Spermatogonial behavior in rats during radiation-induced arrest and recovery after hormone ...

14 Pages·2013·0.68 MB·English

REPRODUCTION
RESEARCH
Spermatogonial behavior in rats during radiation-induced arrest
and recovery after hormone suppression
Amanda VAlbuquerque, Fernanda R C L Almeida, Connie C Weng1, Gunapala Shetty1,
Marvin L Meistrich1 and He´lio Chiarini-Garcia
Laboratoryof StructuralBiologyand Reproduction, Department ofMorphology,Instituteof BiologicalSciences,
FederalUniversityofMinasGerais, AvenidaAntoˆnio Carlos, 6627 -Pampulha,31.270-901 BeloHorizonte,
Minas Gerais,Braziland 1DepartmentofExperimental Radiation Oncology,MDAnderson CancerCenter,
The UniversityofTexas, Houston,Texas, USA
CorrespondenceshouldbeaddressedtoHChiarini-Garcia;Email:[email protected]
Abstract
Ionizingradiationhasbeenshowntoarrestspermatogenesisdespitethepresenceofsurvivingstemspermatogonia,byblocking
theirdifferentiation.Thisblockisaresultofdamagetothesomaticenvironmentandisreversedwhengonadotropinsandtestosteroneare
suppressed,butthemechanismsarestillunknown.WeexaminedspermatogonialdifferentiationandSertolicellfactorsthatregulate
spermatogoniaafterirradiation,duringhormonesuppression,andafterhormonesuppressioncombinedwithLeydigcelleliminationwith
ethanedimethanesulfonate.TheseresultsshowedthatthenumbersandcytoplasmicstructureofSertolicellsareunaffectedbyirradiation,
onlyafewtypeAundifferentiated(A )spermatogoniaandevenfewertypeA spermatogoniaremained,andimmunohistochemicalanalysis
und 1
showedthatSertolicellsstillproducedKITligand(KITLG)andglialcellline-derivedneurotrophicfactor(GDNF).Someofthesecells
expressedKITreceptor,demonstratingthatthefailureofdifferentiationwasnotaresultoftheabsenceoftheKITsystem.Hormonesuppression
resultedinanincreaseinA spermatogoniawithin3days,agradualincreaseinKIT-positivespermatogonia,anddifferentiationmainlytoA
und 3
spermatogoniaafter2weeks.KITL(KITLG)proteinexpressiondidnotchangeafterhormonesuppression,indicatingthatitisnotafactor
inthestimulation.However,GDNFincreasedsteadilyafterhormonesuppression,whichwasunexpectedsinceGDNFissupposedtopromote
stemspermatogonialself-renewalandnotdifferentiation.Weconcludethattheprimarycauseoftheblockinspermatogonialdevelopment
isnotduetoSertolicellfactorssuch(KITL\GDNF)ortheKITreceptor.AseliminationofLeydigcellsinadditiontohormonesuppression
resultedindifferentiationtotheA stagewithin1week,Leydigcellfactorswerenotnecessaryforspermatogonialdifferentiation.
3
Reproduction(2013)146363–376
Introduction countreduction,leadingtotheonsetofinfertilitywithin
several months. Subsequent recovery at later times is
In boys and young men who have been affected by
dependent on thespermatogonialstem cells.
cancer,thesuccessesoftreatmentsthataredeleteriousto
In rodents, the spermatogonial stem cells have been
testicular function have made infertility an important
identified morphologically as individual isolated cells
issue. Over the past 30 years, advances in treatment of
called A single (A) spermatogonia (de Rooij 1998).
several cancers of young men, including testicular s
Whenthesecellsdividewithoutcompleteseparationof
cancer, Hodgkin’s lymphoma, and osteosarcoma, have
theircytoplasm,theyformApairedspermatogonia(A ),
resulted in considerable improvements in the outcomes pr
which undergo further mitotic divisions,formingchains
of these diseases. However, the two main cancer
of cells known as A aligned spermatogonia (A ). All
treatments, chemotherapy and radiotherapy, induce al
thesecellsareoftencalledtypeAundifferentiated(A )
prolonged or permanent azoospermia causing tempor- und
ary or permanent infertility (Fossa & Magelssen 2004, spermatogoniaastheyhavesimilarmorphologytotheAs
Meistrichetal.2005,Shetty&Meistrich2005).Themost cells. When the type Aal spermatogonia differentiate,
sensitive testicular cells to chemotherapeutic drugs and they form A1 differentiating spermatogonia, which then
radiation are the ones that are undergoing constant undergo an extensive and precisely timed sequence of
mitotic activity: the differentiating spermatogonia (Dym mitotic divisions begins producing A1, A2, A3, and A4,
& Clermont 1970, Lu & Meistrich 1979, Kangasniemi intermediate and B spermatogonia. The differentiating
et al. 1990). Death of these cells results in maturation spermatogoniaaremostsensitivetothecytotoxicinsults,
depletion of later stages of germ cells and severesperm but the undifferentiated spermatogonia, particularly the
q2013SocietyforReproductionandFertility DOI:10.1530/REP-12-0494
ISSN1470–1626(paper)1741–7899(online) Onlineversionviawww.reproduction-online.org
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via free access
364 AVAlbuquerqueandothers
A,havelowerratesofdivisionandcansurvivemoderate Materials and methods
s
doses of cytotoxic insults. If they survive the cytotoxic
Animals
insults, spermatogenesis recovery can occur naturally
fromthestemcells.Thus,theeventualrecoveryofsperm LBNF male rats were obtained from Harlan–Sprague Dawley
1
productionafterinsultsdependstoalargeextentonthe (Indianapolis,IN,USA)andhousedattheMDAndersonCancer
survival of the spermatogonial stem cells and their Center facilities under a 12h light:12h darkness photoperiod
functionalabilitytoproliferateanddifferentiate,provid- and allowed food and water ad libitum. All procedures were
ing again the adequate number of type A differentiating approved by the MDAnderson Institutional Animal Care and
spermatogonia for the further steps of the process of Use Committee and the American Association for the
spermatogenesis. AccreditationonLaboratoryAnimalCare.
