The radiation-induced block in spermatogonial differentiation is due to damage to the somatic environment, not the germ cells

ORIGINAL ARTICLE 149

The Radiation-Induced Block inSpermatogonial Differentiation isDue to Damage to the SomaticEnvironment, not the Germ Cells
ZHEN ZHANG,* SHAN SHAO, AND MARVIN L. MEISTRICH
Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

Radiation and chemotherapeutic drugs cause permanent sterility in male rats, not by killing most of the spermatogonial stem cells, but byblocking their differentiation in a testosterone-dependent manner. However, it is not known whether radiation induces this block byaltering the germ or the somatic cells. To address this question, we transplanted populations of rat testicular cells containing stemspermatogonia and expressing the green fluorescent protein (GFP) transgene into various hosts. Transplantation of the stemspermatogonia from irradiated adult rats into the testes of irradiated nudemice, which do not show the differentiation block of their ownspermatogonia, permitted differentiation of the rat spermatogonia into spermatozoa. Conversely transplantation of spermatogonial stemcells from untreated prepubertal rats into irradiated rat testes showed that the donor spermatogonia were able to colonize along thebasementmembrane of the seminiferous tubules but could not differentiate. Finally, suppression of testosterone in the recipient irradiatedrats allowed the differentiation of the transplanted spermatogonia. These results conclusively show that the defect caused by radiation inthe rat testes that results in the block of spermatogonial differentiation is due to injury to the somatic compartment. We also observedcolonization of tubules by transplanted Sertoli cells from immature rats. The present results suggest that transplantation ofspermatogonia, harvested from prepubertal testes to adult testes that have been exposed to cytotoxic therapy might be limited by thesomatic damage and may require hormonal treatments or transplantation of somatic elements to restore the ability of the tissue tosupport spermatogenesis.J. Cell. Physiol. 211: 149–158, 2007. � 2006 Wiley-Liss, Inc.

Contract grant sponsor: NIH;Contract grant number: R01 ES 08075.Contract grant sponsor: Cancer Center Support;Contract grant number: CA 16672.Contract grant sponsor: Lalor Foundation.

Zhen Zhang’s present address is Center for Reproduction andDevelopment, Monash Institute of Medical Research, Clayton, VIC3168, Australia. E-mail: [email protected]

*Correspondence to: Zhen Zhang, Center for Reproduction andDevelopment, Monash Institute of Medical Research, Clayton, VIC3168, Australia. E-mail: [email protected]

Received 15 August 2006; Accepted 15 September 2006

DOI: 10.1002/jcp.20910

With increased rates and durations of cancer survival, especiallyin children and young adults, their quality of life has become animportant concern (Schover and Meistrich, 2005). Theirreproductive ability is a major quality of life issue.Chemotherapy (with alkylating agents or cisplatin) orradiotherapy (either whole body or to local fields close to thereproductive organs) induces prolonged or permanentazoospermia causing temporary or permanent infertility (Fossaand Magelssen, 2004; Shetty and Meistrich, 2005; Meistrichet al., 2005b).The following general observations regarding damage to thereproductive system apply both to model animal systems andhumans (Meistrich and Shetty, 2003b). The prolongedreproductive effects of chemotherapy and radiation aregenerally a result of direct gonadal damage and the germ cellsare considered to be the major target for damage. Thepost-stem cell spermatogonia, which are undergoingdifferentiation, are actively proliferating and are most sensitiveto killing bymost chemotherapy drugs and irradiation (DymandClermont, 1970; Rowley et al., 1974; Meistrich et al., 1982,1997). Stem spermatogonia are more resistant but showmoderate sensitivity to radiation and alkylating agents(Meistrich, 1982; Meistrich et al., 2005b). Killing of all stem cellswill obviously result in permanent azoospermia. If stem cellssurvive, they may in some cases rather quickly initiate therecovery of spermatogenesis and sperm production (Meistrich,1986; Meistrich et al., 1997), or they can remain ‘‘arrested’’ inthe testis as isolated spermatogonia in atrophic tubules(Kreuser et al., 1989; Kangasniemi et al., 1996) and theremay bea long delay before the process is reinitiated or completed(Rowley et al., 1974; Meistrich et al., 1992).In contrast to the sensitivity of the germ cells, there is currentlylittle evidence that moderate doses of radiation orchemotherapy cause prolonged damage to the somaticelements of the testis. Although elevated luteinizing hormoneand low-normal serum testosterone levels have been reported

� 2 0 0 6 W I L E Y - L I S S , I N C .

to indicate compensated Leydig cell failure (Howell et al., 2000),this postulate is unproven, and the change can be explained byreduced testicular blood flow (Wang et al., 1983). Only veryhigh doses of radiation produce prolonged Leydig cell damage(Delic et al., 1986; Shalet, 1993) and no chemotherapeutic drugsproduce more than a transient decrease in testosterone levels(Maines et al., 1990). Neither chemotherapy nor radiation killsSertoli cells, but radiation can increase their production ofcytokines (Guitton et al., 1999).Because the germ cells have contact and a dependentrelationship with the Sertoli cells within the seminiferoustubules and paracrine interactions with neighboring peritubularand interstitial cells, it is difficult to determine whether germ orsomatic cells are the target for damage because injuries toeither could result in similar phenotypes. Although theenhanced recovery of spermatogenesis in rats after irradiationor chemotherapy by androgen and FSH suppression (Meistrich

150 Z H A N G E T A L .

and Kangasniemi, 1997; Udagawa et al., 2001) may seem toindicate that the damage was to the somatic cells because theyexpress androgen and FSH receptors (Kliesch et al., 1992;Meistrich et al., 2005a), models show that either somatic orgerm cell damage can be consistent with these observations(Meistrich and Shetty, 2003a). Fortunately, spermatogonialtransplantation, a powerful functional assay for both defects inspermatogonial stem cells and in the spermatogenicenvironment, makes it possible to determine the damaged celltype or compartment as long as the germ and the somatic cellsare from different sources and are genetically marked (Brinsterand Avarbock, 1994; Ogawa et al., 2000).In the present study, we determined the target forspermatogenic arrest in irradiated rat testes using germ celltransplantation. To test whether the spermatogonia from theirradiated adult rats were capable of differentiation in anenvironment with high testosterone and FSH, they weretransplanted into irradiated mouse testes, which we haveshown to efficiently support spermatogenesis from normaldonor rat spermatogonia (Zhang et al., 2006). To test whetherthe seminiferous tubular environment of irradiated rats wassuitable for development of normal spermatogonia,seminiferous tubular cells from immature rats weretransplanted into these tubules. Possible methods to improvethe environment for transplantation, including suppression ofhormones and Sertoli cell transplantation were examined.

