Complete Spermatogenesis in Orthotopic But Not in Ectopic ...cnc.cj.uc.pt/BEB/private/pdfs/pdfs0809/ReprBiol/Carlos.pdfComplete Spermatogenesis in Orthotopic But Not in Ectopic Transplants

Complete Spermatogenesis in Orthotopic But Not inEctopic Transplants of Autologously Grafted MarmosetTesticular Tissue

C. Marc Luetjens, Jan-Bernd Stukenborg, Eberhard Nieschlag, Manuela Simoni, and Joachim Wistuba

Institute of Reproductive Medicine of the University, 48149 Munster, Germany

Testicular grafting has the potential to become a method topreserve fertility in prepubertal boys undergoing cancertreatment. The possibility of successful germ cell maturationafter autologous grafting should be proven preclinically in anonhuman primate model. Therefore, in two experiments, weanalyzed the potential of autologous testicular grafting in themarmoset model. A first experiment in immature and adulthemi-castrated monkeys addressed the question of whetherfull spermatogenesis in an ectopic graft could be achievedunder a relatively normal endocrine milieu and whether thedonor’s age is of influence. A second experiment in castratedimmature animals examined whether the transplantation site[ectopic (back skin) or orthotopic (scrotum)] influences sper-matogenic progress and whether cryopreserved tissue can besuccessfully transplanted. Grafts were analyzed by histology,immunohistochemistry, and morphometry. Bioactive chori-

onic gonadotropin and serum testosterone were measured. Inthe adults, ectopic grafts degenerated, whereas in the imma-ture animals, grafts survived at the spermatogonial level. Inthe castrates, none of the cryopreserved grafts survived, ec-topic grafts were meiotically arrested, but orthotopic trans-plants completed spermatogenesis. Androgen and bioactivechorionic gonadotropin levels were not decisive for graft de-velopment. When ectopic and orthotopic transplantation siteswere compared, the scrotum has a substantially lower tem-perature. Thus, the higher temperature at the ectopic trans-plantation site may contribute to spermatogenic arrest. Au-tologous grafting of nonhuman primate testicular tissues canresult in complete spermatogenesis. Our findings indicatethat transplantation site and developmental age of the tissueplay a role more important than the endocrine milieu. (En-docrinology 149: 1736–1747, 2008)

TESTICULAR GERM CELL transplantation has become amethod to restore spermatogenesis and to study germ

cell development (for review see Ref. 1). Grafting of testiculartissue offers routes for the experimental generation of ga-metes and might also be applicable to fertility preservationin patients in the future (2, 3). The grafting technique wasoriginally developed in mice and afterward extended toother species (2–11). In the majority of approaches, xenograft-ing of testis tissue has been successfully performed, resultingin mature sperm as the germ cells differentiated in their owntesticular microenvironment (3–5, 7, 12).

Nevertheless, despite success in rodents, interspecies germcell transplantation was not performed successfully so far,and transfer of primate cells into mouse hosts failed (13, 14).Data on germ cell transplantation in nonhuman primatemodels are still preliminary (2), and this technique implies ahigh risk of cancer relapse if applied to patients (15). To date,the methodology for cell sorting before transfer has remainedat the experimental level (16–19). By fluorescent-activatedcell sorting, Fujita et al. (17, 18) demonstrated that all leu-kemic cells can be eliminated, but this could not be confirmedby other groups so far (19). There is a clinical need for pres-ervation of fertility in patients undergoing oncological ther-apies. Unfortunately, therapies not only are aggressive to

forms of cancer but can also damage other tissues, in par-ticular those containing cells with proliferative potential suchas the stem cells of the gonads. Therefore, cancer therapyoften results in infertility (20–22). In addition, boys withmaldescent testes often do not develop complete spermato-genesis, although the pathology has been surgically cor-rected (23, 24). The option of semen cryopreservation is avail-able only for sexually mature men, whereas prepubertal boyscannot preserve mature gametes.

Transplantation of testicular fragments might become an-other technique with clinical applications. The requirementof successful germ cell maturation after autologous grafting,a transplantation method with a clinical perspective, has tobe proven in a preclinical nonhuman primate model. So far,the limited number of studies that performed xenograftingwith human testicular tissue failed to achieve spermatogenicprogress and succeeded only in maintaining a few spermato-gonial cells over a short period, whereas most of the tissuedegenerated (8, 9, 25). A possible reason for the failure couldbe the developmental age of the grafted material (8, 9), butmorphological/structural differences might underlie suchfailure as well (8). Because of the structural similarities withhumans, we chose the marmoset monkey as a preclinicalnonhuman primate model to address autologous testiculargrafting. Interestingly, even testicular grafts of marmosetfailed to complete spermatogenic development in previousstudies (26, 27). Marmoset testicular tissues transplanted intomouse hosts differentiate marginally and cannot increaseserum androgen levels, and spermatogenesis never reachesmeiosis, even if cografted with hamster testicular tissue to

First Published Online January 3, 2008Abbreviations: AUC, Area under the curve; BioCG, bioactive CG; CG,

chorionic gonadotropin; DAB, diaminobenzidine; SCO, Sertoli-cell-only.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/08/$15.00/0 Endocrinology 149(4):1736–1747Printed in U.S.A. Copyright © 2008 by The Endocrine Society

doi: 10.1210/en.2007-1325

1736

maintain high local testosterone concentrations (see below)(2, 4, 6, 7). In a previous experiment, we showed that tes-ticular tissue transplanted heterologously degenerates com-pletely, whereas testis grafts transplanted autologously un-der the back skin of castrated immature animals survived fora long period and matured up to the meiotic stage beforespermatogenesis was arrested (10).

The present study was aimed to understand and overcomemeiotic arrest in autologously grafted material in this non-human primate model. Therefore, we conducted two exper-iments. The first experiment was performed in adult andimmature hemi-castrated monkeys and addressed the ques-tion of whether spermatogenesis in an ectopic graft could beachieved in the presence of a relatively normal male hor-monal milieu and whether transplantation success is influ-enced by the donors’ age. The second experiment performedin fully castrated immature animals examined whether thesite of transplantation influences the outcome of spermato-genesis and whether cryopreserved testis material obtainedbefore puberty can be successfully transplanted.

Materials and MethodsExperimental design

In total, the experimental setting comprised four treatment groupswith five marmosets each (Callithrix jacchus; n � 20). In the first exper-iment, one group of hemi-castrated adult animals (aged 21 months) wascompared with a group of hemi-castrated immature animals (aged 4wk). In the second experiment, two groups of immature animals wereanalyzed (Fig. 1).

Hemi-castrated animals, experiment 1: endocrine milieuand age

In group 1, immature animals were hemi-castrated, and the testiculartissue was divided into fragments that were either ectopically graftedunder the back skin (four transplants per animal) or fixed as pregraftcontrol tissue. The contralateral testis remained to ensure normal en-docrinological development and to raise serum testosterone levels dur-ing puberty. After 14 months, when puberty was completed, the animalswere killed, and tissues were analyzed. We evaluated graft developmentand histology of the remaining testis.

