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Received 17 March 2010; received in revised form 21 May 2010; accepted 21 May 2010

stract

Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lacksufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, wentified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalotis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were alsosent in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were alsoected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradienttrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo

tis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serumthe absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 andU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.2010 Elsevier Inc. All rights reserved.

words: Buffalo; Testis; Spermatogonia; In-vitro Culture

Introduction

Water buffalo (Bubalus bubalis) is indigenous toia and about half of its total world population existsIndia. Production of buffalo milk in India is esti-ted to be around 134 million tons annually whichkes buffalo an important livestock species [1]. Se-tive breeding and improved management have had asitive impact on buffalo production. However, buf-

falo has low reproductive efficiency as a result of de-layed puberty, seasonality, anoestrus, low conceptionrate, and long calving intervals [2]. Although numerousreproductive technologies have been adapted from cat-tle for improvement of buffalo production but, withlimited success. Therefore, there is a need to explore anew area of reproductive technology that can be appliedfor efficient genetic improvement of buffalo.

Germ cells are unique cells that transmit geneticinformation from one generation to another. In mam-permatogonia-specific proteins(Bubalus bubalis) testis and

and in vitro cultivatiSandeep Goela,*, Niranjan Reddya,

Sung-Min Kim

Theriogenology 74 (201ma

epCorresponding author: Tel.: 91-40-23447437; Fax: 91-40-23447441.E-mail address: sandeep@ccmb.res.in (S. Goel).

3-691X/$ see front matter 2010 Elsevier Inc. All rights reserved.:10.1016/j.theriogenology.2010.05.025ressed in prepubertal buffaloir utilization for isolationof spermatogoniaan Mandala, Mayako Fujiharab,roshi Imaib

1232www.theriojournal.comlian embryos, germ cell specification starts in theiblast during gastrulation as primordial germ cells

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1222 S. Goel et al. / Theriogenology 74 (2010) 12211232Cs) [3]. PGCs migrate from the base of the allantoisough the hindgut to the genital ridge [4]. In the malenital ridge, PGCs differentiate to gonocytes and fur-r differentiate to spermatogonia in the testis. Sper-togonia consist of a population of spermatogonialm cells (SSCs) that are capable of self-renewal andlenishing the spermatogonia population. SSCs inents can be genetically modified. After genetic mod-ation, they further differentiate to spermatozoa fol-ing transplantation into a recipient testis, eventuallyducing transgenic offspring [59]. Since no germ-

e competent embryonic stem (ES) cell lines haveen established from any domestic animal so far,Cs could provide an alternative for efficient geneticdification in these species. In goat, transplantation of

netically modified SSCs has succeeded in transmit-g a transgene to the germ-line [10]. Furthermore,rm cell transplantation to bull testis has recentlyulted in the production of donor-derived sperm [11].wever, little is known about male germ-line steml technology in buffalo.The testis consists of different types of cells andntification of germ-cell-specific markers is useful forir isolation, in vitro cultivation and in vivo monitor-following testis transplantation. Germ-cell specific

rkers such as lectin-Dolichos biflorus agglutininA [1214] and UCHL-1 (Ubiquitin Carboxyl-Ter-

nal Esterase L1, previously known as PGP 9.5),16] have been used to isolate germ cells from thetes of domestic animals and to identify them duringvitro culture [14,15,17,18]. To date, no specificrker for testicular germ cells in buffalo has beennd. The domestic water buffalo, Bubalus bubalis, is

rived from the Indian wild buffalo, Bubalus arnee, which is considered an endangered species by theernational Union for Conservation of Nature (www.nredlist.org). Therefore, methods for improving theroduction of domestic buffalo could aid in develop-methods for conserving the wild buffalo. In the

sent study, for the first time, we report spermatogo--specific markers in buffalo testis and use them forrmatogonia isolation and characterization during in

ro culture.

Material and methods

. Collection of the testisTestes were collected from prepubertal (aged 46) and from pubertal (aged 912 mo) water buffalo

ubalus bubalis) calves of Murrha breed from local

ms and a slaughterhouse in India, strictly following miguidelines of the Institutes Bio-Ethical Committee.stes tissues for histochemical analysis were fixedmediately after collection in Bouins fixative. Addi-nal samples were transported on ice within 1 h to theoratory in DMEM/F12 [mixture of Dulbeccosdified Eagle Medium and Nutrient Mixture F-12,

1)] containing 15 mol/L HEPES (Invitrogen, www.itrogen.com). The testes were then washed severales with phosphate buffer saline (PBS), and were

mediately processed for germ-cell isolation.

. Histochemistry of buffalo testesThe fixed testicular tissues were dehydrated, embed-

d in paraffin and sectioned (6 m thick). Sectionsre dewaxed, rehydrated, and stained with DBA andtibodies as described [14]. Dilutions of lectin DBA,mary and secondary antibodies were done in PBSth 1% BSA (Sigma, www.sigmaaldrich.com). Thetion was then incubated with 3% Hydrogen Peroxidesher Scientific, www.fishersci.com) for 10 min,shed with PBS, incubated in 5% BSA in PBS for 15n, incubated with DBA-conjugated horseradish per-idase (DBA-HRP; E.Y. Laboratories, www.eylabs.m; 1:100) for 1h at 37 C in a moist chamber, rinsedee times with PBS, incubated for 35 min in 3,3=-minodbenzidine (DAB) substrate kit (Vector Labo-ories, www.vectorlabs.com) according to the manu-tures instruction, rinsed thoroughly in distilledter, counterstained with Hematoxylin, dehydrated,d mounted in Vectamount (Vector Laboratories, www.torlabs.com) and observed under a Zeiss Axioplan 2

croscope. Negative control sections were incubated inBSA in PBS without lectin DBA.