Materials and MethodsAnimals

Adult nude (Swiss nu-nu/Ncr) mice bred in the M. D. Anderson CancerCenter and LBNF1 (F1 hybrids of Lewis and Brown-Norway) rats(Harlan Sprague-Dawley, Indianapolis, IN) were used as recipients.Donors were transgenic rats expressing green fluorescent protein(GFP) under the control of a CMV enhancer and ubiquitin-C promoter(Lois et al., 2002). The GFP-transgenic rats, originally on aSprague-Dawley genetic background, were backcrossed to the inbredLewis strain (Harlan Sprague-Dawley) and the allele at the majorhistocompatibility complex locus RT1 was monitored using PCR andagarose gel analysis of the closely linked microsatellite locus D20Rat46(Rat Genome Database, http://rgd.mcw.edu/). At the secondgeneration of the backcross, all of the rats used for further breeding hadthe haplotype of the Lewis strain and had lost the Sprague-Dawley allele(data not shown). Therefore, some of the backcrossed GFP-Lewismales from the third generation were used as donors for testicular celltransplantation to LBNF1 rats. Overt inflammation was rarelyobserved, and fibroblastic cells were occasionally observed in recipienttestes after transplantation; however; there were no differences in theoccurrence of these effects when donor cells were from the third orthe sixth backcross generations, indicating that immune response wasnot a factor. GFP-positive females from the fifth Lewis-backcrossgeneration were mated with Brown-Norway male rats (HarlanSprague-Dawley) to obtain LBNF1 rats expressing GFP. All animalswere caged in a controlled environment (12-h light:12-h dark) withunlimited access to food and water. All experiments were approved bythe Institutional Animal Care and Use Committee of the University ofTexas M. D. Anderson Cancer Center.

Preparation of recipients

The nude mice were restrained in plastic chambers without anesthesiaand irradiated with a 3-cm diameter field to the lower abdominal andscrotal areas at a dose rate of 5.6 Gy/min using a dual-source 137Cs g-ray unit (Zhang et al., 2006). Two fractions of irradiation (1.5þ 12 Gy)were given 24 h apart to mouse testes; this dose regimen is effective atdepleting endogenous germ cells without producing severecalcification or other damage to the stroma (Creemers et al., 2002;Zhang et al., 2006). The mice were used as recipients for germ celltransplantation between 4 and 8 weeks after irradiation.In one experiment recipient nude mice were treated with a single doseof busulfan at 44mg/kg as described previously (Zhang et al., 2006). Thiswas done to compare the efficiency of colonization and degree ofdifferentiation of donor spermatogonia from irradiated rats in

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

irradiated recipient mouse testes with that in the more widely usedbusulfan-treated mouse model (Clouthier et al., 1996).Male LBNF1 rats (about 8 weeks old) were anesthetized with aketamine (0.72 mg/kg) and acepromazine (0.022 mg/kg) mixture (i.m.),and irradiated with a 60Co g-ray unit (Eldorado 8; Atomic EnergyCanada Ltd., Ottawa, ON, Canada) as described previously (Shettyet al., 2000). Rats were placed on their backs and 5 mm oftissue-equivalent bolus material (Superflab, Mick Radio-NuclearInstruments, Inc., Bronx,NY)was placedover the scrotum to provide abuild-up layer. The irradiation field extended anteriorly about 6 cmabove the base of the scrotum. A single dose of 6Gywas given at a doserate of about 0.9 Gy/min. The rats were used as recipients for germ celltransplantation between 7 and 21 weeks after irradiation, at whichtimes the tubules contain only a few type A spermatogonia (includingthe A-isolated stem cells, the A-paired, and the A-aligned cells) andalmost no differentiating germ cells at or beyond theA1 spermatogonialstage (Kangasniemi et al., 1996).

Preparation of donor cells

Immature rats (5–21 days old) or irradiated adult rats (10–24 weeksafter irradiation) were used as testicular cell donors. It should be notedthe efficiency of colonization of recipients by spermatogonial stem cellsfrom mice between 5 and 28 days of age varies by only twofold withinthat age range (McLean et al., 2003). To preferentially harvest singlecells from the tubules, the tunica was removed and the tissue wassequentially digested with enzymes at 358C in a shaking water bath asdescribed in detail recently (Zhang et al., 2006). Tubuleswere preparedby two digestions, first with collagenase and then with collagenase andhyaluronidase, in DMEM/F12 medium containing DNase I and fetalbovine serum for 20–30 min each. Tubules were then digested withtrypsin in Dulbecco’s PBS (GIBCO, Grand Island, NY) containing 1mMEGTA and DNase I for 10–15 min. The final pellets were resuspendedin DMEM/F12 containing 10% fetal bovine serum.Because there are only about 1.3 type A spermatogonia per 100 Sertolicells in irradiated adult rat testes (Shuttlesworth et al., 2000), weperformed Percoll gradient centrifugation, which enriches type Aspermatogonia (van Pelt et al., 1996) and increases the relativeabundance of colony-forming stem cells (Kubota et al., 2004). Afterenzymatic digestion, the resuspended cells were loaded onto 30–52%continuous Percoll (Amersham Biosciences, Piscataway, NJ) gradientsand centrifuged for 10min at 10,000 rpm (11,000g) at 48C as describedpreviously (Meistrich et al., 1981). Cells were collected from thefractions determined by phase contrast microscopy to be mostenriched in spermatogonia andwashed once with DMEM/F12medium.This procedure was shown to enrich spermatogonia, identified byGCNA1 immunostaining in cell smears, from about 4% to about 16%(data not shown). The final pellets were resuspended in DMEM/F12with 10% fetal bovine serum.Trypan blue (GIBCO)was added to a concentration of 0.02%. After thecell concentration and viability were determined, the cell suspensionwas kept on ice until transplantation. Cell suspensions from immaturerats contained an average of 7� 107 cells/ml. The cell suspensions fromirradiated rats used for transplantation into irradiated rats, irradiatedmice, and gonadotropin-releasing hormone (GnRH)-antagonist-treated irradiated rats averaged 6� 106, 2� 107, and 4� 107 cells/ml,respectively.