In group 2, adult animals were hemi-castrated to maintain theirnormal endocrinological state. Fragments of the removed testis weregrafted ectopically (four transplants per animal), and pregraft controltissues were fixed. Six months after surgery, grafts were analyzed asdescribed above.

Fully castrated animals, experiment 2: transplantation siteand cryopreservation

In group 3, immature animals were castrated, and fragments of tes-ticular tissue were grafted either ectopically under the back skin (fourtransplants per animal) or orthotopically into the emptied scrotum (fourtransplants per animal). Fragments of each testis were fixed as pregraftcontrols. Tissues were explanted 14 months after transplantation andevaluated as described. This approach aimed at identifying the influenceof the transplantation site on the grafting success.

In group 4, immature animals were castrated, and testicular tissuepieces were either retransplanted under the back skin (four transplantsper animal) or cryopreserved and stored for 4 months at �80 C. Pregraftcontrols were fixed for evaluation of the developmental state beforegrafting and cryopreservation. After 4 months, cryopreserved fragmentswere thawed and washed in cell culture medium before they weretransplanted ectopically (four transplants per animal) to the specificdonors before puberty onset (28). This experiment aimed at investigatingthe influence of cryopreservation on grafting success. All surviving

grafts were analyzed 10 months after the second transplantation surgeryand evaluated as described above.

Animals, tissue collection, and transplantation procedure

Testes were dissected from immature (n � 15, age 1 month) and adult(n � 5, age 21 months) marmosets (C. jacchus) obtained from the insti-tutional breeding facilities. The monkeys were anesthetized with Saffanduring surgery (Provet AG, Lyssach, Switzerland; 0.1 ml/100 g bodyweight). Castration was performed through scrotal incisions, and sub-sequently, the scrotal skin was stitched after removal of the testes.Testicular fragments (size 0.5–1 mm3) were kept in ice-cold DMEM(Invitrogen GmBH, Karlsruhe, Germany) for up to 20 min until grafting(see Ref. 10). For ectopic grafting, skin incisions of 4–5 mm were madeon either side of the dorsal midline, and four grafts per recipient wereautologously placed underneath the shaved back skin. The scrotal graftswere placed in the opening formed during castration before the scrotumwas closed. Incisions were sewed with a single suture. Cryopreservedtestis tissue was grafted after storing and thawing by an identical pro-cedure 4 months after the first surgery. Three of the grafts were selectedat random for cryopreservation according to the protocol in Ref. 2 fortransplantation up to 4 months later. The grafts were equilibrated for 1 hat 4 C in 1.5 m dimethylsulfoxide, 0.1 m sucrose, and 1% human serumalbumin in medium and transferred to 0.25-ml straws (type CBS; Con-sarctic GmbH, Schollkrippen, Germany). The straws were loaded into aprogrammable freezer (Kryoautomat Consarctic BV-65; Consarctic) at 4C and cooled at 2 C/min to �7 C and then nucleated with cold tongs.Cooling proceeded at �0.3 C/min to �40 C and then at 10 C/min to�140 C. The straws were plunged into liquid nitrogen and transferredto a storage Dewar flask. For thawing, the straws were kept at roomtemperature for 1 min to evaporate any remaining liquid nitrogen in thetube and then swirled in a bath of water at 37 C for approximately 1 min.The contents were emptied immediately into a succession of petri dishesthat contained a descending gradient of dimethylsulfoxide (1.0, 0.5, and0.0 m, the first two steps containing 1% human albumin and 0.1 msucrose) in DMEM for 3 min at each step to wash out remainingcryoprotectant.

Throughout the experimental period, animals were kept within theirfamily groups, receiving normal diet and water ad libitum. They wereregularly weighed, and blood samples (500 �l per sample) were drawnover the experimental period. For graft analysis, the animals were anes-thetized with Saffan (0.2 ml/100 g body weight) and killed by exsan-guination. Body weight was recorded, and blood was collected andstored at �20 C. The back skin was removed, and testicular grafts weredissected from the back skin and the scrotum. In groups 1 and 2, theremaining right testis and epididymis were removed. The experimentalwork was performed in accordance with the German Federal Law on theCare and Use of Laboratory Animals (license no. G32/2005).

Sample preparation for histological evaluation

Testicular tissue and pregraft fragments were obtained for controlpurposes at castration, and the explanted grafts were individually fixedin Bouin’s solution for 4 h or snap frozen in liquid nitrogen and storedat �80 C. Tissues were routinely embedded in paraffin, cut into sections(5 �m), and stained with periodic acid Schiff/hematoxylin.

To assess spermatogenic progress and graft development, we used apreviously established scheme (7, 10). In brief, tubular cross-sectionslacking germ cells were determined as Sertoli-cell-only (SCO) tubules,and the most advanced germ cell type in each graft was used to describespermatogenic progress. All seminiferous tubules of a cross-section werescored for the presence of spermatogonia, spermatocytes, round or elon-gating spermatids, and spermatozoa. Histology of all tubules was an-alyzed morphometrically in the most central cross-section of each graftand in material from 4-wk-old testes as well as from the contralateraltestis of the hemi-castrated animals of groups 1 and 2. Pieces of thecontrol tissue were taken from the central portion of the testes. The sizeof the fragment and the number of tubular cross-sections examined wassimilar to those of the transplants recovered from the experimentalanimals. The size of the seminiferous lumen and tubular diameter wereanalyzed with an Axioskop microscope (Zeiss, Oberkochen, Germany)and Axiovision 4.1 (Zeiss) software. Representative images were takenat magnifications of �10, �25, and �40 (Axiocam, Zeiss).

Luetjens et al. • Spermatogenesis in Grafted Testicular Tissues Endocrinology, April 2008, 149(4):1736–1747 1737

Immunohistochemistry

Only primary antibodies validated for Callithrix tissues were used(10). BOULE (boule-like) protein in the sections was analyzed by a rabbitpolyclonal antibody (diluted 1:300) (29, 30) and CDC25A by a rabbitpolyclonal antibody (1:50, sc-97; Santa Cruz Biotechnology, Inc., SantaCruz, CA). All antibodies were applied for 60 min at room temperaturein a blocking buffer. For all staining, Dako-LSAB 2 System (K0672; DakoDiagnostika, Glostrup, Denmark) was added for 30 min after washing,followed by an additional washing step and incubation with diamino-benzidine (DAB) (Dako Diagnostika) for 20 min. Antibodies were vi-sualized by a secondary horseradish peroxidase-labeled mixed anti-mouse and antirabbit IgG (1:100 dilution, K0672; Dako Diagnostika).DAB, used as a substrate, produced a dark brown signal. Briefly, thestaining process was as follows. After washing, the slides were incu-bated in 3% (vol/vol) hydrogen peroxide to suppress endogenous per-

oxidase activity. After washing in Tris-buffered saline buffer (10 mm Trisand 150 mm NaCl, pH 7.6), nonspecific background was blocked byincubation in 5% (vol/vol) normal goat serum diluted in incubationbuffer [0.1% (wt/vol) BSA in washing buffer]. The primary antibody wasincubated in incubation buffer for more than 90 min at room temper-ature. After extensive washing, the secondary antibodies were addedand incubated as a cocktail for more than 90 min at room temperature.DAB was finally added for 8–10 min followed by several washing steps.Control steps were performed by omitting the primary antibody onadjacent sections. In a final step, the slides were counterstained withhematoxylin for 10 sec and mounted under coverslips with Dako Fara-mount (Dako Diagnostika) before observation using a microscope (Ax-ioskop, Zeiss) at different magnifications (objectives, �10 and �25).Digital images of equal exposure were acquired with a CCD camera(Axiocam, Zeiss) controlled by image software (Axiovision, Zeiss).