Sections were also stained with rabbit anti-UCHL-GP 9.5 (Dako, www.dako.com; 1:500), rabbit anti-U5F1/OCT 3/4 (POU Class 5 Homeobox 1, poly-nal, Chemicon, www.chemicon.com; 1:200), rabbit

ti-DDX4/VASA (DEAD (Asp-Glu-Ala-Asp) Boxlypeptide 4, polyclonal, Abcam, www.abcam.com;00), mouse anti-VIMENTIN (clone V9, Sigma,00), goat anti-GATA binding protein 4 (GATA4,ne C-19, Santa Cruz Biotechnology, www.scbt.com;00) and anti--smooth muscle ACTIN (ASMA,ne 1A4, Labvision Corporation, www.labvision., 1:1000) antibodies to assay their expression pattern

the prepubertal and pubertal testes. Briefly, after de-affinization and rehydration, sections were blocked

th 10% fetal bovine serum (Gibco, www.invitrogen.) or 10% rabbit serum (for GATA4 staining) and 3%

A (Sigma, www. sigmaaldrich.com) in PBS for 30

n, incubated with the above mentioned primary anti-

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1223S. Goel et al. / Theriogenology 74 (2010) 12211232dies overnight at 4 C, washed several times with PBS,ubated with 3% H2O2 for 10 min, washed three timesth PBS, incubated with the corresponding HRP-conju-ed secondary antibody, i.e., goat anti-rabbit, goat anti-use IgG, and rabbit anti-goat IgG (all from Calbio-m, www.calbiochem.com; 1: 200) for 30 min at 37 C,

sed three times with PBS. Peroxidase was developedobserved as described earlier. In negative controls,

mary antibody was omitted and instead the section wasubated with 1% BSA in PBS.

. Isolation of spermatogoniaAll chemicals for cell culture were from Calbiochemww.calbiochem.com) unless mentioned otherwise.e collected testes were washed several times withS, minced with scissors, and incubated in DMEM/2 medium supplemented with 15 mol/L HEPES (In-rogen), 100 IU/mL 50 g/mL penicillin-strepto-cin, 40 mg/mL gentamycin, 1.5 mg/mL collagenase

d 5 g/mL DNase at 37 C for 30 min in a shakingter bath operated at 100 cycles/min. After threeshes with DMEM/F12 medium and removal of mostthe interstitial cells, seminiferous tubules fragmentsre incubated in DMEM/12 medium containing 2/mL trypsin and 5 g/mL DNase for 30 min in the

nditions described above. The dispersed cells wereshed twice with medium, suspended in DMEM/F12th 10% fetal bovine serum (FBS; Invitrogen), andered through 100-m and 40-m cell strainer (BDlcon, www.bdbiosciences.com) successively. Col-ted cells were subjected to differential plating andcontinuous Percoll Density Gradient for enrichmentgerm cell population.

. Enrichment of spermatogoniaCollected cells were analyzed for live and deadls by trypan blue dye exclusion and were seededto 100 mm tissue culture dishes (TPP, www.tpp.ch)DMEM/F12 supplemented with 10% FBS for differ-tial plating. Effect of coating culture dishes withA and gelatin were examined for enrichment of

rm cells from testicular cell suspension. Culturehes (TPP, 100 mm) were coated by incubating thehes in 0.5% BSA in PBS or 0.1% gelatin (Sigma) inter overnight (1217 h), washed with PBS twice, andd immediately for differential plating. Isolated cellspension was seeded onto coated dishes at a density2 105 cells/cm2 for 2 h. In control experiments,coated culture dishes (TPP, 100 mm) were used.n-adherent cells were collected following two wash-

s with PBS gently. Adherent cells were dissociated by twubating cells in 0.25% trypsin in PBS for 56 minlowed by strong pipetting. Collected adherent and non-erent cells were analyzed for viability and the presence

spermatogonia.To further enrich the germ cells, collected-floatingls were subjected to Percoll density gradient centrif-ation as described previously by van Pelt et al [19] withnor modifications. Briefly, an iso-osmotic Percoll sus-sion (Pharmacia Biotech, www.gehealthcare.com)

s prepared by the addition of 9 parts of Percoll to 1rt of 1.5 M NaCl (Sigma). A discontinuous densitydient was made by diluting the iso-osmotic Percollpension with HEPES buffered DMEM/F12. Thedients were created by layering 2 mL each of 50, 45,

, 35, 30, 25, and 20% Percoll into a 15 mL centrifugee (TPP). The collected cells were layered on the topthe gradients in 1 mL DMEM/F12 and centrifuged at0x g for 30 min at 25 C. Cells found at the interfacetween the differentdensity suspensions were col-ted as fractions 17 and analyzed for germ cells byHL-1 staining.

. Identification of spermatogonia in separated cellsCollected cells by different methods were fixed inuins fixative for 20 min, washed twice with PBSd attached to poly-l-lysine (Sigma)-coated slidessher Scientific, www.fishersci.com). Cells were stainedth anti-UCHL-1 antibody for identifying germ cellsng methodology described earlier for staining sections.

evaluate the average number of UCHL-1-positivels, 10 random fields from each trail were counted.

. Cultivation and characterization ofrmatogonia in vitro

Cells collected from spermatogonia-enriched frac-n of Percoll density gradient was cultured for a weekvitro. Cells were cultured in 24-well culture dishesPP) with a micro cover glass (Fisher Scientific) at ansity of 2 105 cells/cm2 for staining with germ-l-specific markers. Cover glasses were coated with% gelatin (Sigma) overnight (1217 h) before seed-

cells onto them. The culture medium used wasEM/F12 supplemented with 10 g/mL insulin, 10

/mL apo-transferrin, 100 IU/mL penicillin, 50 g/mLeptomycin, 40 g/mL gentamycin sulfate, singleength non-essential amino acid solution (Gibco, Invitro-), 1 mol/L pyruvate and 10% FBS (Gibco, Invitrogen).

lls were cultured at 37 C in a water-saturated atmo-ere with 95% air and 5% CO2. Medium was changedice per week. Cells were analyzed histochemically

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1224 S. Goel et al. / Theriogenology 74 (2010) 12211232er 67 days of culture when they reached conflu-ce.