Transplantation

Mice were anesthetized with a mixture of ketamine (6.7 mg/ml) andxylazine (1.3 mg/ml) given at 0.15 ml/10-g body weight (i.p). Rats wereanesthetized with a mixture of ketamine (0.72 mg/kg) andacepromazine (0.022 mg/kg) (i.m). For mice, the testis was exposedthrough an abdominal incision, a glass needle (tip inner diameter20–25 mm with a 258 angle) was inserted into the efferent duct, anddonor cells were injected into rat testes using a FemtoJet1

semi-automatic microinjector (Brinkmann Instruments, Inc.,Westbury, NY). Injection volumes were determined from the volumeof suspension with which the needle was filled using a loading pipetteand subtracting the proportion of length of the needle still containingfluid and the estimated amount of leakage. An average of 8ml (minimum1ml, maximum15ml) of cell suspensionwas injected into each recipienttestis. For rats, the testis was exposed in the same way, a 30-G dentalneedle (Terumo Corporation, Tokyo, Japan) was inserted into theefferent duct, and donor cells were injected using a 1-ml syringe.Between 100 ml and 400 ml of cell suspension was injected into each

S O M A T I C D A M A G E B L O C K S G E R M C E L L D E V E L O P M E N T 151

recipient testis, as determined from the graduations on the syringebarrel. Transplantation was considered to be successful if the trypanblue marker dye filled some of the surface seminiferous tubules,however, in most cases the dye filled more than 50% of the tubules.Some of the recipient rats received hormone suppression aftertransplantation. The GnRH antagonist Acyline, was dissolved in sterilewater and subcutaneously injected at a dose of 1.5mg/kg, starting at thetime of transplantation, and then once a week for 12 weeks tocontinuously suppress intratesticular testosterone levels (Shetty et al.,2004). Acyline was obtained from the Contraceptive DevelopmentBranch of the National Institute of Child Health and HumanDevelopment (North Bethesda, MD).

Macroscopic and microscopic assessment of spermatogenesis

Recipients were killed 13weeks after transplantation. After removal ofthe tunica albuginea, the testis was placed in cold PBS, andGFP-positiveregions were identified under fluorescent light. The testes were thenfixed in 4% paraformaldehyde (Sigma, St. Louis, MO) overnight at 48Cand subjected to routine histological processing, paraffin embedding,and sectioning at 4–5mm thickness. The pachytene spermatocytes andspermatids were readily identified by nuclear morphology, size, andlocation within the epithelium (Russell et al., 1990). Because themorphology of cells in this material was not as distinct as previouslyobserved with methacrylate-embedded, Bouin’s-fixed tissue(Shuttlesworth et al., 2000), immunohistochemistry was needed toconclusively identify the earlier stage germ cells.

Immunohistochemical assessment of spermatogenesis

Serial sections were prepared for immunohistochemistry, includingantigen retrieval, inhibition of endogenous peroxidase, and blocking asdescribed previously (Zhang et al., 2006). The rabbit polyclonalanti-GFP antibody (1:5,000 dilution, Novus Biologicals, Littleton, CO)was used to stain donor cells; both Sertoli and germ cells of thetransgenic rats express GFP. The rat anti-mouse monoclonalanti-GCNA1 (germ cell nuclear antigen 1) antibody (1:100 dilution, agift fromDr.George Enders)was used to stain germ cells. The antibodystains mouse germ cells more strongly than rat germ cells, and amongthe rat germ cells the spermatogonia were only very weakly stained(Zhang et al., 2006). The rabbit polyclonal anti-WT1 (Wilms’ tumorgene) antibody (1:1,000 dilution, Santa Cruz Biotechnology, SantaCruz, CA) specifically stains nuclei of Sertoli cells at all ages (Sharpeet al., 2003). The primary antibodies were added onto tissues andincubated overnight at 48C. ABC Elite kits, second antibodies, and3,30-diaminobenzidine (all from Vector Laboratories, Burlingame, CA)were used as recommended by the manufacturer. Sections werecounterstained with hematoxylin.Donor cell-positive tubules were counted in histological sections afteranti-GFP staining. Germ cells were further identified in adjacentsections by staining with anti-GCNA1 or by lack of staining withanti-WT1. The differentiation stages of GFP-positive germ cells weredetermined in each tubule by histological criteria. A tubule wasconsidered to contain differentiated germ cells if there were three ormore germ cells that had reached the early pachytene spermatocytestage within a tubule or if there were five or more germ cells, some ofwhich were in chains, near the basement membrane, since they werelikely type B spermatogonia or very early spermatocytes because thetype A spermatogonia always appear in sections single cells or shortchains (Russell et al., 1990). All data presented are based on thesecriteria for scoring differentiation, although we are aware thatdifferentiated spermatogonia in the type A1 through A4 andintermediate stages will bemissed. The results were based on countingtubules, in most cases, in three sections, which were spaced an averageof 120 mm apart from each other, per testis. All tubules (average of301 for rat recipients; 137 for mouse recipients) were counted in eachsection.

ResultsTest of whether the environment is defective

A population of tubule cells containing stem spermatogoniafrom immature GFP-positive rats was transferred into testes ofirradiated adult LBNF1 rats. A total of 46 recipient testes weresuccessfully injected with donor cells from either the GFP ratsbackcrossed to the Lewis strain for 3–6 generations or from the