FIG. 1. Study design of the two autologous grafting experiments in immature and adult marmosets. Hexagons represent immature monkeys;squares represent the adult animals.

1738 Endocrinology, April 2008, 149(4):1736–1747 Luetjens et al. • Spermatogenesis in Grafted Testicular Tissues

Hormone measurements

Serum testosterone levels were measured using a previously pub-lished RIA (25). Each sample was processed in duplicate after extractionwith diethyl ether. The intra- and interassay coefficients of variationwere 6.3 and 11.5%, respectively. The detection limit of the assay was0.68 nmol/liter.

The reproductive endocrinology of the neotropical primates differsfrom that of other primates owing to peculiarities in the function of theLH/chorionic gonadotropin (CG) system. The marmoset monkey doesnot express the �-subunit of LH but does express the �-subunit of CG,which is the only gonadotropin with LH activity in this species (27, 31).Serum bioactive CG (BioCG) levels of the last three sampling dates weremeasured by an in vitro bioassay based on murine Leydig cells accordingto a previously established method for bioactive LH measurement (28,32) and verified in several studies for male marmosets (10). The intra-and interassay coefficients of variation were 7.6 and 13.0%, respectively.The detection limit of the assay was 5.3 U/liter (WHO 78/549).

Thermography

Temperature measurements of the shaved back skin and the furlessscrotum were performed on three different untreated animals. We usedan infrared camera (Variotherm; Jenoptik AG, Jena, Germany) con-trolled by image software (Irbis; Jenoptik).

Statistical analysis

Data were analyzed by applying one-way ANOVA. Values of tubularand luminal diameter were compared by ANOVA (all pairwise, multiplecomparison by Tukey test). Computations were performed using thestatistical software package SIGMASTAT 2.03 (SPSS Inc., Chicago, IL).Values were considered significantly different if P � 0.05. Analysis of thetestosterone and BioCG values were performed for testosterone by uni-variate variance analysis for repeated measurements over the entireexperimental period and for BioCG over the last three blood samplingdates.

ResultsBody weight gain over the study period observed in bothexperiments

Groups 1, 3, and 4: immature animals. The body weight of allanimals increased over the study period (Table 1). At the age

of 4 wk, the marmosets had an average weight of 67.3 � 2.8 g.After surgery, they were housed within their families.Twelve weeks later, when the immature animals reached asufficient developmental state (225.2 � 8.0 g mean bodyweight), consecutive blood samples up to the end of thestudy period were drawn twice weekly. The average bodyweight at the end of the study period was 317.9 � 13.9 g. Theaverage weight gain in the 60 wk of the study did not varysignificantly among the three animal groups (Table 1).

Group 2: adult animals. The monkeys had an average bodyweight of 408.4 � 20.1 g at the time of surgery and were keptwithin their families after surgery for another 26 wk. Nosignificant weight change (425.6 � 14.0 g) was observed overthe study period.

The significant weight difference among the animals ofgroups 1, 3, and 4 compared with group 2 at the end of thestudy is caused by the different age at the end of the studyperiod (groups 1, 3, and 4 were about 65 wk; group 2 wasabout 90 wk).

Graft survival, spermatogenic progress, and endocrine state

In all 20 treated animals, the grafts and remaining testesand epididymides were explanted and weighed, and thenumber of surviving grafts was recorded. No significantweight differences among the analyzed grafts were found,nor were significant organ weight differences found betweenthe groups. The grafts were analyzed for the stage of sper-matogenic progress (Table 1). In none of the analyzed graftswere any signs of inflammation found, such as macrophagesor other leukocytes.

Hemi-castrated animals, experiment 1: endocrine milieuand age

Group 1. In total, nine grafts of 20 transplanted fragmentswere recovered from four individuals (Table 1). In one mon-

TABLE 1. Summary of the treated animals at the transplantation and analysis time point

Animal no.Body weight at

start ofimplantation

(g)

Body weight aftercryopreservation

(g)No. ofgrafts

Body weight atexplantation

(g)

Survivinggrafts inback skin

Grafts inscrotum

Grafts aftercryo-

preservation

Efficiency ofsurviving

grafts(%)

Most advancedgerm cell stage

in back skin

Most advancedgerm cell stage

in scrotum

Group 1 (4 wk hemi-castrated), back: fresh grafts51718 65 4 353 3 3/4 (75%) Spermatogonia38031 78 4 384 3 3/4 (75%) Spermatogonia47171 58 4 319 2 2/4 (50%) SCO36941 82 4 311 0 0/4 (0%)31338 77 4 288 1 1/4 (25%) SpermatogoniaMean 72 � 5 20 331 � 17 9 9/20 (45%) 3/4 Spermatogonia

Group 2 (adult hemi-castrated), back: fresh grafts43285 408 4 421 0 0/4 (0%)49849 357 4 374 2 2/4 (50%) Fibrotic45699 475 4 515 3 3/4 (75%) Spermatocytes48574 380 4 397 1 1/4 (25%) Fibrotic45010 422 4 424 1 1/4 (25%) SCOMean 408 � 20 20 426 � 24 7 7/20 (35%) 1/4 Spermatocytes

Group 3 (4 wk castrated), back and scrotum: fresh grafts46660 67 4 � 4 336 4 4 8/8 (100%) Spermatocytes Spermatozoa46231 62 4 � 4 308 4 4 8/8 (100%) Spermatocytes Spermatozoa49483 47 4 � 4 164 4 3 7/8 (87.5%) Spermatocytes Spermatozoa51810 52 4 � 4 350 3 3 6/8 (75%) Spermatocytes Spermatozoa32391 80 4 � 4 277 3 2 5/8 (62.5%) Spermatocytes SpermatozoaMean 62 � 6 20 � 20 287 � 33 18 16 34/40 (85%) 5/5 Spermatocytes 5/5 Spermatozoa

