Effect of NMDA Receptor Agonist and Antagonist on Spermatogonia Stem Cells Proliferation in 2- and 3- Dimensional Culture Systems

Effect of NMDA Receptor Agonist and Antagonist on Spermatogonia Stem Cells Proliferation in 2- and 3- Dimensional Culture Systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of NMDA Receptor Agonist and Antagonist on Spermatogonia Stem Cells Proliferation in 2- and 3- Dimensional Culture Systems Amir Hessam Eskafi Noghani, Reza Asadpour, Adel Saberivand, Zohreh Mazaheri, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-840124/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801or Mk) on the proliferation of SSCs in two-dimensional (2D) and three-dimensional (3D) culture systems. The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was confirmed by anti- Plzf and anti- GFR-α1 antibodies via immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated via qRT-PCR and flow cytometry methods, respectively. The results showed that the proliferation rate of SSCs in three-dimensional culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from other groups after 14 days treatment. mRNA expression levels of Plzf in 3D-cultures supplemented by 20 mM D-serine and 50 mM glutamic acid were considerably higher than the 3D control group (p<0.001). The flow cytometry analysis revealed that the amount of Plzf in the 2D-culture groups of SSCs with 20mM MK-801 was considerably lower compared to the 2D-culture control group (p<0.001). This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs. Molecular Biology NMDARs Spermatogonia stem cells Two-dimensional culture system Three-dimensional culture system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Spermatogonia Stem Cells (SSCs) in the testis have self-renewal, proliferation, and differentiation capacities and provide a spermatogenesis process in the seminiferous tubules [1]. The process of spermatogenesis is impaired by chemotherapeutic agents and radiotherapy procedures in cancer patients [2]. Therefore, in these patients, obtaining cells from testicular tissue and the in-vitro proliferation of SSCs are critical steps to enhance the rate of SSC transplantation in patients [3]. The most critical issue for the proliferation of in-vitro SSCs is to have an environment close to the microenvironment of seminiferous tubules [4]. Some experiments indicated that three-dimensional (3D) culture systems provide an appropriate niche for cells, like seminiferous tubules [5]. Recently, DTM hydrogel scaffold introduces a different approach to the study of SSCs which improves cell-to-cell connectivity and facilitates the propagation of SSCs [6, 7]. N-methyl d-aspartate type glutamate receptor (NMDAR) is an ionotropic receptor consisting of two obligatory GluN1(NR1) subunits, with high binding affinity and agonist effect of glycine or D-serine combined with two subunits of GluN2 and /or two GluN3 subunits that all bind glutamate [8]. Recent studies indicated that several subtypes of ionotropic glutamate receptor (GluRs) are expressed in rat testis [9] . NMDARs were found in germinal epithelium and interstitial spaces [10, 11]. Previous studies have revealed that the agonist NMDA subtype of glutamate receptors directly affects spermatogonia mitotic activity [10, 12], spermatogonia proliferation [13], and spermatozoa maturation via the modulation of testosterone and estrogen amounts in the epididymis [14]. Recently, the D-amino acids were detected in various types of testis cells, including Sertoli cell, Leydig cell, spermatogonia, spermatid, and spermatozoa [15, 16]. It was reported D-serine plays a key role in brain tissue physiology [17], cell development, and cell death signaling [18]. D-serine binds to the NMDA subtype of glutamate receptors and acts like D-aspartate on the hypothalamic-pituitary axis or directly on testicular Leydig cells and promotes testosterone synthesis [13]. Moreover, D-serine may upregulate spermatogenesis by enhancing the activity of NMDA receptors [12]. Dizocilpine (MK-801) is a non-competitive glutamate receptor antagonist which affects the central nervous system. MK-801-induced schizophrenia models describe injuries in the testis in response to reactive oxygen species (ROS) [19]. Moreover, it was shown testosterone declines after the blockade of NMDA receptors by a noncompetitive NMDA receptor antagonist (MK-801), and it is attributed to the reduction of sperm motility [9]. Consequently, cell-scaffold interaction may enhance the cell survival rate. Since there is no available data on the proliferative effect of agonists NMDA receptors on SSCs. The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801or Mk) on the proliferation of SSCs in two-dimensional (2D) and three-dimensional (3D) culture systems. Materials And Methods Animals and Drugs Male Naval Medical Research Institute [NMRI) mice were used in this examination. The animals were obtained from Animal Resources Department, University of Tabriz (Tabriz, Iran). The mice were kept in animal houses under standard conditions (at 22 ± 2 °C room temperature, 50–55% humidity, light / dark cycles: 12/12 hours per day). The chemicals that were used in this study were purchased from Sigma–Aldrich unless otherwise mentioned. Testis dissociation and SSCs isolation Ten male NMRI mice (6 days old; n =10) were euthanized with intraperitoneal injection 100 mg/Kg ketamine and 10 mg /Kg xylazine. Their testis was removed and placed into a petri dish containing culture medium and antibiotic. Testicular tissue cells were isolated with slight modifications according to the methods presented by [20]. In summary, after washing the testicular tissue several times in PBS solution for cell isolation, the testicular capsule was removed, and then the testicular tissue was divided into approximately 1 mm³ using the mechanical procedure. Thereafter, the dissociated tissues were placed in a solution containing trypsin (0.5 mg / ml, Sigma-Aldrich, St Louis, MO, USA), collagenase I (0.5 mg / mL Sigma-Aldrich, St Louis, MO, USA), and hyaluronidase (0.5 mg / mL, Sigma-Aldrich, St Louis, MO, USA). Then, a cell was centrifuged (1500 rpm for 5 min) and washed in DMEM/F12 (Gibco, Grand Island, NY, USA) medium. Finally, the next digestion was accomplished for 15 min in a solution containing collagenase I (1mg/mL, Sigma-Aldrich, St Louis, MO, USA) DNase (0.5 mg/mL Sigma-Aldrich, St Louis, MO, USA,), and hyaluronidase (1.5 mg/mL, Sigma-Aldrich, St Louis, MO, USA) in the water bath at 37°C. The obtained cells were washed and prepared for culture after passing via a 70 µm cell strainer. SSCs culture and proliferation After enzymatic digestion, cell viability was evaluated with 0.4 % trypan blue solution in a 6-well plate with the culture medium DMEM/F12 containing 10% FBS (Gibco, Grand Island, NY, USA) with penicillin (100 U/mL) and streptomycin (100 µg/mL; Gibco, Grand Island, NY, USA). To isolate the SSCs which usually attach to the bottom of the culture dish later than the somatic cells, the supernatant was removed after 24 hours and centrifuged (1000 rpm for 5 minutes) to separate the non-attached cells. Then cells were cultured in DMEM/F12 medium containing 5 % Knockout serum replacement (KSR) (Gibco, Grand Island, NY, USA) GDNF (10ng/mL, Sigma Alderich St.Louis.MO, USA), LIF (10 3 U/mL, Sigma Alderich St.Louis.MO, USA) EGF (20ng/mL, Sigma Alderich St.Louis.MO, USA) bFGF (10ng/mL, Sigma Alderich St.Louis.MO, USA) in gelatin (0.2 %; Sigma Alderich St.Louis.MO, USA) coated dishes, and then incubated at 35°C in a humidified atmosphere with 5 % CO 2. The medium replaces every 48h . After two weeks since the initial culture, SSCs colonies were observed. After two weeks in culture, the Plzf protein was traced as a positive marker to confirm the identity of the colonies derived from the SSCs [21]. Immunocytochemistry for characterization of SSCs colonies To characterize SSC colonies, Plzf expression was determined by immunocytochemistry (ICC). After fixation with paraformaldehyde (4%) permeability with 0.4 % Triton X100 (Sigma-Aldrich, St Louis, MO, USA) and blocking with 10% goat serum (Sigma-Aldrich, St Louis, MO, USA), cells were treated with rabbit polyclonal antibody anti-Plzf (sc28319;mouse monoclonal, 1:500, Santa Cruz, Houston, USA) and anti-GFR-α1 (pa519873;Rabbit polyclonal, 1:100, Thermo Fisher, Altrincham, UK) for 2 h at 37°C. After washing with PBS, a secondary antibody, Donkey Anti-Rabbit, labeled with fluorescent isothiocyanate (FITC) 1: 100 (Sigma-Aldrich, St Louis, MO, USA), was added for 3 h. Control cells were treated under similar conditions except for the removal of primary antibodies. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 1μg / mL; Sigma-Aldrich, St Louis, MO, USA) [22]. Tissue decellularization In this experimental study, testicular scaffolds were generated using the testes obtained from 6 mice models (6-8 weeks old). The tunica albuginea was removed from the testes of the mice using a 29-gage insulin syringe and washed with phosphate buffer saline [PBS, Sigma, USA) to remove the residual blood. The sections of 100 µm diameters of testis were immersed in % 0.5 (v/v) of sodium dodecyl sulfate (SDS, Sigma-Aldrich, St Louis, MO, USA) and % 0.5 (v/v) triton X-100 (TX-100; Sigma-Aldrich, St Louis, MO, USA) and then incubated for 18 h in water. Then, they were placed by PBS for 2 h to remove detergents. After washing, the decellularized tissue sections were sterilized in 70% ethanol for 1 hour and immersed in PBS for 2 hours. Before usage, the scaffolds were immersed in a culture medium for 24 hours [23]. Histological examination Hematoxylin-eosin (H&E) staining was used to confirm the quality of DTM and evaluate cell migration to the scaffold after its recellularization. Each sample was fixed in formaldehyde [10%), dehydrated through increasing ethanol concentration, after clearing by xylen and embedding in paraffin, 5-μm thick sections were prepared with a microtome (Leica, Germany). Sections were deparaffinized, rehydrated, and stained via Hematoxylin & eosin (all from Merck Germany). Alcian blue staining (Merck, Germany) was conducted to assess the amount of glycosaminoglycan's (GAGs) based on previous described protocols [24]. Masson's Trichrome staining was performed according to guidelines to determine the existence of collagen [25]. Slides were observed under a light microscope (Labomed, USA), and images (nuclei are stained dark blue, cytoplasm are stained bright-red, as well as collagen is stained green or blue) were taken with a Ziess Camera. DAPI staining for nuclei staining of decellularized tissue Both confirmed the intact and decellularization of the testis by 4, 6-diamidino-2- phenylindol (DAPI) staining and DNA quantification. To assess the homogeneity of decellularization, the samples were collected from different sites of the scaffolds. In brief, DAPI solution (1μg/mL; Sigma-Aldrich, St Louis, MO, USA) was pipetted directly to each tissue section after fixing the tissues in formalin. They were kept in the dark room for 15 min. After washing with PBS, the samples were evaluated with an inverted fluorescence microscope. DNA content in decellularized testicular tissue For quantitative testing, the total amount