A variety of testicular toxic insults, such as radiation
(Kangasniemietal. 1996),procarbazine (Meistrich etal.
Irradiation
1999),andothers(reviewedinMeistrich&Shetty(2003)),
induce seminiferous tubule atrophy in rats despite the Rats,atw10weeksofage,wereanesthetizedandall,except
presence and persistence of undifferentiated type A for unirradiated controls, were irradiated to the lower part of
spermatogonia that appear to be blocked from further thebodywithasingledoseof6Gy60Cogammaradiationas
described previously (Meistrich & Kangasniemi 1997, Porter
differentiation.Inmanyofthesecases,thesuppressionof
etal.2006).
testosterone and follicle-stimulating hormone (FSH)
with gonadotropin-releasing hormone (GNRH) analogs
stimulated spermatogonial differentiation, resulting in Treatments
spermatogenic progression (Meistrich & Shetty 2003). In
The hormonal suppression treatment was initiated 10 weeks
addition,althoughspermatogonialstemcellstransplanted
afterirradiation.Acyline,aGNRH-ant(obtainedfromNational
into irradiated rats can colonize along the basement
Institutes of Health-National Institute of Child Health and
membrane of seminiferous tubules, they fail to differ-
HumanDevelopment,Bethesda,Maryland,USA),wasfreshly
entiate, unless hormones are suppressed (Zhang et al.
prepared in sterile water before each use and a dose of
2007). Such studies demonstrated that some of the
1.5mg/kgwasinjecteds.c.weeklyfor6weeks.Furthermore,
somatic cells forming the stem cell niche that controls
to reduce action of residual intratesticular testosterone levels,
manyspermatogonialeventscanbedamagedbyradiation
theseirradiatedratsreceivedfours.c.silasticimplants(20cm
andtheirfunctionrescuedbyhormonesuppression.
totallength)containingflutamide(Sigma–Aldrich;Porteretal.
In previous studies (Meistrich et al. 1999, Shuttles-
2009,Zhouetal.2010).Additionally,toevaluatetheeffectsof
worth et al. 2000), the block in type A spermatogonial
absence and Leydig cell factors, the Leydig cells were
differentiation and spermatogenesis recovery following
destroyed after a single i.p. dose (75mg/kg) of ethane
hormone suppression was evaluated considering cell dimethanesulfonate(EDS;Bartlettetal.1986),synthesizedat
proliferation, clonal size, apoptosis, and appearance of MDAndersonbyDrWilliamBornmann).
differentiated cells as well as increases in testis weight,
spermcount,andfertility.Geneexpressionprofilesinthe
irradiatedratsandirradiatedratswithhormonesuppres- Experimentaldesigns
sion have also been analyzed (Zhou et al. 2010, 2011). Two separate experiments were performed as illustrated
In this study, we have extended the characterization of inFig.1.
the kinetics and molecular events of spermatogonial
differentiation in rats during spermatogenic recovery ExperimentI:cellcountsandmoleculareventsof
after testis irradiation and hormone suppression with a spermatogonialdifferentiation
GNRH antagonist (GNRH-ant), using morphological
To analyze the events of spermatogonial differentiation at
(lightandtransmissionelectronmicroscopy)andstereo-
differenttimesafterstimulationofdifferentiationwithhormone
logical approaches and molecular markers (e.g., KIT).
suppression, 26 rats were given a single dose of 6Gy
The light microscopy techniques that we developed
irradiation. As a control (group C, nZ5), age-matched,
were able to morphologically distinguish the spermato-
untreated,unirradiatedratswerekilledatthesametimepoints
gonialsubtypes–fromtypeAundifferentiateduptotypeB
as the irradiated, hormone-suppressed rats. Six rats that were
spermatogonia (Chiarini-Garcia et al. 2003). Addition- irradiated (group X, nZ6) were not given any hormone
ally,molecularexpressionofknownfactorsproducedby
treatment and were assessed at 11 and 16 weeks after
Sertolicells,suchasglialcellline-derivedneurotrophic
irradiation, which corresponded to 1 and 6 weeks after the
factor (GDNF) and Kit ligand (KITL (KITLG), formerly beginning of treatment in the hormone-suppressed group.
known as stem cell factor, SCF), which regulate stem Intermediate time points were not taken because previous
spermatogonial self-renewal (Meng et al. 2000) and studies demonstrated that no changes in testicular histology,
spermatogonial differentiation (Ohta et al. 2000), repopulation indexes, labeling indexes, and serum hormone
respectively, were evaluated and related to the sperma- levels occurred between 10 and 30 weeks after irradiation
togonial counts. (Kangasniemietal.1996).Theresultsofthisstudyalsoshowed
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Spermatogonialbehaviorafterhormonesuppression 365
Experiment I
Davidson’s fluid fixative: methacrylate (light microscopy morphometry)
and paraffin (immunohistochemistry)
Week 0 Week 10 Week 16
10wk LBNF1 rats
Group C Non-irradiated control (n=5)
No treatment
3d (1) 1w (1) 2w (1) 4w (1) 6w (1)
Group X 6 Gy irradiation (n=6)
No treatment
1w (3) 6w (3)
Group XAF 6 Gy irradiation (n=20)
Acyline + flutamide
3d (4) 1w (4) 2w (4) 4w (4) 6w (4)
Experiment II
Glutaraldehyde fixative: Araldite (light microscopy morphometry and electron microscopy)
Week 0 Week 10 Week 14
10wk LBNF1 rats
Group C Non-irradiated control (n=3)
No treatment
4w (3)
6 Gy irradiation (n=3)
Group X No treatment
3w (3)
6 Gy irradiation (n=3)
Group X(EDS)AF
X(EDS)AF
1w (3)
6 Gy irradiation (n=6)
Group XAF Acyline + flutamide
2w (3) 4w (3) Figure1Experimentaldesigns.
no significant differences between the rats assayed at 11 and calibratedwithamicrometerslide.Whenthetubularsections
16 weeks after irradiation, and hence, the results for the two wereslightlyoval,onlythesmallerdiameterwasmeasured.
time points were averaged. Ten weeks after irradiation
Absolute counts of Sertoli cells and type A spermatogonia.