Group 4 (4 wk castrated), back: fresh and cryopreserved grafts42257 66 237 4 � 3 375 2 0 2/7 (28.6%) Spermatocytes44225 78 241 4 � 3 324 2 0 2/7 (28.6%) Spermatocytes45655 62 250 4 � 3 352 3 0 3/7 (42.9%) Spermatocytes45926 60 205 4 � 3 315 4 0 4/7 (57.1%) Spermatocytes49700 76 255 4 � 3 267 3 0 3/7 (42.9%) SpermatocytesMean 69 � 4 238 � 9 20 �15 327 � 18 14 0 14/35 (40.0%) 5/5 Spermatocytes


key, no graft survived. In the grafts, spermatogonia wereobserved as the most advanced germ cell stage (28.7 � 30.9%of the tubules; Table 2). In the pregraft control tissues, onlygonocytes positioned in the tubular lumen and a few earlyspermatogonia at the basal membrane were observed. Themature contralateral control testis showed complete sper-matogenesis up to the level of elongated spermatids in al-most every tubular cross-section (Table 2). Thus, the graftedmaterial differentiated only up to spermatogonial arrest. Oc-casionally, in the ectopic grafts, seminiferous tubules lackingall germ cells (SCO) were observed (53.4 � 27.1% of tubules;Table 2). On average, serum testosterone levels had alreadyrisen over 20 nmol/liter during the sampling period butremained very variable until shortly before the end of thestudy (Fig. 2A).

Group 2. Seven ectopic grafts were explanted from four an-imals (Table 1). In one animal, none of the grafted fragmentssurvived the study period. In general, the ectopic grafts ex-planted from the back skin sites contained fibrotic or SCOtubules that had lost all germ cells. In only one ectopic graft,a single tubule with some degenerating spermatocytes (0.4 �1.2% of the tubules of the section; Table 2) was found. Allother tubules of this transplant exhibited only spermatogoniaas the most advanced germ cell type or were SCO. As ex-pected, the average testosterone levels remained normal overthe entire sampling phase (Fig. 2A).

Serum testosterone values of group 2 were also signifi-cantly elevated (P � 0.001) compared with the postpubertallevels of the hemi-castrated monkeys of group 1. Both groupshad similar testosterone values only in the last six studyweeks (Fig. 2A).

The different ages of the monkeys at the time point oftransplantation resulted in different explantation rates (inimmature animals, 45% of the grafts were found at study endvs. 35% in the adults). Although in only 20% (one of five) ofthe adult animals did the transplanted material contain germcells, in 60% of the juvenile animals, grafts with spermato-gonia were found. The majority of the tissue grafted in the

adult state degenerated to SCO or fibrotic tubules. Never-theless, in principle, survival and development even up tomeiosis was found in at least one tubule of one ectopic graftof an adult recipient, indicating that successful graft supportis possible but obviously much more difficult with materialderived from a mature testis.

Fully castrated animals, experiment 2: transplantation siteand cryopreservation

Group 3. We analyzed 18 of 20 originally ectopically graftedfragments from the back skin and 16 of 20 orthotopic graftsfrom the scrotum, respectively (Table 1 and Fig. 3). In theectopic grafts (Fig. 3, A and B), degenerating spermatocytes(32.1 � 28.9% of the tubules per section; Table 2) but nofurther advanced spermatogenic cell stages were found (Ta-ble 2 and Fig. 4, A and B). However, the orthotopic grafts inthe scrotum exhibited (Fig. 3C) complete spermatogenesis(12.4 � 12.2% of the tubules; Table 2) in all animals, and sometubules contained morphologically normal spermatozoa (Ta-ble 2 and Fig. 4, C–F). Such spermatozoa were analyzed bysmear preparation immediately after explantation and werefound to be immotile (Fig. 4G). In the pregraft control tissues,only gonocytes and early spermatogonia localized at thebasal membrane were observed. In this group of castratedanimals, the average testosterone values remained low overthe entire study period, just above the detection limit(Fig. 2A).

Group 4. Fourteen of 20 of the transplanted fresh grafts andnone of the cryopreserved grafts survived (Table 1). Tissuefragments could be analyzed from all five animals; theycontained tubules with meiotic arrest and spermatocytes asthe most advanced germ cell type (23.5 � 27.3% of the tu-bules; Table 2). The remaining sample showed arrest at thespermatogonial level, SCO, or fibrotic degeneration. In thepregraft control tissue, only gonocytes and early spermato-gonia were found. The testosterone levels remained low,comparable to group 3 (Fig. 2A).

TABLE 2. Histological summary of the transplants grouped according to treatment

LocationTotal no.of tubulesper section

Most advanced cell stage

Elongatingspermatids

Roundspermatids

Spermato-cytes

Spermatogonia/gonocytes

Sertolicells

Fibrosis

Group 1 (4 wk), back: fresh graftsTestis juvenile 231 0 � 0 0 � 0 0 � 0 100 � 0 0 � 0 0 � 0Back skin graft 258 0 � 0 0 � 0 0 � 0 28.7 � 30.9 53.4 � 27.1 18 � 21.7Testis adult 408 94 � 6.1 4 � 5.8 1.8 � 1.7 0 � 0 0.2 � 0.4 0 � 0

Group 2 (adult), back: fresh graftsTestis adult before 337 92.4 � 8.5 2.6 � 2.6 4.5 � 6.5 0.6 � 1.3 0 � 0 0 � 0Back skin graft 196 0 � 0 0 � 0 0.4 � 1.2 1.7 � 3.2 11.7 � 18.1 86.2 � 20.1Contralateral testis adult 469 89.5 � 9.9 8.2 � 8.4 2.2 � 2.1 0 � 0 0 � 0 0 � 0

Group 3 (4 wk), back and scrotum:fresh grafts

Testis juvenile 242 0 � 0 0 � 0 0 � 0 94.3 � 10.9 5.7 � 10.9 0 � 0Back skin graft 1027 0 � 0 0 � 0 32.1 � 28.9 18.4 � 14.2 33.1 � 26.3 16.4 � 17.4Scrotum graft 842 12.4 � 12.2 19.3 � 22.2 17.9 � 15.2 6.2 � 8.3 31 � 31.9 13.2 � 19

Group 4 (4 wk), back: fresh &cryopreseverd grafts

Testis juvenile 206 0 � 0 0 � 0 0 � 0 100 � 0 0 � 0 0 � 0Back skin graft 692 0 � 0 0 � 0 23.5 � 27.3 26.1 � 19.5 30.5 � 12.6 19.9 � 24.2Back skin graft (cryopreseverd)

The columns describe the percentage � SD of tubules with the most advanced cell type found per transplantation site.


Spermatogenic development was successfully completedup to spermatozoa in orthotopic grafts in group 3 but wasarrested at the meiosis stage (spermatocytes) in the ectopi-cally transplanted tissue, indicating that the transplantationsite influences maturation of the grafts.

In group 4, the subsequent transplantation of cryopre-served tissue did not affect those transplants that were al-ready freshly engrafted several months before and that de-veloped meiotic arrest, but these stored grafts failed tosurvive the remaining study period. None of these graftswere found at the time point of analysis. This might becaused either by the increased age of the animals at the timeof the second transplantation procedure or by damage of thetissue during cryopreservation.