of DNA from native tissue and decellularized testicular scaffold were extracted by a genomic DNA purification kit (Qiagen, UK). The purity and concentration of DNA were calculated by Nanodrop spectrophotometers (UV-visible; Thermo Nanodrop ND-1000) at 260 nm and agarose gel [24]. Synthesis of dual-crosslinked hydrogels: Natural ECM of decellularized testis and Hyaluronic acid hydrogel For composite-cross-linked hydrogels, 10 mg of sodium hyaluronate powder was dispensed into a circular silicone rubber mold. The same volume of lyophilized testicular tissue extract was sandwiched between two rubber molds and tightly secured. Crosslinking solutions consisted of 0.2 M NaOH containing butanediol diglycidyl ether (BDDE). The concentration of cross linkers was measured in units of µL BDDE/g HA [25]. This fluid was inserted into each rubber mold by a syringe equipped with a 25-gage needle. A second needle created an air escape, and caution was considered to stop bubble trapping. A volume of crosslinking fluid was applied to fill each mold. Molds were roughly 100 µL in volume and had an HA concentration of 10%. HA and crosslink solution were then incubated at 40ºC for 8 h. The cross-linked hydrogels were separated from the molds and relocated to a large amount of distilled deionized water or saline (0.9% NaCl) for swelling. Cytocompatibility of scaffold by MTT assay Following the scaffold was produced, its toxicity was tested to confirm that it did not contain any hazardous substances during the scaffold preparation and decellularization stages. After the culture of SSCs at a concentration of 5 ×10 4 in each well of 96-well plate, scaffold extract was added to cells in the culture medium, and after 24 h, 48 h, and 72 h since the inception of the culture, the cell was evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In brief, add 200 µl of MTT solution (0.5ng/ml) to each well and incubated for 4 h at 37 °C in a humidified atmosphere 5 % CO 2 incubator, and after that, cells lysed and purple formazan crystal dissolved by adding 400 μL of DMSO (D8418, Sigma-Aldrich, St Louis, MO, USA) in each well for 30 min at room temperature. Finally, the purple formazan formed during the test step was measured at 540 nm with the microplate reader [1]. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) evaluated the 3D structure of scaffold, quality, decellularization, and cell migration after recellularization. The tissues were fixed with 2.5% glutaraldehyde for 2 h, and then with 1% osmium tetroxide and dewatering by increasing the degrees of ethanol (30 to 100%). After dehydration, the tissues were placed on a grid, covered with a gold-palladium coating, and seen via SEM (FEI Quanta 200). The number and diameter of SSCs colonies in each field and pores size of the scaffold were analyzed using SEM [26]. Preparation of DTM and Reconstitution of testicular cells on it Reconstituted testicular artificial tissue using a hyaluronic gel matrix 2% and 50 mg of Decellularized testes extract (1:1 v/v ratio) were placed in a 24-well culture dish in a thin layer form. Then, 1×10 5 cells were added to each well plate (Nunc, Roskilde, Denmark) to which collagen gel matrix and somatic cells adhere poorly. After gently agitating the gels for 1 minute, they were overlaid with DMEM/F12 medium supplemented with 5 % KSR, 5 % FBS. During culture, the period medium was replaced every 2 days. After two weeks of culture, the matrix gel was re-dissolved with 0.25% trypsin to release the embedded cells, and then cell viability and proliferation were determined after two weeks of initiation of culture [25]. Experimental Design The culture groups were classified into 2 categories include (1) 2D culture and (2) 3D culture. Each group were defined in a subgroup with different treatments include 1a (2D- D-Serine) or 2a (3D- D-Serine); D-Serine treatment, 1b [2D- Glutamic acid) or 2b (3D- Glutamic acid); Glutamic acid, 1c (2D- MK-801) or 2c; MK-801 (3D- MK-801). D-Serine and MK-801 were used 20 µM, and Glutamic acid was used 50 µM in a final concentration. SSCs cultured in each experimental group for 2 weeks for assessment of SSCs proliferation in different culture medium situations. Two normal groups included 2D and 3D without D-serine, and MK-801 were designed as control groups in the whole study phases. Gene expression analysis In the second weeks of proliferation culture, the total RNA of the cells cultured in 2D and 3D culture was extracted using guanidine / phenol solution (Qiazol-Qiagene USA). Then RNA was treated with DNase I (Fermentase, USA) to eliminate genomic contamination. The purity and concentration of RNA were determined using Nano Drop 2000 (Thermo scientific). For Complementary DNA (cDNA) synthesis, 1 µg total RNA was carried out by reverse transcription kit, using a Prime Script RT reagent kit (Hyper script RT-PCR- GeneAll) according to the manufacture instructions. PCR was carried out using mixed Master Mix and SYBR green in the stage one of thermal cycle (ABI stage one, USA). PCR program began with melting cycle of 15 min at 95°C. The stage was followed by 40 cycles: initial denaturation 30s at 95°C, annealing 30s at 60ºC, and extension 30s at 60ºC. The specific primers used to determine the expression levels of pre-meiotic genes including Plzf [Forward: 5' AGT GGG ATT GAT GAG GAG ATG G 3' and Reverse 5' AGT GGA GTG TAG GGA GAA GGA 3’) as SSCs specific markers and βactin (Forward 5' TCA GAG CAA GAG AGG CAT CC 3'and Reverse: 5' GGT CAT CTTCTC ACG GTT GG 3') as internal control gene. To investigate the presence of non-specific products and primer dimers, a melting curve analysis was performed. Furthermore, this process was replicated in triplicates for target and reference genes. All samples were normalized to the βactin as a reference gene using (∆∆CT) method [27]. Protein evaluation by Flow cytometry The process of cell staining for flow cytometry was done based on pervious researchers by Kanatsu et al. [28]. Furthermore, the cells fixed at 4% paraformaldehyde and permeabilized at 0.5% Triton X-100 (Invitrogen, UK) for 5 minutes before being blocked by a combination of 10% goat serum and Bovine serum albumin [BSA, 1 mg/ml). The incubation of cells was performed with the primary antibodies, including rabbit anti Plzf at 4°C overnight. Cells were incubated with a certain number of secondary antibodies (FITC, conjugated goat anti rabbit IgG) 60 min at 37° C in the dark. After washing with PBS, flow cytometric analysis was accomplished with a Becton Dickinson (BD) and was analyzed with Flowjo 7.6 software. Statistical analysis The data presented are expressed as mean ± standard error (mean ± SEM). Kolmogorov-Smirnov test was used to determine the normal distribution of data. An independent sample t-test was used for DNA content and MTT analysis. For multiple comparisons of data, One-way analysis of variance (ANOVA) followed by Tukey’s test used. Graph Pad Prism software version 8 (Graph Pad Software San Diego, CA) was used for data analysis. Data with less than < 0.05 were considered statically significant. Results SSCs colonies characterization SSCs were isolated using a protocol involving two enzymatic digestion steps. SSCs proliferating colonies were observed approximately two week after obtaining the culture of cells derived from testicular tissues. The small colonies formed from them were observed in the culture medium, and the number of these colonies increased. To confirm the identity of SSCs, the colonies were investigated using two specific markers of SSCs ( Plzf and GFRα1 ) via ICC staining (Fig.1 A,B ). The results showed that the majority of colonies are positive with both SSCs markers. For further confirmation, the gene expression related to SSCs was measured by qRT-PCR prior to the initiation of the differentiation phase. Evaluation of decellularization The first stage of analyzing the effectiveness of decellularization is to validate the removal of cellular components. The histological evaluation indicated that the cells were destroyed with SDS and Triton x100 in the testis. H&E staining demonstrated well preserved structural architecture of the testis with no cell fragments remained in the tissue. The empty seminiferous tubules are clearly distinguishable. The total amount of glycosaminoglycans (GAGs) in Alcian blue and Masson's trichrome staining were tested and showed strong retention in decellularized testis relative to normal tissue (Fig.2,A). The remaining GAG in the scaffold was stained with Alcian blue, indicating that more than 80% of GAG remained. No significant difference was observed in decellularized testis compared to the normal (p=0.1964) (Fig.2,C). The amount of DNA was measured to evaluate the efficiency of the decellularization method and compared to DNA amount in native tissue. Analyzing DNA contents showed that DNA was successfully removed from the testis compared to the normal group (p<0.0001) (Fig.2, B). The pictures of the SEM assessment were showed a tubular structure in the tissue without any cells. Besides, the structure was remained normal in some parts of tissue (Fig. 2, D). Proliferation of SSC on scaffold after 14 days culture MTT results demonstrated that the decellularized scaffolding was non-toxic. SSCs were cultured inside the decellularized testicular scaffold, and cell viability was assessed after 24 h and 48 h. The proliferation rate of SSCs in the three-dimensional culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from the other groups after 14 days of treatment. Nevertheless, the proliferation rate of SSCs in the 3D-culture with MK-801 was significantly lower than in other groups (Fig. 3). Molecular evaluation of Plzf in 3-D cultured SSC Our results indicated that the expression of Plzf gene in the 3D group of spermatogonia stem cell cultures with 20 Mm D-serine and 50 mM glutamic acid was significantly greater than the control group (p<0.001). The expression of desired gene in the 2D and 3D group of SSC culture with 20mM MK-801 (MK20) was, decreased compared to each of the control groups, respectively. However, the alteration in gene expression was not statistically significant (p=0.294, p=1.064 respectively) (Fig. 4). Protein level PLZF marker in SSCs in different experimental groups The detection of germ cells at the protein level was performed via one in all experimental groups. The flow cytometric results revealed that the expression of Plzf protein in the 3D culture group of SSCs with D-serine and glutamic was substantially more than the 3D culture control group (p<0.001). The expression of Plzf in the 2D culture groups of SSCs with MK-801 was lower than the 2D culture control group (p<0.001). No significant difference was observed in the 3D culture of SSC with MK-801 compared to 3D culture control (p=0.187) Discussion For the first time, we developed the SSCs culture in the DTM hydrogel scaffold and indicated the role of D-serine, and MK-801 as the agonist and antagonist of NMDA receptors, respectively, in the spermatogenesis process. Decellularized testicular matrix [DTM) hydrogel scaffold is suitable for the surrounded SSCs and Sertoli cells to enhance the proliferation and differentiation of SSCs [ 29 ]. Our results, like