The testicular cells in culture were stained withtin DBA and antibodies against POU5F1, UCHL-1d DDX4. Briefly, after fixing cells in Bouins solu-n and permeablizing with 0.1% Triton X-100gma), cells were washed with PBS several times,ubated in 0.3% H2O2 in methanol for 10 min,shed 3 times with PBS, incubated in 10% FBS orth 5% BSA (for DBA staining) in PBS. Other pro-ures were similar to immunostaining of testes sec-

ns described earlier. In negative controls, primarytibody and lectin were omitted and instead the cellsre incubated with 1% BSA in PBS. To ascertain thesence of germ cells in focal colonies, cells were

uble stained with DBA and an antibody (anti-U5F1 or anti-DDX4). Briefly, fixed and permeabil-d cells were incubated with 10% FBS and 5% BSAPBS for 1 h to block non-specific binding sites.llowing overnight incubation in an antibody at theen dilution (1:200), cells were washed several times

th PBS, incubated with goat anti-rabbit-Cy3 (Molec-r probes; 1:200) and DBA-FITC (Vectors Laborato-s; 1:100) for 1 h at 37 C, rinsed three times withS, stained with 100 ng/mL DAPI (Molecular Probes;w.invitrogen.com) for 30 min, mounted in slow-

de (Molecular Probes, Eugene, USA) and observedder an Axioplan 2 microscope fitted with an epifluo-cent lamp. In negative controls, primary antibodyd lectin were omitted and instead the sections wereubated with 1% BSA in PBS.To assay the proliferative ability of spermatogoniaculture, cells attached to poly-l-lysine-coated coverss were incubated with single strength 5-bromo-2-

oxyuridine (BrdU, Roche, www.roche.com) for 6 hfore cells reached confluence, fixed in Bouins solu-n for 10 min at room temperature, washed severales with PBS, permeabilized with 0.1% Triton-X

0, washed with PBS several times, incubated in 2Nl for 30 min at 37 C, neutralized with 0.1 M borate

ffer, pH 8.5, incubated in 10% FBS with 5% BSA inS for 30 min to block non-specific binding sites,ubated with mouse anti-BrdU antibody (Chemicon,w.chemicon.com; 1:100) for 1 h at 37 C, rinsederal times with PBS, incubated with goat anti-use-Cy3 (Invitrogen, 1:200) and DBA-FITC (1:100)1 h at 37 C, washed several times with PBS,

unted in SlowFade and observed proliferating germls that stained both with DBA and anti-BrdU anti-

dy (nucleus). the. RT-PCR analysis of cultured spermatogoniaTo examine the presence of differentiated germ cellsculture, total RNA was prepared from cells (pooledm 4 trials) and analyzed for the presence of meiosis-cific transcripts. RNAs were also isolated from pu-

rtal (912 mo old) and prepubertal buffalo testes asntrols. RNAs was isolated using a ToTally RNA kitmbion, Inc. www.ambion.com) according to thenufacturers protocol. The isolated RNAs wereoled from the testes of respective age groups forparing cDNA. Extracted RNAs were diluted withPC-water and incubated with 2 units of RNase free-ase (Roche, www.roche.com) for 30 min at roomperature. The samples were heated at 70 C for 15

n to inhibit DNase activity and were stored on ice.ndom Primers and RNase OUT (both from Invitro-n) were added to the samples, incubated for 5 min at

C and kept on ice. For reverse transcription,LV high performance reverse transcriptase (Epi-

tre biotechnologies, www.epibio.com) was added tosamples and incubated for 10 min at 30 C, for 60

n at 42 C and for 5 min at 99 C (RT ). At thee time, the reactions without the addition of reverse

nscriptase were done to check genomic DNA con-ination (RT ). PCR amplification was carried out1 l of the above cDNA solution per 19 l of PCRction mixture containing 2 mM MgCl2, 0.25 mMTPs, 1 PCR buffer, 5 pmol of each primers and 1of Taq DNA polymerase (AmpliTaq GoldTM, Ap-ed Biosystems, www.appliedbiosystems.com). Thelowing primers were used for amplification of specifices: PHOSPHOGLYCERATE KINASE 2 (PGK2) 5=-CCATGAAGAAGAACCAGA-3=, 5=-TCAGCAG-ACAGGCTCTAA-3=, 161 bp ([GeneBank] access. no._138733.3) (annealing at 57 C, 30 cycles); PRO-

MINE 2 (PRM2) 5=-TCCGTTAACAGCAAGAG-G-3=, 5=-CCTCTGTGAGTCCTCCCGTA-3=, 245([GeneBank] access. no. NM_164157.2) (annealingC, 30 cycles); -ACTIN (ACTB) 5=-CGATCCA-CAGAGTACTTGCG-3=, 5=-CGAGCGTGGCTA-GTTCACC-3=, 451 bp ([GeneBank] access. no._001101) (annealing at 58 C, 30 cycles). The PCR

ducts were separated and visualized by 2% agarose gelctrophoresis containing 0.5 g/mL ethidium bromide.l PCR products were sequenced to confirm identity.

. Statistical analysis

Testes samples from at least three animals from eache group were used for immunohistochemistry analy-, for the isolation of spermatogonia (n 8) and for

characterization of germ-cell colonies (n 5). The

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1225S. Goel et al. / Theriogenology 74 (2010) 12211232ults are presented as mean SEM. The statisticalalysis was conducted using ANOVA. Differencesre determined by analyzing the data with FishersLD test for significance between the means. Theel of significance was set at P 0.05.

Results

. Histochemistry of buffalo testesLectin DBA affinity in the prepubertal buffalo testiss restricted to germ cells and no somatic cells wereined (Fig. 1A). At this age, germ cells are easilyntified by their large size, topological position andrphology in the seminiferous tubules of testis. DBAining was localized on the surface of germ cells. Theined cells had a large nucleus with 13 nucleoli. A

germ cells showed weak affinity for DBA (Fig.). UCHL1 expression was present exclusively in

rm cells in stained prepubertal testes sections (Fig.