Comparing the results with regard to the status of castra-tion, evaluation of the ectopic grafts revealed less spermat-ogenic development in animals with a remaining testis, i.e. inmonkeys with normal serum testosterone levels comparedwith those with very low serum testosterone (Tables 1 and

FIG. 2. Hormone levels of the four animal groups. A, Average � SEMserum testosterone levels of the four treatment groups over the treat-ment period. Testosterone levels of fully castrated animals (groups 3and 4) remained low until the end of the study, whereas the hemi-castrated immature animal group showed high testosterone varia-tions around puberty and a steady increase after reaching maturityup to the levels of the hemi-castrated mature animals (group 2). Thisgroup presented testosterone levels around 40–60 nmol/liter over theentire treatment phase. B, Correlation of the average � SEM BioCGvalues with the corresponding average � SEM testosterone levels. Thetwo castrated study groups clustered together, demonstrating no as-sociation with spermatogenic success. All animals of group 3 exhibitedcomplete spermatogenesis, whereas none of group 4 had overcome themeiotic arrest in their grafts.

FIG. 3. Transplantation sites, with arrows marking the testiculargrafts. A, Marmoset shaved on the back to show in vivo the cica-trices and possible humps with underlying growing testis tissue; B,marmoset back skin seen from the inside after removal from theanimal; C, opened marmoset scrotum of a castrated animal (group3) to observe the testicular transplants. Two grafts are positionednext to the penis (P).


FIG. 4. Histology of the grafted testicular tissue and smears of tissue recovered from the scrotum. A, Ectopic graft with tubules containing alumen (arrow). No cell stages later than spermatocytes were found in this tissue. B, Tubules were mainly fibrotic, and only the tubule wallsremained in the inside of the back skin (arrow). Scale bars, 500 �m (A and C) and 200 �m (B and D). C–F, Histology of a scrotal transplantof group 3: C, overview of a transplant dissected in the middle; D, the tubules show different stages of development with some having onlyspermatocytes and others showing complete spermatogenesis (arrow); E and F, different tubules containing elongating and elongated sper-matozoa. Scale bar, 20 �m. G, Three micrographs of marmoset spermatozoa after an orthotopic graft was spread onto a slide. Scale bar, 20 �m.


2 and Fig. 2A). The area under the curve (AUC) values of thecastrated animals (groups 3 and 4) were significantly differ-ent from group 1 (P � 0.001) (Fig. 2A). The AUC of group 1(AUCgroup1 � 2606; P � 0.05) was higher than the values ofthe two immature groups (AUCgroup3 � 338; AUCgroup4 �239).

In contrast, the BioCG values obtained from the last threesamples did not differ significantly among the monkeys (Fig.2). When BioCG values were correlated with the testosteronelevels, the two castrated groups appeared clustered (Fig. 2B).Furthermore, the two hemi-castrated groups differed fromthe immature animal groups but did not form a cluster. Theadult group exhibited higher BioCG values and higher tes-tosterone values, although group 1 had only higher testos-terone levels (Fig. 2B).

Morphometry of the transplants in both experiments

In accordance with the arrested developmental state of thegrafts, tubular and luminal diameters in the transplants wereat an intermediate state compared with those of immaturecontrols and adult testes. All differences were statisticallysignificant. Although in all immature control tissue, 98.1 �6.5% of the tubules contained gonocytes or early spermato-gonia as the most advanced germ cell type, complete sper-matogenesis up to elongated spermatids was found in 92.4 �8.5% of all the tubules from the adult testes (Table 2). Thetubular (84.6 � 1.8 �m) and luminal (48.2 � 2.0 �m) diam-eters in the seminiferous tubules of the ectopic grafts ob-tained from immature treatment groups were similar,whereas the diameters of the seminiferous tubules (34.6 � 1.7vs. 29.2 � 7.4 �m) of the ectopic grafts obtained from theadult treatment group were significantly smaller (P � 0.001).The tubular (111.9 � 2.3 �m) and luminal (61.3 � 2.0 �m)diameters of the orthotopic grafts from the scrotum in group3 were significantly (P � 0.001) larger than those found in thetubules of ectopic grafts from the back skin but significantlysmaller (P � 0.001) than those in the adult testes (200.4 � 2.8vs. 77.8 � 2.6 �m). However, the situation in the orthotopicgrafts (38.7 � 5.0 vs. 14.4 �m) obtained from the adult ani-mals of group 2 was comparable to that of the ectopic graftsof the other groups, but diameters of the tubules were sig-nificantly smaller than in transplants recovered from thescrotal transplantation sites of the immature groups (P �0.001).

Thermography

Surface temperature of the scrotal and the back skin wasmeasured with a Variotherm infrared camera. The meantemperature of the scrotum was 5 C lower compared with theshaved back skin surface at the site for ectopic transplanta-tion (31.0 � 0.3 C vs. 35.8 � 0.3 C; n � 3; P � 0.001; Fig. 5A).

Immunohistochemistry

In group 3, immunostaining for BOULE and CDC25A oforthotopic and ectopic grafts of the same animal were com-pared. These proteins are markers for the meiotic progress ofthe germ cells into haploidization. Although BOULE expres-sion determines entry into meiosis, the expression of

CDC25A is related to genomic reduction (Fig. 5, B and C).BOULE staining was detected in the primary spermatocytesof the transplants at both transplantation sites, indicatingthat the germ cells can enter meiosis (Fig. 5, B1 and C1).However, CDC25A staining was negative in the ectopicgrafts but positive in orthotopically transplanted tissue (Fig.5, B2 and C2). Because BOULE activates CDC25A expression,this finding indicated an early meiotic arrest in the ectopicgrafts.

Discussion

In a previous approach, we autologously grafted testiculartissue in marmosets into an ectopic position under the backskin of immature, fully castrated animals, and after puberty,we obtained developed tissue but with meiotic arrest (10).Therefore, we designed two experiments to address this mat-uration arrest of the germline in autologously grafted ma-terial. The first experiment used hemi-castrated monkeysconserving a natural endocrine profile over the experimentalperiod to assess the meaning of hormone milieu, in particularof testosterone. In addition, we asked whether the donor’sage might play a role as observed in other transplantationstudies (1). In the second experiment, we performed a studydesign similar to our previous study (10) in immature cas-trated animals, but here we analyzed whether the site oftransplantation influences the outcome of spermatogenesisand, in addition, whether cryopreserved testis material canbe autologously grafted after storage over a longer period.

In the first experiment, the hemi-castrates of group 2 werealready adult when they received the grafts derived fromtheir fully matured and functional testis with complete sper-matogenesis. The survival and spermatogenic progress ofthese grafts was marginal; they were almost completely de-generated. In contrast, in immature animals, a higher per-centage of explants and a better survival rate of the graftswere observed. Although only on a spermatogonial level, themajority of the grafts contained living germ cells. The de-velopmental age of the grafts at the time point of transplan-tation might have caused this difference, very likely becauseof the higher sensitivity to periods of ischemia known toaffect mature material more strongly than immature material(33, 34).

