All protocols involving the use of animals were performed in accordance with the approved Guidelines for Animal Experiments of Gansu Agricultural University,and were approved by the Animal Experimental Ethical Inspection Committee of Gansu Agricultural University.
Experimental animals and test reagent
A total of 11 male unmated about two-year-old Bactrian camels were collected in winter from breeding farmers in Laohe County,Wuzhong City,Ningxia Hui Autonomous Region.These home-bred animals were divided into two groups:the normal(five of samples)and cryptorchidism groups(six of samples)for comparative analyses.The cryptorchid testes were located in the abdominal cavity (Intra-abdominal cryptorchidism).Tissue specimens were collected by orchiectomy.
The samples were treated according to different test schemes.Fresh testes were processed into small pieces and then divided into three samples.One sample was frozen in liquid nitrogen for western blotting hybridization reaction,while the other sample was fixed with 4% paraformaldehyde solution for histochemical analysis,and the third fixed in glutaraldehyde for transmission electron microscopy observation.The study design was approved by the academic committee of the College of Veterinary Medicine of Gansu Agricultural University.
All antibodies were purchased from commercial suppliers. Rabbit polyclonal antibody NSE(bs-10445R-HRP,anti- NSE/HRP), SP(bs-0065R-HRP, anti-SP/HRP), NFH(bs-10680R-HRP, anti-NFH/HRP) and DβH(bs-0596R-HRP,anti-DβH /HRP), anti-rabbit IgG antibody(bs-0295G-AF488) and DAB chromogenic kit(ZLI-9018)were purchased from Beijing ZSGB-BIO Co.,Ltd., Beijing,China,Immunohistochemical staining kit(SP-0023),produced by ZYMED Laboratories Co.,Ltd.,San Diego,USA, purchased from Jiamay Biotech Co.,Ltd.,Beijing,China.And other chemicals were obtained commercially and of reagent grade.
Experiment 1: H&E staining,histochemical staining,and observation of tissue samples
Testicular tissue samples (1×1× 0.6 cm) were fixed with paraformaldehyde and rinsed in running water for 24h before conventional gradient ethanol dehydration. Afterwards, they were made transparent with xylene, embedded in paraffin,and 5μm serial sections were cut every fiveth section was mounted and stained either with standard haematoxylin and eosin(H&E) to examine the general morphology, followed by Alician blue(AB pH=2.5,30 min)and periodic acid Schiff reaction(PAS) and examined for the presence of positive staining cells,the acidic mucin and proteoglycan were blue,with the nuclei being reddish[18].
Experiment 2: transmission electron microscopic technique
The testes were cut into 0.1cm×0.1cm×0.1cm, fixed in the 2.5% glutaraldehyde in 0.1M phosphate- buffered saline(pH=7.4) or 48h,and then post fixed in 1% osmium tetroxide at 4°C for 1h.The pieces were dehydrated with a graded ethanol series,and then embedded in epoxy resin (Epon812, American). Ultrathin sections were cut,stained with uranyl acetate and lead citrate,and then examined under a JEM-100CX electron microscope (NEC, Japan).
Experiment 3: immunofluorescence staining
Green immunofluorescence staining:Paraffin sections of testicular tissues were prepared following the same procedure as demonstrated in section 1.2.1.Slice thickness was 5μm,high-pressure antigen retrieval was performed,30g/L H2O2 aqueous solution was added to block peroxidase for 10min,Sections were incubated with goat serum albumin for 15min.A volume of 50μL of primary antibody(i.e.,rabbit polyclonal anti-NSE,anti-SP,anti-NFH and anti- DβH)was added dropwise at a dilution ratio of 1:1,000.For the negative control group,0.01mol.L-1 phosphate buffer solutionbuffered saline(PBS)was used as the substitute for the primary antibody.Incubation was carried out for 2h at 37°C before the fluorescent secondary antibody was added dropwise.Alexa Fluor488-labeled goat anti-rabbit IgG antibody(1:1,000,bs-0295G-AF488) was used as the secondary antibody.After incubation at 37°C for 2h,observations were made directly under the microscope.Positive cells displayed fluorescent green and the immunofluorescence images were collected by an ECHO fluorescence microscope(REVOLVE RVL-100-G).