(considered as time zero), the remaining 20 ratswere treated
Stereological studies were performed to obtain the absolute
with weekly injections of GNRH-ant and implantation of
numbers of Sertoli cells and type A spermatogonia in GMA-
flutamidecapsules (groupXAF)andwerekilledattimesfrom
embedded,Davidson’sfluid-fixedtestesfromtheirradiated(X)
3daysto6weeksafterstartinghormonesuppression.
andcontrol(C)groups.Asitwasnotpossibletodistinguishthe
Testes were fixed by vascular perfusion (Chiarini-Garcia &
A spermatogonial subtypes in glycol methacrylate-embedded
Meistrich2008)withDavidson’sfluidmodifiedasdescribedby
material, they were considered as a whole. The numbers of
Latendresseetal.(2002).Aftertheperfusionprocedure,eachtestis
each cell type were calculated, as described previously
wasweighedwiththetunicaalbugineaintact.Theywerecarefully
(Drumondetal.2011).
sliced into two pieces and immersed in 70% ethanol and kept
First,theweightofthetesticularparenchymawascalculated
under48Cuntiltheembeddingprocedure.Forhistomorphome-
basedonthemeasurementstakenonotheranimalsofthesame
tricalevaluationofgermandSertolicellnumbers,halfofthetissue
strain obtained after removal of interstitial fluid and tunica
was embedded in glycol methacrylate (GMA) resin (JB4,
albuginea (Porter et al. 2006). For control animals, the
Polysciences,Inc.,Warrington,PA,USA),and4mm-thicksections
parenchymal weight was 96.8% of the testicular weight, and
were stained with toluidine blue–borate (Chiarini-Garcia et al.
for the irradiated rats, it was only 64.6% (M L Meistrich,
2011).Theotherhalfwasembeddedinparaffinandsectionedat
unpublished results) because of extensive edema after
5mmthicknessforimmunohistochemicalanalysis.
irradiation. Then, the absolute volume of the testicular
Tubulardiameter.SlidesofGMA-embedded,Davidson’sfluid- parenchyma was calculated based on a testicular density of
perfused tissues were used for measurement of 20 nearly 1.0g/cm3(SinhaHikimetal.1988).
circulartubularcrosssectionsperanimal,randomlychosenat Next,usingthepointcountingmethod,w4410intersections
400! magnification, using a ruler fitted in a 10! eyepiece, distributed in ten randomly selected fields per animal (two to
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366 AVAlbuquerqueandothers
threefieldsperhistologicalsection)werecountedasbeingover same exposure time for all frames. Photomicrographs of ten
theseminiferousepitheliumortherestoftesticularparenchyma. randomlychosencross-sectionedseminiferoustubulesperanimal
Thevolumedensityoftheseminiferousepitheliumwasobtained were analyzed using the NIH Image J (Image Processing and
by dividing the sum of points falling on the seminiferous Analysis in Java, v1.45s) Software. The average fluorescence
epithelium by the total number of points over the tissue, and intensity (mean pixel intensities) of the Sertoli cell cytoplasm,
the volume of the seminiferous epithelium was estimated by identifiedastheregionwithinthetubuleexcludingthenucleiof
multiplyingthisbythevolumetothetestisparenchyma. thecells,thelumen,germcells,andvacuolarareas,wasmeasured
Point counting of w18000 intersections in 40 randomly (SupplementaryFigure2,seesectiononsupplementarydatagiven
selected fields for each animal (four to five fields per at the end of this article). Next, the fluorescence due to non-
histological section), as being over Sertoli cell nucleoli, type specificbindingofantibodytotissueswasmeasuredbytakingthe
Aspermatogonialnuclei,oroverotherareasoftheseminifer- averagefluorescenceintensityovertheregionoftheperitubular
ous epithelium was similarly employed to determine the myoid cells and basement membrane (PM). As the variations
volume densities of these cell components. The total volume between slides appeared to be a result of background fluor-
of all the Sertoli cell nucleoli and spermatogonial nuclei was escence(BG)overareaswithnotissue(spacesintheinterstitium
calculated by multiplying their volume densities by the due totissue contraction), mostprobably due to fluctuations in
absolutevolumeoftheseminiferousepithelium. lamp intensity, both the Sertoli cell (SF) and myoid/basement
The average volumes of individual Sertoli cell nucleoli or membrane (PM) fluorescence levels were normalized between
spermatogonial nuclei were determined from their diameters slides by dividing them by the background fluorescence (BG).
measuredwitharulerintheeyepiece,asdescribedpreviously. Then the difference between the normalized Sertoli cell
Finally, the total number of these cells was calculated by fluorescence and the normalized non-specific myoid/basement
dividing the total volumes of Sertoli cell nucleoli or type A membrane fluorescence was calculated to determine the
spermatogonia by the individual volumes of each Sertoli corrected,specificSertolicellimmunofluorescenceintensity.
nucleolusorspermatogonialnucleusrespectively. MCM7CcellsandKITCcellswerecountedin50randomly
chosen,nearlycircularseminiferoustubulesperanimalinthe
Sertoli and germ cell numbers per tubule cross section.
irradiated-onlyanimals(X)andthehormone-suppressed(XAF)
The numbers of Sertoli cell nucleoli and germ cell nuclei,
group.