The hemi-castrated animals showed normal testosteronevalues; i.e. in the young animal group, the testosterone valueswere already elevated before puberty but highly variable. Inan earlier study, consecutive blood sampling revealed ex-treme fluctuations in testosterone concentrations, suggestingan erratic secretion (28, 35). The onset of puberty was ascer-tained in intact animals by an increase in serum testosteronelevels from values of less than 10 nmol/liter to values above80 nmol/liter (10). Lunn et al. (36) demonstrated higher levelsof biologically active testosterone, and Pugeat et al. (37) re-ported that plasma testosterone-estradiol-binding protein inNew World monkeys has a low affinity resulting in highlevels of plasma testosterone (20–40 nmol/liter), comparedwith those in Old World monkeys or humans (3–9 nmol/liter). The difference in binding characteristics may explainthe high testosterone values in marmosets. This seems to bea specific feature of marmosets and is in accordance with


FIG. 5. Thermographic and immunohistological images. A, Infrared measurement of the shaved back skin (left side, C01) and scrotum (rightside, C01) together with a reference region (C02) of a male marmoset. The arrows indicate the region of interest. The average temperatureswithin a circle (diameter 5 mm) are given at the top right corner. B and C, BOULE and CDC25A immunostaining of grafts from the back skin(B) and the scrotum (C). The images demonstrate that BOULE (B1 and C1) is expressed at both transplantation sites, whereas CDC25A (B2and C2) is expressed only in late and postmeiotic germ cells (arrow). Scale bar, 200 �m.


earlier observations in normal prepubertal animals (28). Thetestosterone levels were at a postpubertal state at the end ofthe experiment. Nevertheless, spermatogenesis in the sur-viving grafts did not progress beyond a very early devel-opmental stage, and it has been demonstrated that a blockadeof neonatal activation of the pituitary-gonadal axis has onlya minor effect on future proliferation of germ cells in mar-mosets (38). Obviously, normal postpubertal testosteronevalues did not affect spermatogenic development positivelyin the grafts. In the hemi-castrated animals, the functionalcontralateral testis may inhibit or at least not support sper-matogenesis of the grafts via serum testosterone or otherfactors (e.g. inhibins). Reliable assays for FSH and inhibin arenot available for the marmoset, but in fully castrated animals,FSH must be high compared with hemi-castrated marmosets.Treatment of gonadotropin-deficient boys with recombinantFSH leads to testis growth and full spermatogenesis (39). Theendogenous elevated FSH values in the fully castrated ani-mals may better support the remaining Sertoli cells to matureand support the adjacent germ cells of the grafts. The or-thotopic grafts in the fully castrated animals reach full sper-matogenesis, which implies that, independent of the implantposition, spermatogenesis is initiated, but the back skin tem-perature at the ectopic grafts prevents their meiotic matu-ration. Insufficient FSH levels in the grafts of hemi-castratedanimals might prevent spermatogenic onset.

In accordance with our previous study (10), in the cas-trated animals, serum testosterone levels remained at base-line. It is likely that the Leydig cells in the grafts were func-tional but unable to raise the serum testosterone levels tonormal values because of their limited number (10). As in ourprevious study, we analyzed the Leydig cells of grafts fromall four animal groups for their 5�-reductase expression todetermine their maturity. The results of this study in allanimal groups were similar and confirmed our earlier find-ings (data not shown). Thus, in the marmoset, high circu-lating serum testosterone levels are not essential for the or-thotopic grafts to complete spermatogenesis. Even localtestosterone might be of minor significance for the matura-tion of marmoset germ cells. When xenologously cograftedwith hamster tissue, marmoset germ cells were also arrestedat spermatogonial level, although the surrounding tubules ofrodent origin matured to haploid cells (7).

High gonadotropin (CG) levels in the castrated animals(groups 3 and 4) are not necessary to induce early events ofspermatogenesis up to a meiotic state because the hemi-castrated animals also had high serum CG. The levels wereeven higher than observed in other animals at the onset ofpuberty (28). It might be that the hemi-castrated immatureanimals had a different endocrine profile compared withthose castrated during puberty. If so, testosterone feedbackcould have lowered the CG peaks in these animals. To eval-uate this question, our study design would have required anextra animal group of hemi-castrated orthotopically trans-planted animals. The contralateral testis of these animalswould have normal spermatogenesis and relatively normalsex hormone values throughout their lifetime to support thetransplants in the scrotum. However, CG alone cannot beresponsible for differences in spermatogenic progress be-cause the animals of group 3 were all castrated, and thus,

independent of the transplantation site, the grafts in thecastrates, which were all found to be fully vascularized, musthave had similar systemic gonadotropin levels to supportlocal testosterone production for maintenance and stimula-tion of germline differentiation. Nevertheless, the ectopicallytransplanted tissue exposed to high gonadotropin levelswere also found to be arrested at meiosis.

Testicular tissue obtained from immature monkeys cancomplete spermatogenesis when grafted autologously intothe scrotal position. In contrast, testicular fragments trans-planted ectopically under the back skin were arrested at themeiotic level. Testicular maturation of the orthotopically po-sitioned grafts also occurred when ectopic grafts in the sameindividuals failed to develop beyond meiosis (4, 7, 10, 40).This observation suggested that local factors influence de-velopmental progress at the different transplantation sitesthat either support the grafts (scrotal) or block their fullmaturation (back skin).

When we compared the ectopic transplantation site underthe back skin with the orthotopic scrotal position by ther-mographic imaging of three normal male animals, we founda substantial difference of almost 5 C. The temperature of thescrotal skin has been demonstrated to also be a suitablemarker for testicular temperature (41) Furthermore, our find-ing is consistent with results in men determining the scrotaltemperature to be around 32–35 C (reviewed in Ref. 42).

The higher temperature at the ectopic transplantation siteon the back of the animals may contribute to the develop-mental arrest of germ cells. Such impact on germ cell mat-uration has been reported in other species and was also usedas an experimental method for male contraception (43, 44).In early contraception attempts for men, many different tech-niques were performed to increase testicular temperature(reviewed in Ref. 42). In an efficacy study, even a mild tem-perature increase of 2 C resulted in infertility in all volun-teering subjects (45). In bulls, a clear correlation was dem-onstrated, showing that the higher the gradient betweenbody and scrotum temperature, the better the sperm quality(46). Only transplantation into an orthotopic scrotal positionresulted in fully matured grafts. The most prominent differ-ence between the scrotum and the back is the local temper-ature, and thus, this local factor might be a good candidateto explain spermatogenic arrest in the ectopic transplantationsites (10, 25).

