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Glycylglycine Plays Critical Roles in the Proliferation of Spermatogonial Stem Cells

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Glycylglycine Plays Critical Roles in the Proliferation of Spermatogonial Stem Cells 3802 MOLECULAR MEDICINE REPORTS 20: 3802-3810, 2019 Glycylglycine plays critical roles in the proliferation of spermatogonial stem cells BO XU1,2*, XIANG WEI1*, MINJIAN CHEN1,2*, KAIPENG XIE3,4, YUQING ZHANG1,2, ZHENYAO HUANG1,2, TIANYU DONG1,2, WEIYUE HU1,2, KUN ZHOU1,2, XIUMEI HAN1,2, XIN WU1 and YANKAI XIA1,2 1State Key Laboratory of Reproductive Medicine, Institute of Toxicology; 2Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166; 3Nanjing Maternity and Child Health Care Institute, 4Department of Women Health Care, Nanjing Maternity and Child Health Care Hospital, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu 210004, P.R. China Received May 9, 2018; Accepted July 9, 2019 DOI: 10.3892/mmr.2019.10609 Abstract. Glial cell line-derived neurotrophic factor (GDNF) properties that distinguish stem cells from somatic cells, is critical for the proliferation of spermatogonial stem cells and SSCs are the only germline stem cells that can undergo (SSCs), but the underlying mechanisms remain poorly under- self-renewal division (1). The balance between proliferation stood. In this study, an unbiased metabolomic analysis was and differentiation is therefore essential to the normal function performed to examine the metabolic modifications in SSCs of SSCs and to maintain male fertility (2). following GDNF deprivation, and 11 metabolites were observed Glial cell line-derived neurotrophic factor (GDNF) is to decrease while three increased. Of the 11 decreased metab- secreted by Sertoli cells, and is an important factor in the olites identified, glycylglycine was observed to significantly cell fate determination of SSCs, which was identified in rescue the proliferation of the impaired SSCs, while no such the year 2000 (3). While GDNF+/− mice have depleted stem effect was observed by adding sorbitol. However, the expres- cell reserves (3), the overexpression of GDNF results in the sion of self-renewal genes, including B-cell CLL/lymphoma accumulation of undifferentiated spermatogonia and in 6 member B, ETS variant 5, GDNF family receptor α1 and the development of testicular tumors (4), indicating that the early growth response protein 4 remained unaltered following right concentration of GDNF is critical for the proliferation glycylglycine treatment. This finding suggests that although of SSCs (5). Although GDNF has no significant effect on the glycylglycine serves an important role in the proliferation of activity of SSCs at concentrations in the range 1-100 ng/ml, SSCs, it is not required for the self-renewal of SSCs. GDNF at concentrations <1 ng/ml is insufficient to maintain the proliferation of SSCs in vitro over a period of 7 days (6). Introduction However, the mechanisms underlying the GDNF-dependent proliferation of SSCs remain elusive. Spermatogenesis is a complex developmental process that Metabolomics is the study of the final products of gene has spermatogonial stem cells (SSCs) at its foundation. The expression and, as a high-throughput analysis, is considered SSCs niche is in the basal compartment of the seminiferous to be more reflective of the biological phenotype compared tubules. SSCs undergo spermatogenesis to produce sperma- to genomic, transcriptomic and proteomic studies (7). Thus, tozoa. Self-renewal and the potential to differentiate are two metabolomics may be particularly well-suited to detect the dynamic modifications that occur during complex biological processes. Metabolomics has been used in the discovery of small molecules that are potential biomarkers of self-renewal (8), reprogramming (9) and differentiation (10), Correspondence to: Professor Yankai Xia or Professor Xin Wu, and it has also been previously used to reveal metabolic State Key Laboratory of Reproductive Medicine, Institute of mechanisms related to spermatogenesis (11,12). Toxicology, Nanjing Medical University, 101 Longmian Road, In the present study, metabolomics was used to evaluate the Nanjing, Jiangsu 211166, P.R. China alterations in SSCs metabolites, including glycylglycine and E-mail: [email protected] sorbitol, following GDNF deprivation, in order to elucidate the E‑mail: [email protected] underlying mechanisms of GDNF-dependent proliferation. *Contributed equally Materials and methods Key words: spermatogonial stem cells, glycylglycine, proliferation, metabolomics, glial cell-derived neurotrophic factor Chemicals and reagents. The following chemicals and reagents were purchased from the respective chemical suppliers: Glycylglycine (cat. no. G1002; purity ≥99%; Sigma‑Aldrich; XU et al: GLYCYLGLYCINE PLAYS CRITICAL ROLES IN SPERMATOGONIAL STEM CELL PROLIFERATION 3803 Merck KGaA, Darmstadt, Germany); sorbitol (cat. no. S6021; sonicated for 3 min (frequency, 20 kHz; power, 60%; pulses, purity ≥98%; Sigma‑Aldrich; Merck KGaA); TRIzol® 6/4) and centrifuged at 16,099 x g at 4˚C for 15 min to remove (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, cellular debris. The supernatant was subjected to metabolomic USA); the Cell Counting Kit-8 (CCK-8) assay kit (Beyotime analysis. Quality control (QC) samples were prepared by Institute of Biotechnology, Haimen, China); and the cDNA mixing equal volumes of each SSCs sample. Synthesis kit and SYBR® Green Master Mix kit (Takara The metabolomic analysis was performed on an UltiMate™ Bio, Inc., Otsu, Japan). The Cell Light™ EdU kit (cat. 3000 ultra high-performance liquid chromatography system no. C10310) was purchased from Guangzhou RiboBio Co., (Dionex; Thermo Fisher Scientific, Inc.), coupled to an Ltd. (Guangzhou, China). Orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific, Inc.) in both positive and negative modes simultane- SSCs culture. SSCs were cultured in vitro following a previ- ously (15). The detailed operating procedures was conducted ously described protocol (13), and the cells were characterized according to a previous study (16). Through this approach, as in a previous study (14). All experiments involving mice >70% of differential metabolites observed in the QC sample were approved by the Institutional Animal Care and Use had a percent relative standard deviation (%RSD) of <30%, Committee of Nanjing Medical University (IACUC:1601247). and the internal standard had a %RSD of <20%, indicating the The primary cells were isolated from 6-8 day old male reliability of the metabolomic analysis (17). C57BL/6 mice obtained from Nanjing Medical University. The mice were housed in groups in a polypropylene cages Cell viability assay and proliferation. Cellular viability was at 21±2˚C, a humidity of 50±10% and a 12 h light/dark cycle evaluated using the CCK-8 Kit. Cells were plated at a density (lights on at 7:00 a.m.), and THY1-positive cells were enriched of 1.5x104 cells/well in 96-well plates and incubated overnight using magnetic-activated cell separation (Miltenyi Biotech (37˚C, 5% CO2). The SSCs treatment groups were: Complete GmbH, Bergisch Gladbach, Germany). Cells were plated at a medium; GDNF deprivation; GDNF deprivation with glycylg- density of 1.5-2.0x105 cells/well on 12-well plates coated with lycine rescue; and GDNF deprivation with sorbitol rescue. On mitotically inactivated SIM mouse embryo-derived thiogua- the 2nd, 3rd, 4 and 5th days, 10 µl CCK‑8 solution was added nine- and ouabain-resistant feeder layers (cat. no. SNLP76/7-4; to each well, and the cells were incubated for 2 h at 37˚C in 5% American Type Culture Collection, Manassas, VA, USA). CO2. The medium was replaced every 2 days. The absorbance Long-term cultures of SSCs were supported in serum-free was determined using a TECAN infinite M200 plate reader Minimum Essential Medium-α (Thermo Fisher Scientific, (Tecan Group, Ltd., Mannedorf, Switzerland) at 450 nm. Inc.) and supplied with 20 ng/ml GDNF (R&D Systems The Cell Light™ EdU kit was used to assess SSCs prolif- China Co., Ltd., Shanghai, China), 1 ng/ml basic fibroblast eration. SSCs (~8x103 cells/well) were seeded in a 96-well growth factor 2 (BD Biosciences, San Jose, CA, USA), and plate and treated for 5 days as aforementioned. EdU (25 µM) 150 ng/ml GDNF family receptor α1 (GFRA1; R&D Systems was added to the culture medium for an additional 10 h. The China Co., Ltd.). In addition, 2 mM L‑glutamine (Thermo cells were fixed with 4% formaldehyde for 30 min at room Fisher Scientific, Inc.), 2% bovine serum albumin, 10 µg/ml temperature and neutralized using glycine (2 mg/ml) for transferrin, 50 µM free fatty acid mixture (5.6 mM linolenic 10 min at room temperature, according to the manufacturer's acid, 13.4 mM oleic acid, 2.8 mM palmitoleic acid, 35.6 mM instructions. Then the cells were permeabilized using 0.5% linoleic acid, 31 mM palmitic acid, 76.9 mM stearic acid;), Triton X-100 in PBS for 20 min, and staining with Hoechst 30 nM Na2SeO3, 50 µM 2‑mercaptoethanol, 5 µg/ml insulin, 33342 was performed. Cell images and data were automati- 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid cally obtained using High Content Screening (HCS; Cellomics and 60 µM putrescine, all purchased from Sigma‑Aldrich; ArrayScan VTI HCS Reader; Thermo Fisher Scientific, Inc.). Merck KGaA, were added to the medium. Cells were main- Appropriate filter sets for the detection of two fluorophores tained in a humidified atmosphere at 5% CO2 and 37˚C. The were used, and different fluorescent signals were recorded in medium was replaced every 2 days and cells were passaged two different image collection channels. Channel 1 contained every 5-6 days. For GDNF deprivation, the concentration the blue nuclear images; channel 2 contained the red images of GDNF was reduced to 0.1 ng/ml for 12 h or 24 h when indicating where EdU was incorporated and which cells were SSCs were ~50% confluent. For the rescue assay, two doses proliferating. For each treatment, three independent wells were (1 and 10 µM) of each metabolite (glycylglycine and sorbitol) measured. The x20 objective was used to collect images, and were chosen for optimization (according to http://www.hmdb.
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