from spermatogonia up to diplotene primary spermatocytes,
were counted in 50 randomly chosen seminiferous tubule
ExperimentII:kineticsofspermatogonialdifferentiationafter
crosssectionsincontrolandin100seminiferoustubulecross
hormonesuppressionandultrastructuralstudies
sections per animal for each time point of the hormone
suppression(XAF),inirradiated-only(X)andcontrol(C)groups, Tomorphologicallydistinguishthespermatogonialtypes(A ,A ,
und 1
using the Davidson’s fluid-perfused, GMA-embedded tissues. A ,A ,A ,In,Bspermatogonia)andassessultrastructuralchanges
2 3 4
Resultswereexpressedascountspertubulecrosssection. ingermandSertolicellsandtubulestructure,12ratsweretreated
withasingledoseof6GyirradiationasinexperimentI(Fig.1).
Immunohistochemistry. In order to analyze some molecular Age-matchedcontrolrats(groupC,nZ3),withoutanytreatment,
changes after irradiation and during subsequent hormone
were included for comparison. Three irradiated rats (group X),
suppression,slidesoftheparaffin-embedded,Davidson’sfluid-
without hormone suppression, were killed at 3 weeks after the
perfused tissues were immunostained for the proliferation
beginningofthehormonaltreatmentofthesuppressedgroup.To
marker, minichromosome maintenance protein 7 (MCM7
evaluate the combined effects of Leydig cell elimination and
mouseMAB,Abcam,Cambridge,MA,USA,ab79802),theSertoli
hormonesuppressiononspermatogonialdifferentiation,threeof
cell juxtacrine factors GDNF (rabbit polyclonal, Santa Cruz,
theirradiatedrats(X(EDS)AFgroup)weretreatedwithEDSat10
Dallas,TX,USA,D-20,sc:328)andKITLG(rabbitpolyclonal
weeks after irradiation and then hormonally suppressed with
antibody against C-terminus, gift of Dr Shayu Deshpande,
acylineandflutamideandanalyzed1weeklater.Theremainingsix
Deshpande et al. (2010)), and the KIT receptor (c-kit, goat
irradiatedratsweretreated,starting10weeksafterirradiation,with
polyclonal Santa Cruz, M-14, sc: 1494). The sections were acyline and flutamide (XAF group, nZ3/time point), as in
subjectedtoantigenretrievalandthenincubatedwithablocking
experimentI,for2or4weeks.Animalsinallthesegroupswere
solution. The slides were incubated with primary antibodies
perfusion fixed with 5% glutaraldehyde in 0.05M cacodylate
overnightat48Cinahumidifiedchamber.AfterwashinginPBS,
buffer (pH 7.4). Testes were removed, weighed, and sliced into
the slides were incubated with Alexa Flour 488-labeled anti-
small slabs. Fragments were post-fixed in osmium tetroxide/
rabbitIgGforKITLGandGDNF,withAlexaFluor594-labeled
potassiumferrocyanidemixtureandembeddedinaplasticresin,
anti-goatIgGforKIT,orwithasecondarybiotinylatedantibody
Araldite502(EMS,Hatfield,PA,USA).
forMCM7.Thebiotinylatedsecondaryantibodywasdetectedby
the avidin–biotin–peroxidase complex method with diamino- Spermatogonialdifferentiationkinetics.Toassessthekineticsof
benzidineusingtheVectastaineliteABCkit(VectorLaboratories, spermatogonial differentiation, 1mm-thick sections were
Burlingame,CA,USA).Controlslideswerepreparedwithoutthe obtained from testis fixed with glutaraldehyde/osmium and
addition of primary antibodies (Supplementary Figure 1, see embedded in Araldite and stained with toluidine blue–borate
sectiononsupplementarydatagivenattheendofthisarticle). forlightmicroscopystudies.Thismethod,whichwepreviously
ForquantificationofGDNFandKITLconcentrationinSertoli called high-resolution light microscopy, allowed us to
cells, fluorescence images from a set of slides from different recognize the different spermatogonial subtypes by their
animals,stainedatthesametime,werephotographedusingthe morphologicalfeatures.
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Spermatogonialbehaviorafterhormonesuppression 367
Nucleiofallspermatogonialsubtypes(A toB)andSertoli diameters in the irradiated-only group (192G2mm)
und
cell nucleoli were counted in 50 randomlychosenseminifer- were significantly lower than those of the untreated
oustubulecrosssectionsineachanimal.Consideringthatthe controls (355G3mm). The hormone suppression pro-
nuclei(andnucleoli)havedifferentdiameters,theAbercrombie duced a further progressive decrease in the tubular
(1946)correctionforbiasintheestimationofnuclearnumbers diameter (3 days: 192G1mm, 1 week: 188G3mm,
in cross sections was applied. For that, ten diameters of each 2weeks:183G2mm,4weeks:174G3mm,and6weeks:
germ cell subtype nuclei and Sertoli cell nucleoli were 166G4mm)(P!0.05).
measured. The Abercrombie (1946) formula was applied to
Todeterminewhethertherewasaneffectofirradiation
obtaincorrectedcounts,whichwereusedtocalculatetheratio
on Sertoli cell number, a stereological approach was
ofgermcellsper100Sertolicellnucleoli,anacceptedmethod
applied. The total Sertoli cell number per testis was
forcomparisonofgermcellcounts(Dym&Clermont1970).
shown not to be changed significantly as a result of
Transmission electron microscopy. To evaluate the spermato- irradiation; in the control animals, it was 49G4!106
gonialandSertolicellultrastructureafterirradiationandduring andin theirradiated-onlyanimals,40G2!106.