GFP-LBNF1 rats.No statistically significant differences betweenthe results obtained using donors from the different backcrossgenerations or the different genetic backgrounds existed in anyof the individual recipient types, so they were pooled (Table 1).Since the immature rat donor cells were from rats in the5–21 days age range, we subdivided them into three groups,aged 5–10, 11–16, and 17–21 days. There were no significantdifferences in the ability of cells to form colonies and thepercentage of different classes of colonies with age, and hencethe results were pooled. Transplanted testes examined under afluorescence microscope (Fig. 1A) showed short and longerregions of strong green fluorescence, both indicating aggregatesof cells with cytoplasmic staining, weak green fluorescencelining the basement membrane, and small, localized spots ofbright staining (not shown but similar to those in Fig. 1D).Donor cells, identified in sections of recipient seminiferoustubules by anti-GFP immunostaining, were found in threemajormorphologic patterns: cells in contact with the basementmembrane showing continuous cytoplasmic staining (12% ofGFP-positive tubules, Fig. 2D,E), cells forming a structure in thelumen (71%, Fig. 2F), and isolated cells or colonies of only a fewcells along the basement membrane (17%, Fig. 2A). In general,the isolated cells or short chains of cells along the basementmembrane did not immunostain with anti-WT1 (Fig. 2B) but didstain weakly with anti-GCNA1 (Fig. 2C). Based on criteria suchas morphology, location, numbers, and immunostaining, theywere identified as spermatogonia. Differentiated germ cellswere never observed in any of the 46 irradiated LBNF1 testestransplantedwith immature rat germ cells (Table 1). In contrast,almost all of the cells showing continuous strong cytoplasmicGFP-staining were conclusively identified as Sertoli cells byWT1 immunostaining (Fig. 2D,F, insets). Furthermore themorphology of the donor cells along the basement membranewas typical of Sertoli cells in irradiated rats (Fig. 2D). In somecases fibroblastic or peritubular-like cells divided the tubulesfilledwith donor Sertoli cells into separateminitubules (Fig. 2E).The luminal Sertoli cells surrounded a core of mostly GFP-negative cells (Fig. 2F) that could not be stained by GCNA1 andsometimes appeared to be degenerating.To test whether the donor spermatogonial stem cells fromimmature rats were indeed functional, they were injected intoirradiatedmouse testes, which are permissive recipients for thesurvival and differentiation of rat spermatogonial stem cells.Under fluorescence microscopy, transplanted testes showedlong regions of tubules with strong green fluorescence andsome tubules with only chains of cells along the basementmembrane (Fig. 1B). Immunohistochemical analysis of sectionswith anti-GFP antibody showed that 35% of the host’s tubulecross-sections were colonized with donor germ cells (Table 1,Fig. 2G).Donor spermatogenesis had developed in 76%of thesedonor cell-colonized tubules, as confirmed by anti-GCNA1staining of donor cells reaching the spermatocyte and spermatidstage in many of the tubules (Fig. 2H, asterisks). Some tubules(24%) had only a few isolated donor cells along the basementmembrane. Most of them appeared to be type Aspermatogonia, although they were not detected with theGCNA1 staining under conditions inwhich the backgroundwaskept low (Fig. 2H, arrowhead). Donor Sertoli cells were rarelyobserved either on the basement membrane or in the lumen ofthe tubules of recipient irradiated mouse testes. These resultsdemonstrate that the donor stem spermatogonia from theimmature rats were fully functional and capable ofdifferentiating in a supportive environment. Similar resultswerealso obtained when these seminiferous tubule cells fromimmature rats were transplanted into busulfan-treated mice(Zhang et al., 2006).To further test whether the donor spermatogonial stem cellsfrom immature rats were functional in rat testes that werecapable of supporting endogenous spermatogenesis, cells from

TABLE

1.Testifenvironmentisdefective:Colonizationanddevelopmentofcells

from

immature

GFP-transgenicrats

indifferentirradiatedadultrecipienttestes

Recipients

Meandonor

age(days)c

Injected

live

cells

(T106)

GFP-positive

tubules(%)

TypeofGFP-positive

cells

inhost

tubules(%)a

Type(number

oftestes)b

Injectiontimeafter

irradiation(w

eeks)c

Cytoplasm

icstaining,along

basem

entmem

brane

Inlumen

Individualcells,at

basem

entmem

brane

(typeA

spermatogonia)

Differentiated

germ

cells

IrradiatedLB

NF 1

rats

(n¼46)

10.8

(7.3–21)

11.9d(5–21)

19W

1e

18W

2e

12W

271W

317W

20

Irradiatednudemice(n¼14)

7.7

(6.7–8)

10.9f(10–11)

0.7W

0.1

35W5

N.D.g

N.D.g

24W

476W

4IrradiatedLB

NF 1

rats,

GnRH-antagonisttreated(n¼7)

9.0

(7.6–10)

10.0h(6–12)

17W

422W5

72W

10i

9W

219W

11

Dataarepresentedas

mean�standarderrorofthemeanunless

otherwisenoted.

a Types

ofdonorcells

intubules(see

detailin

text)as

apercentage

oftotalGFP-positive

tubules.

bSamplesize

(n)represents

number

ofrecipienttestes

inwhichdonorcells

weresuccessfully

injected

into

thelumen.

c Values

givenas

meanandrange.

dThirty-twotestes

received

donorcells

from

backcrossed

GFP-Lew

israts

ofgenerations3–6,and14received

donorcells

from

GFP-LBNF 1

rats.

eFewer

cells

wereobtained

from

5-to

10-day-old

rats

than

from

11-to

21-day-old

rats

andhence

fewer

cells

wereinjected

(12�106vs.26�106)andthepercentage

ofGFP-positive

tubulecross-sectionswas

proportionatelylower

(12%vs.23%).

f Donorcells

werefrom

backcrossed

GFP-Lew

israts

ofgenerations2–3.

g Notdetermined.GFP-positive

cells

withthesecharacteristicswererarelyobserved

inthisgroup.

hFive

testes

wereinjected

withdonorcells

from

backcrossed

GFP-Lew

israts

ofgenerations5–6,andtw

owithdonorcells

from

GFP-LBNF 1

rats.

i Tubuleswithdonorcells

withcytoplasm

icstainingalongthebasem

entmem

braneandtubuleswithdonorcells

inthelumen

werenotdistinguished

during

thecounting.


