (EHM see below) before being loaded by aspiration into the straw.
Each 0.25 mL empty straw was loaded with up to 2 embryos in a volume range of 20–30 μL, taking care to leave an air bubble at the end of straw. Next, the capsule was inserted by pressure into the straw end (Fig. 2). The insertion of the capsule allowed the removal of excess HM medium; the solution that contains the embryo is reduced to 5 μL. The straw loaded with the embryos and with the inserted capsule is transferred between the following solutions (Fig. 3); the volume of the solution in the straw is increased by capillary force and according to the increasing volume of the different vials (Fig. 3). The straws are evacuated by gentle blotting on sterile gauze in two times: the first after loading the 100% ES and the second after loading with 100% VS solution.
Three replicates of each embryonic stage and vitrification system were performed. The experiment was performed at room temperature (20-25 °C) and all media were at 25 °C using either:
- a)
Two-step system (TS), with the exposure of the embryos at only one equilibration and one vitrification solution or;
- b)
Multi-step system (MS), based on the exposure to 4 increasing concentrations of cryoprotectants for the equilibration procedure and two different concentrations for the vitrification solution.
Embryo handling medium (EHM) consisted of TCM-199 + HEPES supplemented with 0.6% bovine serum albumine (BSA) and 0.5 mol/L trehalose.
Equilibrium solution (ES) consisted of EHM supplemented with 7.5% of ethylene glycol (EG) and 7.5% of dimethyl sulphoxide (DMSO) .
Vitrification solution (VS) consisted of EHM supplemented with 18% of EG and 18% DMSO.
Two-step system (TS) (Fig. 3a)
The TS involves the use of a single equilibrium solution (ES: 100%) and a single vitrification solution (VS: 100%). The embryos were loaded into the E.Vit device (as described above) and then the E.Vit device lowered into ES (1 mL of ES in 1.5 mL micro-centrifuge tube) for 5 min. After which the ES was gently removed from the E.Vit device before being lowered into VS (1 mL of VS in 1.5 mL micro-centrifuge tube) for 30 s and then plunged into liquid nitrogen. Between the passage from the VS and the immersion in liquid nitrogen, excess VS was quickly removed by gentle blotting on sterile gauze, from the E.Vit device.
Multi-step system (MS) (Fig. 3b)
After loading embryos into the E.Vit device (described above), excess handling medium was removed from the E.Vit device before being exposed, in a step-wise manner, to increasing concentrations of ES cryoprotectants (1 mL of each of the following ES concentrations diluted with EHM) set up in 1.5 mL micro-centrifuge tubes; ES 1: 25%, ES2: 50%, ES3: 75%, ES4: 100%). The E.Vit device with the embryos was exposed to each ES concentration (with increasing volume) for 90 s for a total of 6 min. Between the equilibrium phase and the exposure to the VS1 medium, excess ES4 was removed from the E.Vit device by gentle blotting on sterile gauze. The E.Vit device containing the embryos was subsequently plunged into 1 mL of 75% of final VS concentration (VS1) for 30 s (1.5 mL micro-centrifuge tube), followed immediately by plunging into 100% VS concentration (VS2) for 30 s (1 min total exposure for the two VS concentrations). Excess V2 medium was removed from the E.Vit device by gently blotting on sterile gauze before being immersed into liquid nitrogen.
Warming of the embryos
Embryos were left in LN2 storage for a minimum 7 days. Warming for both the TS and MS systems was carried out as follows. The E.Vit device, with the embryos inside, immediately after removing from the liquid nitrogen, was plunged stepwise into decreasing concentrations of sucrose (1 mol/L, 0.5 mol/L and 0.25 mol/L in TCM-199 + 20% FCS at 38.5 °C). The E.Vit device was left immersed in each solution for 5 min.
Embryo recovery and post warming in vitro culture
After the warming procedure, the capsule was removed by cutting the end of straw and embryos released in HM. The number of embryos recovered after the removing the capsule from each straw was recorded. The embryos were washed 2-3 times with HM and incubated in IVC at 38.5 °C at 5% CO2 at maximum humidity for the subsequent evaluations after 2, 24 and 48 h of culture.