hormone suppression, sections were obtained at 70nm Studies of changes in germ cell numbers were
thickness from the Araldite resin blocks used for light performedusingcountsofcellspertubularcrosssection,
microscopy analysis. These ultrathin sections were collected
which is valid when there is not much longitudinal
incoopergrids,stainedwithuranylacetateandleadcitrate,and
shrinkageorexpansionofthetubules.Therewasonlya
observed under the transmission electron microscope (Zeiss
modestshrinkageafterirradiationasindicatedbya22%
EM-10).Thespermatogonialidentificationattheultrastructural
increase in numbers of Sertoli cell nucleoli per cross
levelwasbasedonthatofChiarini-Garcia&Russell(2002).
section in irradiated-only compared with control rats
(Table 1). Radiation produced dramatic effects on germ
Statisticalanalysis cells with a 20-fold reduction in the numbers of
spermatogonia per tubule cross section and a block in
All variables measured were tested for normality prior to
their differentiation so that no spermatocytes were
analyses, using the univariate procedure of the Statistical
observed even at 11–16 weeks after irradiation
AnalysisSystem(SASInstitute,Cary,NC,USA).Thevaluesof
(Table 1). Stereological methods confirmed that there
variableswerecomparedbetween thecontrol andirradiated-
only group with a t-test with P!0.05 being considered was a marked decrease in the numbers of type A
significant. Comparisons between the irradiated-only and spermatogonia per testis to 3.1G0.4!106 in the
irradiated hormone-suppressed groups were done by irradiated-onlyanimals compared with 20.0G2.6!106
ANOVA. In the event that significant treatment effects were in thecontrolgroup (P!0.05).
established, multiple comparisons were performed using When hormones were suppressed with GNRH-ant
probability of differences adjusted by Tukey–Kramer (SAS and flutamide in the irradiated animals, the number of
2001)withP!0.05beingconsideredsignificant.Inthetables Sertoli cell nucleoli per tubule cross section did not
andgraphs,dataarereportedasmeansandS.E.M. change significantly so that spermatogonial numbers
couldbedirectlycompared.Hormonesuppressionwith
acyline and flutamide produced an increase in the
Results numberofspermatogoniaat3daysandthisnumberwas
Experiment I maintained at twice the level of that in the irradiated-
onlyratsforthefirst2weeks(Table1).Differentiationto
Bodyand testisweight
the spermatocyte stage was first observed at 4 weeks
The body weight did not change after irradiation or afterstartingthehormonesuppression,andby6weeks,
after hormonal suppression. Meanwhile, radiation had anappreciablenumberofpachyteneprimaryspermato-
adramaticeffectontestisweight,reducingitfrom1.88G cyteswerefoundasthemostadvancedgermcellsinthe
0.01g in the untreated control group to 0.56G0.02g repopulated seminiferous tubules(Table1).
(similar at both time points assessed), about 30% of the
weightforcontrols(P!0.05),intheirradiated(X)group. Immunohistochemical analysis
During the subsequent hormone suppression with
To test whether the spermatogonia observed after
acyline plus flutamide, the weights of irradiated testes
irradiation were expressing a proliferation marker; we
progressively declined further (3 days: 0.52G0.02g,
performed immunohistochemistry for MCM7 after
1week:0.47G0.01g,2weeks:0.39G0.02g,4weeks:
irradiation and during hormone suppression (Fig. 2). In
0.36G0.01g,and6weeks:0.34G0.01g).
control rats, all spermatogonia and primary spermato-
cytes until the mid-pachytene stage were MCM7-
Morphologicaland stereological analysis
positive;aweak signal was observed in later pachytene
Testicular atrophy after irradiation or during the sub- spermatocytes and no signal in Sertoli cells, round, and
sequent hormone suppression was first analyzed using elongated spermatids (Fig. 2A). In irradiated-only rats
seminiferous tubule diameters. The mean tubular and those with hormone suppression as well,
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368 AVAlbuquerqueandothers
Table1NumbersofSertoliandgermcellspertubularcrosssectionincontrol(C),irradiated(X)andhormone-suppressed(XAF)ratsafterdifferent
timesofGnRH-antCflutamideadministration.Sertolicellswereonlyscoredifthenucleoluswaspresentinthesection.
Total
Treatment Sertoli spermatogonia PL L Z P D
C 12.28G0.45 8.10G0.21 5.28G0.14 6.50G0.78 5.74G0.86 33.84G1.58 6.70G2.96
X 13.94G0.53 0.52G0.03* 0*,a 0*,a 0*,a 0*,a 0*,a
XAF3days 12.24G0.49 0.73G0.04 0a 0a 0a 0a 0a
XAF1w 12.67G0.67 1.08G0.18 0a 0a 0a 0a 0a
XAF2w 12.97G0.53 1.14G0.03 0a 0a 0a 0a 0a
XAF4w 14.15G0.60 3.05G0.46 0.52G0.27a 0.08G0.05a 0.14G0.04a 0.13G0.06a 0a
XAF6w 13.92G0.60 2.98G0.29 4.46G0.37b 1.16G0.10b 0.91G0.10b 1.76G0.66b 0.08G0.03b
ValuesaremeanGS.E.M.Asterisksindicateasignificantdifference(P%0.05)betweentheCandXgroups(t-test).Differentlettersinthesamecolumn
indicateasignificantdifference(P%0.05)betweenXandindividualXAFgroups,usingTukey’stest.Totalspermatogonia(typeAspermatogoniaC
typeintermediateCtypeBspermatogonia);PL,preleptotene;L,leptotene;Z,zygotene;P,pachytene;D,diplotene.
all spermatogonia were also MCM7-positive, indicating increased during hormone suppression (Fig. 2E), paral-
that they were proliferative (Fig. 2B). The clones of leling the increase in spermatogonia and primary
spermatogonia observed after 2 weeks of treatment had spermatocytes (Table1).