152 Z H A N G E T A L .

immature GFP-transgenic rats were injected into irradiated rattestes, and the recipients were given GnRH-antagonisttreatment after the transplantation. Thirteen weeks aftertransplantation, two patterns of GFP fluorescence wereobserved in the tubules (Fig. 1C). Localized short regions ofbright fluorescence were indicative of aggregates of cells withcytoplasmic GFP and longer regions of tubules with moderatespots of fluorescence were indicative of nuclear GFP. Whenhistological sections were analyzed with anti-GFPimmunostaining, 22% of the host’s tubules contained donorcells (Fig. 2I,K, Table 1). Of the GFP-positive tubules, 28%contained donor germ cells: 9% contained only isolated or a fewcells at the basement membrane, which we believe were type Aspermatogonia and 19% showed differentiated donor germcells (Fig. 2I,J). The GnRH-antagonist treatment aftertransplantation had therefore allowed many of the donorspermatogonia that had colonized the irradiated adult LBNF1testes to differentiate, often to the spermatocyte stage, andoccasionally to round spermatids. Thus, these results not onlyconfirm that the donor spermatogoniawere functional, but alsothat the hormone suppression restored the ability of theenvironment of the irradiated LBNF1 rat testes to supportdonor spermatogonial differentiation. The remainder of theGFP-positive tubules (72%) were colonized by donor Sertolicells (determined by anti-WT1, data not shown) in the lumen orat the basement membrane with moderate or strongcytoplasmic GFP staining (Fig. 2K, Table 1).

Test of whether spermatogonia are defective

To test the functionality of the irradiated spermatogonia, wetransferred a population of tubule cells enriched inspermatogonia by Percoll gradient centrifugation fromGFP-positive irradiated adult Lewis or LBNF1 rat testes intoirradiated, nude mouse testes, which are capable of supportingdifferentiation of normal spermatogonial stem cells fromimmature rats. Because testis cells from the donor rats bred onthe two genetic backgrounds showed no significant differencein numbers and patterns of GFP-positive tubules, the resultswere pooled (Table 2). Long regions of tubules in recipienttestes had strong GPF-green fluorescence (Fig. 1E). Anti-GFPstaining of histological sections showed that 14% of the mouseseminiferous tubules were colonized by rat donor cells(Fig. 3D,F). Donor spermatogenesis had clearly developed fromstem cells in 57% of these tubules (Table 2), and even latespermatids with the morphology of rat sperm nuclei wereobserved (Fig. 3D,E). Very similar results were obtained byinjection of irradiated rat tubule cells into busulfan-treatedmouse testes, in which 12% of tubules were GFP positive and54% of the GFP-positive tubules showed differentiating ratgerm cells. Another 16% of GFP-positive tubules in theirradiated mouse recipients had only a few isolated donor cellsalong the basement membrane, which is typical of type Aspermatogonia. Clusters of GFP-positive cells were alsoobserved adjacent to the lumen in 22% of the GFP-positivetubules (Table 2). Many of these clusters also appeared toconsist of germ cells (Fig. 3F, arrow), which was confirmed bypositive staining with anti-GCNA1 (not shown). The overallconclusion is that donor spermatogonial stem cells from theirradiated adult rats were functional in that they colonized anddifferentiated in irradiated and busulfan-treated adult mousetestes.To confirm that these irradiated stem spermatogonia behavedsimilarly to normal spermatogonia from immature rats and tofurther test the effect of environment on the differentiation ofthese spermatogonia, we transferred them into irradiatedLBNF1 rat testes, without or with GnRH-antagonist treatment,and analyzed the tissues 13 weeks later. Without theGnRH-antagonist treatment, the recipient testes showed only

Fig. 1. Green fluorescence observed in intact testes of recipient animals transplanted with GFP-labeled rat seminiferous tubular cells.A–- C: Cells from immature GFP-transgenic rats were transplanted into an irradiated adult rat (A), an irradiated adult mouse (B), or aGnRH-antagonist-treated, irradiated adult rat (C) testis. Regions showing aggregates of donor cells (arrows) and donor cells forming a layer orarranged as chains along the basement membrane (arrowheads) are indicated. D–F: Cells from irradiated GFP-transgenic LBNF1 rats weretransplanted into an irradiated adult rat (D), an irradiated adult mouse (E), or a GnRH-antagonist-treated, irradiated adult rat (F) testis.Bar¼ 300 mm.


punctate staining over short regions of the tubule (Fig. 1D).Histological analysis showed that 8% of the cross-sectionscontained GFP-positive cells (Table 2). A significant fraction(38%) of the GFP-positive tubules contained germ cells alongthe basement membrane; these generally appeared as isolated,single cells, but sometimes they formed pairs or small groups(Fig. 3A). These stained weakly for GCNA1 (Fig. 3B) and wereconsidered to be donor-derived type A spermatogoniacolonies that were unable to further differentiate in theirradiated rat testes. Another 30% of the GFP-positive tubuleshad small clusters of somatic (GCNA1-negative) donor cells inthe lumen (Fig. 3C). In contrast to the structures observed withimmature rat donors, these cells did not stain with WT1. Also31% of the GFP-positive tubules showed cytoplasmic anti-GFPstaining of Sertoli cells (not shown), but this differed from thetransplanted cells from immature rats in that the nucleus wasGFP-negative, whereas immature rat donor cells producedstrong nuclear and cytoplasmic GFP staining (Fig. 2D).However, when the recipient irradiated rats were treated withGnRH antagonist, brightly GFP-fluorescent cells, whichsometimes appeared as chains, were found along the basementmembrane, and less bright cells, which appeared as diffusespots, were found throughout some of the tubules (Fig. 1F).Immunohistochemistry of histological sections showedGFP-positive cells in 26% of tubule cross-sections (Table 2).Many GFP-positive tubules (38%) contained donor cells withnuclear GFP staining, found singly or in pairs or small groupsalong the basementmembrane (not shown). Sincemost of themhad the morphology of spermatogonia and stained weakly withGCNA1, these must have been derived from stem cells thatcolonized but failed to differentiate beyond the Aspermatogonia stage; a few others appeared to have themorphology of Sertoli cells. However, a significant number(15%) of GFP-positive tubule cross-sections did showdifferentiated donor germ cells up to the primary spermatocytestage (Fig. 3G,H). Thus, the GnRH-antagonist treatmentrestored the ability of the somatic environment to support


differentiation of transplanted spermatogonial stem cells fromother adult irradiated rats, just as it does with the endogenousstem cells. In addition, nearly half (46%) of these GFP-positivetubules were due to somatic cells either forming sphericalstructures in the lumen (Fig. 3I) or associated with theendogenous Sertoli cell epithelium (not shown). Thesestructures differed both from those observed with immaturerat donors and those in untreated irradiated recipient rats, inthat they were loosely organized and contained a few Sertolicells identified by WT1 staining (Fig. 3I, inset).

