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An interplay of NOX1-derived ROS and oxygen determines the spermatogonial stem cell self-renewal efficiency under hypoxia

In this study, Morimoto et al. sought to investigate how reactive oxygen species (ROS) are regulated in spermatogonial stem cells (SSCs). Using in vivo and genomic approaches, the authors report that hypoxic niche sets the ROS levels in SSCs, and spermatogenesis depends on an intricate balance between NOX1 and mitochondria-derived ROS.

Cdkn1a KO mice were kindly provided by Dr. O. Niwa (Radiation Effects Research Foundation, Hiroshima/Nagasaki, Japan). For transplantation experiments, we collected 5to 10-day-old pup testes, and the cells were dissociated into single cells by a two-step enzymatic procedure using type IV collagenase and trypsin, as described previously (Ogawa et al. 1997). To delete Hif1a gene, the testis cells were cultured overnight with AxCANCre [multiplicity of infection (moi) = 2; RIKEN BRC, Tsukuba, Japan], as described previously (Takehashi et al. 2007). These cells were transplanted into B6 ´ DBA/2 F1 (BDF1) mice that had been injected intraperitoneally with busulfan (44 mg/kg; Japan SLC, Shizuoka, Japan). The mice were used at least 4 weeks after busulfan treatment.
For spermatogonial transplantation, testis cells were suspended in Dulbecco's modified Eagle's medium/10% fetal bovine serum. For colony counting, approximately 10 µl of donor cells (5,000 per testis) were microinjected into the seminiferous tubules of busulfan-treated BDF1 mice via the efferent duct (Ogawa et al. 1997). For serial transplantation experiments, recipient testes were dissociated into single cells and a portion of the total cell suspension from one primary recipient testis was microinjected into two secondary recipient testes. For production of offspring, cells were microinjected into the seminiferous tubules of 5-to 10-day-old W mice (Japan SLC). Each injection filled 75-85% of the seminiferous tubules. The Institutional Animal Care and Use Committee of Kyoto University approved all of the animal experimentation protocols.

Flow cytometry
To analyze ROS levels, GS cells were dissociated into single cells using Trypsin/EDTA (Nacalai Tesque, Kyoto, Japan) for 5 min. Cells were next incubated with  Table   S8). All flow cytometric analyses were carried out three times and representative data are shown.

Western blot analysis
Samples were separated by SDS-PAGE and transferred to Hybond-P membranes (Amersham Biosciences, Buckinghamshire, UK), and incubated with the primary antibodies.
For HIF1A analysis, nuclear extract was prepared, as described previously (Schreiber et al. 1989). Band intensity was measured using Image Gauge (Fuji Film, Tokyo Japan). The antibodies used are listed in Supplemental Table S8. The sequenced reads were mapped to the mm10 mouse reference genome using STAR (version 2.6.0c) (Dobin et al. 2013), with the GENCODE M21 annotation gtf file after trimming adaptor sequences and low-quality bases with cutadapt-1.16 (Martin et al. 2011).
To identify hypoxia-regulated genes, the uniquely mapped reads were counted and normalized to calculate the gene expression levels using HTSeq (version 0.11.2)(Anders et al. 2014), with GENCODE M21 annotation gtf file (protein-coding genes) and DESeq2 (version 1.22.2) after filtering low expressed genes (cpm =< 5) across paired conditions in each experiment (Love et al. 2014). An IPA (Qiagen; https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis) was performed to identify upstream regulators that could explain the changes in gene expression.
The upstream regulators categorized as transcription regulators in the molecular type were 20 chosen for the upstream analysis.

Immunostaining
For immunostaining, samples were fixed in 4% paraformaldehyde for 2 h. After immersion in blocking buffer (0.1%Tween 20, 1% bovine serum albumin, and 10% donkey (for GFRA1 or BCL6B staining) or goat (other antibodies) serum in phosphate-buffered saline for > 1 h, the samples were incubated with the primary antibodies at 4 °C overnight.
Next, the secondary antibodies were added, and the samples incubated for 1 h at room temperature. The samples were counterstained with Hoechst 33342 (Sigma), and visualized using a confocal microscope (Fluoview FV1000D; Olympus, Tokyo, Japan). The antibodies used are listed in Supplemental Table S8.

Analysis of recipient testes
Recipient mice were killed 8 weeks post-transplantation. Donor cell colonies were counted under UV light. Donor cell clusters were defined as colonies when the entire basal surface of the tubule was occupied and measured at least 0.1 mm in length (Nagano et al. 1999).
For quantification of tubules with spermatogenesis, tbe sections were viewed at a magnification of ´ 400 to determine the extent of spermatogenesis. The number of tubule cross-0sections with spermatogenesis (defined as the presence of multiple layers of germ cells in the seminiferous tubule) or no spermatogenesis was recorded for one section from each testis.

Southern blot analysis
Genomic DNA was collected from cultured cells by phenol/chloroform extraction and ethanol precipitation on the day following AxCANCre infection. To estimate the deletion efficiency, a 400-bp fragment of Hif1a was amplified by PCR using the primers 5'-TTACCTTTACTAGCCCACCTGC-3' and 5'-TGAAAACATCTGCTTTGGAAATG-3', which was used as a hybridization probe. Eighteen micrograms of DNA were digested with EcoRI and PstI, and separated on a 1.0% agarose gel. Hybridization was performed, as described previously (Takehashi et al. 2007). The intensity of bands was estimated using NIH Image 1.63.

Staining of testes
For immunostaining, testis samples were fixed in 4% paraformaldehyde for 2 h at 4°C. After being embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan), the samples were processed for cryosectioning. Sections of 10-µm thickness were prepared.  Supplemental Table S9.

Microinsemination
Testes were collected and refrigerated overnight before microinsemination, as described previously (Ogonuki et al. 2006). Germ cells were collected by mechanically dissociating the seminiferous tubule segments. These cells were microinjected into BDF1 oocytes using a Piezo-driven micropipetter (PrimeTech, Ibaraki, Japan). Embryos at the twocell stage after 24 h in culture were transferred to the oviduct of ICR recipient females.