Testicular cell isolation:
Amol University of Special Modern Technologies, Amol, Iran, approved the protocols for animal care and surgical intervention of adult mice in this study. Pasteur Institute (Iran) provided male mice (7 weeks old, C57BL/6 mice). Phosphate-buffered saline with 2% BSA and 0.1 percent Triton X100 was used to collect mouse testis. In Dulbecco's modified Eagle's medium, the testis seminiferous tubules were minced into minute pieces (DMEM, Invitrogen, USA). Enzymatic digestion solution containing Dispase (0.5 mg/ml) (Sigma Aldrich, USA), collagenase IV (0.5 mg/ml) (Sigma Aldrich, USA), and DNase (0.5mg/ml) (Sigma Aldrich, USA) in HBSS buffer with Ca2+ and Mg2+ (PAA, USA) was used to collect testicular cells at 37°C for 10 minutes. The cell solution was then rinsed with DMEM/F12 (Invitrogen, USA), passed through a 70m mesh filter, and centrifuged at 1500 rpm for 10 minutes. The isolated testicular cells were maintained at 37 degrees Celsius in 5% CO2 in the air. Every three days, the culture media was replaced (Azizi, Asgari et al. 2019).
Generation and culture of ES-Like cells from testis:
As we previously stated, ES-like cells were generated from SSCs (Azizi, Asgari et al. 2019). Oct4 promoter-reporter GFP transgenic mice from the C57BL/6 adult mouse strains were cultured in a mouse spermatogonia stem cells culture medium. About 6 weeks after culture initiation, ES-like cells with a high level of Oct4-GFP were generated and subcultured in an embryonic stem cells medium for growth (Azizi, Conrad et al. 2016).
Formation of embryoid bodies from ES-like:
In our study, 2–3 ES-like cells were grown in 10-cm2 bacterial-grade petri plates with low attachment. For 3–4 days, the EBs were formed spontaneously in suspension culture in a sterile bacterial petri dish containing a DMEM with 15% fetal bovine serum (FBS), 1% NEAA solution, 1% L-glutamine, 1% Pen-Strep, and 0.1 % -mercaptoethanol (Bojnordi, Azizi et al. 2017).
Differentiation of neurons from ES-Like cells:
Three stages of neuronal differentiation of ES-like cells were induced. As previously discussed, stage 1 was related to EB development (Bojnordi, Azizi et al. 2017). In stage 2, EBs were grown in suspension in low-attachment 10-cm2 bacterial-grade petri plates with 1% L-glutamine, DMEM-F12, , 1% N2, 1% NEAA, 10 ng/ml bFGF, and 500 nM retinoic acid (RA) to generate neural progenitor cells. During the second stage, the FBS concentration was lowered to 10% (days 1–2), 5% (days 3–4), and 3% (days 5–6). Following that, about 10 days after the start of suspension culture, the EBs were plated (which were coated with poly-L-ornithine) on 6- or 24-well tissue culture plates with neurobasal medium, 1% N2, 5% FBS, 1% NEAA, 1% L glutamine, and 2% B27 for an additional 10 days to allow the development of the neuronal progenitor (Azizi, Skutella et al. 2017, Azizi, Hamidabadi et al. 2019).
Patch-Clamp Recording in neural cells cultures:
Type I and II cells were fixed as a pellet in PIPES with paraformaldehyde/ glutaraldehyde for electron microscopy. The fixative was changed after 10 minutes. Pellets were then post-fixed for 40 minutes in OsO4/potassium hexacyanoferrate, washed in uranyl acetate buffer and sodium maleate buffer (pH 6.0), and block-stained with uranyl acetate. Following dehydration with increasing ethanol concentrations (5 min each, 4 x 20 min), the tissue was Epon-embedded (60 °C polymerizations, 36 h). A Zeiss EM10 was used to examine ultrathin slices (50 nm) (Azizi, Skutella et al. 2017, Bojnordi, Azizi et al. 2017).