Discussion

The present results demonstrate that the spermatogonia inirradiated LBNF1 rats, whose differentiation is blocked in thisenvironment, are capable of differentiation aftertransplantation. We previously showed that they couldundergo differentiation in situ when intratesticulartestosterone and FSH levels were suppressed with GnRHanalogs (Shetty et al., 2000). Here, we show that they are evencapable of efficient differentiation when transplanted intoirradiated or busulfan-treated mouse testes, which also havehigh intratesticular testosterone levels and likely high FSH levelsdue to the loss of germ cells and the resistance of Leydig cells tothese agents.To more quantitatively assess the functional capabilities of thetype A spermatogonia from irradiated rats (Table 2), we shallcompare them with results obtained with immature rats(Table 1); the comparison is summarized in Table 3. The tubularpreparations used for transplantations in both cases containedlargely Sertoli cells, peritubular cells, and early stage germ cells.The colonization efficiency in irradiated rats for cells fromirradiated rat donors, measured by the percentage of tubulecross-sections containing germ cells per 106 injected cells, was0.59%, which is the same order of magnitude as the value of0.15% for cells from immature rats. Similar efficiencies (0.37%and 0.29% of tubules per 106 cells, respectively) were observed

Fig. 2. Immunohistochemical analysis of testicular sections after transplantation of seminiferous tubule cells from GFP-transgenic immaturerats into the following recipients: (A–F) irradiated adult rat testes, (G, H) irradiated adult mouse testes, or (I–K) GnRH-antagonist-treated,irradiated adult rat testes. A–C: Donor type A spermatogonia (arrows) identified as isolated cells stained with anti-GFP (A), unstained withanti-WT1 (B), and weakly stained with anti-GCNA1 (C). An endogenous spermatogonium (arrowhead) is unstained with anti-GFP. Note thatbecause rat spermatogoniaweaklywithanti-GCNA1, thedurationof the staining reaction in (C)had tobeextended, resulting inhighbackground.D:DonorSertolicellsshowingcytoplasmic,aswellasnuclear,anti-GFPstainingformanepithelial layeronthebasementmembranecoveringabouthalf of the tubule. Inset is a serial section stained using anti-WT1. E:Donor Sertoli cells showing cytoplasmic and nuclear anti-GFP staining fill thetubule, which is sometimes divided into segments with fibroblastic or peritubular cells. F: Donor Sertoli cells showing cytoplasmic and nuclearanti-GFP-positivestaining forminthe lumenandsurroundaclumpofunidentified,possiblydegeneratingcells. Inset isa serial sectionstainedusinganti-WT1.G,H: (�) Tubules showing differentiation of donor rat spermatogenic stemcells to the spermatid stage, identified by anti-GFP staining(G). (Arrowheads) Tubule showing donor type A spermatogonium, identified by anti-GFP staining (G) but no detectible anti-GCNA1 stainingunder the short staining reaction time conditions used (H). Tubule showing endogenous recovery of mouse spermatogenesis (#) identified byabsence of anti-GFP staining (G) and strong anti-GCNA1 staining (H). I, J: Tubules in irradiated rat treated with GnRH antagonist showingdifferentiation of donor stem spermatogonia to the spermatocyte stage, identified by positive anti-GFP staining (�) or recovery of endogenousspermatogenesis identified by negative anti-GFP staining (#). K: Seminiferous tubules in irradiated rat treated with GnRH antagonist showingtransplantedSertoli cellswith cytoplasmic anti-GFP staining along thebasementmembrane (arrow)or in a clumpof cells in the lumenof a tubule(arrowhead).TheseGFP-positivecellswerealso stainedbyanti-WT1antibody inaserial section (datanot shown).Differentiatinggermcells (#) inthis part are endogenous. Bar¼ 50 mm in A–F, I–K; ¼37.5 mm in G and H.

154 Z H A N G E T A L .

for transplantation of cells from irradiated and immature rats,respectively, into GnRH-antagonist-treated irradiated rats.This indicates that the GnRH-antagonist treatment started atthe time of transplantation did not markedly affect thecolonization efficiency. Also in mouse testes, the normalizedpercentages of tubules showing donor germ cells was roughlycomparable with cells derived from irradiated or immature rats(92% vs. 55% per 106 cells). Overall, we conclude that


seminiferous tubule cells obtained from irradiated adults havesimilar, if not slightly higher, concentrations of functionalspermatogonial stem cells than do similar preparations fromimmature rats. Others have shown that, in testes of immaturerats, the concentration of stem spermatogonia capable ofcolonization after transplantation is about 3.3-fold higher thanin those of adults (Ryu et al., 2003). It appeared that theelimination of (non-stem) differentiating germ cells by

TABLE

2.Testifspermatogoniaaredefective:Colonizationanddevelopmentofcells

from

irradiatedadultGFP-transgenicrats

indifferentirradiatedadultrecipienttestes

Recipients

Transplanttime

afterirradiation

ofdonor(w

eeks)c

Injected

live

cells

(T106)

GFP

positive

tubules(%)

TypeofGFP-positive

cells

inhost

tubules(%)a

Type(number

oftestes)b

Injectiontimeafter

irradiation(w

eeks)c

Cytoplasm

icstaining,along

basem

entmem

brane

Inlumen

Individualcells

atbasem

entmem

brane

(typeA

spermatogonia)

Differentiated

germ

cells

IrradiatedLB

NF 1

rats

(n¼4)

12.1

18.7d(18.7–18.7)

1.2W

0.1

8W3

31W

15

30W

10

38W

90

Irradiatednudemice(n¼10)

4.2

(4.0–5.6)

16.2e(12–23.9)

0.11W

0.05

14W4

5W

422W

916W

557W

10

IrradiatedLB

NF 1

rats,

GnRH-antagonisttreated(n¼4)

14.3

(14–14.4)

10.2d(10–10.4)

12W

326W4

46W

5f

38W

215W

7

Dataarepresentedas

mean�standarderrorofthemeanunless

otherwisenoted.

a Types

ofdonorcells

intubules(see

detailin

text)as

apercentage

oftotalGFP-positive

tubules.

bSamplesize

(n)represents

number

ofrecipienttestes

inwhichdonorcells

weresuccessfully

injected

into

thelumen.

c Values

givenas

meanandrange.

dDonorcells

werefrom

GFP-positive

LBNF 1

rats.