previous findings, showed that the DTM hydrogel scaffold is appropriate for the proliferation of SSCs [ 30 ]. DTM hydrogel scaffolds contain laminin and collagen, which can regulate the proliferation of SSCs and so, the purity of SSCs increases [ 29 , 31 ]. The other constituents of DTM are GAGs which surround the Sertoli cells and Leydig cells and affect cell adhesion, and proliferation [ 32 ]. These results showed that DTM contains some growth factors which can maintain SSCs activity and gene expression. Moreover, this study's results revealed that the supplementation of a 3D culture medium with glutamic acid improved spermatogonia proliferation. Similar to these results, Santillo et al. revealed that the addition of glutamate to spermatogonia germ cell 1 (GC1) medium increased spermatogonia proliferation [ 33 ]. The previous study showed that glutamate-induced the phosphorylation of ERK1/2 protein in spermatogonia GC1 cells [ 33 ]. Moreover, a surge of Akt activity in spermatogonia cells treated with glutamate was observed [ 10 ]. It has already been shown that extracellular-regulated kinase (ERK1) and Akt regulate various biological functions such as gene expression and cell cycle [ 34 , 35 ], and play an important role in germ cell proliferation [ 36 ]. Besides, it was declared that L-glutamine receptors are functionally expressed in rat testis which might promote stem cell proliferation by increasing glutathione levels inside and outside the cell as well as cysteine/cysteine balance [ 9 , 37 , 38 ]. Another mechanism is that glutamic acid stimulates the proliferation of SSCs via anti-apoptotic pathways and by increasing testosterone in Leydig cells [ 39 ]. Based on the results, we found that enriched 3D culture medium with D-serine enhances SSCs proliferation. In agreement with our results, stated that enriched media with D-serine had significantly increased the proliferation and survival of SSC [ 18 ]. On the other hand, it was indicated that treatment with D-serine increased the number of germ cells in silkworms via ERK phosphorylation in the testis [ 39 ]. After treating testes with D-serine, phosphorylated ERK was identified in spermatocytes and spermatids [ 40 ] which is consistent with an earlier finding that the meiosis of rat spermatocytes depended on the ERK activation of rat spermatocytes [ 41 ]. Furthermore, agonist NMDA receptors can stimulate the proliferation of stem cells and increase their population in culture by supporting the intracellular antioxidant glutathione synthesis and reducing intracellular ROS [ 42 ]. This study's findings revealed that the survival rate of SSCs in the 3D culture decreased after treating 3D and 2D cultures with MK-801. In line with our previous results, the experiments showed that the administration of MK-801 to rat testis resulted in increased oxidative stress in rat testis [ 43 ]. Similarly, Saleh et al. demonstrated that the administration of MK-801 to rabbits increased the atrophy of the tubular structure and the degeneration of germinal cells in the lumen of the seminiferous tubule in histopathological evaluation [ 44 ]. Moreover, it was reported that the level of total antioxidant status and sperm kinematics parameters have decreased following the administration of MK-801 [ 45 , 46 ]. Histological data showed that using MK- 801 as antagonist NMDA, due to the testicular seminiferous tubule atrophy, degenerative changes in the epithelial cells, and a decrease in the number of the spermatozoa and necrotic [ 47 ]. MK-801 reduces SSCs proliferation by increasing ROS production and increasing the expression of Bax and Bcl2 in SSCs [ 18 ]. These results showed that the presence of D-serine alone or along with glutamic acid in the 3D culture of mouse SSCs creates a testis-like microenvironment. Conclusions In summary, this study indicated that the DTM hydrogel scaffold culture system provided a simple and alternative method for culturing SSCs that eliminates potential diversity and contamination caused by feeder cells. Moreover, our results showed that supplemented culture medium with 20mM D-serine and 50mM glutamic acid could be considered a promising vehicle to support cells and provide a suitable niche for the proliferation of SSCs. The results of this study could be a way to further study the process of spermatogenesis in vitro and provide novel therapeutics for a higher proliferation rate of SSCs in male infertility to achieve an acceptable SSCs number. Declarations Acknowledgments This study is part of the Ph.D. thesis in the Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, and is sponsored financially by the research deputy of the University of Tabriz. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflicts of interest The authors have no conflicts of interest to declare which are relevant to the content of this article. Availability of data and material The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Code availability Not applicable' for that section. Authors' contributions Amir Hessam Eskafi Noghani: performed the research and prepared manuscript Reza Asadpour : contributed to study design, wrote the draft, and interpreted the data. AS: contributed in performing study and revised the manuscript Zohreh Mazaheri: contributed in the construction of 3D culture system, interpret the data, Gholamreza Hamidian: revise the manuscript and interpret histology data; all authors approved the submitted version. Ethics approval Ethics Committee approved all experimental procedures of the University of Tabriz, Tabriz, Iran, to use animals (Permission No. IR.TABRIZU.REC.1399.045). Consent to participate Not applicable' for that section. Consent for publication Not applicable' for that section. References Movassagh SA, Movassagh SA, Dehkordi MB, Pourmand G, Gholami K, Talebi A et al (2020) Isolation, identification and differentiation of human spermatogonial cells on three-dimensional decellularized sheep testis. Acta Histochem 122(8):151623 AbuMadighem A, Solomon R, Stepanovsky A, Kapelushnik J, Shi Q, Meese E et al (2018) Development of spermatogenesis in vitro in three-dimensional culture from spermatogonial cells of busulfan-treated immature mice. Int J Mol Sci 19(12):3804 Sanjo H, Komeya M, Sato T, Abe T, Katagiri K, Yamanaka H et al (2018) In vitro mouse spermatogenesis with an organ culture method in chemically defined medium. PLoS One 13(2):e0192884 Huleihel M, Nourashrafeddin S, Plant TM (2015) Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta). Asian J Androl 17(6):972 Gholami K, Pourmand G, Koruji M, Ashouri S, Abbasi M (2018) Organ culture of seminiferous tubules using a modified soft agar culture system. Stem Cell Res Ther 9(1):249 Baert Y, Rombaut C, Goossens E (2017) Scaffold-based and scaffold-free testicular organoids from primary human testicular cells. InOrganoids Humana, New York, pp 283–290 Murdock MH, David S, Swinehart IT, Reing JE, Tran K, Gassei K et al (2019) Human testis extracellular matrix enhances human spermatogonial stem cell survival in vitro. Tissue Eng Part A 25(7–8):663–676 Huang X-T, Li C, Peng X-P, Guo J, Yue S-J, Liu W et al (2017) An excessive increase in glutamate contributes to glucose-toxicity in β-cells via activation of pancreatic NMDA receptors in rodent diabetes. Sci Rep 7:44120 Takeshi T, Balcar Vladimir J, Hideo T (2004) Possible expression of functional glutamate transporters in the rat testis. J Endocrinol 181:233–244 Santillo A, Falvo S, Chieffi P, Burrone L, Chieffi Baccari G, Longobardi S et al (2014) D-aspartate affects NMDA receptor-extracellular signal-regulated kinase pathway and upregulates androgen receptor expression in the rat testis. Theriogenology 81(5):744–751 Storto M, Sallese M, Salvatore L, Poulet R, Condorelli D, Dell'Albani P et al (2001) Erratum: Expression of metabotropic glutamate receptors in the rat and human testis. J Endocrinol 170(2):71–78 Di Fiore MM, Santillo A, Falvo S, Longobardi S, Chieffi Baccari G (2016) Molecular mechanisms elicited by D-aspartate in Leydig cells and spermatogonia. Int J Mol Sci 17(7):1127 Santillo A, Falvo S, Chieffi P, Di Fiore MM, Senese R, Chieffi Baccari G (2016) D-Aspartate induces proliferative pathways in spermatogonial GC-1 cells. J Cell Physiol 231:490–495 Falvo E, Tremante E, Arcovito A, Papi M, Elad N, Boffi A et al (2016) Improved doxorubicin encapsulation and pharmacokinetics of ferritin–fusion protein nanocarriers bearing proline, serine, and alanine elements. Biomacromol 17:514–522 D’Aniello G, Ronsini S, Guida F, Spinelli P, D’Aniello A (2005) Occurrence of D-aspartic acid in human seminal plasma and spermatozoa: possible role in reproduction. Fertil Steril 84(5):1444–1449 Sakai K, Homma H, Lee J-A, Fukushima T, Santa T, Tashiro K et al (1998) Localization ofd-Aspartic Acid in Elongate Spermatids in Rat Testis. Arch Biochem Biophys 351(1):96–105 Matsui Ta, Sekiguchi M, Hashimoto A, Tomita U, Nishikawa T, Wada K (1995) Functional comparison of D-serine and glycine in rodents: the effect on cloned NMDA receptors and the extracellular concentration. J Neurochem 65(1):454–458 Modirshanechi G, Eslampour MA, Abdolmaleki Z (2020) Agonist and antagonist NMDA receptor effect on cell fate during germ cell differentiation and regulate apoptotic process in 3D organ culture. Andrologia e13764 Turkmen R, Akosman MS, Demirel HH (2019) Protective effect of N-acetylcysteine on MK-801-induced testicular oxidative stress in mice. Biomed Pharmacother 109:1988–1993 Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T (2003) Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69(2):612–616 Sadri-Ardekani H, Mizrak SC, van Daalen SK, Korver CM, Roepers-Gajadien HL, Koruji M, Hovingh S, de Reijke TM, de la Rosette JJ, van der Veen F, de Rooij DG (2009) Propagation of human spermatogonial stem cells in vitro. Jama 302(19):2127–2134 Mahaldashtian M, Naghdi M, Ghorbanian MT, Makoolati Z, Movahedin M, Mohamadi SM (2016) In vitro effects of date palm (Phoenix dactylifera L.) pollen on colonization of neonate mouse spermatogonial stem cells. J Ethnopharmacol 186:362–368 Majidi Gharenaz N, Movahedin M, Mazaheri Z (2020) Three-Dimensional Culture of Mouse Spermatogonial Stem Cells Using A Decellularised Testicular Scaffold. Cell J 21(4):410–418 Daryabari S, Kajbafzadeh A-M, Fendereski K, Ghorbani F, Dehnavi M, Rostami M et al (2019) Development of an efficient perfusion-based protocol for whole-organ decellularization of the ovine uterus as a human-sized model and in vivo application of the bioscaffolds. J Assist Reprod Genet 36:1211–1223 Zawko SA, Suri S, Truong Q, Schmidt CE (2009) Photopatterned anisotropic swelling of dual-crosslinked hyaluronic acid hydrogels. Acta Biomater 5(1):14–22 Schindelin J, Rueden CT, Hiner MC, Eliceiri KW (2015) The ImageJ ecosystem: An open platform for biomedical image analysis. Mol Reprod Dev 82:518–529 Ziloochi Kashani M, Bagher Z, Asgari HR, Najafi M, Koruji M, Mehraein F (2020) Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold. Syst Biol Reprod Med 66(3):202–215 Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M (2004) Generation of pluripotent stem cells from neonatal mouse testis. Cell 119(7):1001–1012 Yang Y, Lin Q, Zhou C, Li Q, Li Z, Cao Z et al (2020) A testis-derived