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1Open AccessAsian Australas. J. Anim. Sci.[Epub ahead of as.infopISSN 1011-2367 eISSN 1976-5517Isolation and Identification of Prepubertal Buffalo (Bubalus bubalis)Spermatogonial Stem CellsWanyou Feng1,a, Shibei Chen1,2,a, Dagiang Do1,3, Qinyou Liu1,*, Yanfei Deng1, Xiaocan Lei1,Chan Luo1, Ben Huang1, and Deshun Shi1,*1State Key Laboratory of Conservation and Untilization of Subtropical Agro-Bioresources,Guangxi University, Nanning 530005, ChinaABSTRACT: Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. Inthe present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell markergenes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubuleswere enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes inthis stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, weredetermined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining inbuffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalospermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derivedneurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups(p 0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-likecells, and accelerating the generation of genetic modified buffaloes. (Key Words: Buffalo, Spermatogonial Stem Cell, Germ CellMarker, Proliferation)INTRODUCTIONWater buffalo is an economically important livestock,which partially distributes in south China, providing highquality of milk, meat and work power (Shi et al., 2012).Buffalo is low fertility species, characterized by lowconception rate, seasonality estrus and delayed puberty, andis in urgent need of improvement through propagation of*Corresponding Authors: Qinyou Liu. Tel: 86-771-3239202,Fax: 86-771-3239202, E-mail: [email protected] / DeshunShi. Tel: 86-771-3239202, Fax: 86-771-3239202, E-mail:[email protected] of Reproduction of Nanxishan Hospital, Guilin 541002,China.3Bacgiang Agriculture and Forestry University, Bacgiang 220000Vietnam.aThese authors contributed equally to this work.Submitted Jul. 13, 2015; Revised Sept. 30, 2015; Accepted Nov. 14, 2015superior germ plasma by modern reproductive technologies(Shi et al., 2007). At present, several technologies fromcattle have been adapted for improvement of buffaloproduction, but there are various limitations. Therefore,developing of stem cell technology in buffalo hassignificance in the genetic improvement of this species (Shiet al., 2007; Mahla et al., 2012). Spermatogonial stem cells(SSCs) have received a great deal of attention in recentyears, as they have unique capacity for self-renewal andproduce large number of differentiating haploid germ cellswhich could transmit paternal genetic information to nextgenerations (Kanatsu-Shinohara and Shinohara, 2013).SSCs can also be genetically modified and furtherdifferentiated to spermatozoa in vitro, subsequentlycontribute to produce genetic modified offspring throughthe method of intracytoplasmic sperm injection (ICSI) orSSCs transplantation into a recipient testis (KanatsuShinohara et al., 2011; Sehgal et al., 2014). Recently,Copyright 2016 by Asian-Australasian Journal of Animal SciencesThis is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License hich permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2Asian Australas. J. Anim. Sci. [Epub ahead of print]successful germ cell transplantation into bull testis hadresulted in production of donor-derived sperm cells(Stockwell et al., 2009). In case of establishment livestockspecies male germ cells line, researchers can facilely createtargeted gene editing animals using TALEN orCRISPR/Cas9 technology (Gaj et al., 2013; Chapman et al.,2015). Nevertheless, no SSC lines have yet been establishedfor livestock species. The most likely reason is lack ofsufficient understanding of expression of related markers inthe male stem cell, especially water buffalo.To efficiently isolate and purify SSCs, the availability ofSSCs specific expressing markers was of great importance.Oct-4 (also as knows Pou5fl), THY-1, c-kit, PGP9.5 (also asknows UCHL-1) and Dolichos biflorus agglutinin (DBA)are used to identify bovine (Izadyar et al., 2002; Oatley etal., 2004; Reding et al., 2010), goat (Heidari et al., 2012;Abbasi et al., 2013), sheep (Rodriguez-Sosa et al., 2006),pig (Lee et al., 2013; Lee et al., 2014), dog (Harkey et al.,2013), mouse and human (von Kopylow et al., 2010) andchicken SSCs (Momeni-Moghaddam et al., 2014;Sisakhtnezhad et al., 2015). Recently, Oct-4 and c-kit werereported to be experessed in buffalo testis andspermatogonail stem cells (Xie et al., 2010; Yu et al., 2014).In the further, it is essential for determination themorphological, physiological and stem cell potentialthrough various markers and different biological methods.So the aim of this study was to determine expression patternof different markers and transcripts in prepubertal buffalotestis and SSCs, and optimize the in vitro culture medium ofbuffalo SSCs.seminiferous tubes by the pinhead of 1 mL syringe. Thenseminiferous tubes were incubated for 15 min at 37 C inPBS containing 1% collagenase (Type V; Sigma, USA), anddigested into fragments by beating upon during incubation.Most of the Leydig cells were discarded after washingseveral times with Dulbecco modified eagle medium(DMEM; Invitrogen Corporation, Carlsbad, CA, USA).Seminiferous cord fragments were centrifuged three times(each time 1,000 rpm for 3 min) with DMEM containing10% fetal bovine serum (Hyclone, Logan, UT, USA).Following incubation in DMEM containing 2.5% trypsinase(Sigma, USA) for 6 to 8 min, when the cells were separatedinto a single pellet from the remaining tube fragments,digestion was ceased with DMEM containing 10% fetalbovine serum (FBS; Hyclone, USA). To remove debris, cellsuspension was filtered using a 200 μm nylon cell strainer(Falcon, Becton Dickinson Labware, NY, 10010/USA).Next, the collected cell suspension was removed bycentrifugation at 1,200 rpm for 5 min, and cells werecultured in DMEM containing 10% FBS for 4 to 6 hours.Non-adherent cells were poured over a Percoll densitygradient (11%, 19%, 21%, 35%) and centrifuged at 1,800rpm for 40 min. Then the cells were cultured in DMEMwith different concentrations of FSB (0%, 1.5%, 2.5%,3.5%) and different concentrations of glial cell-derivedneurotrophic factor (GDNF) (0 ng/mL, 20 ng/mL, 40ng/mL, 80 ng/mL) overnight at 37 C in a high humidity 5%(v/v) CO2 in air atmosphere. Cell proliferation was assessedby Brdu incorporation assay.Hematoxylin-eosin staining and histochemistryFixed testicular tissues from 3 to 7 ages of monthMATERIALS AND METHODSbuffalo testes were dehydrated, and then embedded inparaffin and sectioned 5 µm thick using standardCollection of buffalo testisTestes from 3 to 7 month old prepubertal buffalo procedures. Sections were processed through xylene and(Bubalus bubalis) calves were collected from Nanning ethanol into water, stained with hematoxylin and eosin, orslaughterhouse. A small piece of testis tissue was used for immunohistochemistry. These sections weresubmerged immediately after collection in liquid nitrogen washed by PBS-Tween-20 for 3 min, and exposed 3%frozen for isolation of RNA. For histochemical analysis, H2O2-methanol for 30 min. All sections were placed intotestes tissue was immediately fixed in 4% formalin fixative sodium citrate-hydrochloric acid buffer solution (pH 6.0),following collection. For SSC isolation, the testes were and were heated three times (6 min each time). After heated,transported to the laboratory in phosphate buffer saline sections were washed by PBS-tween-20 for 3 to 5 min,(PBS) containing penicillin (100 U/mL), and streptomycin blocked with 5% FBS in PBS for 1 hour at roomtemperature, and incubated with primary antibody for(100 μg/mL) (Sigma, St Louis, MO, USA, P4333).overnight at 4 C, including anti-Oct4 (Abcam, ab18976,1:100), anti-THY-1 (Abcam, ab3105, 1:100), anti-PGP9.5Cell isolation and purificationCell isolation was performed as described previously (Abcam, ab10404, 1:50) and anti-c-kit (Abcam, ab5506,(Xie et al., 2010). Briefly, testes from 3 to 7 age of months 1:100) respectively. After washing three times with PBSbuffalo were immediately washed several times by PBS tween-20 for 5 min each, these sections were incubated withcontaining penicillin (100 U/mL), and streptomycin (100 3% H2O2 for 10 min, washed with PBS-tween-20 for 3 min.μg/mL). Then the tunica albuginea of testes was manually These sections were exposed for 45 min at roomdecapsulated, and parenchyma was used for cell isolation. A temperature to secondary antibody (HRP-conjugated goatsmall piece of testis parenchyma was cut and separated into anti-rabbit IgG, Abcam, ab672, 1:500), washed with PBS-

3Asian Australas. J. Anim. Sci. [Epub ahead of print]tween-20 as above. The sections were also stained withDBA substrate kit (Vector Laboratories, Burlington, ON,Canada) according to the manufacture’s instructions.ImmunofluorescencePrimary culture of buffalo spermatogonial stem-likecells was treated under different concentrations of FBS andGDNF in cultures plates for 12 days. After washing, cellcolonies were fixed with 4% paraformaldehyde in PBS at4 C for 1 hour. Following, cell colonies were washed threetimes with PBS for 5 min each, and permeabilized with 1%Triton-100 for 30 min. The colonies were again washedthree times with PBS for 5 min each, and blocked 1%bovine serum albumin (BSA) in PBS for 1 h. Then, thecolonies were incubated with anti-Oct4 (Abcam, ab18976,1:200), anti-THY-1 (Abcam, ab3105, 1:200), anti-PGP9.5(Abcam, ab10404, 1:100), and anti-c-kit (Abcam, ab5506,1:200) respectively overnight at 4 C. Then the incubatedcolonies were washed three times with PBS for 5 min each,following incubation with secondary antibody: FITCconjugated goat anti-rabbit IgG for 90 min at 37 C. For thenuclear staining, colonies were counterstained withHochest33342 (Life Technology, 62249, NY, NY, USA) for5 min, washed three times with PBS for 5 min each, andanalyzed under fluorescence microscope (N-STORM,Nikon).Analysis of cell viability by alkaline phosphatasestainingExpression of alkaline phosphatase (AP) activity in cellcolonies was