eEighttestes

received

donorcells

from

backcrossed

GFP-Lew

israts

ofgenerations3–4,andtw

otestes

received

donorcells

from

GFP-LBNF 1

rats.

f Tubuleswithdonorcells

withcytoplasm

icstainingalongthebasem

entmem

braneandtubuleswithdonorcells

withcytoplasm

icstainingin

thelumen

werenotdistinguished

duringthecounting.



irradiation counteracted this decline with age and that thefunctional ability of the stem spermatogonia in the irradiated rattestes was not reduced.We assessed the differentiation ability of these stem cells bydetermining the percentages of tubule cross-sections in whichthe donor cells showed differentiation. Similar fractions ofGFP-positive germ-cell-containing tubules had differentiated germcells (67% vs. 76%, respectively) when irradiated or immaturerat stem cells were injected into irradiated nude mice. Thefractions of GFP-positive germ-cell-containing tubule cross-sections in which the germ cells differentiated were also notsignificantly different when donor stem cells from irradiatedrats or from immature rats (26% vs. 42%) were injected intoGnRH-antagonist-treated irradiated rats. Overall, we concludethat there is little or no quantitative or qualitative damage to thespermatogonial stem cells in the irradiated rats.In contrast, the environment for supporting differentiation inthe irradiated testis was indeed defective. Whenspermatogonial stem cells from normal immature rat testeswere transferred into irradiated adult rat testes, donorspermatogonia survived and likely proliferated in the tubulesbut could not initiate differentiation beyond the Aspermatogonial stage in the recipient testes (Fig. 2A–C,Tables 1and 3). These spermatogonia had the capability of becomingfunctional as demonstrated by their ability to differentiate wheninjected into irradiated or busulfan-treated mouse testes(Zhang et al., 2006) or GnRH-antagonist-treated rat testes(Table 1, Fig. 2I,J). Likewise, stem spermatogonia fromirradiated rat testes, which were indeed capable of furtherdifferentiation after transplantation, as demonstrated in mousetestes (Fig. 3D–F) or in GnRH-antagonist-treated rat testes(Fig. 3G–I), did not differentiate past theA spermatogonial stagewhen transplanted into another irradiated rat’s testes(Fig. 3A–C, Tables 2 and 3).However, the capability to support differentiation oftransplanted stem spermatogonia up to the spermatocyte(Fig. 3G,H) or round spermatid (not shown) stages can berestored by suppressing gonadotropins and testosterone.Development of these transplanted cells could not progressbeyond the round spermatid stage, because testosterone orFSH are required to complete spermatid differentiation, as isthe case in normal rats. We had previously shown that afterstopping the GnRH-antagonist treatment, spermatogenesisderived from endogenous stem cells continues to completion,and mature fertile sperm are produced (Meistrich et al., 2001);the same may be true for the transplanted stem cells, but thisproposition has not yet been tested.The identity of the defect in the environment in the irradiatedrat testis that prevents the differentiation of the type Aspermatogonia is still unknown. The intratesticulartestosterone levels are primarily responsible for theunfavorable environment, but FSH also contributes (Shettyet al., 2000; Shetty and Meistrich, 2005). One possiblemechanism by which testosterone inhibits spermatogonialdifferentiation is by increasing interstitial edema in the testis(Porter et al., 2006), but how the edema blocks spermatogonialdifferentiation is still unknown. The somatic cells that expressandrogen receptor (Sertoli, Leydig, peritubular myoid, orvascular smooth muscle) (Meistrich et al., 2005a) or FSHreceptor (Sertoli) (Kliesch et al., 1992) are candidates formediating the hormone-induced block in spermatogonialdifferentiation. The Sertoli cells are in direct contact with thegerm cells and produce proteins, such as stem cell factor(Tajima et al., 1991) that regulate spermatogonialdifferentiation. The peritubular myoid cells in the basementmembrane are in close proximity to the type A spermatogoniaand respond to androgens to affect Sertoli cell function (Tanet al., 2005). The Leydig cells and closely associatedmacrophages (Hedger et al., 2005) and vascular cells may be

Fig. 3. Immunohistochemical analysis of recipient testicular sections after transplantation of seminiferous tubule cells from irradiated adultGFP-transgenic rats into an irradiated adult LBNF1 rat (A–C), an irradiated adultmouse (D–F), and aGnRH-antagonist-treated, irradiated adultLBNF1rat testis (G–I).A,B:TypeAdonor spermatogonia (arrows) identifiedas isolatedcells stainedwithanti-GFP(A)andwithanti-GCNA1(B).EndogenoustypeAspermatogoniumwasnotstainedbyanti-GFPbutstainedbyanti-GCNA1(arrowheads).C:SphericalGFP-positivestructureinthe lumenof a tubule (arrowhead).D–F: Spermatogenesis derived from irradiatedadult rat spermatogonial stemcells in recipientmouse tubules(�) stained by anti-GFP (D). Mature spermatids with the morphology of rat sperm nuclei were observed (arrowhead). Endogenous mousespermatogenesis (#)anddonor rat spermatogenesis (arrow)co-exist in thetubuleatright (D)confirmedbyanti-GCNA1staining,whichstains themousegermcellsmore strongly than the rat cells (E). F:Donorgermcells stainedbyanti-GFPantibodywere sometimesobservedadjacent to thelumen(arrow),not inapparentassociationwith thebasementmembrane.G,H:Tubulesorregionsof tubules in irradiatedrats treatedwithGnRHantagonistshowingdifferentiationofdonorstemspermatogoniatothespermatocytestage, identifiedbypositiveanti-GFPstaining(�)orrecoveryof endogenous spermatogenesis identified by negative anti-GFP staining (#) (G). All differentiated germ cells (endogenous and donor-derived)werestainedbyanti-GCNA1(H). I:Clusterofdonorcells stainedbyanti-GFPandaninsetof its serial sectionshowingpositivestainingbyanti-WT1(arrowheads). Endogenousmouse spermatogenesis (#) is also shown to co-exist with donor rat spermatogenesis (�). (A) and (B), (D) and (E), (G)and (H) are serial sections. Bar¼ 50 mm.