hydrogel as an efficient feeder-free culture platform to promote mouse spermatogonial stem cell proliferation and differentiation. Front Cell Dev Biol 8:250 Topraggaleh TR, Valojerdi MR, Montazeri L, Baharvand H (2019) A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids. Biomater Sci 7(4):1422–1436 Spang MT, Christman KL (2018) Extracellular matrix hydrogel therapies: in vivo applications and development. Acta Biomater 68:1–14 Yang L, Li X, Wu Y, Du P, Sun L, Yu Z et al (2020) Preparation of PU/Fibrin Vascular Scaffold with Good Biomechanical Properties and Evaluation of Its Performance in vitro and in vivo. Int J Nanomedicine 15:8697–8715 Santillo A, Falvo S, Di Fiore MM, Di Giacomo Russo F, Chieffi P, Usiello A et al (2019) AMPA receptor expression in mouse testis and spermatogonial GC-1 cells: A study on its regulation by excitatory amino acids. J Cell Biochem 120(7):11044–11055 Cooper TG, Yeung CH, Nashan D, Nieschlag E (1988) Epididymal markers in human infertility. J Androl 9(2):91–101 Vogiatzi P, Giordano A (2007) Following the tracks of AKT1 gene. Cancer Biol Ther 69(10):1521–1524 Chieffi P, Chieffi S (2013) Molecular biomarkers as potential targets for therapeutic strategies in human testicular germ cell tumors: an overview. J Cell Physiol 228(8):1641–1646 Zhang W, Zhang K, Li G, Yan S, Cui L, Yin J (2018) Effects of large dimensional deformation of a porous structure on stem cell fate activated by poly(l-glutamic acid)-based shape memory scaffolds. Biomater Sci 6(10):2738–2749 Li B, He X, Zhuang M, Niu B, Wu C, Mu H et al (2018) Melatonin ameliorates busulfan-induced spermatogonial stem cell oxidative apoptosis in mouse testes. Antioxid Redox Signal 28(5):385–400 Hu JC-C, Zhang C, Sun X, Yang Y, Cao X, Ryu O et al (2000) Characterization of the mouse and human PRSS17 genes, their relationship to other serine proteases, and the expression of PRSS17 in developing mouse incisors. Gene 251(1):1–8 Suzuki C, Tanigawa M, Tanaka H, Horiike K, Kanekatsu R, Tojo M et al (2014) Effect of d-serine on spermatogenesis and extracellular signal-regulated protein kinase (ERK) phosphorylation in the testis of the silkworm, Bombyx mori. J Insect Physiol 67:97–104 Godet M, Sabido O, Gilleron J, Durand P (2008) Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells. Dev Biol 315(1):173–188 Huang X, Kong H, Tang M, Lu M, Ding JH, Hu G (2012) D-Serine regulates proliferation and neuronal differentiation of neural stem cells from postnatal mouse forebrain. CNS Neurosci Ther 18(1):4–13 Parlaktas BS, Ozyurt B, Ozyurt H, Tunc AT, Akbas A (2008) Levels of oxidative stress parameters and the protective effects of melatonin in psychosis model rat testis. Asian J Androl 10(2):259–265 Saleh SY, Sawiress FA, Tony MA, Hassanin AM, Khattab MA, Bakeer MR (2015) Protective role of some feed additives against dizocelpine induced oxidative stress in testes of rabbit bucks. J Agric Sci 7(10):239 Turkmen R, Akosman MS, Demirel HH (2019) Protective effect of N-acetylcysteine on MK-801-induced testicular oxidative stress in mice. Biomed Pharmacother 109:1988–1993 Zhang X, Wang L, Zhang X, Ren L, Shi W, Tian Y et al (2017) The use of KnockOut serum replacement (KSR) in three dimensional rat testicular cells co-culture model: An improved male reproductive toxicity testing system. Food Chem Toxicol 106:487–495 Ozyurt B, Ozyurt H, Akpolat N, Erdogan H, Sarsilmaz M (2007) Oxidative stress in prefrontal cortex of rat exposed to MK-801 and protective effects of CAPE. Prog Neuropsychopharmacol Biol Psychiatry 31(4):832–838 Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 12 Sep, 2021 Reviewers invited by journal 27 Aug, 2021 Editor assigned by journal 23 Aug, 2021 First submitted to journal 23 Aug, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-840124","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":48700254,"identity":"2fd51cc8-b959-4131-88ea-9760a74c3299","order_by":0,"name":"Amir Hessam Eskafi Noghani","email":"","orcid":"","institution":"University of Tabriz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amir","middleName":"Hessam Eskafi","lastName":"Noghani","suffix":""},{"id":48700255,"identity":"62485d45-6095-4482-943d-ad6e6ea2d295","order_by":1,"name":"Reza Asadpour","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYLACHjB5+ACQkJAhQQvjsQSQFh4StDCfMUDYiA+Ytx9/+OHtDpvE7W1nPr+6UWPBw8B++OgGfFpkzuQYS849k5Y458zZbdY5x4AO40lLu4FPiwRDDoM0b9vhxBkSZ7cZ57ABtUjwmOHXwv/88W+wFvk3z4xz/hGjRSLBDGILwxnmx7ltRGl5Y2YJ9IvxDIZjZsy5fRI8bAT9wp/++AYwxGRnMBx+/DnnW50cP/vhY3i1gAFjA5hikwCTBJUjaWH+QJTqUTAKRsEoGHEAABhJSVBFl6y1AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4717-9952","institution":"University of Tabriz","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Reza","middleName":"","lastName":"Asadpour","suffix":""},{"id":48700256,"identity":"0c4ad878-b233-45ef-81a6-02caae6d76a3","order_by":2,"name":"Adel Saberivand","email":"","orcid":"","institution":"University of Tabriz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Adel","middleName":"","lastName":"Saberivand","suffix":""},{"id":48700257,"identity":"3b4d8c30-baca-4df0-901f-3d7835e77b3b","order_by":3,"name":"Zohreh Mazaheri","email":"","orcid":"","institution":"Institute of Basic Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zohreh","middleName":"","lastName":"Mazaheri","suffix":""},{"id":48700258,"identity":"c04357e0-534d-49b2-9a93-2eea16551764","order_by":4,"name":"Gholamreza Hamidian","email":"","orcid":"","institution":"University of Tabriz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gholamreza","middleName":"","lastName":"Hamidian","suffix":""}],"badges":[],"createdAt":"2021-08-23 11:08:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-840124/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-840124/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":12904373,"identity":"9459a801-add7-4230-8021-7979a075263f","added_by":"auto","created_at":"2021-08-30 18:43:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":388268,"visible":true,"origin":"","legend":"Characterization of SSCs formation. Phenotypic characteristics of SSCs revealed oval shape. Immunostaining of SSCs using Plzf. The ICC results indicated the presence of Plzf \n(A) and GFRα1 (B) under a fluorescence microscope. Nuclei are stained with DAPI. Scale bar, 20 µm.\n","description":"","filename":"Fig01.png","url":"https://assets-eu.researchsquare.com/files/rs-840124/v1/e37d631868dccb6ae406641d.png"},{"id":12904372,"identity":"486193c2-3039-474c-9e1a-fa54503c3378","added_by":"auto","created_at":"2021-08-30 18:43:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":451860,"visible":true,"origin":"","legend":"Histological evaluation of the testis after SDS-based decellularization process. A) H\u0026E staining evidenced preservation of structure. Alcian blue and Masson’s trichrome staining showed the presence of GAGs. Scale bar, 100 µm. B) DNA quantification manifested remarkable cell eliminated by decellularization procedure compared to the normal testis. C) GAGs concentration of decellularized testicular tissue was not significantly altered in comparison with normal tissue. D) SEM assessment of decellularized testicular tissue. The results are presented as means ± SD. different letters: Significant difference with other experimental group (p\u003c0.05).","description":"","filename":"Fig02.png","url":"https://assets-eu.researchsquare.com/files/rs-840124/v1/c61a3a6d4c6956592fdccb10.png"},{"id":12904706,"identity":"45e9a512-f811-4607-955a-f7124aafd3ee","added_by":"auto","created_at":"2021-08-30 18:46:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32707,"visible":true,"origin":"","legend":"Cell compatibility assay of SSCs in different experimental groups after A) 24 h. and B) 48 h. C) Proliferation rate of SSCs on 3D and 2D culture after 14 days cultivation in different culture media. MTT assay represents the toxicity effect of scaffold during 24 \u0026 48 h culture of SSCs. Forth teen days after SSCs cultivation in 2D and 3D condition into different medium indicates a cell proliferation in 3D culture medium treated by Gu50 and DS20. Although, the results showed a proliferation rate decrease in 3D culture medium treated by MK20. SSC: Spermatogonial Stem Cell; DS: D-Serine; Gu: Glutamic acid; MK: MK-801. Different letters: Significant difference with other experimental groups (p\u003c0.05).","description":"","filename":"Fig03.png","url":"https://assets-eu.researchsquare.com/files/rs-840124/v1/6a4cea40474f1d60895059a3.png"},{"id":12904371,"identity":"85495e89-fc0f-4643-b0fd-f179c68d1751","added_by":"auto","created_at":"2021-08-30 18:43:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12952,"visible":true,"origin":"","legend":"Expression level of Plzf in 2D and 3D cultured SSCs with 20 mM D-Serine and 50 mM glutamic acid or MK-801. The real time PCR results were normalized to a reference gene (β-actin gene). The letters shown in the figure 4 have statistically significant differences (p\u003c0.001). SSCs: Spermatogonial Stem Cell; MK: MK-801. The results are presented as means ± SD.","description":"","filename":"Fig04.png","url":"https://assets-eu.researchsquare.com/files/rs-840124/v1/cb0939b552b8758aafbd8e1f.png"},{"id":12904374,"identity":"12466784-b237-4ef1-a132-481d300fc032","added_by":"auto","created_at":"2021-08-30 18:43:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":125749,"visible":true,"origin":"","legend":"Protein level of Plzf in 2D and 3D cultured SSC with 20mM D- serine and 50 mM glutamic acid or MK-801. Intensity of FITC dye was used for analyzing of Plzf protein in comparison with isotype control sample by flow cytometric assay. The different letters shown in the figure 5 have statistically significant differences (p\u003c0.001). SSCs: Spermatogonial Stem Cells; DS: D-Serine; MK: MK801. The results are presented as means ± SD. ","description":"","filename":"Fig05.png","url":"https://assets-eu.researchsquare.com/files/rs-840124/v1/a16d241537ea10636fa48183.png"},{"id":13711978,"identity":"4b655615-9a21-4079-aaac-dada0ab3d5e6","added_by":"auto","created_at":"2021-09-17 14:25:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1389999,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-840124/v1/059f5819-945d-4c28-99fe-27177f893584.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEffect of NMDA Receptor Agonist and Antagonist on Spermatogonia Stem Cells Proliferation in 2- and 3- Dimensional Culture Systems\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpermatogonia Stem Cells (SSCs) in the testis have self-renewal, proliferation, and differentiation capacities and provide a spermatogenesis process in the seminiferous tubules\u0026nbsp;[1]. The process of spermatogenesis is impaired by chemotherapeutic agents and radiotherapy procedures in cancer patients\u0026nbsp;[2]. Therefore, in these patients, obtaining cells from testicular tissue and the in-vitro proliferation of SSCs are critical steps to enhance the rate of SSC transplantation in patients\u0026nbsp;[3]. The most critical issue for the proliferation of in-vitro SSCs is to have an environment close to the microenvironment of seminiferous tubules\u0026nbsp;[4]. Some experiments indicated that three-dimensional (3D) culture systems provide an appropriate niche for cells, like seminiferous tubules\u0026nbsp;[5]. Recently, DTM hydrogel scaffold introduces a different approach to the study of SSCs which improves cell-to-cell connectivity and facilitates the propagation of SSCs\u0026nbsp;[6, 7].