Mice
The donors were 3-week-old immature male FVB/N-Tg (PolII-luc) Ltc transgenic mice with an H-2 haplotype (H2q). These mice were created by the transgenic service of Level Biotechnology (New Taipei City, Taiwan), and the generation process was described in our previous studies [11, 13, 14]. In brief, after pronuclear microinjection of the PolII-Luc transgene into the FVB/N embryos, they could encode a 712-bp mouse RNA polymerase II promoter (PolII) and a modified firefly luciferase cDNA (Promega pGL-2). The animals were hemizygotes, and could express the transgene for luciferase (luc) and transmit this gene to their offspring. The recipients were 3-week-old immature FVB/NJNarl wild-type male mice with an H2q. We obtained these mice from the National Laboratory Animal Center (Taipei City, Taiwan).
A total of 12 pairs of donor and recipient mice were included in our experiments. They were all bred in the animal house of Taipei Medical University at a temperature of 22–24 °C and 12/12 h light/dark regimen. All procedures were reviewed and approved by the Animal Experimental Committee at the Taipei Medical University, in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Experimental design
The study was divided into three parts (Figure 1). First, we removed the 3-week-old donors’ gonads, and a total of 24 donor mice were divided into two groups. In the vitrification group, the testis from the donor mice were vitrified and thawed before transplantation. For the other 12 mice in the control group, the ITT was transplanted to the recipients immediately. After thawing, we measured the level of expression of caspase-3 by real-time reverse transcription polymerase chain reaction (RT-PCR). Each transplanted testicular graft was measured volume 10 µg. After washing in Dulbecco’s phosphate buffered saline (DPBS; Gibco®; Thermo Fisher Scientific, USA), the grafts were implanted in the scrotum of each recipient. We monitored all the transplanted grafts in vivo using BLI technology for 31 days until adulthood. On day 31, we removed the grafts and evaluated the tissue by hematoxylin and eosin (H&E) staining and IHC.
Preparation of donor ITT
The immature male FVB/N-Tg (PolII-luc) Ltc transgenic donor mice were euthanized by cervical dislocation under isoflurane anesthesia. Their testes was isolated, and the tunica albuginea was immediately removed into medium and maintained at 4 °C. The seminiferous tubules were isolated and cut into pieces less than 0.5 mm. Tissues were transferred to a 1.5 mL microfuge tube and washed three times with DPBS before cryopreservation or transplantation.
Cryopreservation protocol
The vitrification protocol was based on previous studies [15–17]. The vitrification solution used consisted of 1.5 M dimethyl sulfoxide (DMSO, D2650, Sigma, USA), 0.1 mol/L sucrose (S1888, Sigma, USA), 10% fetal bovine serum (Biological Industries, Israel), 1% penicillin streptomycin in L-15 medium (20183-027, Gibco®; Thermo Fisher Scientific). To achieve equilibration, 1 mL of cryoprotectant was gently introduced every minute with orbital shaking for 10 min. The tissues were incubated for 5 min at room temperature until precipitation of the ITT. The tissues were divided into 30 mL for each centrifuge and transferred to a foil (CoolRack®; Corning Inc., New York, USA) at 4 °C. Finally, the foil was immersed in liquid nitrogen and the ITT was transferred to a cryovial (Biomate, Taiwan) for 1 month. The warming procedure involved removing the ITT from the liquid nitrogen and quickly plunging it into a prewarmed 37 °C solution containing sucrose (1 M). The ITT was placed on an orbital shaker and DPBS was introduced at the rate of 1 mL per minute to lower the concentration of DMSO to 0.15 M. Before further assessment, the ITT was washed twice with DPBS.
Assessment of thawed ITT recovery
After general anesthesia with Zoletil (20–40 mg/kg, Virbac) and Xylazine (5–10 mg/kg, Bayer), we proceeded to the transplantation procedure and the skin was shaved around the testicular region. We performed unilateral orchiectomy and refilled the space with 10 mL of fresh or thawed seminiferous tubules. Finally, we closed the wound and a dose of Carprofen (5 mg/kg, Selleck Chemicals, USA) was given.
Real-time polymerase chain reaction
Total RNA was extracted with the RNA extraction kit (Qiagen, USA) and synthesis of the cDNA was done with the SuperScript III synthesis kit (Invitrogen, USA). All experimental steps were performed according to the manufacturer instructions. Caspase-3 mRNA expression was determined using StepOnePlus Real-Time PCR Systems (Thermo Fisher Scientific, USA). The relative mRNA was normalized with b-actin. All experiments were repeated three times [18].
Transplantation and in vivo BLI imaging assessment
Bioluminescence imaging was obtained using the In Vivo Imaging System 200 (Xenogen Corp., USA). The recipients were injected with D-luciferin intraperitoneally (150 mg/kg, BSAL-8220; Biosynth Carbosynth®, USA) 10 min before imaging, anesthetized (1%–3% isoflurane, Abbott, USA), and placed into a light-tight camera box on the stage of the imaging chamber. An overlay image (black-and-white picture) was taken with the aid of a light inside the imaging chamber. Luminescence was quantified using Living Image software. Luminescence was quantified by summing pixel intensities within the region of interest, as described [10].
H&E staining
For H&E staining, the testes of mice were fixed in Bouin’s solution (HT10132, Sigma-Aldrich). Then, the tissues were embedded in paraffin and the sections were cut at 5 mm. Histological and morphological changes of the testicular structures were examined under light microscopy [19].
Immunohistochemistry
Testes were collected and the tissue sections were incubated in 0.01 M citrate buffer (pH 6.0) at 95 °C for 30 min. Then, the sections were washed three times in PBS. Endogenous peroxidase was blocked with H2O2 for 10 min. Next, unspecific staining was blocked with 0.5% non-fat milk in PBS for 1 h. We used the following antibody probes: proliferating cell nuclear antigen (PCNA antibody 10205-2-AP; Proteintech), anti-sperm acrosome associated 1 (Spaca1 antibody 12829-1-AP; Proteintech), luciferase (ab181640; Abcam), sex determining region Y-box 9 (Sox 9 A19710; ABclonal), 3b-hydroxysteroid dehydrogenase (3b-HSD A1823; ABclonal), and DEAD (Asp–Glu–Ala–Asp)-box polypeptide-4 (DDX4, 51042-1-AP; Proteintech). The sections were placed in a humidified chamber overnight at 4 °C and then incubated for 15 min with rabbit/mouse horseradish peroxidase-labeling secondary antibody. After washing with PBS three times, the sections were mounted on glass slides and analyzed by light microscopy.
Statistical analysis
We performed all the statistical analysis using Statistical Package for the Social Sciences version 25.0 (SPSS; IBM, USA). The changes of the photons after transplantation were measured by the following equation: (measurement on a certain day – measurement at baseline) / measurement at baseline. Differences between the vitrification group and control group were compared using Student’s t-test, and a P value < 0.05 was considered statistically significant.