a tendency to appear as clusters (Fig. 2C), but after TheexpressionofKIT,adifferentiatingspermatogonial
4weeks,theywereobservedspreadoutalongthebasal marker,wasexaminednext. Incontrolanimals,mostof
membrane (Fig. 2D). As expected, the MCM7-positive the spermatogonia were stained for KIT; however, few
cellnumberwaslowinirradiatedratsandprogressively unstained spermatogonia, usually in stages II–VI,
A B
C D
E 6 b
ction 5 Fciognutrreol2raItmsm(Au),niorsratadiinaitnegd-oofnMlyCraMts7(-Bp)o,saitnivdeincerlalstsin
e
s s 4 afterirradiationandtreatmentwithacylineplus
os b flutamidefor2(C)and4(D)weeks.Arrowheads
s/cr 3 pointtoMCM7-positivecells.At2weeks,the
ell a,b X spermatogoniasometimesappeartobeclustered
7+ c 2 a a XAF walhoenrgeathseabta4sewmeeeknstmtheemyabrreanmeo.rBeagr:e2n5ermamlly.spread
M
C 1 (E)MCM7-positivecellspertubularcrosssectionin
M
irradiated-onlyrats(X)andafteracylineplus
0 flutamidetherapy(XAF).Differentlettersindicatea
0 3d 1w 2w 4w significantdifference(P!0.05)betweentheXand
Duration of hormone suppression XAFgroupsusingTukey’stest.
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Spermatogonialbehaviorafterhormonesuppression 369
recognizedusingDAPIfluorescence,wereobserved(not ofthecycle,withthehighestintensityobservedatstages
shown).Intheratsreceivingonlyirradiation,abouthalf XIII–I.Inirradiatedanimalswithtubularatrophy,thereis
of the spermatogonia were KIT-negative and the rest nolongeraseminiferousepithelialcycleandtheGDNF
were KIT-positive (Fig. 3A and B). The presence of KIT- protein concentrations appeared to be the same in all
positive spermatogonia in the seminiferous epithelium tubules (Fig. 4B). Following the initiation of hormone
after the insult indicates that the spermatogonial suppression, the GDNF protein concentrations over
differentiation block after irradiation is not due to the Sertoli cells appeared to increase progressively from
absence of the KIT receptor protein. The hormonal 3 days of treatment up to 4 weeks of hormone
suppression gradually increased the numbers of KIT- suppression(Fig.4C),atwhichtimetheincreasebecame
positivecellsfrom1upto4weeksoftreatment(Fig.3C). significant(P!0.05, Fig.4D).
Comparing the KIT-positive spermatogonial cells with SimilarstudiesoftheexpressionofKITLprotein,using
the morphological findings, we observed an increasing anantibodyspecificforthecytoplasmicterminalregion,
percentage of KIT-positive cells among the spermato- confirms that in control animals, it accumulates in the
goniaduring hormone suppression (Fig.3D). basolateralregion of Sertoli cells(Fig.5A), as described
To evaluate the effects of irradiation and acyline plus recently (Deshpande et al. 2010). In irradiated-only
flutamide treatment on the expression of Sertoli cell animals,theKITLproteinisfullymaintainedandappears
factors affecting spermatogonial development, GDNF to be uniformly distributed over Sertoli cell cytoplasm
and KITL protein concentrations were assessed by (Fig. 5B), but after 4 weeks of hormone suppression, it
immunofluorescence.Incontrolanimals,GDNFprotein appearsagaintobestrongerbasally(Fig.5C).Hormone
fluorescencewasspecifictotheSertolicellcytoplasmic suppression did not significantly affect the concen-
domain(Fig.4A).Thefluorescenceintensitychangedas trations of KITL protein in the Sertoli cells of irradiated
thesurroundinggermcellsprogressedthroughthestages rats(Fig.5D).
Ai Bi DAPI
S
Aii Bii KIT
S
Figure3(AandB)KITimmunofluorescenceof
Aiii Biii Merge spermatogoniainirradiated-onlyanimals.(AiandBi)
DAPI;(AiiandBii)KITimmunofluorescence;(Aiiiand
Biii)merge.KIT-positive(arrowheads)andKIT-negative
(doublearrowhead)spermatogoniaareindicated;
S,Sertolicell.Bars:AandB10mm.(C)KIT-positivecells
S
pertubularcrosssectioninirradiated-only(X,whitebar)
andafterdifferenttimesoftreatmentwithacylineC
flutamide(XAF,greybars).Nosignificantdifferences
werefoundbetweentheirradiated-onlyanimals
C D analyzedattwodifferenttimepointsandthevaluesare
nia/ 1.2 b b mber/ 11..42 Morphology cDoimffebreinnetdleattnedrspilnodttiecdataeta‘0s’ig(nnoifichaonrmtdoinffeerseunpcperession).
+ spermatogocross section 00..48 a a,b b matogonial nucross section 0001....8640 KIT+ Aoc(Pobf!tuseenr0rtv4.s0e.w5d(D)ewa)emekCrsooeonmafglptsXraoeraiabstnuomdtnewXnbAete,rtFewthgeeerexofnceulwputhsdepeuadtsocinfthragyoltmTenunutkemheecybe’etsolrlttseaoslft.
KIT 0 Sper 0.02 KITCspermatogoniapercrosssectionandthetotal
0 3d 1w 2w 4w 0 3d 1w 2w numberofmorphologicallyidentifiedinstained
Duration of hormone suppression Duration of hormone suppression GMA-embeddedsections.