. 1. Immunohistochemical analysis of germ- and pluripotent-cell-specricted to germ cells (arrows) in a 4 month old testis section. Howevning of the testis from 4-month-old buffalo. An intense UCHL1 expr

stronger than DBA-affinity in stained section. (C) POU5F1-stailusively present in undifferentiated germ cells of prepubertal buffalo

falo. DDX4 antibody specifically bound to undifferentiated germ cells (arro). Unlike DBA binding, germ cells stained intenselyth anti-UCHL-1 antibody in prepubertal testes sec-ns. UCHL-1 protein was localized both in nuclei andtoplasm of stained cells. Expression of POU5F1 pro-n was also present in germ cells of prepubertal buf-o testis and no somatic cells were stained (Fig. 1C).the stained germ cells, the localization of POU5F1tein was largely cytoplasmic and occasionally it was

served in the nucleus. Expression of DDX4 wastected specifically in germ cells of the prepubertalffalo testis (Fig. 1D). DDX4 staining was predomi-ntly cytoplasmic and weak in the nuclei. POU5F1pression (Supplemental Fig. 1A) and DDX4 expres-n (Supplemental Fig. 1B) were present mostly inferentiated germ cells such as spermatocytes andrmatids and rarely in spermatogonia. In the stained

rm cells, POU5F1 protein was mainly localized tocytoplasm and occasionally to the nucleus. VI-

NTIN staining was restricted to somatic cells, al-

kers in the prepubertal buffalo testis. (A) Lectin DBA binding wase of the germ cells (arrowheads) showed no affinity. (B) UCHL1as present in germ cells (arrows). Note that the UCHL-1 staining

tis section of a 4-month-old buffalo. POU5F1 expression wasarrows). (D) DDX4 staining of a testis section from 5-month-old1BwitiocyteifalInproobdebuna

ex

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1226 S. Goel et al. / Theriogenology 74 (2010) 12211232ugh a few germ cells were also stained in prepuber-buffalo testes sections (Fig. 2A). GATA4 protein

pression was present in a few Sertoli cells and in theerstitial cells of stained testes sections (Fig. 2B).erestingly, some of the germ cells were also stainedth anti-GATA4 antibody. Staining of GATA4 wasmarily detected in the nuclei of stained cells, al-ugh in some cells a weak cytoplasmic staining was

served. ASMA expression was present in the peritubu-myoid cells and in the interstitial cells of stained testestions (Fig. 2C). In sections where primary antibody andtin was omitted, no positive cells were observed (Fig.).. Germ cell separation and enrichment

We next attempted to isolate a pure population ofrmatogonia from the prepubertal buffalo testis. SinceHL-1 stained spermatogonia intensely with lowerkground staining than lectin DBA, UCHL-1 stainings further utilized for the estimation of purity of germ

. 2. Immunohistochemical analysis of somatic-cell-specific markers innth old testis section. VIMENTIN and GATA4 expression was present in

ENTIN (arrowheads, A) and GATA4 (arrowheads, B) expression wass (yellow arrows). (C) -Smooth muscle ACTIN (ASMA) staining of 4-body (arrows). (D) In a negative control where primary antibody and lls during the isolation and enrichment procedures. Af- centwo-step enzymatic digestion, the dissociated testicularls consisted of 9095% viable cells as determined bypan blue dye exclusion. In the isolated cell population,.3 3.1% of total cells were UCHL-1-positive.Isolated testicular cells were subjected to differentialting for 2h in culture dishes coated with BSA and

latin. In the non-adherent cell population, the num-rs of UCHL-1-positive cells in the BSA coated dishes.6 2.1%), gelatin-coated dishes (36.3 2.4%),

d uncoated dishes (38.1 2.2%) were not signifi-tly different (Fig. 3A). Dishes that were coated with

latin had a higher number of adherent UCHL-1-sitive cells (12.8 1.4%, P 0.05) than BSA coatedhes (6.5 0.4%) or uncoated dishes (5.4 0.2%)g. 3A). There was no difference in cell viability and% cells were viable in all groups. Because the coating

not affect spermatogonia enrichment, uncoatedhes were used in subsequent experiments.Non-adherent cells collected after differential plat-

were subjected to discontinuous Percoll density

rtal buffalo testis. (A) VIMENTIN and (B) GATA4 staining of a 4cells (red arrows) and in cells of the interstitium (asterisk). Note that

sent in germ cells. GATA4 expression was absent from many Sertolild testis section. Peritubular myoid cells stained positive with ASMA

ere omitted, no positive cells are present. Scale bar 20 m.terceltry16

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1227S. Goel et al. / Theriogenology 74 (2010) 12211232llected in fraction 4 (between the 30 and 35% Percollers) were rich in spermatogonia consisting of 55.3% UCHL-1-positive cells (Fig. 3C). UCHL-1 stain-

was intense and uniform within the cells andHL-1-negative cells were comprised of somaticls (Fig. 3D). Enrichment of spermatogonia was moren 2-fold (P 0.05) following differential plating oflated cells and almost 3-fold (P 0.05) after Percollnsity gradient centrifugation with no difference inl viability (Fig. 3B).. In vitro cultivation and characterization ofrmatogonia