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; N-methyl d-aspartate type glutamate receptor (NMDAR) is an ionotropic receptor consisting of two obligatory GluN1(NR1) subunits, with high binding affinity and agonist effect of glycine or D-serine combined with two subunits of GluN2 and /or two GluN3 subunits that all bind glutamate\u0026nbsp;[8]. Recent studies indicated that several subtypes of ionotropic glutamate receptor (GluRs) are expressed in rat testis\u0026nbsp;[9]\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e NMDARs were found in germinal epithelium and interstitial spaces\u0026nbsp;[10, 11]. Previous studies have revealed that the agonist NMDA subtype of glutamate receptors directly affects spermatogonia mitotic activity\u0026nbsp;[10, 12], spermatogonia proliferation\u0026nbsp;[13], and spermatozoa maturation via the modulation of testosterone and estrogen amounts in the epididymis\u0026nbsp;[14].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Recently, the D-amino acids were detected in various types of testis cells, including Sertoli cell, Leydig cell, spermatogonia, spermatid, and spermatozoa\u0026nbsp;[15, 16]. \u0026nbsp;It was reported D-serine plays a key role in brain tissue physiology \u0026nbsp;[17], cell development, and cell death signaling\u0026nbsp;[18]. D-serine binds to the NMDA subtype of glutamate receptors and acts like D-aspartate on the hypothalamic-pituitary axis or directly on testicular Leydig cells and promotes testosterone synthesis\u0026nbsp;[13]. Moreover, D-serine may upregulate spermatogenesis by enhancing the activity of NMDA receptors\u0026nbsp;[12].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; Dizocilpine (MK-801) is a non-competitive glutamate receptor antagonist which affects the central nervous system. MK-801-induced schizophrenia models describe injuries in the testis in response to reactive oxygen species (ROS)\u0026nbsp;[19]. Moreover, it was shown testosterone declines after the blockade of NMDA receptors by a noncompetitive NMDA receptor antagonist (MK-801), and it is attributed to the reduction of sperm motility\u0026nbsp;[9].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; Consequently, cell-scaffold interaction may enhance the cell survival rate. Since there is no available data on the proliferative effect of agonists NMDA receptors on SSCs. The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801or Mk) on the proliferation of SSCs in two-dimensional (2D) and three-dimensional (3D) culture systems.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals and Drugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Male Naval Medical Research Institute [NMRI) mice were used in this examination. The animals were obtained from Animal Resources Department, University of Tabriz (Tabriz, Iran). The mice were kept in animal houses under standard conditions (at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026thinsp;\u0026deg;C room temperature, 50\u0026ndash;55% humidity, light / dark cycles: 12/12 hours per day). The chemicals that were used in this study were purchased from Sigma\u0026ndash;Aldrich unless otherwise mentioned.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTestis dissociation and SSCs isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTen male NMRI mice (6 days old; \u003cem\u003en\u003c/em\u003e=10) were euthanized with intraperitoneal injection 100 mg/Kg ketamine and 10 mg /Kg xylazine. Their testis was removed and placed into a petri dish containing culture medium and antibiotic. Testicular tissue cells were isolated with slight modifications according to the methods presented by [20]. In summary, after washing the testicular tissue several times in PBS solution for cell isolation, the testicular capsule was removed, and then the testicular tissue was divided into approximately 1 mm\u0026sup3; using the mechanical procedure. Thereafter, the dissociated tissues were placed in a solution containing trypsin (0.5 mg / ml, Sigma-Aldrich, St Louis, MO, USA), collagenase I (0.5 mg / mL Sigma-Aldrich, St Louis, MO, USA), and hyaluronidase (0.5 mg / mL, Sigma-Aldrich, St Louis, MO, USA). Then, a cell was centrifuged (1500 rpm for 5 min) and washed in DMEM/F12 (Gibco, Grand Island, NY, USA) medium. Finally, the next digestion was accomplished for 15 min in a solution containing collagenase I (1mg/mL, Sigma-Aldrich, St Louis, MO, USA) DNase (0.5 mg/mL Sigma-Aldrich, St Louis, MO, USA,), and hyaluronidase (1.5 mg/mL, Sigma-Aldrich, St Louis, MO, USA) in the water bath at 37\u0026deg;C. The obtained cells were washed and prepared for culture after passing via a 70 \u0026micro;m cell strainer.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eSSCs culture and proliferation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter enzymatic digestion, cell viability was evaluated with 0.4 % trypan blue solution in a 6-well plate with the culture medium DMEM/F12 containing 10% FBS (Gibco, Grand Island, NY, USA) with penicillin (100 U/mL) and streptomycin (100 \u0026micro;g/mL; Gibco, Grand Island, NY, USA). \u0026nbsp;To isolate the SSCs which usually attach to the bottom of the culture dish later than the somatic cells, the supernatant was removed after 24 hours and centrifuged (1000 rpm for 5 minutes) to separate the non-attached cells. Then cells were cultured in DMEM/F12 medium containing 5 % Knockout serum replacement (KSR) (Gibco, Grand Island, NY, USA) GDNF (10ng/mL, Sigma Alderich St.Louis.MO, USA), LIF (10\u003csup\u003e3\u003c/sup\u003eU/mL, Sigma Alderich St.Louis.MO, USA) EGF (20ng/mL, Sigma Alderich St.Louis.MO, USA) bFGF (10ng/mL, Sigma Alderich St.Louis.MO, USA) in gelatin (0.2 %; Sigma Alderich St.Louis.MO, USA) coated dishes, and then incubated at 35\u0026deg;C in a humidified atmosphere with 5 % CO\u003csub\u003e2.\u0026nbsp;\u003c/sub\u003eThe medium replaces every 48h\u003csub\u003e. \u0026nbsp;\u003c/sub\u003eAfter two weeks since the initial culture, SSCs colonies were observed. After two weeks in culture, the \u003cem\u003ePlzf\u0026nbsp;\u003c/em\u003eprotein was traced as a positive marker to confirm the identity of the colonies derived from the SSCs \u0026nbsp;[21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunocytochemistry for characterization of SSCs colonies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize SSC colonies, \u003cem\u003ePlzf\u0026nbsp;\u003c/em\u003eexpression was determined by immunocytochemistry (ICC). After fixation with paraformaldehyde (4%) permeability with 0.4 % Triton X100\u0026nbsp;(Sigma-Aldrich, St Louis, MO, USA) and blocking with 10% goat serum (Sigma-Aldrich, St Louis, MO, USA), cells were treated with rabbit polyclonal antibody \u003cem\u003eanti-Plzf\u003c/em\u003e (sc28319;mouse monoclonal, 1:500, Santa Cruz, Houston, USA) and anti-GFR-\u0026alpha;1 (pa519873;Rabbit polyclonal, 1:100, Thermo Fisher, Altrincham, UK) for 2 h at 37\u0026deg;C. After washing with PBS, a secondary antibody, Donkey Anti-Rabbit, labeled with fluorescent isothiocyanate (FITC) 1: 100 (Sigma-Aldrich, St Louis, MO, USA), was added for 3 h. Control cells were treated under similar conditions except for the removal of primary antibodies. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 1\u0026mu;g / mL; Sigma-Aldrich, St Louis, MO, USA)\u0026nbsp;[22].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue decellularization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In this experimental study, testicular scaffolds were generated using the testes obtained from 6 mice models (6-8 weeks old). The tunica albuginea was removed from the testes of the mice using a 29-gage insulin syringe and washed with phosphate buffer saline [PBS, Sigma, USA) to remove the residual blood. The sections of 100 \u0026micro;m diameters of testis were immersed in % 0.5 (v/v) of sodium dodecyl sulfate (SDS, Sigma-Aldrich, St Louis, MO, USA) and % 0.5 (v/v) triton X-100 (TX-100; Sigma-Aldrich, St Louis, MO, USA) and then incubated for 18 h in water. Then, they were placed by PBS for 2 h to remove detergents. \u0026nbsp;After washing, the decellularized tissue sections were sterilized in 70% ethanol for 1 hour and immersed in PBS for 2 hours. Before usage, the scaffolds were immersed in a culture medium for 24 hours\u0026nbsp;[23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Histological examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHematoxylin-eosin (H\u0026amp;E) staining was used to confirm the quality of DTM and evaluate cell migration to the scaffold after its recellularization. Each sample was fixed in formaldehyde [10%), dehydrated through increasing ethanol concentration, after clearing by xylen and embedding in paraffin, 5-\u0026mu;m thick sections were prepared with a microtome (Leica, Germany). Sections were deparaffinized, rehydrated, and stained via Hematoxylin \u0026amp; eosin (all from Merck Germany). Alcian blue staining (Merck, Germany) was conducted to assess the amount of glycosaminoglycan\u0026apos;s (GAGs) based on previous described protocols\u0026nbsp;[24]. Masson\u0026apos;s Trichrome staining was performed according to guidelines to determine the existence of collagen\u0026nbsp;[25]. Slides were observed under a light microscope (Labomed, USA), and images (nuclei are stained dark blue, cytoplasm are stained bright-red, as well as collagen is stained green or blue) were taken with a Ziess Camera.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDAPI staining for nuclei staining of decellularized tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth confirmed the intact and decellularization of the testis by 4, 6-diamidino-2- phenylindol (DAPI) staining and DNA quantification. To assess the homogeneity of decellularization, the samples were collected from different sites of the scaffolds. In brief, DAPI solution (1\u0026mu;g/mL; Sigma-Aldrich, St Louis, MO, USA) was pipetted directly to each tissue section after fixing the tissues in formalin. They were kept in the dark room for 15 min. After washing with PBS, the samples were evaluated with an inverted fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA content in decellularized testicular tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor quantitative testing, the total amount of DNA from native tissue and decellularized testicular scaffold were extracted by a genomic DNA purification kit (Qiagen, UK). The \u0026nbsp;purity and concentration of DNA were calculated by Nanodrop spectrophotometers (UV-visible; Thermo Nanodrop ND-1000) at 260 nm and agarose gel\u0026nbsp;[24].