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370 AVAlbuquerqueandothers
A Control B X-only C XAF-4W
D 0.8
*
0.7
n
o Figure4ImmunofluorescenceofGDNFwithin
si 0.6
s tubularcrosssectionsincontrol(A),irradiated-only
e
pr 0.5 (B),and4-weekhormone-suppressedanimals(C).
x
e (D)QuantificationoftheaveragespecificGDNF
ein 0.4 * XAF immunofluorescenceintensityoverSertolicytoplasm
F prot 0.3 X idnurtuinbguhleorcmroosnsesescutpiopnressisnioinrrwaditihateadcyolinnleyC(Xfl)uatanmdide
N 0.2 (XAF).Nosignificantdifferenceswerefoundbetween
D
G 0.1 theirradiated-onlyanimalsattwodifferenttime
pointsandthevaluesarecombinedandplottedat‘0’
0 (nohormonesuppression).Asterisksindicatevalues
0 3d 1w 2w 4w 6w
amongXandXAFgroupsthatshowasignificant
Duration of hormone suppression difference(P!0.05)usingTukey’stest.
Experiment II of the control value (P!0.05) inthe irradiated (X) group
(C:1.86G0.03g;X:0.56G0.01g).TheadditionofEDSto
Based on the results from experiment I, in which
the hormone suppression regimen decreased the testis
progressive increases in spermatogonial numbers were
weight more rapidly than hormone suppression alone,
observed after the initiation of hormone suppression, a
reducingitto0.42G0.02gafter1weekoftreatment.
secondexperimentwasdesignedtoassessthekineticsof
spermatogonialdifferentiationusingalightmicroscopic
Spermatogonialkinetics
techniquecapableof identifying thedifferentspermato-
gonial types. Sections of glutaraldehyde/osmium-fixed, Araldite-
embedded tissues were analyzed by counting the
Bodyand testisweight numbers of different subtypes of spermatogonia and
AsinexperimentI,thebodyweightdidnotchangeafter applying appropriate normalization and correction
irradiation or after hormonal suppression. Radiation had factors to obtain the relative numbers of those cells
thesamedramaticeffectontestisweightreducingitto30% (Table 2). The remaining type A spermatogonia in the
A Control B X-only C XAF-4W
D
n
sio 2.5 Figure5ImmunofluorescenceofKITLwithintubular
es crosssectionsincontrol(A),irradiated-only(B),
xpr 2.0 and4-weekhormone-suppressedanimals(C).
e
ein 1.5 XAF (flDu)oQreuscaennticfiecaintitoennsoitfythineSaevretoraligecyKtoITpLlaismmmiunntou-bule
prot 1.0 X crosssectionsinirradiated-onlyanimals(X)and
G duringhormonesuppression(XAF).Nosignificant
L
T 0.5 differenceswerefoundbetweentheirradiated-only
KI animalsattwodifferenttimepointsandthevaluesare
0 combinedandplottedat‘0’(nohormonesuppression).
0 3d 1w 2w 4w 6w Therewerenosignificantdifferencesbetweenthe
Duration of hormone suppression differenttreatmentgroups(PR0.05).
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Spermatogonialbehaviorafterhormonesuppression 371
Table2NumberofSertolicells,scoredifthenucleoluswasobserved,pertubularcrosssectionandnumbersofspermatogonialsubtypesper100
Sertolicellnucleoliincontrolrats(C),irradiatedrats(X)andratstreatedwithGnRH-antCflutamide,without(XAF)orwithEDS(X(EDS)AF).Cells
werescoredinglutaraldehyde/osmium-fixed,Araldite-embeddedtissuesandvalueshavebeenalreadycorrectedfordifferencesindiameters
(Abercrombie1946).
Groups Se A A A A A In B
und 1 2 3 4
C 1.67G0.05 10.19G0.15 1.60G0.45 3.38G0.61 3.65G0.56 4.65G0.51 8.28G0.86 20.12G2.67
X 2.41G0.10 0.92G0.32*,a 0.24G0.07*,a 0*,a 0* 0* 0* 0*
X(EDS)AF–1w 2.25G0.17 1.91G0.34a 0.77G0.06a 0.27G0.03a,b 0.35G0.16 0.11G0.06 0.14G0.12 0
XAF–2w 2.31G0.13 2.02G0.32a 0.75G0.01a 0.32G0.08a,b 0.26G0.05 0.08G0.06 0.27G0.22 0.09G0.08
XAF–4w 2.73G0.08 4.50G0.11b 2.87G0.25b 1.74G0.12b 0.99G0.23 0.92G0.16 0.07G0.03 0.22G0.09
ValuesaremeanGS.E.M.Asterisksindicateasignificantdifference(P!0.05)betweenCandXgroup(t-test).Differentlettersinthesamecolumn
indicateasignificantdifference(P!0.05)betweenXandindividualtreatedgroups,usingTukey’stest.A ,undifferentiatedtypeAspermatogonia;
und
A1–A4,differentiatingtypeAspermatogonia;In,intermediatespermatogonia;B,typeBspermatogonia.
irradiated rats were primarily A spermatogonia and irradiation only and also with hormone suppression
und
their numbers were still drastically decreased from showed that it, sometimes along with an associated
control levels (Table 2); only few cells differentiated to myoid cell, protruded into the seminiferous epithelium
theA subtype (Fig.6B). forminganinvaginationinthebasalaspectofSertolicells
1
TheadditionofasingledoseofEDSatthestartofthe andsomespermatogonia(Fig.7DandE).Ultrastructural
hormone-suppressive treatment (X(EDS)AF group) effec- examination of the spermatogonia along the basement
tively eliminated the all Leydig cells (Fig. 6C) and membrane of irradiated and hormone-suppressed rats
resulted in rapid spermatogenesis recoveryas indicated (Fig. 7B, D, and F) showed that, except for the
by the increase in spermatogonial subtypes observed invaginations described above, the cells apparently had
after 1 week (Table2). normal nuclear and cytoplasmic morphology, as
Hormone suppression for 2 weeks increased the describedpreviouslybyChiarini-Garcia&Russell(2002).