The spermatogonia-enriched cell population col-

. 3. Purification of spermatogonia from the prepubertal buffalo testis.difference in germ cell purity was observed in the floating cell popuowing 2 hr incubation. However, a significantly higher number of UCes. (B) Viability and purity of germ cells at various stages of the ce

rease in purity following differential plating and Percoll density gradups. (C) Relative numbers of UCHL-1-positive cells present in differisolation, (b) differential plating, and (c) Percoll density gradient ceresults represent pooled data obtained from 8 different experiments

h the same color represent significant differences (P 0.05). Bar ted after Percoll density gradient was cultivated in celro. Cultured cells showed a high nucleus-to-cyto-sm ratio and each cell had 23 nucleoli when ob-ved at high magnification (Fig. 4A, 4B). These cellsmed flat focal colonies within 45 days of cultureg. 4A). These colonies could be easily identified asatch of epithelial-type cells surrounded by fibroblas-cells in culture and grew in size, reaching an averagemeter of 145 3.6 m by 57 days of culture.me of the colonies reached 190 4.6 m in diam-r. These focal colonies contained DBA-positive cellsg. 4B) and UCHL-1-positive cells (Fig. 4C). DBAining was variable in the stained cells and some ofcells stained weakly or not at all. UCHL-1 expres-

n in the stained cells was also variable and a few

ect of coating on purification of germ cells by differential plating.dishes coated with either BSA or gelatin and in uncoated dishes

ositive cells could be collected as adherent cells in gelatin-coatedtion and purification procedures. Note that there was a significanttrifugation. No difference in cell viability was observed betweentions of Percoll gradient. (D) UCHL-1-stained cells (arrows) aftertion. (d) Negative control, where primary antibody was omitted.presented as mean SEM. In the graphs, different letters on bars.vitplaser

for(Fia pticdiaSoete(Fistathesio

(A) Efflation inHL-1-pll isolaient cenent fracntrifugaand are

50 mls remained unstained (Fig. 4C). Cultured cells ex-

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1228 S. Goel et al. / Theriogenology 74 (2010) 12211232ssed a pluripotency-specific marker (POU5F1, Fig.) and germ cell-specific marker (DDX4, Fig. 4E).U5F1 expression was mostly in the nuclei andX4 was expressed mostly in the cytoplasm. No

sitive cells were detected in the negative controlere primary antibody was omitted (Fig. 4F).Double staining with lectin DBA and anti-BrdU

tibody revealed that spermatogonia could survive

. 4. Buffalo testicular cells in culture. (A) Spermatogonia grew as focaal colonies were positive for DBA binding after 7 days culture. Notenity from some cells. Spermatogonia growing as a chain (arrows) anony expressing UCHL-1 (C), DDX4 (D) and POU5F1 (E). Staineditted in immunostaining of DDX4 (D) and POU5F1 (E). Arrowhead

omitted, no positive cells are seen (F). Bar 50 m.d proliferate in culture for a week (Supplemental Fig. PGApproximately 35.3 3.2% of the DBA-positivels were also BrdU-positive. DBA-positive cells inlture expressed POU5F1 (Fig 5A) and DDX4 (Fig). Interestingly, the expression of POU5F1 andX4 was not restricted to DBA-positive cells and

s also detected in DBA-negative cells (Fig. 5Ad B).Meiotic stage-specific transcripts such as PRM2 and

ies of adherent cells with epithelial morphology (asterisk). (B) Thee staining intensity among the stained cells with the loss of DBAarrowhead) are positive for DBA staining. A focal spermatogoniappear reddish-brown (arrows). Hematoxylin counterstaining wassent unstained cells. In negative control, where primary antibody2).celcu

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1229S. Goel et al. / Theriogenology 74 (2010) 12211232ular cells that were cultured for one week in vitropplemental Fig. 3). However, PRM2 and PGK2

nscripts were detected in pubertal testis.

Discussion

. Histochemistry of buffalo testesTo our knowledge, this is the first report that de-ibes specific markers for buffalo spermatogonia. Thentified markers can be utilized for the identificationbuffalo spermatogonia during isolation and for theiraracterization in culture. In the present study, wend that DBA and UCHL-1 specifically bound to

ffalo spermatogonia in the prepubertal testis. DBAs specific affinity for -D-N-acetyl-galactosamined is known to bind to bovine gonocytes and sper-togonia for the first 30 weeks after birth [20]. In pig,A has been found to be a specific marker for pri-

. 5. Immunohistochemical characterization of cultured spermatogotured cells with DBA and antibodies. DBA-positive cells stained greenning of cultured cells. POU5F1 expression was seen in most DBA-ression (asterisk). POU5F1 expression was also seen in DBA-negativs. (B) DDX4-DBA double-stained cells. DBA-positive cells showression was weak or lost (asterisk). DDX4 staining was also seen in Dm.rdial germ cells (PGCs) [21] and gonocytes in the exonatal testis [14]. UCHL-1 is specifically expressedspermatogonia of bull [22], pig [15], monkey [23],ep [16,24], and human [25]. In bull (Bos taurus),A and UCHL-1 were used to identify spermatogonia

the prepubertal testis [26]. Here we show that DBAd UCHL-1 also specifically bind to buffalo spermato-nia in the prepubertal testis. These markers make itssible to isolate and characterize spermatogonia inlture.Pou5f1 is essential for maintaining the pluripotentialenotype [27]. Pou5f1 is expressed in cells of morulad inner cell mass (ICM), epiblasts, and PGCs [28]. Inle embryos, Pou5f1 expression persists in germ cellsoughout fetal development. After birth, it is main-ned in proliferating gonocytes, pro-spermatogoniad later in undifferentiated spermatogonia [29,30]. Indition, ES cells, embryonic germ (EG) cells, embry-ic carcinoma (EC) cells, epiblast and PGCs also

erlapping images after double-fluorescence staining of 7 dayslls positive for antibodies appeared red. (A) POU5F1-DBA double

cells (arrows), however, a few DBA-positive cell lost POU5F1(arrowheads). Note the nuclear localization of POU5F1 in stainedession of DDX4 (arrows). In some DBA-positive cells, DDX4ative cells (arrowheads). (A=, B=) Cells stained with DAPI. Barne