detected by AP staining. The purified andcultured Buffalo spermatogonia were washed three timeswith PBS before they were fixed with 4% (w/v)paraformaldehyde for 30 min. Following these colonieswere washed three times with PBS for 5 min each, indolylphosphate toluidine (NBT/BCIP; Amresco, Solon,OH, USA) were used as substrates for 15 to 30 min at 37 Cin dark.Analysis of cell proliferation by Brdu incorporationassayThe 5-bromode-oxyuridine (Brdu; Sigma, USA) andDBA were used to determine buffalo spermatogoniaproliferation. For Brdu incorporation assay, the number ofcells in each 24-well plate was counted under invertedmicroscope (CKX41; Olympus, Tokyo, Japan), and wereseeded at a density of 5.0 104 cells/mL, with three wells agroup. On 0, 1, 2, 3, 4, 5, and 6 days after culture, Brdu (0.3µg/µL) was added to each well of the parallel samples, andcultured to 7 days, fixed with 4% paraformaldehyde for 15min at room temperature. Then the cells were permeabilizedwith 1% Triton-100 for 30 min at room temperature,washed three times with PBS for 3 min each. In thefollowing the cells were denaturalized with 4 Mhydrochloric acid for 30 min at room temperature, andwashed again three times using PBS for 3 min each. Thecells were blocked with 1% BSA in PBS for 1 h at roomtemperature, and incubated anti-Brdu (2 μg/mL) overnightat 4 C. Next day, the cells were heated for 30 min at 37 C,and washed two times with PBS for 5 min each. Then thecells were incubated with secondary antibody:FITCconjugated goat anti-rabbit IgG (1:200) for 90 min at 37 Cin dark, and washed again three times with PBS for 5 mineach. For the nuclear staining, the cells were counterstainedwith Hochest33342 (Life Technology, 62249, USA) for 5min, washed three times with PBS for 5 min each, andanalyzed under fluorescence microscope (N-STORM,Nikon, Tokyo, Japan).Reverse transcription polymerase chain reactionanalysisTotal RNA was purified from testes and culturespermatogonia, respectively. The stored testes tissue pieceswere picked out from liquid nitrogen, and the in vitroculture spermatogonia were washed twice with precoolingPBS, then processed for RNA isolation using TRizolreagent according to the manufacture’s instructions. Theextracted RNA were diluted with DEPC-water andincubated with 10 units of RNase-free DNase for 30 min at37 C, following inactivation of DNase by adding 1 μLeathylene diamine tetraacetic acid. The first strand cDNAwas synthesized according with M-MLV reversetranscriptase manufacture’s instructions (GenScriptCorporation, Piscataway, NJ, USA). The primers sequenceswere list as in Table1. The reaction mixture for polymerasechain reaction (PCR) contained 2 PCR reaction mixtureTable 1. Primers for amplification of germ cell marker genesGeneOct4THY-1PGP9.5c-kitβ-actinForward ATGCTTCTAGGPCR, polymerase chain reaction.Reverse ACTGCTGTCPCR size (bp)/Gene No.312/NM 174580.2502/NM 001034765.1358/XM 005207872.1195/NM 174375.2199/NM 001290932.1

4Asian Australas. J. Anim. Sci. [Epub ahead of print](A)(B)Figure 1. Detection of germ cell marker transcripts expressed in the prepubertal buffalo testis (A) and spermatogonial stem-like cellscolonies (B). M, DNA ladder; Oct-4 (line 1, 312 bp); c-kit (line 3, 195 bp); THY-1 (line 5, 502 bp); PGP9.5 (line 7, 358 bp). Negativecontrol (line 2, 4, 6, 8, 10); β-actin (line 9, 199 bp), positive control. In the (B), the position of PGP9.5 (line 5, 358 bp) and THY-1 (line7, 502 bp) was exchanged.buffer 10 μL, cDNA 1 μL and specific primers (20 µm eachprimer), Oct4 and c-kit (annealing at 52 C, 35 cycles),THY-1 and PGP9.5 (annealing at 56 C, 35 cycles). ThePCR products were separated and visualized by 2% agarosegel eletrophoresis containing ethidium bromide.Statistical analysisThe results are presented as mean standard error of themean and statistical analysis was performed by analysis ofvariance or Student’s t-test after arcsine transformation ofthe proportional data of spermatid-like cell formation andviability. Duncan’s multiple comparisons test was used tocompare mean values among treatments.RESULTSAnalysis of germ cell marker genes expression inprepubertal buffalo testisThe reverse transcription polymerase chain reaction(RT-PCR) analysis revealed that Oct-4, PGP9.5, THY-1 andc-kit were all expressed in testes of prepubertal buffalo,with PCR product fragments of 312 bp for Oct-4, 195 bpfor c-kit, 502 bp for THY1, 358 bp for PGP9.5 and 199 bpfor β-actin (Figure 1A), respectively. No bands were visiblein the case of negative controls, where the cDNA wasreplaced with purified water. The Hematoxylin eosin (HE)stained germ cells in the testicular sections from the 3 to 7months old buffalo were easy to identify by their large size,Figure 2. Analysis of germ cell markers expressed in the prepubertal buffalo testis sections by histochemistry. Histological sections ofprepubertal buffalo testis were stained with Hematoxylin and Eosin (A). Antibodies against germ cell markers; (B) DBA, (C) PGP9.5,(D) Oct-4, (E) THY-1, and (F) c-kit in histological sections of prepubertal buffalo testis . Scale bar, 100 µm.