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of dual-crosslinked hydrogels: Natural ECM of decellularized testis and Hyaluronic acid hydrogel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;For composite-cross-linked hydrogels, 10 mg of sodium hyaluronate powder was dispensed into a circular silicone rubber mold. The same volume of lyophilized testicular tissue extract was sandwiched between two rubber molds and tightly secured. Crosslinking solutions consisted of 0.2 M NaOH containing butanediol diglycidyl ether\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e(BDDE). The concentration of cross linkers was measured in units of \u0026micro;L BDDE/g HA\u0026nbsp;[25]. This fluid was inserted into each rubber mold by a syringe equipped with a 25-gage needle. A second needle created an air escape, and caution was considered to stop bubble trapping. A volume of crosslinking fluid was applied to fill each mold. Molds were roughly 100 \u0026micro;L in volume and had an HA concentration of 10%. HA and crosslink solution were then incubated at 40\u0026ordm;C for 8 h. The cross-linked hydrogels were separated from the molds and relocated to a large amount of distilled deionized water or saline (0.9% NaCl) for swelling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytocompatibility of scaffold by MTT assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Following the scaffold was produced, its toxicity was tested to confirm that it did not contain any hazardous substances during the scaffold preparation and decellularization stages. After the culture of SSCs at a concentration of 5 \u0026times;10\u003csup\u003e4\u003c/sup\u003e in each well of 96-well plate, scaffold extract was added to cells in the culture medium, and after 24 h, 48 h, and 72 h since the inception of the culture, the cell was evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In brief, add 200 \u0026micro;l of MTT solution (0.5ng/ml) to each well and incubated for 4 h at 37 \u0026deg;C in a humidified atmosphere 5 % CO\u003csub\u003e2\u003c/sub\u003e incubator, and after that, cells lysed and purple formazan crystal dissolved by adding 400 \u0026mu;L of DMSO (D8418, Sigma-Aldrich, St Louis, MO, USA) in each well for 30 min at room temperature. Finally, the purple formazan formed during the test step was measured at 540 nm with the microplate reader\u0026nbsp;[1].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Scanning electron microscopy (SEM) evaluated the 3D structure of scaffold, quality, decellularization, and cell migration after recellularization. \u0026nbsp;The tissues were fixed with 2.5% glutaraldehyde for 2 h, and then with 1% osmium tetroxide and dewatering by increasing the degrees of ethanol (30 to 100%). After dehydration, the tissues were placed on a grid, covered with a gold-palladium coating, and seen \u003cem\u003evia\u003c/em\u003e SEM (FEI Quanta 200). The number and diameter of SSCs colonies in each field and pores size of the scaffold were analyzed using SEM\u0026nbsp;[26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Preparation of DTM and Reconstitution of testicular cells on it\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReconstituted testicular artificial tissue using a hyaluronic gel matrix 2% and 50 mg of Decellularized testes extract (1:1 v/v ratio) were placed in a 24-well culture dish in a thin layer form. Then, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were added to each well plate (Nunc, Roskilde, Denmark) to which collagen gel matrix and somatic cells adhere poorly. After gently agitating the gels for 1 minute, they were overlaid with DMEM/F12 medium supplemented with 5 % KSR, 5 % FBS. During culture, the period medium was replaced every 2 days. After two weeks of culture, the matrix gel was re-dissolved with 0.25% trypsin to release the embedded cells, and then cell viability and proliferation were determined after two weeks of initiation of culture\u0026nbsp;[25].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe culture groups were classified into 2 categories include (1) 2D culture and (2) 3D culture. Each group were defined in a subgroup with different treatments include 1a (2D-\u0026nbsp;D-Serine) or 2a (3D- D-Serine); D-Serine treatment, 1b [2D- Glutamic acid) or 2b (3D- Glutamic acid); Glutamic acid, 1c (2D- MK-801) or 2c; MK-801 (3D- MK-801). D-Serine and MK-801 were used 20 \u0026micro;M, and Glutamic acid was used 50 \u0026micro;M in a final concentration. SSCs cultured in each experimental group for 2 weeks for assessment of SSCs proliferation in different culture medium situations. Two normal groups included 2D and 3D without D-serine, and MK-801 were designed as control groups in the whole study phases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the second weeks of proliferation culture, the total RNA of the cells cultured in 2D and 3D culture was extracted using guanidine / phenol solution (Qiazol-Qiagene USA). Then RNA was treated with DNase I (Fermentase, USA) to eliminate genomic contamination. \u0026nbsp;The purity and concentration of RNA were determined using Nano Drop 2000 (Thermo scientific). For Complementary DNA (cDNA) synthesis, 1 \u0026micro;g total RNA was carried out by reverse transcription kit, using a Prime Script RT reagent kit (Hyper script RT-PCR- GeneAll) according to the manufacture instructions. PCR was carried out using mixed Master Mix and SYBR green in the stage one of thermal cycle (ABI stage one, USA). PCR program began with melting cycle of 15 min at 95\u0026deg;C. The stage was followed by 40 cycles: initial denaturation 30s at 95\u0026deg;C, annealing 30s at 60\u0026ordm;C, and extension 30s at 60\u0026ordm;C. The specific primers used to determine the expression levels of pre-meiotic genes including \u003cem\u003ePlzf\u003c/em\u003e [Forward: 5\u0026apos; AGT GGG ATT GAT GAG GAG ATG G 3\u0026apos; and Reverse 5\u0026apos; AGT GGA GTG TAG GGA GAA GGA 3\u0026rsquo;) as SSCs specific markers and \u0026beta;actin (Forward 5\u0026apos; TCA GAG CAA GAG AGG CAT CC 3\u0026apos;and Reverse: 5\u0026apos; GGT CAT CTTCTC ACG GTT GG 3\u0026apos;) as internal control gene. To investigate the presence of non-specific products and primer dimers, a melting curve analysis was performed. Furthermore, this process was replicated in triplicates for target and reference genes. All samples were normalized to the \u0026beta;actin as a reference gene using (∆∆CT) method\u0026nbsp;[27].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein evaluation by Flow cytometry\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe process of cell staining for flow cytometry was done based on pervious researchers by Kanatsu et al. [28]. Furthermore, the cells fixed at 4% paraformaldehyde and permeabilized at 0.5% Triton X-100 (Invitrogen, UK) for 5 minutes before being blocked by a combination of 10% goat serum and Bovine serum albumin [BSA, 1 mg/ml). The incubation of cells was performed with the primary antibodies, including rabbit anti \u003cem\u003ePlzf\u003c/em\u003e at 4\u0026deg;C overnight. Cells were incubated with a certain number of secondary antibodies (FITC, conjugated goat anti rabbit IgG) 60 min at 37\u0026deg; C in the dark. After washing with PBS, flow cytometric analysis was accomplished with a Becton Dickinson (BD) and was analyzed with Flowjo 7.6 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented are expressed as mean \u0026plusmn; standard error (mean \u0026plusmn; SEM). Kolmogorov-Smirnov test was used to determine the normal distribution of data. An independent sample t-test was used for DNA content and MTT analysis. For multiple comparisons of data, One-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s test used. Graph Pad Prism software version 8 (Graph Pad Software San Diego, CA) was used for data analysis. \u0026nbsp;Data with less than \u0026lt; 0.05 were considered statically significant.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSSCs colonies characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSSCs were isolated using a protocol involving two enzymatic digestion steps. SSCs proliferating colonies were observed approximately two week after obtaining the culture of cells derived from testicular tissues. \u0026nbsp;The small colonies formed from them were observed in the culture medium, and the number of these colonies increased. To confirm the identity of SSCs, the colonies were investigated using two specific markers of SSCs (\u003cem\u003ePlzf\u003c/em\u003e and \u003cem\u003eGFR\u0026alpha;1\u003c/em\u003e) \u003cem\u003evia\u003c/em\u003e ICC staining (Fig.1 A,B ). The results showed that the majority of colonies are positive with both SSCs markers. \u0026nbsp;For further confirmation, the gene expression related to SSCs was measured by qRT-PCR prior to the initiation of the differentiation phase.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of decellularization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The first stage of analyzing the effectiveness of decellularization is to validate the removal of cellular components. The histological evaluation indicated that the cells were destroyed with SDS and Triton x100 in the testis. H\u0026amp;E staining demonstrated well preserved structural architecture of the testis with no cell fragments remained in the tissue. The empty seminiferous tubules are clearly distinguishable. The total amount of glycosaminoglycans (GAGs) in Alcian blue and Masson\u0026apos;s trichrome staining were tested and showed strong retention in decellularized testis relative to normal tissue (Fig.2,A). The remaining GAG in the scaffold was stained with Alcian blue, indicating that more than 80% of GAG remained. No significant difference was observed in decellularized testis compared to the normal (p=0.1964) (Fig.2,C). \u0026nbsp;The amount of DNA was measured to evaluate the efficiency of the decellularization method and compared to DNA amount in native tissue. Analyzing DNA contents showed that DNA was successfully removed from the testis compared to the normal group (p\u0026lt;0.0001) (Fig.2, B). \u0026nbsp;The pictures of the SEM assessment were showed a tubular structure in the tissue without any cells. Besides, the structure was remained normal in some parts of tissue (Fig. 2, D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProliferation of SSC on scaffold after 14 days culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;MTT results demonstrated that the decellularized scaffolding was non-toxic. SSCs were cultured inside the decellularized testicular scaffold, and cell viability was assessed after 24 h and 48 h. The proliferation rate of SSCs in the three-dimensional culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from the other groups after 14 days of treatment. Nevertheless, the proliferation rate of SSCs in the 3D-culture with MK-801 was significantly lower than in other groups (Fig. 