156 Z H A N G E T A L .

responsible for the edema that appears to be correlated withthe spermatogonial arrest (Porter et al., 2006).The colonization of the irradiated rat testes by transplantedSertoli cells obtained from immature rats, which was extensive,and that by Sertoli cells from adult rats, which was limited, wereboth unexpected. In the rat, Sertoli cells normally proliferateonly until postnatal Day 15. The large size of some of thetransplanted Sertoli cell colonies, even those produced from15-day-old donor rats, suggests that additional proliferationoccurred after transplantation. Smaller structures containingSertoli cells derived from irradiated adult tubule cells wereobserved only in GnRH-antagonist-treated, irradiated ratrecipients, and may have been formed by aggregation oftransplanted Sertoli cells.Minitubules containing Sertoli cells and differentiatingspermatogenic cells have been reported previously in the lumenafter transplantation of immature rat testes cells intobusulfan-treated rats (Jiang and Short, 1995). Structurescontaining donor-derived Sertoli cells along the basementmembrane were observed after transplantation of perinatalmouse tubule cells into germ-cell-depleted mice, particularlyafter endogenous Sertoli cells were depleted with cadmium(Shinohara et al., 2003). Since we did not expect aradiation-induced reduction in the number of endogenous


Sertoli cells, it was surprising that donor Sertoli cells colonizedalong the basement membranes of irradiated rat testis tubules.The colonies of donor-derived Sertoli cells observed in ourstudy failed to directly support the development of germ cells.Although complete spermatogenesis was reported in theluminal minitubules of busulfan-treated rat testes, the origin ofthese cells could not be unequivocally identified since nomarker was employed to distinguish donor cells (Jiang andShort, 1995). But after transplantation of perinatal mousetubular cells into germ-cell-depleted mouse testes, some of theminitubules produced in the mouse did contain donor Sertoliand germ cells, particularly when the host Sertoli cells weredepleted with cadmium or when mice with a defect in theirSertoli cells were used as recipients (Shinohara et al., 2003;Kanatsu-Shinohara et al., 2005). Still development of germ cellsoccurred rarely, in at most 3% of tubules when there was nodeliberate removal of endogenous Sertoli cells with cadmium.Also cultured immature rat tubular cells grafted into nude miceformed tubular structures that supported a few spermatogonia,but these did not differentiate (Gassei et al., 2006). Thus, ourresults are not inconsistent with previous reports; perhapsfurther studies of our rat model are necessary to produce andidentify such infrequent examples of donor Sertoli-cellsupported germ cells and their development.

TABLE

3.Comparisonofspermatogonialstem

cellpotentialoftubularcells

from

immature

rats

versusthose

from

irradiatedrats

Donor

Recipient

Cellsinjected

(T106)

%oftubules

withdonor

germ

cells

%oftubuleswith

donorgerm

cells

/106cells)

%oftubule

cross-sectionswith

donorgerm

cells

containingonly

typeA

spermatogonia

%oftubule

cross-sectionswith

donorgerm

cells

containing

differentiated

cells

Immature

rat

Irradiatedrat

19W

12.4W

0.3%

0.15W

0.02

100W

0%

0W

0%

Irradiatedrat

Irradiatedrat

4.9W

0.4a

2.8W

0.7%

0.59W

0.19

100W

0%

0W

0%

Immature

rat

Irradiatedmouse

0.7W

0.1

35W

5%

55W

924W

4%

76W

4%��

Irradiatedrat

Irradiatedmouse

0.4W

0.2a

12W

4%

92W

26

33W

11%

67W

11%�

Immature

rat

GnRH-antagonist-treatedirradiatedrat

17W

43.5W

0.7%

0.29W

0.10

58W

12%

42W

12%��

Irradiatedrat

GnRH-antagonist-treatedirradiatedrat

47W

9a

14W

2%

0.37W

0.14

74W

9%

26W

9%�

Dataarepresentedas

mean�standarderrorofthemean.

Values

arecalculatedfrom

thedataonindividualanimalsusedforTables1and2.

a Actualnumbersmultipliedby4,since

thespermatogoniawereenriched

anaverageoffourfold

inPercollgradients.

� Significantlydifferentpercentage

oftubulecross-sectionsshowingdifferentiationforsametypeofdonorcells

compared

toirradiatedratrecipient,

� P<0.05,usingMann–W

hitney

non-param

etrictest.

��Significantlydifferentpercentage

oftubulecross-sectionsshowingdifferentiationforsametypeofdonorcells

compared

toirradiatedratrecipient,

� P<0.001usingMann–W

hitney

non-param

etrictest.



Cryopreservation of spermatogonia and autologoustransplantation is considered as a potential method toregenerate spermatogenesis and possibly rescue fertility afterchemo- or radiotherapy and clinical trials have been initiated(Radford et al., 1999; Brook et al., 2001; Radford, 2003; Orwigand Schlatt, 2005). Our observations in the rat model thatfunctional transplanted spermatogonia may not be able todifferentiate in the damaged somatic environment, suggest thathormonal or somatic cell transplantation methods forpreventing or reversing that somatic damage should beconsidered. Although suppression of testosterone andgonadotropins with GnRH-analogs alone has been unsuccessfulin protecting or restoring endogenous spermatogenesis in non-human primates (Boekelheide et al., 2005) or in patients (Shettyand Meistrich, 2005), hormonal suppression could restore thesomatic environment sufficiently to allow transplanted stemspermatogonia to develop. Alternatively, transplantation offunctional Sertoli (Kanatsu-Shinohara et al., 2005) or Leydigcells (Lo et al., 2004) might be used to compensate for damageto endogenous cells of these types.

Acknowledgments

We thank Dr. G. Enders for kindly supplying anti-GCNA1antibody, Dr. R. Behringer for supplying the breeder GFP ratand opening his laboratory for making glass pipettes, and Dr.Hyun K. Kim and Dr. Richard Blye of the NICHD for supplyingthe Acyline. We are also thankful to Dr. C.C.Y. Weng formaintenance of GFP mice, Jun Ju and Kuriakose Abraham fortechnical assistance, Dr. Kate Loveland for critical suggestionson the manuscript, and Walter Pagel for editorial assistance.The work was supported by NIH (R01 ES 08075 to Dr.Meistrich) and Cancer Center Support Grant CA 16672.Contract grant sponsor: Lalor Foundation, Fellowship(to Dr. Zhang).

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The radiation-induced block in spermatogonial differentiation is due to damage to the somatic environment, not the germ cells