Experiment 4: immunohistochemical staining
The streptavidin-peroxidase method was used for immunohistochemical staining.The paraffin sections were routinely dewaxed and differentiated by gradient ethanol;3% H2O2 in methanol solution was used to block peroxidase for 10min.Normal goat serum albumin was incubated for 15min; 50μL of rabbit polyclonal anti-mouse NSE(bs-10445R),SP (bs-0065R),NFH(bs-10680R)or DβH(bs-0596R) antibodies were diluted at 1:400 and added dropwise to each section separately.After incubation at 37°C for 2h and washing by PBS shaking,50μL of biotinylated goat anti-rabbit IgG working solution was added dropwise to each section,followed by the addition of 50μL of horseradish-labeled streptavidin working solution.T-he sections were then incubated at 37°C for 15min.Freshly prepared DAB substrate soluteion was then added for microscopic examination,followed by routine dehydration,making transparent,and mounting.Positive cells displayed as brown-yellow,with nuclei showing as blue after counterstaining.The negative control group was stained with 0.01mol·L-1 of PBS instead of the primary antibody.
Experiment 5: western blot analysis
Total protein was extracted from testicular tissue was extracted.100mg of liquid nitrogen-preserved testicular tissue was weighed and crushed.Radioimmunoprecipitation assay buffer containing phenylmethylsulfonyl fluoride was added proportionally.The mixture was then homogenized with a glass homogenizer until fully lysed,and then subjected to hypothermal centrifugation at 14,000×g for 5min.The supernatant was transferred to another tube,and reagent with half of the volume of the solution was used to dissolve the precipitate.After standing still at 25°C for 30 min,the mixture was then centrifuged at 4°C and 19,575×gfor 20min.The supernatant was analyzed for protein concentration according to the instructions of the BCA protein assay kit(Beyotime Biotechnology Research Institute),dispensed,and stored at -70°C for use.
The protein was extracted and loaded following the routine procedure:the protein was separated by polyacrylamide gel electrophoresis(SDS-PAGE);5% stacking gel and 12% separating gel were prepared.30μg of protein was loaded to the gel sample well for electrophoresis,and the protein in the gel with the target band was subjected to wet transfer to the membrane.The membrane was incubated with primary antibody NSE,SP,NFH or DβH(1:1,000)at 4°C overnight,and then was washed by Tris-buffered saline+Tween 20(TBST).The horseradish peroxidase-labeled cow anti-rabbit IgG was used as the secondary antibody for incubation for 2h at 25°C,and TBST was used to wash the membrane three times for 10min each.The polyvinylidene fluoride membrane was then subjected to color development.The chemiluminescent substrate solutions A and B were mixed at a ratio of 1:1,and were allowed to react at 25°C.The transfer membrane was photographed for analysis.β-actin was used as the internal reference.
Measurement and statistical analysis
Tissue sections were imaged using a Nikon Eclipse 80i microscope camera system.Leydig cell characteristic index:ten sections were randomly selected from each group,and six non-repeating fields were randomly selected from each section(bar=20μm,400×).The transverse and longitudinal diameters of Leydig nuclei,as well as the mean area of Leydig nuclei in the triangular and quadrangular mesenchymal tissues of each field were randomly counted(n=40).Statistical analyses were performed by Image Pro Plus 6.0 software.
The intensity of immunofluorescence results was scored as follows:-:no positive expression;+/-:occasional positive expression;+:positive expression;++:medium-intensity positive expression;+++:strong positive expression;++++:high-density strong positive expression[19].
The sections were imaged using a NIKON ECLIPSE 80i microscope camera system.Five sections were selected randomly from each group for immunohistochemical staining.Six non-repetitive fields(bar=20μm,400×)were randomly selected for each section.The mean positive signal intensity and positive area of each field were statistically analyzed by Image Pro Plus 6.0 software to evaluate the average light absorbance.A total of 30 statistical data were collected for each group,and the results were expressed as mean±standard deviation(mean ± SD).SPSS15.0 software was used for statistical analysis,and the expression difference of NFH,SP,NSE and DβH between normal and cryptorchid testes was analyzed by one-way analysis of variance.Paired t-tests were carried out,and the level of statistical significance was set at p<0.05.
The western Blot expression band was first selected,the gray curve of which was then analyzed using Image J 1.48.The area under the peak was calculated as the band density value.The density of β-actin was taken as the base value,and the relative densities were obtained by comparing the densities of NSE,SP,NFH or DβH expression bands in the normal testis group(Simply marked as N-NSE,N-SP,N-NFH or N-DβH)and the cryptorchidism group(Simply marked as C-NSE,C-SP,C-NFH or C-DβH).The relative density values were then statistically processed by SPSS 21.0 statistical software.All data were expressed as mean±standard deviation.Difference between variables were analyzed by t-tests difference between variables were analyzed using paired t-tests,and the level of statistical significance was set at p< 0.05.