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular evaluation of \u003cem\u003ePlzf\u003c/em\u003e in 3-D cultured SSC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results indicated that the expression of \u003cem\u003ePlzf\u003c/em\u003e gene in the 3D group of spermatogonia stem cell cultures with 20 Mm D-serine and 50 mM glutamic acid was significantly greater than the control group (p\u0026lt;0.001). The expression of desired gene in the 2D and 3D group of SSC culture with 20mM MK-801 (MK20) was, decreased compared to each of the control groups, respectively. However, the alteration in gene expression was not statistically significant (p=0.294, p=1.064 respectively) (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein level PLZF marker in SSCs in different experimental groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe detection of germ cells at the protein level was performed via one in all experimental groups. The flow cytometric results revealed that the expression of \u003cem\u003ePlzf\u003c/em\u003e protein in the 3D culture group of SSCs with D-serine and glutamic was substantially more than the 3D culture control group (p\u0026lt;0.001). The expression of \u003cem\u003ePlzf\u003c/em\u003e in the 2D culture groups of SSCs with MK-801 was lower than the 2D culture control group (p\u0026lt;0.001). No significant difference was observed in the 3D culture of SSC with MK-801 compared to 3D culture control (p=0.187)\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor the first time, we developed the SSCs culture in the DTM hydrogel scaffold and indicated the role of D-serine, and MK-801 as the agonist and antagonist of NMDA receptors, respectively, in the spermatogenesis process. Decellularized testicular matrix [DTM) hydrogel scaffold is suitable for the surrounded SSCs and Sertoli cells to enhance the proliferation and differentiation of SSCs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our results, like previous findings, showed that the DTM hydrogel scaffold is appropriate for the proliferation of SSCs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. DTM hydrogel scaffolds contain laminin and collagen, which can regulate the proliferation of SSCs and so, the purity of SSCs increases [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The other constituents of DTM are GAGs which surround the Sertoli cells and Leydig cells and affect cell adhesion, and proliferation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These results showed that DTM contains some growth factors which can maintain SSCs activity and gene expression.\u003c/p\u003e \u003cp\u003eMoreover, this study's results revealed that the supplementation of a 3D culture medium with glutamic acid improved spermatogonia proliferation. Similar to these results, Santillo et al. revealed that the addition of glutamate to spermatogonia germ cell 1 (GC1) medium increased spermatogonia proliferation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The previous study showed that glutamate-induced the phosphorylation of ERK1/2 protein in spermatogonia GC1 cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, a surge of Akt activity in spermatogonia cells treated with glutamate was observed [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It has already been shown that extracellular-regulated kinase (ERK1) and Akt regulate various biological functions such as gene expression and cell cycle [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and play an important role in germ cell proliferation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Besides, it was declared that L-glutamine receptors are functionally expressed in rat testis which might promote stem cell proliferation by increasing glutathione levels inside and outside the cell as well as cysteine/cysteine balance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Another mechanism is that glutamic acid stimulates the proliferation of SSCs via anti-apoptotic pathways and by increasing testosterone in Leydig cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on the results, we found that enriched 3D culture medium with D-serine enhances SSCs proliferation. In agreement with our results, stated that enriched media with D-serine had significantly increased the proliferation and survival of SSC [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. On the other hand, it was indicated that treatment with D-serine increased the number of germ cells in silkworms via ERK phosphorylation in the testis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. After treating testes with D-serine, phosphorylated ERK was identified in spermatocytes and spermatids [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] which is consistent with an earlier finding that the meiosis of rat spermatocytes depended on the ERK activation of rat spermatocytes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Furthermore, agonist NMDA receptors can stimulate the proliferation of stem cells and increase their population in culture by supporting the intracellular antioxidant glutathione synthesis and reducing intracellular ROS [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study's findings revealed that the survival rate of SSCs in the 3D culture decreased after treating 3D and 2D cultures with MK-801. In line with our previous results, the experiments showed that the administration of MK-801 to rat testis resulted in increased oxidative stress in rat testis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Similarly, Saleh et al. demonstrated that the administration of MK-801 to rabbits increased the atrophy of the tubular structure and the degeneration of germinal cells in the lumen of the seminiferous tubule in histopathological evaluation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, it was reported that the level of total antioxidant status and sperm kinematics parameters have decreased following the administration of MK-801 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Histological data showed that using MK- 801 as antagonist NMDA, due to the testicular seminiferous tubule atrophy, degenerative changes in the epithelial cells, and a decrease in the number of the spermatozoa and necrotic [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. MK-801 reduces SSCs proliferation by increasing ROS production and increasing the expression of Bax and Bcl2 in SSCs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These results showed that the presence of D-serine alone or along with glutamic acid in the 3D culture of mouse SSCs creates a testis-like microenvironment.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study indicated that the DTM hydrogel scaffold culture system provided a simple and alternative method for culturing SSCs that eliminates potential diversity and contamination caused by feeder cells. Moreover, our results showed that supplemented culture medium with 20mM D-serine and 50mM glutamic acid could be considered a promising vehicle to support cells and provide a suitable niche for the proliferation of SSCs. The results of this study could be a way to further study the process of spermatogenesis in \u003cem\u003evitro\u003c/em\u003e and provide novel therapeutics for a higher proliferation rate of SSCs in male infertility to achieve an acceptable SSCs number.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eAcknowledgments\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is part of the Ph.D. thesis in the Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, and is sponsored financially by the research deputy of the University of Tabriz.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eThe authors have no conflicts of interest to declare which are relevant to the content of this article.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u0026nbsp; The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u0026nbsp; Not applicable\u0026apos; for that section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e \u003cstrong\u003eAmir\u003c/strong\u003e Hessam Eskafi Noghani: performed the research and prepared manuscript Reza Asadpour : contributed to study design, wrote the draft, and interpreted the data. AS: contributed in performing study and revised the manuscript Zohreh Mazaheri: contributed in the construction of 3D culture system, interpret the data, Gholamreza Hamidian: revise the manuscript and interpret histology data; all authors approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval \u0026nbsp;\u0026nbsp;\u003c/strong\u003e Ethics Committee approved all experimental procedures of the University of Tabriz, Tabriz, Iran, to use animals (Permission No. IR.TABRIZU.REC.1399.045).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e Not applicable\u0026apos; for that section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Not applicable\u0026apos; for that section.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMovassagh SA, Movassagh SA, Dehkordi MB, Pourmand G, Gholami K, Talebi A et al (2020) Isolation, identification and differentiation of human spermatogonial cells on three-dimensional decellularized sheep testis. Acta Histochem 122(8):151623\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbuMadighem A, Solomon R, Stepanovsky A, Kapelushnik J, Shi Q, Meese E et al (2018) Development of spermatogenesis in vitro in three-dimensional culture from spermatogonial cells of busulfan-treated immature mice. Int J Mol Sci 19(12):3804\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanjo H, Komeya M, Sato T, Abe T, Katagiri K, Yamanaka H et al (2018) In vitro mouse spermatogenesis with an organ culture method in chemically defined medium. PLoS One 13(2):e0192884\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuleihel M, Nourashrafeddin S, Plant TM (2015) Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta). Asian J Androl 17(6):972\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGholami K, Pourmand G, Koruji M, Ashouri S, Abbasi M (2018) Organ culture of seminiferous tubules using a modified soft agar culture system. Stem Cell Res Ther 9(1):249\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaert Y, Rombaut C, Goossens E (2017) Scaffold-based and scaffold-free testicular organoids from primary human testicular cells. InOrganoids Humana, New York, pp\u0026nbsp;283\u0026ndash;290\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurdock MH, David S, Swinehart IT, Reing JE, Tran K, Gassei K et al (2019) Human testis extracellular matrix enhances human spermatogonial stem cell survival in vitro. Tissue Eng Part A 25(7\u0026ndash;8):663\u0026ndash;676\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang X-T, Li C, Peng X-P, Guo J, Yue S-J, Liu W et al (2017) An excessive increase in glutamate contributes to glucose-toxicity in β-cells via activation of pancreatic NMDA receptors in rodent diabetes. Sci Rep 7:44120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeshi T, Balcar Vladimir J, Hideo T (2004) Possible expression of functional glutamate transporters in the rat testis. J Endocrinol 181:233\u0026ndash;244\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantillo A, Falvo S, Chieffi P, Burrone L, Chieffi Baccari G, Longobardi S et al (2014) D-aspartate affects NMDA receptor-extracellular signal-regulated kinase pathway and upregulates androgen receptor expression in the rat testis. Theriogenology 81(5):744\u0026ndash;751\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStorto M, Sallese M, Salvatore L, Poulet R, Condorelli D, Dell'Albani P et al (2001) Erratum: Expression of metabotropic glutamate receptors in the rat and human testis. J Endocrinol 170(2):71\u0026ndash;78\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Fiore MM, Santillo A, Falvo S, Longobardi S, Chieffi Baccari G (2016) Molecular mechanisms elicited by D-aspartate in Leydig cells and spermatogonia. Int J Mol Sci 