Fluidigm Dynamic Arrays for Gene Expression Analysis:
Quantity of the Oct4 gene expression (Mm03053917_g1) in SSCs, ES-like, EBs, Sertoli cells, and Mouse Embryonic Fibroblasts (MEF, for control) was assayed by Fluidigm dynamic array chips. Glyceraldehyde-3-phosphate dehydrogenase (Mm99999915_g1) was utilized as a housekeeping gene for normalization. All cells were selected with a micromanipulator, lysed with a lysis buffer solution containing 1.3 μl TE buffer, 9 μl RT-PreAmp Master Mix, 0.2 μl R.T./Taq Superscript III (Invitrogen, USA), 2.5 μl 0.2× assay pool, and 5.0 μl Cells Direct 2× Reaction Mix (Invitrogen, USA). The targeted transcripts were quantified using TaqMan real-time PCR on the Biomark Real-Time quantitative PCR system (Fluidigm-PCR)(Azizi, Conrad et al. 2016, Conrad, Azizi et al. 2016).
Immunocytochemical and immunohistofluorescence staining:
The testicular cell was fixed with 4% paraformaldehyde and then permeably with 0.1% Triton/PBS. The testicular cell was blocked with 1% BSA/PBS and incubation with primary antibody vimentin. Then, We used secondary antibodies specific for incubation fluorochrome species, and the labeled cells were nuclear-counterstained treatment with 0.2 μg/ml of 4', 6-diamidino-2-phenylindole (DAPI) dye. The Labeled positive testicular cells were studied under confocal microscopy Zeiss LSM 700 (Germany), and images were used a Zeiss LSM-TPMT camera (Germany).
In IMH staining, cells were washed with PBS and fixed in 4%paraformaldehyde. Dehydrated tissues were located in Paraplast Plus and chop with a microtome device at 10 μm thickness. Testis tissues section were mounted on Superfrost Plus slides and kept at 25°C until used. In the process of IMH staining, samples were washed with xylene and gradually replaced with water in ethanol. For tissues, antigens retrieval was performed by heat-induced epitope retrieval at 94˚C for 18 minutes. The non-specific binding site of tissue samples was blocked with 10% serum and 0.3% Triton in PBS. As explained above, the experiment of immunofluorescence staining for these samples was continued.
Search strategy and data preparation for protein-protein interactions (PPI) network analysis:
Gene databases (NCBI: https://www.ncbi.nlm.nih.gov/ gene/ and KEGG: https://www.genome.jp/ Kegg/genes/) were searched for gene-related datasets. (Spermatogonia stem cell), (ES-like cell), (Embryonic bodies), (Neurons), and "Mus musculus" [porgn: __txid10090] were the keyword phrases. The gene expression profile was then saved in an excel file. In this section, P< 0.04 was used to select gene interactions and clusters.
PPI network analysis:
STRING (Szklarczyk, Gable et al. 2019) (version 11.5) online tool was applied to predict protein-protein biological and functional interactions (https://string-db.org/ ). The gene-related with a significant role in Oct4 (Pou5f1) were uploaded in the STRING online tool. The master regulator of Pou5f1, gene co-expression, and signaling pathways were highlighted, To identify. The highlighted genes were imported to Cytoscape (Doncheva, Morris et al. 2018) (version 3.9.0) for further analysis and protein-protein interactions network visualization.
Gene set enrichment analysis:
To confirm the biological roles of the genes involved in the PPI network of first co-expression nodes with Oct4 (Pou5f1), we have performed functional gene enrichment analysis using the STRING enrichment analysis in the Cytoscape software.
Statistical analysis
The tests were performed at least three times. The average gene expression in each group was assessed, and the experimental data were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post-hoc testing.
Ethical statement:
In this study, all animal experiments were approved (Approval ID: Ir.ausmt.rec.1398.03.07) to the local and international guidelines for the use of experimental animals and were approved by the ethics committee of Amol University of Special Modern Technologies and all sections of this report adhere to the ARRIVE Guidelines for reporting animal research.