Testicular grafting in animal models aims at new routestoward fertility preservation in prepubertal boys threatenedby infertility due to disease treatments and destroying thegermline. To date, the most advanced methodology for tissuepreparation is to cryopreserve testicular biopsies, and inexperimental studies, successful transplantation experi-ments have been reported (11, 21, 47). Therefore, we addi-tionally examined the development of cryopreserved testistissue to analyze the survival and progression of such ma-terial autologously grafted onto the back skin of animals thatpreviously had received fresh testis tissue. Although morethan 60% of the fresh grafts could be recovered and somedeveloped up to the premeiotic stage, none of the cryopre-served transplants survived. Because our surgery procedureworked well in the material grafted freshly, we exclude thattissue loss occurred because of a surgical problem or an


infection. Although we could clearly determine the trans-plantation sites by the surgical scars, neither was tissuefound nor were any signs of inflammation seen. This failuremay have been caused by an insufficient cryopreservationprotocol, although a similar protocol has been successfullyused in other experiments (2, 9). Alternatively, the recipientsaged between both time points of transplantation. Thus, in-creased age at the second grafting date, the transplantationof the cryopreserved material, might also have had an ad-verse effect on the transplanted tissue. Testicular grafting forclinical purposes could be an option but only if the possiblerisks can be resolved (48).

In summary, we demonstrate for the first time that autol-ogous grafting of nonhuman primate testicular tissues or-ganized similarly to human testes resulted in full spermat-ogenesis in grafts retransplanted into the scrotum ofimmature animals. Our findings indicate that orthotopictransplantation and the developmental age of the tissue playmost important roles for grafting success. Factors involvedmight be the temperature as well as the resistance against aperiod of hypoxia. Factors such as position of the graft, sizeof the graft, angiogenesis, and adequate cryopreservationprotocols of testicular tissue material have to be carefullyevaluated further in this nonhuman primate model.

Acknowledgments

We thank O. Damm, J. Salzig, H. Kersebom, and R. Sandhowe fortechnical assistance, R. Chandolia for veterinarian advice, and S. Ni-eschlag, M.A., for language editing of the manuscript. The authors areindebted to T. Mondritzki of Bayer Healthcare AG in Elberfeld, Ger-many, for thermographic imaging of the marmosets.

Received September 25, 2007. Accepted December 26, 2007.Address all correspondence and requests for reprints to: Prof.

Dr. Eberhard Nieschlag, Institute of Reproductive Medicine of theUniversity, Domagkstrasse 11, D-48149 Munster, Germany. E-mail:[email protected].

The project was supported by a Grant from the Medical Faculty of theUniversity Munster for Innovative Medical Research (IMF LU 1 106 08).

Disclosure Statement: All authors have nothing to declare.

References

1. Dobrinski I 2005 Germ cell transplantation. Semin Reprod Med 23:257–2652. Schlatt S, Kim SS, Gosden R 2002 Spermatogenesis and steroidogenesis in

mouse, hamster and monkey testicular tissue after cryopreservation and het-erotopic grafting to castrated hosts. Reproduction 124:339–346

3. Honaramooz A, Snedaker A, Boiani M, Scholer H, Dobrinski I, Schlatt S 2002Sperm from neonatal mammalian testes grafted in mice. Nature 418:778–781

4. Schlatt S, Honaramooz A, Boiani M, Scholer HR, Dobrinski I 2003 Progenyfrom sperm obtained after ectopic grafting of neonatal mouse testes. BiolReprod 68:2331–2335

5. Ohta H, Wakayama T 2004 Full-term development of offspring using roundspermatids produced ectopically from fetal male germ cells. J Reprod Dev50:429–437

6. Snedaker AK, Honaramooz A, Dobrinski I 2004 A game of cat and mouse:xenografting of testis tissue from domestic kittens results in complete catspermatogenesis in a mouse host. J Androl 25:926–930

7. Wistuba J, Mundry M, Luetjens CM, Schlatt S 2004 Cografting of hamster(Phodopus sungorus) and marmoset (Callithrix jacchus) testicular tissues intonude mice does not overcome blockade of early spermatogenic differentiationin primate grafts. Biol Reprod 71:2087–2091

8. Geens M, De Block G, Goossens E, Frederickx V, Van Steirteghem A, Tour-naye H 2006 Spermatogonial survival after grafting human testicular tissue toimmunodeficient mice. Hum Reprod 21:390–396

9. Schlatt S, Honaramooz A, Ehmcke J, Goebell PJ, Rubben H, Dhir R, Do-brinski I, Patrizio P 2006 Limited survival of adult human testicular tissue asectopic xenograft. Hum Reprod 21:384–389

10. Wistuba J, Luetjens CM, Wesselmann R, Nieschlag E, Simoni M, Schlatt S

2006 Meiosis in autologous ectopic transplants of immature testicular tissuegrafted to Callithrix jacchus. Biol Reprod 74:706–713

11. Hermann BP, Sukhwani M, Lin CC, Sheng Y, Tomko J, Rodriguez M,Shuttleworth JJ, McFarland D, Hobbs RM, Pandolfi PP, Schatten GP, OrwigKE 2007 Characterization, cryopreservation, and ablation of spermatogonialstem cells in adult rhesus macaques. Stem Cells 25:2330–2338

12. Honaramooz A, Li MW, Penedo MC, Meyers S, Dobrinski I 2004 Acceleratedmaturation of primate testis by xenografting into mice. Biol Reprod 70:1500–1503

13. Reis MM, Tsai MC, Schlegel PN, Feliciano M, Raffaelli R, Rosenwaks Z,Palermo GD 2000 Xenogeneic transplantation of human spermatogonia. Zy-gote 8:97–105

14. Nagano M, McCarrey JR, Brinster RL 2001 Primate spermatogonial stem cellscolonize mouse testes. Biol Reprod 64:1409–1416

15. Jahnukainen K, Hou M, Petersen C, Setchell B, Soder O 2001 Intratesticulartransplantation of testicular cells from leukemic rats causes transmission ofleukemia. Cancer Res 61:706–710

16. von Schonfeldt V, Krishnamurthy H, Foppiani L, Schlatt S 1999 Magnetic cellsorting is a fast and effective method of enriching viable spermatogonia fromDjungarian hamster, mouse, and marmoset monkey testes. Biol Reprod 61:582–589

17. Fujita K, Ohta H, Tsujimura A, Takao T, Miyagawa Y, Takada S, MatsumiyaK, Wakayama T, Okuyama A 2005 Transplantation of spermatogonial stemcells isolated from leukemic mice restores fertility without inducing leukemia.J Clin Invest 115:1855–1861

18. Fujita K, Tsujimura A, Miyagawa Y, Kiuchi H, Matsuoka Y, Takao T, TakadaS, Nonomura N, Okuyama A 2006 Isolation of germ cells from leukemia andlymphoma cells in a human in vitro model: potential clinical application forrestoring human fertility after anticancer therapy. Cancer Res 66:11166–11171

19. Geens M, Van de Velde H, De Block G, Goossens E, Van Steirteghem A,Tournaye H 2007 The efficiency of magnetic-activated cell sorting and fluo-rescence-activated cell sorting in the decontamination of testicular cell sus-pensions in cancer patients. Hum Reprod 22:733–742