17(7):1127\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantillo A, Falvo S, Chieffi P, Di Fiore MM, Senese R, Chieffi Baccari G (2016) D-Aspartate induces proliferative pathways in spermatogonial GC-1 cells. J Cell Physiol 231:490\u0026ndash;495\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalvo E, Tremante E, Arcovito A, Papi M, Elad N, Boffi A et al (2016) Improved doxorubicin encapsulation and pharmacokinetics of ferritin\u0026ndash;fusion protein nanocarriers bearing proline, serine, and alanine elements. Biomacromol 17:514\u0026ndash;522\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026rsquo;Aniello G, Ronsini S, Guida F, Spinelli P, D\u0026rsquo;Aniello A (2005) Occurrence of D-aspartic acid in human seminal plasma and spermatozoa: possible role in reproduction. Fertil Steril 84(5):1444\u0026ndash;1449\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakai K, Homma H, Lee J-A, Fukushima T, Santa T, Tashiro K et al (1998) Localization ofd-Aspartic Acid in Elongate Spermatids in Rat Testis. Arch Biochem Biophys 351(1):96\u0026ndash;105\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsui Ta, Sekiguchi M, Hashimoto A, Tomita U, Nishikawa T, Wada K (1995) Functional comparison of D-serine and glycine in rodents: the effect on cloned NMDA receptors and the extracellular concentration. J Neurochem 65(1):454\u0026ndash;458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eModirshanechi G, Eslampour MA, Abdolmaleki Z (2020) Agonist and antagonist NMDA receptor effect on cell fate during germ cell differentiation and regulate apoptotic process in 3D organ culture. Andrologia e13764\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurkmen R, Akosman MS, Demirel HH (2019) Protective effect of N-acetylcysteine on MK-801-induced testicular oxidative stress in mice. Biomed Pharmacother 109:1988\u0026ndash;1993\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T (2003) Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69(2):612\u0026ndash;616\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadri-Ardekani H, Mizrak SC, van Daalen SK, Korver CM, Roepers-Gajadien HL, Koruji M, Hovingh S, de Reijke TM, de la Rosette JJ, van der Veen F, de Rooij DG (2009) Propagation of human spermatogonial stem cells in vitro. Jama 302(19):2127\u0026ndash;2134\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahaldashtian M, Naghdi M, Ghorbanian MT, Makoolati Z, Movahedin M, Mohamadi SM (2016) In vitro effects of date palm (Phoenix dactylifera L.) pollen on colonization of neonate mouse spermatogonial stem cells. J Ethnopharmacol 186:362\u0026ndash;368\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMajidi Gharenaz N, Movahedin M, Mazaheri Z (2020) Three-Dimensional Culture of Mouse Spermatogonial Stem Cells Using A Decellularised Testicular Scaffold. Cell J 21(4):410\u0026ndash;418\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaryabari S, Kajbafzadeh A-M, Fendereski K, Ghorbani F, Dehnavi M, Rostami M et al (2019) Development of an efficient perfusion-based protocol for whole-organ decellularization of the ovine uterus as a human-sized model and in vivo application of the bioscaffolds. J Assist Reprod Genet 36:1211\u0026ndash;1223\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZawko SA, Suri S, Truong Q, Schmidt CE (2009) Photopatterned anisotropic swelling of dual-crosslinked hyaluronic acid hydrogels. Acta Biomater 5(1):14\u0026ndash;22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindelin J, Rueden CT, Hiner MC, Eliceiri KW (2015) The ImageJ ecosystem: An open platform for biomedical image analysis. Mol Reprod Dev 82:518\u0026ndash;529\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiloochi Kashani M, Bagher Z, Asgari HR, Najafi M, Koruji M, Mehraein F (2020) Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold. Syst Biol Reprod Med 66(3):202\u0026ndash;215\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M (2004) Generation of pluripotent stem cells from neonatal mouse testis. Cell 119(7):1001\u0026ndash;1012\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Lin Q, Zhou C, Li Q, Li Z, Cao Z et al (2020) A testis-derived hydrogel as an efficient feeder-free culture platform to promote mouse spermatogonial stem cell proliferation and differentiation. Front Cell Dev Biol 8:250\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTopraggaleh TR, Valojerdi MR, Montazeri L, Baharvand H (2019) A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids. Biomater Sci 7(4):1422\u0026ndash;1436\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpang MT, Christman KL (2018) Extracellular matrix hydrogel therapies: in vivo applications and development. Acta Biomater 68:1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Li X, Wu Y, Du P, Sun L, Yu Z et al (2020) Preparation of PU/Fibrin Vascular Scaffold with Good Biomechanical Properties and Evaluation of Its Performance in vitro and in vivo. Int J Nanomedicine 15:8697\u0026ndash;8715\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantillo A, Falvo S, Di Fiore MM, Di Giacomo Russo F, Chieffi P, Usiello A et al (2019) AMPA receptor expression in mouse testis and spermatogonial GC-1 cells: A study on its regulation by excitatory amino acids. J Cell Biochem 120(7):11044\u0026ndash;11055\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCooper TG, Yeung CH, Nashan D, Nieschlag E (1988) Epididymal markers in human infertility. J Androl 9(2):91\u0026ndash;101\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVogiatzi P, Giordano A (2007) Following the tracks of AKT1 gene. Cancer Biol Ther 69(10):1521\u0026ndash;1524\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChieffi P, Chieffi S (2013) Molecular biomarkers as potential targets for therapeutic strategies in human testicular germ cell tumors: an overview. J Cell Physiol 228(8):1641\u0026ndash;1646\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Zhang K, Li G, Yan S, Cui L, Yin J (2018) Effects of large dimensional deformation of a porous structure on stem cell fate activated by poly(l-glutamic acid)-based shape memory scaffolds. Biomater Sci 6(10):2738\u0026ndash;2749\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi B, He X, Zhuang M, Niu B, Wu C, Mu H et al (2018) Melatonin ameliorates busulfan-induced spermatogonial stem cell oxidative apoptosis in mouse testes. Antioxid Redox Signal 28(5):385\u0026ndash;400\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu JC-C, Zhang C, Sun X, Yang Y, Cao X, Ryu O et al (2000) Characterization of the mouse and human PRSS17 genes, their relationship to other serine proteases, and the expression of PRSS17 in developing mouse incisors. Gene 251(1):1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki C, Tanigawa M, Tanaka H, Horiike K, Kanekatsu R, Tojo M et al (2014) Effect of d-serine on spermatogenesis and extracellular signal-regulated protein kinase (ERK) phosphorylation in the testis of the silkworm, Bombyx mori. J Insect Physiol 67:97\u0026ndash;104\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodet M, Sabido O, Gilleron J, Durand P (2008) Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells. Dev Biol 315(1):173\u0026ndash;188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang X, Kong H, Tang M, Lu M, Ding JH, Hu G (2012) D-Serine regulates proliferation and neuronal differentiation of neural stem cells from postnatal mouse forebrain. CNS Neurosci Ther 18(1):4\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParlaktas BS, Ozyurt B, Ozyurt H, Tunc AT, Akbas A (2008) Levels of oxidative stress parameters and the protective effects of melatonin in psychosis model rat testis. Asian J Androl 10(2):259\u0026ndash;265\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaleh SY, Sawiress FA, Tony MA, Hassanin AM, Khattab MA, Bakeer MR (2015) Protective role of some feed additives against dizocelpine induced oxidative stress in testes of rabbit bucks. J Agric Sci 7(10):239\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurkmen R, Akosman MS, Demirel HH (2019) Protective effect of N-acetylcysteine on MK-801-induced testicular oxidative stress in mice. Biomed Pharmacother 109:1988\u0026ndash;1993\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Wang L, Zhang X, Ren L, Shi W, Tian Y et al (2017) The use of KnockOut serum replacement (KSR) in three dimensional rat testicular cells co-culture model: An improved male reproductive toxicity testing system. Food Chem Toxicol 106:487\u0026ndash;495\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzyurt B, Ozyurt H, Akpolat N, Erdogan H, Sarsilmaz M (2007) Oxidative stress in prefrontal cortex of rat exposed to MK-801 and protective effects of CAPE. Prog Neuropsychopharmacol Biol Psychiatry 31(4):832\u0026ndash;838\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NMDARs, Spermatogonia stem cells, Two-dimensional culture system, Three-dimensional culture system","lastPublishedDoi":"10.21203/rs.3.rs-840124/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-840124/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801or Mk) on the proliferation of SSCs in two-dimensional (2D) and three-dimensional (3D) culture systems. The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was confirmed by anti-\u003cem\u003ePlzf\u003c/em\u003e and anti-\u003cem\u003eGFR-α1\u003c/em\u003e antibodies \u003cem\u003evia\u003c/em\u003e immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated \u003cem\u003evia\u003c/em\u003e qRT-PCR and flow cytometry methods, respectively.\u0026nbsp;\u003c/p\u003e\u003cp\u003eThe results showed that the proliferation rate of SSCs in three-dimensional culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from other groups after 14 days treatment. mRNA expression levels of \u003cem\u003ePlzf\u003c/em\u003e in 3D-cultures supplemented by 20 mM D-serine and 50 mM glutamic acid were considerably higher than the 3D control group (p\u0026lt;0.001). The flow cytometry analysis revealed that the amount of \u003cem\u003ePlzf\u003c/em\u003e in the 2D-culture groups of SSCs with 20mM MK-801 was considerably lower compared to the 2D-culture control group (p\u0026lt;0.001). This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs.\u003c/p\u003e","manuscriptTitle":"Effect of NMDA Receptor Agonist and Antagonist on Spermatogonia Stem Cells Proliferation in 2- and 3- Dimensional Culture Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-30 18:43:07","doi":"10.21203/rs.3.rs-840124/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-09-13T02:52:03+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-08-27T11:37:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-08-23T14:40:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2021-08-23T07:08:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"045df97b-a9aa-4340-aa80-ef2dd636b06a","owner":[],"postedDate":"August 30th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":6826965,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2021-12-01T12:29:45+00:00","versionOfRecord":[],"versionCreatedAt":"2021-08-30 18:43:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-840124","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-840124","identity":"rs-840124","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}