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1230 S. Goel et al. / Theriogenology 74 (2010) 12211232this study, POU5F1 expression was present in germls in the prepubertal buffalo testis as it is in mice,31]. The presence of POU5F1 expression in sper-togonia is suggestive of their stem cell nature, al-ugh testis transplantation assay could provide fur-r validation. In the pubertal buffalo testis, the

pression of POU5F1 protein was observed in differ-tiated germ cells, such as spermatocytes and roundrmatids and in a few undifferentiated spermatogo-. A similar expression pattern is observed in thetis of prepubertal pigs [34]. However, these resultsagain in disagreement with the findings in mice, whereexpression of Pou5f1 was restricted to undifferentiatedrmatogonia in adult mice testis [30,31]. In conclusion,U5F1 expression in the buffalo testis is rather dynamicis observed both in undifferentiated and differentiated

rm cells.DDX4 (also known as VASA) is a member of theAD-box family of genes and encodes an ATP-de-

ndent RNA helicase. Ddx4 is expressed in PGCs juster their colonization of embryonic gonads and inrm cells undergoing gametogenic processes until thest-meiotic stage in both male and female mice [35].wever, Ddx4 protein is not detected in pluripotentls such as ES cells and EG cells [36]. DDX4 expres-n is present in spermatogonia of pig [37], bull [38],mate [39], and sheep [24]. In the prepubertal testis,X4 and POU5F1 expression was localized in sper-togonia. POU5F1 and DDX4 expression in the pu-

rtal testis were detected in spermatogonia and differ-iated germ cells such as spermatocytes and spermatidspplemental Figure 1). Pou5f1 expression in PGCs of

2.5 was down regulated in Ddx4 null mice [40], whichses the possibility that Pou5f1 expression is under theect control of Ddx4 function in a stage-specific man-r. It is therefore likely that DDX4 expression may be-regulated with POU5F1 expression in the buffalotis.VIMENTIN expression was present in somatic cellsh as Sertoli and interstitial cells, but interestingly, its also detected in a few germ cells in the prepubertaltis. A similar observation was made in the prepuber-sheep testis [41], where VIMENTIN was present incytoplasm of prespermatogonia-I and disappeared

en they changed to prespermatogonia-II. In the neo-tal pig testis, gonocytes also stained positive forMENTIN expression [14]. However, VIMENTINpression in the bull [26] testis was restricted to Ser-i cells and was absent from germ cells. This incon-tency could be attributed to species-specific charac-

istics in buffalo or to the antibody used in this study. groilar to VIMENTIN staining, GATA4 expressions present in interstitial cells, Sertoli cells and in somethe germ cells in the prepubertal buffalo testis.TA4 is a specific marker for Sertoli cells in sheep], bull [26], mouse [42], human [43,44] and pig [45]tes and is required for Sertoli cell development and

subsequent steps in testicular organogenesis [46].e expression of GATA4 in germ cells of the buffalotis is rather unique. The reason for this species-cific characteristic is unclear.

. Purification of germ cellsGerm cells from the buffalo testis could be isolatedng 2-step enzymatic digestion. Similar methods haveen used previously for the isolation of germ cellsm bull [12,13], pig [14], and sheep testes [16,24].ti-UCHL-1 antibody could effectively identify germls during the isolation and purification from the pre-bertal buffalo testis. UCHL-1 has been used previ-sly for germ-cell identification during the isolation ofls from pig [15] and sheep [16,24] testes. In earlierorts, lectin DBA affinity was used for purifying

rm cells from the bull testis [12,13]. In the presentdy, UCHL-1 expression was more intense than DBAnity therefore; we utilized it for the analysis of germl purity in the isolation procedure. BSA-coated cul-e dishes showed no difference in the recovery ofHL-1-positive germ cells from control group (Fig). This finding differs from an earlier report, whereA coating could significantly enhance the purity ofll germ cells in the differential plating method [13].is could be due to the difference in adhering abilitybuffalo germ cells compared to cattle. Gelatin-coatedlture dishes had higher numbers of adherent germls, which suggests that gelatin coating could facili-e the attachment of buffalo germ cells to culturehes. Approximately 55% UCHL-1-positive germ cellsre obtained by the combination of differential plating

Percoll density-gradient centrifugation methods. Inr hand, germ cell purity in isolated cells was lower thant reported in bull using similar procedures [13]. The

purity may be due to different characteristics of buf-o germ cells and the difference in reagents used in thisdy.

. Spermatogonia cultivation, proliferation, andaracterization

Buffalo germ cells from the prepubertal testis couldvive and proliferate in vitro in DMEM/F12 supple-nted with 10% FBS without any supplemental

wth factors on gelatin-coated cover glasses. Sper-

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1231S. Goel et al. / Theriogenology 74 (2010) 12211232togonia grew as focal colonies, which were positivegerm-cell specific markers (DBA and DDX4) and aripotent-cell specific marker (POU5F1). Somaticl proliferation was also significant due to high serumncentration in the culture medium. DBA affinity hasen used to identify bull spermatogonia [17,18] andrcine gonocytes [14,47] in culture. Isolated germls remained viable during culture and could prolif-te, as determined by DBA labeling, BrdU incorpo-ion and colony formation, suggesting buffalo sper-togonia can be sustained in the present culture

nditions. The high viability of the DBA-positive cellsbe attributed to the presence of FBS, which con-

ns numerous undefined factors. Our enriched sper-togonia culture contained approximately 50% so-tic cells. It is, therefore, likely that secretions fromatic cells in culture, especially Sertoli cells may

ve a beneficial effect on proliferation of spermatogo-. Similar findings have been reported for pig gono-

tes [14,47] and bull spermatogonia [17,18] culturedvitro.Cultured germ cells that were positive for DBA also

pressed DDX4 and POU5F1. The expressions ofX4 and POU5F1 were also present in DBA-nega-

e cells in culture. This finding is in agreement withtestis histochemistry analysis, in which a few sper-

togonia did not stain with DBA. Another possibilitythat some of the cells that still expressed POU5F1d DDX4 lost their affinity for DBA. Although intis sections, POU5F1 expression was predominantlytoplasmic, its expression was largely nuclear in cul-ed cells. Similarly, in a few cultured cells, DDX4pression was detected in the nuclei. It is likely thatre is trans-localization of POU5F1 and DDX4 in theclei of the cultured cells. Further studies are neededcheck this possibility. Since germ cells of the testiso expressed somatic cell-specific markers such asMENTIN and GATA4, double staining with theserkers and germ-cell specific markers could not be useddistinguish germ cells from somatic cells in the culture.vertheless, the presence of DBA-positive cells thatined with DDX4 and POU5F1 confirmed the presencebuffalo spermatogonia in culture. The absence ofnscripts specific to differentiated germ cells fromltured cells confirmed the presence of undifferen-ted spermatogonia in culture (Supplemental Figure 3).pendix. Supplementary data

Supplementary data associated with this article can bend, in the online version, at doi:10.1016/j.theriogenology.

