Immature rat testis sustained long-term development using an integrative model

Sample collection

Specific pathogen free (SPF) experimental animals including pregnant female Sprague–Dawley (SD) rats and BALB/c male nude mice were obtained from the Experimental Animal Center of Xi’an Jiaotong University. Six sets of models from six different litter were performed and each litter of neonatal rats was randomly divided into control and experimental group. Part of the male offspring was executed on postnatal day 8 (PND 8) and the testes were removed aseptically and then immediately transferred into a culture medium on ice for later culture. The other were fed normally until PND 69 as control.

Organ culture protocol

Before organ culture, a 5 mm thick agarose gel formed by 1.5% hot agarose solution composed of agarose powder and double-distilled water was prepared. The next step was to cut agarose gel into 10 ×5 ×5mm3 pieces with a sterile blade and then soak those pieces in culture medium overnight. After sliced into small pieces (1–2 mm3) on ice, the isolated neonatal testes were transferred onto the surface of agarose gel half-soaked in the 24-well plate in which 600 μL of culture medium was added (Fig. 1A). The culture medium was DMEM/F12 (cat# 11039-021, Gibco, USA) supplemented with 10% KSR (cat# A31815-01, Gibco, USA), penicillin (100 IU/mL) and streptomycin (100 μg/mL) (cat# P1400, Solarbio, China). One piece of testis was held on each gel brace, and a medium was changed every 2 days. The testes were incubated at 37 °C in a humidified incubator containing 5% CO2 and 95% air for 4 days. Part of the tissues was used for xenografting 4 days after culture and the rest was fixed for histology.

Fig. 1figure 1

The organ culture system (A) and the testes (↑) after xenotransplantation for 57 days (B, C)

Xenografting

Six BALB/c male nude mice aged 4 weeks were acclimated for 3 days in experimental animal center and then were castrated under anesthesia and the testicular tissue that had been cultured for 4 days was transplanted under the dorsal skin of recipient nude mice 2 weeks after castration. Three grafts per rat were transplanted on either side of the midline. Keeping warm after surgery of castration and xenotransplantation and giving antibiotics in drinking water for 3 days were necessary. Experimental animals kept in the experimental animal center were treated with prepared water and food under 12 h light/dark cycle.

The xenografted testes on the dorsal skin of recipient were resected on the 57th day after xenotransplantation (Fig. 1B–C). For the control group, the testes of male rats were harvested on the same day, namely PND 69. The collected testes were divided into two parts, one was used to be fixed for histology and ultrastructural study, another was used for gene and protein analysis.

Testicular histology

For fixation, testicular tissue was immersed in 4% paraformaldehyde fixative solution at 4 °C for 6 h. After dehydration in ethanol and xylene, the samples were embedded in paraffin and cut into 5 μm sections. At last, sample staining was done with hematoxylin for 1–2 min and eosin for 1 min. Diameter of seminiferous tubules was measured by using Image J version 1.8.0 (National Institutes of Health, USA). Fifty tubule cross sections of testicular tissue before and after culture, xenografted testes and control were randomly selected in each setting. Observation and evaluation under the microscope were performed by an independent investigator.

Immunohistochemistry

Immunohistochemical staining used the sections made above. Sections were deparaffinized in xylene and rehydrated in graded ethanol series. After antigen retrieval with sodium citrate buffer (cat# C02-02002, Bioss, China) at 98 °C for 10 min and endogenous peroxidase blocking with normal goat serum, the sections were incubated in a humidified box at 4 °C overnight with rabbit anti-8-OH-dG polyclonal antibody (1:500, cat# bs-1278R, Bioss, China). The secondary antibody was biotin-labeled goat anti-rabbit IgG (cat# SP-0023, Bioss, China), and 3,3′-diaminobenzidine (DAB, cat# C-0010, Bioss, China) staining was performed under the microscope. Diluting solution was used to replace primary antibody for the negative control.