20. Colpi GM, Contalbi GF, Nerva F, Sagone P, Piediferro G 2004 Testicularfunction following chemo-radiotherapy. Eur J Obstet Gynecol Reprod Biol113(Suppl 1):S2–S6

21. Orwig KE, Schlatt S 2005 Cryopreservation and transplantation of spermato-gonia and testicular tissue for preservation of male fertility. J Natl Cancer InstMonogr 34:51–56

22. Robertson JA 2005 Cancer and fertility: ethical and legal challenges. J NatlCancer Inst Monogr 34:104–106

23. Cortes D, Thorup JM, Visfeldt J 2001 Cryptorchidism: aspects of fertility andneoplasms. A study including data of 1,335 consecutive boys who underwenttesticular biopsy simultaneously with surgery for cryptorchidism. Horm Res55:21–27

24. Cortes D, Thorup J, Lindenberg S, Visfeldt J 2003 Infertility despite surgeryfor cryptorchidism in childhood can be classified by patients with normal orelevated follicle-stimulating hormone and identified at orchidopexy. BJU Int91:670–674

25. Wyns C, Curaba M, Martinez-Madrid B, Van Langendonckt A, Francois-Xavier W, Donnez J 2007 Spermatogonial survival after cryopreservation andshort-term orthotopic immature human cryptorchid testicular tissue graftingto immunodeficient mice. Hum Reprod 22:1603–1611

26. Millar MR, Sharpe RM, Weinbauer GF, Fraser HM, Saunders PT 2000 Mar-moset spermatogenesis: organizational similarities to the human. Int J Androl23:266–277

27. Luetjens CM, Weinbauer GF, Wistuba J 2005 Primate spermatogenesis: newinsights into comparative testicular organisation, spermatogenic efficiency andendocrine control. Biol Rev Camb Phil Soc 80:475–488

28. Chandolia R, Luetjens CM, Wistuba J, Yeung CH, Nieschlag E, Simoni M2006 Changes in endocrine profile and reproductive organs during puberty inthe male marmoset monkey (Callithrix jacchus). Reproduction 132:355–363

29. Handel MA 1998 Monitoring meiosis in gametogenesis. Theriogenology 49:423–430

30. Tung JY, Luetjens CM, Wistuba J, Xu EY, Reijo-Pera RA, Gromoll J 2006Evolutionary comparison of the reproductive genes, DAZL and BOULE, inprimates with and without DAZ. Dev Gen Evol 216:158–168

31. Muller T, Simoni M, Pekel E, Luetjens CM, Chandolia R, Amato F, NormanRJ, Gromoll J 2004 Chorionic gonadotropin �-subunit mRNA but not lutein-izing hormone �-subunit mRNA is expressed in the pituitary of the commonmarmoset (Callithrix jacchus). J Mol Endocrinol 32:115–128

32. Wickings EJ, Qazi MH, Nieschlag E 1979 Determination of biologically activeLH in the serum of male rhesus monkeys (Macaca mulatta). J Reprod Fertil57:497–504

33. Rathi R, Honaramooz A, Zeng W, Turner R, Dobrinski I 2006 Germ celldevelopment in equine testis tissue xenografted into mice. Reproduction 131:1091–1098

34. Schmidt JA, de Avila JM, McLean DJ 2006 Grafting period and donor ageaffect the potential for spermatogenesis in bovine ectopic testis xenografts. BiolReprod 75:160–166

35. Kelnar CJ, McKinnell C, Walker M, Morris KD, Wallace WH, Saunders PT,Fraser HM, Sharpe RM 2002 Testicular changes during infantile ‘quiescence’


in the marmoset and their gonadotrophin dependence: a model for investi-gating susceptibility of the prepubertal human testis to cancer therapy? HumReprod 17:1367–1378

36. Lunn SF, Cowen GM, Fraser HM 1997 Blockade of the neonatal increase intestosterone by a GnRH antagonist: the free androgen index, reproductivecapacity and postmortem findings in the male marmoset monkey. J Endocrinol154:125–131

37. Pugeat M, Rocle B, Chrousos GP, Dunn JF, Lipsett MB, Nisula BC 1984Plasma testosterone transport in primates. J Steroid Biochem 20:473–478

38. Sharpe RM, Fraser HM, Brougham MF, McKinnell C, Morris KD, Kelnar CJ,Wallace WH, Walker M 2003 Role of the neonatal period of pituitary-testicularactivity in germ cell proliferation and differentiation in the primate testis. HumReprod 18:2110–2117

39. Raivio T, Wikstrom AM, Dunkel L 2007 Treatment of gonadotropin-deficientboys with recombinant human FSH: long-term observation and outcome. EurJ Endocrinol 156:105–111

40. Dobrinski I 2005 Germ cell transplantation and testis tissue xenografting indomestic animals. Anim Reprod Sci 89:137–145

41. Coulter GH, Senger PL, Bailey DR 1988 Relationship of scrotal surface tem-perature measured by infrared thermography to subcutaneous and deep tes-ticular temperature in the ram. J Reprod Fertil 84:417–423

42. Mieusset R, Bujan L 1995 Testicular heating and its possible contributions tomale infertility: a review. Int J Androl 18:169–184

43. Jia Y, Sinha-Hikim AP, Lue YH, Swerdloff RS, Vera Y, Zhang XS, Hu ZY,Li YC, Liu YX, Wang C 2007 Signaling pathways for germ cell death in adultcynomolgus monkeys (Macaca fascicularis) induced by mild testicular hyper-thermia and exogenous testosterone treatment. Biol Reprod 77:83–92

44. Schwalm A, Gauly M, Erhardt G, Bergmann M 2007 Changes in testicularhistology and sperm quality in llamas (Lama glama), following exposure to highambient temperature. Theriogenology 67:1316–1323

45. Shafik A 1992 Contraceptive efficacy of polyester-induced azoospermia innormal men. Contraception 45:439–451

46. Brito LF, Silva AE, Barbosa RT, Kastelic JP 2004 Testicular thermoregulationin Bos indicus, crossbred and Bos taurus bulls: relationship with scrotal, tes-ticular vascular cone and testicular morphology, and effects on semen qualityand sperm production. Theriogenology 61:511–528

47. Jahnukainen K, Ehmcke J, Hergenrother SD, Schlatt S 2007 Effect of coldstorage and cryopreservation of immature non-human primate testicular tis-sue on spermatogonial stem cell potential in xenografts. Hum Reprod 22:1060–1067

48. Hou M, Anderson M, Eksborg S, Soder O, Jahnukainen K 2007 Xenotrans-plantation of testicular tissue into nude mice can be used for detecting leukemiccell contamination. Hum Reprod 22:1899–1906

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.


Documents

Complete Spermatogenesis in Orthotopic But Not in Ectopic ...cnc.cj.uc.pt/BEB/private/pdfs/pdfs0809/ReprBiol/Carlos.pdfComplete Spermatogenesis in Orthotopic But Not in Ectopic Transplants