Asian Australas. J. Anim. Sci. [Epub ahead of print]5Figure 3. Observation of in vitro culture buffalo spermatogonial stem-like cells. Percoll density gradient isolation of buffalospermatogonial stem-like cells (A); in vitro culture of Percoll density gradient isolated cells (B); 24 hours after in vitro culture (C); 3 daysafter in vitro culture (D); 6 days after in vitro culture (E); 9 days after in vitro culture (F); Scale bar, 100 µm.topological position and morphology (Figure 2A). Theaffinity of the specific antibodies Oct-4, PGP9.5, THY-1, ckit, and DBA was restricted to germ cells without stainingthe somatic cells (Figure 2B-F). Five specific antibodiesstaining was localized on the surface of germ cells, and thecells showed weak affinity for Oct4 (Figure 2D) and strongaffinity for PGP9.5 (Figure 2C).Observation of in vitro cultured buffalo spermatogonialstem-like cellsSpermatogonial stem-like cells were isolated andcultured from prepubertal buffalo through differentialplating and subsequent Percoll gradient separation (Figure3A). The buffalo spermatogonial stem-like cells wereobserved as round cells with a high ratio ofnucleus:cytoplasm (Figure 3B). After 24 h culture, SSCswere observed to grow with adherence by morphology(Figure 3C). With the extension of in vitro culture, SSCclusters were observed in the following 3 to 9 days (Figure3D-F). On the 9 day, the spermatogonial stem-like cellcolonies (AP-positive) appeared (4A) and had differentforms as single (Figure 4B), cluster (Figure 4C) and alignedFigure 4. Buffalo spermatogonial stem-like cells colonies formed after 9 days of culture and were alkaline phosphatase (AP)-positive.After 9 days in vitro culture, the spermatogonial stem-like cells colonies were identify by alkaline phosphatase staining (A) and haddifferent forms: single (B); cluster (C) and aligned (D). Scale bar, 100 µm.

6Asian Australas. J. Anim. Sci. [Epub ahead of print](Figure 4D).Determination of germ cell marker genes expression inbuffalo spermatogonial stem-like cells coloniesWith total RNA of buffalo spermatogonial stem-like cellcolonies as template, we found that germ cell marker Oct-4,PGP9.5, THY-1, and c-kit mRNA were all expressed by RTPCR (Figure 1B). In the further, the results ofimmunocytochemistry analysis of colonies revealed thatgerm cell markers PGP9.5, THY-1, Oct-4, and c-kit werealso expressed in in vitro culture buffalo spermatogonialstem-like cell colonies (Figure 6A-D). It was observed thatOct-4 expression was weaker than other markers both inSSC colonies and in the testis (Figure 2D and Figure 6D).Table 2. The number of DBA-positive cells at differentconcentrations of fetal bovine serum groupsFBS concentration (%)01.52.53.5Times3333Average number ofpositive DBA1.33 0.58c7.67 1.53b12.00 2.22a8.67 1.53bDBA, Dolichos biflorus agglutinin; FBS fetal bovine serum.The different superscripts in the same table indicate significant difference(p 0.05).DISCUSSIONIn vitro culture SSCs provide an opportunity as anOptimization of in vitro culture medium for buffalo alternative approach to promote the production of geneticmodification animals. The key points to advance thisspermatogonial stem-like cellTo optimize the in vitro culture medium of buffalo research is acquisition of enough quantities of SSCs andspermatogonial stem-like cells, different concentrations of determination of specific gene markers, thus the use ofFBS and GDNF were added to DMEM. After 9 days in different isolation methods and various markers forvitro culture, the proportion of DBA-positive cells in 2.5% enrichment and identification of these cells are ofconcentration of FBS group