For the evaluation we have used the following morphometric criteria [24]:
Time of the start of re-expansion (the first appearance of the blastocoele cavity or increase in size).
Time of completion of re-expansion (the blastocyst occupies the whole perivitelline space).
Time of hatching (the trophectoderm blebbs out of the zona pellucida).
In particular, the following embryo development parameters were recorded:
after 2 h: number of embryos that showed a start of re-expansion of the blastocoel cavity;
after 24 h: number of embryos with completion of expansion of blastocoel cavity;
after 48 h: number of hatched blastocyst.
Analysis of apoptosis through the use of the TUNEL technique and confocal microscope
Apoptotic cell death in the embryos was evaluated by TUNEL using an In situ Cell Death Detection kit reaction mixture (Fluorescein; Roche Diagnostics Corp., Indianapolis, IN, USA). This method is used to detect cells that contain single and double strand breaks (nick) more or less extensive along the nuclear DNA molecule.
The TUNEL kit consists of an enzyme, the TdT (Terminal deoxynucleotidyltransferase), which catalyzes the polymerase reaction of nucleotides (always added to the mixture) labeled with fluorescein [25], at the free end 3´OH of fragmented DNA molecules both at the single and double stranded levels. The labeled nucleotides “fill” the single or double strand breaks on the DNA and emit a typical fluorescence green light; the more intense the more the DNA breaks are extended. To determine apoptosis of embryos a subset of embryos from Control fresh IVP embryos (EB n = 13; FEB n = 15), and from vitrified/warmed TS (EB n = 14; FEB n = 12) and MS (EB n = 12; FEB n = 20) after 24 h of IVC were fixed in 4% paraformaldehyde in PBS at 37 °C for 1 h. Following fixation, they were washed 3 times per 15 min in PBS + 0.1% PVA, to remove the residual fixative. The embryos were then permeabilized with 0.1% Triton X 100 in 0.1% sodium citrate for 5 min at 4 °C. The permeabilization aims to make permeable the zona pellucida at the entrance of the reagents that will be added later. The samples were incubated in TUNEL (Enzyme Solution + Label Solution) for 1 h at 38.5 °C in the dark. As a negative control, embryos (EB n = 2; FEB n = 3) from the control group were incubated in the presence of Label Solution and subsequently processed, as described below for the other groups of vitrified embryos.
Following incubation, the embryos were washed 3 times for 15 min in PBS+ 0.1% PVA, stained with a Glycerol-Hoechst 33342 solution (10 μg/mL) for the evaluation of the nuclear chromatin of the blastomeres. A drop of dye solution was placed on a glass slide and the embryos were transferred (3-5 embryos) to the drop. A coverslip was placed on the drop to slightly compress the embryos in order to facilitate the visualization of the nuclei. The slide was kept for 1 h in the dark at 4 °C and then read with the confocal microscope. The images were acquired with a laser scanning confocal microscope (Leica TCS SP5), equipped with 543 nm HeNe, 488 nm Argon and 405 nm 405-diode laser using an immersion objective (in oil) 40 × (NA = 1.25). The parameters related to fluorescence intensity (laser energy, gain, offset and pinhole size) were maintained with constant values during all the acquisitions of images.
TUNEL’s green fluorescence was determined using excitation wavelengths of 488 and 543 nm and emission spectra of 515-565 nm (green).
The number of TUNEL positive nuclei in embryos was determined and the apoptotic index (No. of apoptotic cells/ total No. of cells × 100) was calculated [26].
Statistical analysis
Data were analyzed by StataIC 11.2 software (Stata Corp LP, USA). Recovery rate, blastocoel re-expansions after 24 h post warming and hatching (survival) after 48 h post warming were compared between embryonic stage EB and FEB and between the two methods of vitrification procedures (TS and MS) using Chi-square χ2 test with post hoc Bonferroni test. Values of P < 0.05 were considered statistically different.