numbers of A spermatogonia by twofold and also
und
supported initiation offurtherdifferentiation resulting in
occasional cells of up to B spermatogonial stage
Discussion
(Fig. 6D). Clones of type A /A spermatogonia (Fig. 6E)
al 1
were frequently seen in the cross sections starting after In this study, we have further characterized the
2weeksofhormonaltreatment.Thenumbersofcellsin spermatogonia present after radiation induces a block
all stages of spermatogonial development progressively in their differentiation in LBNF1 rats and after their
increasedat4weeksofhormonesuppression(P!0.05). differentiationisrestoredbyhormonesuppression.Here,
Although the A spermatogonial number reached weusedglutaraldehydefixationandAralditeembedding
1
control levels at this time point, the numbers of to recognize each spermatogonial subtype by
subsequent spermatogonial subtypes, from type A to B morphology at the light microscope level. Radiation or
2
radiationfollowedbyhormonesuppressiondidnotalter
spermatogonia(Fig.6Fand G), did not.
the morphology (Chiarini-Garcia et al. 2003) or
ultrastructure (Chiarini-Garcia & Russell 2002) of the
Ultrastructureof germ and Sertoli cells
spermatogoniaastheyappearedasdescribedpreviously.
Ultrastructural studies showed that after irradiation and We noted that after irradiation alone, most of the
theinitiationofhormonetreatment,theSertolicellnuclei spermatogonia were undifferentiated type A but about
became more elongated and highly infolded and had 20% were A . As it is the longer chains of undiffer-
1
more heterochromatin close to the nuclear envelope entiated clones that transform into A spermatogonia,
1
(Fig. 7C and E) compared with those from control rats this is consistent with and supports a previous obser-
(Fig. 7A). No other Sertoli cell organelles displayed vation in whole-mounted tubules that most of the
significant abnormal morphological changes. The Ser- spermatogonia were type A as single cells or as pairs
toli–Sertoli junctions characterized by ectoplasmic andonlyafewwereseenaslongerclones(Shuttlesworth
specializations normally observed in control rats were et al. 2000). Hormone suppression induced differen-
still observed after irradiation. These junctions were tiationtotheBspermatogonialstagewithin2weeksand
observed more basally in the epithelium (Fig. 7C) when to thespermatocytestagewithin 4 weeks.
only Sertoli cells and no type A spermatogonia were Although stem spermatogonia do notexpressKIT,the
present within that region of the seminiferous tubule. undifferentiatedtypeAspermatogonianormallybecome
However,theectoplasmicspecializationappearedabove KIT-positive cells just before they initiate their differen-
the cells, when spermatogonia were present after tiation to A spermatogonia. In irradiated rat testes
1
hormonesuppression(Fig.7E),indicatingthatthebarrier withoutanyhormonesuppression,weobservedappreci-
between basal and adluminal compartments may be able numbers of KIT-positive spermatogonia, some of
preserved. Observations of the basal lamina after whichcouldbeA andothersmustbeA .Inaprevious
1 und
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372 AVAlbuquerqueandothers
Figure6Lightmicrographsofspermatogoniainglutaraldehyde/osmium-fixed,Araldite-embeddedtissuefrom(A)control,showingundifferentiated
spermatogonia(A )andalldifferentiatedgermcellsand(B)anirradiatedrat,inwhichtypeA1spermatogonia(A)arethemostadvancedgermcells.
und 1
TheA spermatogoniapresentalargerandmoreroundednucleusthantheA ,withmorefinelygranularnucleoplasmandamorereticulatednucleolus
1 und
protrudingcentrallyfromnuclearenvelope.Afterirradiationandhormonalsuppression,withEDSfor1week(C)orwithoutEDSfor2weeks(D),themost
advancedgermcellswereintermediateandtypeBspermatogoniarespectively.After4weeks,clonesofA spermatogoniawerefrequent(E,asterisks)and
al
allspermatogonialsubtypesuptotypeBspermatogoniawereseen(FandG).A spermatogonia(F)weredistinguishedfromearlier-stagecellsbythe
2
increaseinflecksofheterochromatinalongnuclearenvelopeandnucleolusbeingmorereticulatedandirregularthanA spermatogonia.A ,
1 und
undifferentiatedtypeAspermatogonia;A ,A ,typeAdifferentiatingspermatogonia;In,intermediatespermatogonia;B,typeBspermatogonia;
1 2
P,pachyteneprimaryspermatocyte;S,Sertolicell;L,Leydigcell;Ma,macrophage;ap,apoptosis;BV,bloodvessel;V,vacuole;Int,interstitium.Bar:12mm.
study(Prabhuetal.2006).KITproteinwasnotdetectable As the presence of healthy A spermatogonia after
und
in the type A spermatogonia of LBNF rats, although irradiation must be the source of the subsequent
1
insituhybridizationanalysisshowedthatKitmRNAwas spermatogenesis recovery in the rat testis and radiation
present.Prabhuetal.(2006)diddetectKITproteininthe canproducespermatogonialDNAdamage,itisessential
spermatogoniastimulatedtodifferentiateafter4weeksof to verify whether the remaining spermatogonial cells
hormonesuppression,correlatingwith ourobservations after irradiation are healthy and proliferative. We
of more KIT-positive cells at that time and the presence therefore assessed the presence of MCM7, a protein
of more B spermatogonia. We attribute the differences thatispartofacomplexessentialforchromosomalDNA
betweenthestudiestothegreatersensitivitythepresent replication (Pacek & Walter 2004). In control animals,
method.Fromourresults,weconcludethatsomeofthe we confirmed that MCM7 is primarily expressed in
type A spermatogonia in the irradiated testis have KIT, spermatogonia and early primary spermatocytes (Com
andtherefore,theblocktospermatogonialdifferentiation et al. 2006). In irradiated animals, the numbers of
is not due to the absence of a defect in spermatogonia MCM7-positive spermatogonia indicated that all the
involvingtheabsenceoftheKITreceptor. surviving spermatogonia were expressing MCM7 and
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