0.05.025.knowledgements

This work was supported by grants from DepartmentBiotechnology (BT/PR 10908/MED/31/35/2008)

d Department of Science and Technology (DST/INT/P/P-72/09) in India and by Japanese Society for themotion of Science. The authors would like to thank

Prabhu and Chan Pasha for assistance in collectingtes and T Avinash Raj for help in section cutting. Dr.epali Garg is acknowledged for a critical reading ofmanuscript.

ferences

] Borghese A, Mazzi M. Buffalo population and strategies in theworld. In: Borghese A, editor. Buffalo production and research.Food and Agriculture Organization of the United Nations,Rome. 2006. p. 140.

] Perera BM. Reproduction in domestic buffalo. Reprod DomestAnim 2008;43:[Suppl 2]20006.

] Capel B. The battle of the sexes. Mech Dev 2000;92:89103.] De Rooji, DG, Russell LD. All you wanted to know about sper-

matogonia but were afraid to ask. J Androl 2000;21:77698.] Nagano M, Shinohara T, Avarbock MR, Brinster RL. Retrovi-

rus-mediated gene delivery into male germ line stem cells.FEBS Letter 2000;475:710.

] Nagano M, Brinster CJ, Orwig KE, Ryu BY, Avarbock MR,Brinster RL. Transgenic mice produced by retroviral transduc-tion of male germ-line stem cells. Proc Natl Acad Sci USA2001;98:130905.

] Hamra FK, Gatlin J, Chapman KM, Grellhesl DM, Garcia JV,Hammer RE, Garbers DL. Production of transgenic rats bylentiviral transduction of male germ-line stem cells. Proc NatlAcad Sci USA 2002;99:149316.

] Kanatsu-Shinohara M, Toyokuni S, Shinohara T. Transgenicmice produced by retroviral transduction of male germ line stemcells in vivo. Biol Reprod 2004;71:12027.

] Kanatsu-Shinohara M, Toyokuni S, Shinohara T. Genetic se-lection of mouse male germLine stem cells in vitro: offspringfrom single stem cells. Biol Reprod 2005;72:23640.

] Honaramooz A, Behboodi E, Megee SO, Overton SA, Ga-lantino-Homer H, Echelard Y, Dobrinski I. Fertility and germ-line transmission of donor haplotype following germ cell trans-plantation in immunocompetent goats. Biol Reprod 2003;69:12604.

] Stockwell S, Herrid M, Davey R, Brownlee A, Hutton K, HillJR. Microsatellite detection of donor-derived sperm DNA fol-lowing germ cell transplantation in cattle. Reprod Fertil Dev2009;214:628.

] Izadyar F, Spierenberg GT, Creemers LB, Den Ouden K, deRooij DG. Isolation and purification of type A spermatogoniafrom the bovine testis. Reproduction 2002;124:8594.

] Herrid M, Davey RJ, Hutton K, Colditz IG, Hill JR. A com-parison of methods for preparing enriched populations of bovinespermatogonia. Reprod Fertil Dev 2009;21:3939.

] Goel S, Sugimoto M, Minami N, Yamada M, Kume S, Imai H.Identification, isolation, and in vitro culture of porcine gono-

cytes. Biol Reprod 2007;77:12737.

[15] Luo J, Megee S, Rathi R, Dobrinski I. Protein gene product 9.5is a spermatogonia-specific marker in the pig testis: application toenrichment and culture of porcine spermatogonia. Mol Reprod Dev

[16

[17

[18

[19

[20

[21

[22

[23

[24

[25

[26

[27

[28

[29

[30

[31

[32

[33

[34] Goel S, Fujihara M, Minami N, Yamada M, and Imai H. Expres-sion of NANOG, but not POU5F1, points to the stem cell poten-tial of primitive germ cells in neonatal pig testis. Reproduc-

[35

[36

[37

[38

[39

[40

[41

[42

[43

[44

[45

[46

[47

1232 S. Goel et al. / Theriogenology 74 (2010) 122112322006;73:153140.] Rodriguez-Sosa JR, Dobson H, Hahnel A. Isolation and trans-

plantation of spermatogonia in sheep. Theriogenology 2006;66:2091103.

] Izadyar F, Den Ouden K, Creemers LB, Posthuma G, ParvinenM, De Rooij DG. Proliferation and differentiation of bovinetype A spermatogonia during long-term culture. Biol Reprod2003;68:27281.

] Aponte PM, Soda T, Teerds KJ, Mizrak SC, van de Kant HJ, deRooij DG. Propagation of bovine spermatogonial stem cells invitro. Reproduction 2008;136:54357.

] van Pelt AMM, Morena AR, van Dissel-Emiliani FMF, BoitaniC, Gaemers IC, de Rooij DG, Stefanini M. Isolation of thesynchronized A spermatogonia from adult vitamin A-deficientrat testes. Biol Reprod 1996;55:43944.

] Ertl C, Wrobel KH. Distribution of sugar residues in the bovinetestis during postnatal ontogenesis demonstrated with lectin-horseradish peroxidase conjugates. Histochemistry 1992;97:16171.

] Takagi Y, Talbot NC, Rexroad CE, Jr, Pursel VG. Identificationof pig primordial germ cells by immunocytochemistry and lec-tin binding. Mol Reprod Dev 1997;46:56780.

] Wrobel KH, Bickel D, Kujat R, Schimmel M. Configuration anddistribution of Bovine Spermatogonia. Cell Tissue Res 1995;279:27789.

] Tokunaga Y, Imai S, Torii R, Maeda T. Cytoplasmic liberationof protein gene product 9.5 during the seasonal regulation ofspermatogenesis in the monkey (Macaca fuscata). Endocrinol-ogy 1999;140:187583.