Immunofluorescence

After deparaffinization, rehydration and antigen retrieval, sections were incubated with Triton X-100 for 10 min and blocking buffer (cat# P0102, Beyotime, China) for 1 h at room temperature. Primary antibody (ACROSIN, 1:100, cat# NBP2-14260, Novus Biologicals, USA) was added to tissue sections at 4 °C for a night, followed by incubating with secondary antibody (1:1000, cat# P0176, Beyotime, China) without light at room temperature for 1 h. After counterstaining with DAPI (cat# C1005, Beyotime, China), sections were observed under fluorescence microscope. The seminiferous tubules were randomly selected for each group and the number of spermatids in the ACROSIN-positive tubules was counted and compared between the control and new integrative model groups.

Apoptosis assay

A TUNEL apoptosis assay kit (cat# C1098, Beyotime, China) was used to detect apoptosis according to the manufacturer’s instructions. After deparaffinized and hydrated, the sections were incubated with 20 μg/mL DNase-free proteinase K at 37 °C for 20 min, followed by washing with PBS for 3 times and blocking endogenous peroxidase with 3% H2O2 in PBS at 25 °C for 20 min. Subsequently, the sections were incubated with working solution containing biotin-dUTP and TdT enzyme at 37 °C away from light for 60 min. Then the sections were washed again and incubated with streptavidin–HRP solution, followed by DAB solution for color developing. Two or more TUNEL-positive cells existed in a seminiferous tubule was considered as positive. The ratio of number of apoptotic positive tubules and total number of tubules in a cross section was counted as apoptosis index (AI).

Ultrastructural study

Washing tissue promptly with PBS after harvested and then immersing in 4% formaldehyde and 2.5% glutaraldehyde in PBS at 4 °C for 2 h was the first step, followed by post-fixed in 1% osmium tetroxide at 4 °C for 2 h. Subsequently, the sections were double stained with uranyl acetate for 15 min and lead citrate for 5 min after dehydrating, embedding, and sectioning. Observation under H-7650 transmission electron microscope (TEM) at 80 kV (Hitachi, Japan) was done by an independent researcher.

Real-time quantitative PCR

The detailed procedure of RNA extraction was based on the protocol of TaKaRa MiniBEST Universal RNA Extraction Kit (cat# 9767, Takara, Japan), and the PrimeScript™ RT Master Mix (cat# RR036A, Takara, Japan) was used to synthesize cDNA. Real-time quantitative PCR was done using TB Green Premix Ex Taq II (cat# RR820A, Takara, Japan) with total reaction volume of 20 μl on the Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). GAPDH was assayed in all samples as an internal control. The amplification process was initiated with a denaturation cycle at 95 °C for 2 min, followed by 39 cycles of 95 °C for 10 s and 60 °C for 30 s. All analyses were performed in triplicate samples. Quantitative analysis of gene expression was evaluated by the method of 2−ΔCt algorithm. The gene names and primer sequences are listed in Table 1.

Table 1 The genes and primer sequencesWestern blotting

The total proteins in the testicular tissue were extracted by using lysis buffer (cat# P0013C, Beyotime, China). Three different samples were performed in each group. Then the protein lysates were separated by 8–12% SDS-PAGE (25 min: 90 V, and 100 min: 120 V, room temperature), and electro-transferred (90 min: 200 mA, 4 °C) onto the polyvinylidene fluoride membranes. After incubating in blocking buffer for 2 h, the membranes were incubated overnight in primary antibodies (Nrf2, 1:1250, cat# 16396-1-AP, Proteintech, USA; Keap1, 1:2500, cat# 10503-2-AP, Proteintech, USA; NQO1, 1:5000, cat# ab80588, Abcam, UK; SOD1, 1:5000, cat# ab51254, Abcam, UK; GAPDH, 1:2500, cat# AF1186, Beyotime, China) at 4 °C, and then incubated for 2 h in HRP-conjugated secondary antibodies (1:2000, cat# SA00001-2, Proteintech, USA). The final detection was performed by using ECL reagent (cat# P0018FS, Beyotime, China). Image gray value was analyzed using Image J version 1.8.0.

Statistical analysis

Data were expressed as mean ± SEM. Significant differences were identified by unpaired two-tailed t-test or Welch’s t-test using GraphPad Prism version 8.0 (GraphPad, USA). Differences were considered as significant at P < 0.05.

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