] Herrid M, Olejnik J, Jackson M, Suchowerska N, Stockwell S,Davey R, Hutton K, Hope S, Hill JR. Irradiation enhances theefficiency of testicular germ cell transplantation in sheep. BiolReprod 2009;81:898905.

] He Z, Kokkinaki M, Jiang J, Dobrinski I, Dym M. Isolation,characterization, and culture of human spermatogonia. Biol Re-prod 2009;82:36372.

] Herrid M, Davey RJ, Hill JR. Characterization of germ cellsfrom pre-pubertal bull calves in preparation for germ cell trans-plantation. Cell Tissue Res 2007;330:3219.

] Brehm A, Ovitt CE, Schler HR. Oct-4: more than just aPOUerful marker of the mammalian germLine? Acta PatholMicrobiol Immunol Scand A 1998;106:11424.

] Ovitt CE, Schler HR. The molecular biology of Oct-4 in theearly mouse embryo. Mol Hum Reprod 1998;4:102131.

] Pesce M, Gross MK, Schler HR. In line with our ancestors:Oct-4 and the mammalian germ. Bioessays 1998a;20:72232.

] Tadokoro Y, Yomogida K, Ohta H, Tohda A, Nishimune Y.Homeostatic regulation of germinal stem cell proliferation bythe GDNF/FSH pathway. Mech Dev 2002;113:2939.

] Pesce M, Wang X, Wolgemuth DJ, Scholer H. Differentialexpression of the Oct-4 transcription factor during mouse germcell differentiation. Mech Dev 1998b;71:8998.

] Surani MA. Reprogramming of genome function through epi-genetic inheritance. Nature 2001;414:1228.

] Donovan PJ, de Miguel MP. Turning germ cells into stem cells.Curr Opin Genet Dev 2003;13:46371.tion 2008;135:78595.] Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M,

Noce T. Expression and intracellular localization of mouseVasa-homologue protein during germ cell development. MechDev 2000;93:13949.

] Fujiwara Y, Komiya T, Kawabata H, Sato M, Fujimoto H,Furusawa M, Noce T. Isolation of a DEAD-family protein genethat encodes a murine homolog of Drosophila vasa and itsspecific expression in germ cell lineage. Proc Natl Acad SciUSA 1994;91:1225862.

] Lee GS, Kim HS, Lee SH, Kang MS, Kim DY, Lee CK, KangSK, Lee BC, Hwang WS . Characterization of pig vasa homologgene and specific expression in germ cell lineage. Mol ReprodDev 2005;72:3208.

] Bartholomew RA, Parks JE. Identification, localization, andsequencing of fetal bovine vasa homolog. Anim Reprod Sci2007;101:24151.

] Hermann BP, Sukhwani M, Lin CC, Sheng Y, Tomko J, Ro-driguez M, Shuttleworth JJ, McFarland D, Hobbs RM, PandolfiPP et al. Characterization, cryopreservation, and ablation ofspermatogonial stem cells in adult rhesus macaques. Stem Cells2007;25:23308.

] Tanaka SS, Toyooka Y, Akasu R, Katoh-Fukui Y, Nakahara Y,Suzuki R, Yokoyama M, Noce T. The mouse homolog ofDrosophila Vasa is required for the development of male germcells. Dev Genes Evol 2000;14:84153.

] Steger K, Wrobel KH. Immunohistochemical demonstration ofcytoskeletal proteins in the ovine testis during postnatal devel-opment. Anat Embryol 1994;189:52130.

] Viger RS, Mertineit C, Trasler JM, Nemer M. Transcription factorGATA-4 is expressed in a sexually dimorphic pattern duringmouse gonadal development and is a potent activator of the Ml-lerian inhibiting substance promoter. Development 1998;125:266575.

] Ketola I, Rahman N, Toppari J, Bielinska M, Porter-Tinge SB,Tapanainen JS, Huhtaniemi IT, Wilson DB, Heikinheimo M.Expression and regulation of transcription factors GATA-4 andGATA-6 in developing mouse testis. Endocrinology 1999;140:147080.

] Ketola I, Pentikinen V, Vaskivuo T, Ilvesmki V, Herva R,Dunkel L, Tapanainen JS, Toppari J, Heikinheimo M. Expres-sion of transcription factor GATA-4 during human testiculardevelopment and disease. J Clin Endocrinol Metab 2000;85:392531.

] McCoard SA, Wise TH, Fahrenkrug SC, Ford JJ. Temporal andspatial localization patterns of GATA-4 during porcine gona-dogenesis. Biol Reprod 2001;65:36674.

] Tevosian SG, Albrecht KH, Crispino JD, Fujiwara Y, EicherEM, Orkin SH. Gonadal differentiation, sex determination andnormal Sry expression in mice require direct interaction be-tween transcription partners GATA4 and FOG2. Development2002;129:462734.

] Goel S, Fujihara M, Tsuchiya K, Takagi Y, Minami N, YamadaM, Imai H. Multipotential ability of primitive germ cells fromneonatal pig testis cultured in vitro. Reprod Fertil Dev 2009;21:696708.

Spermatogonia-specific proteins expressed in prepubertal buffalo (Bubalus bubalis) testis and their utilization for isolation and in vitro cultivation of spermatogonia1. Introduction2. Material and methods2.1. Collection of the testis2.2. Histochemistry of buffalo testes2.3. Isolation of spermatogonia2.4. Enrichment of spermatogonia2.5. Identification of spermatogonia in separated cells2.6. Cultivation and characterization of spermatogonia in vitro2.7. RT-PCR analysis of cultured spermatogonia2.8. Statistical analysis

3. Results3.1. Histochemistry of buffalo testes3.2. Germ cell separation and enrichment3.3. In vitro cultivation and characterization of spermatogonia

4. Discussion4.1. Histochemistry of buffalo testes4.2. Purification of germ cells4.3. Spermatogonia cultivation, proliferation, and characterization

Appendix. Supplementary dataAcknowledgementsReferences