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Theriogenology 77 (2012) 1008–1013

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Vitrification of non-human primate immature testicular tissueallows maintenance of proliferating spermatogonial cells after

xenografting to recipient mice

J. Poelsa, A. Van Langendonckta, J.P. Dehouxb, J. Donneza, C. Wynsa,*a Department of Gynecology, Medical School, Institute of Experimental and Clinical Research, Université catholique de Louvain, 1200

Brussels, Belgiumb Experimental Surgery Unit, Medical School, Institute of Experimental and Clinical Research, Université catholique de Louvain, 1200

Brussels, Belgium

Received 26 August 2011; received in revised form 7 October 2011; accepted 8 October 2011

Abstract

This study demonstrates preservation of tissue integrity, maintenance of proliferating spermatogonia and Leydig cell func-tionality after vitrification and transplantation of non-human primate immature testicular tissue. The objective was to assess thepotential of vitrification of non-human primate immature testicular tissue (ITT) in an in vivo xenotransplantation model. Testicularissue was obtained from one immature rhesus monkey (Macaca mulatta) aged 4 years. Collection and vitrification of testicularissue, followed by short-term xenografting (3 wks) to nude mice were performed to evaluate and compare vitrified/warmed andresh tissue. Fresh ungrafted tissue was used for control purposes. Cell density and seminiferous tubule (ST) integrity weressessed by light microscopy. Presence of spermatogonia (SG) (MAGE-A4), proliferation (Ki-67) and Leydig cell (LC)unctionality (3�-hydroxysteroid dehydrogenase; 3�-HSD) were evaluated by immunohistochemistry (IHC). Qualitative analysisevealed preservation of the histologic characteristics of SG and Sertoli cells (SCs), as well as cell-cell cohesion and cell adhesiono the basement membrane, in both vitrified and fresh grafted tissues. Survival of SG able to proliferate and functional LCs wasonfirmed by IHC in fresh and vitrified grafts. In conclusion, vitrification appears to be a promising approach, representing anlternative strategy to slow-freezing in the emerging field of ITT cryopreservation and cryobanking.

2012 Elsevier Inc. All rights reserved.

Keywords: Non-human primate; Immature testicular tissue; Cryopreservation; Vitrification; Fertility preservation; Xenografting

www.theriojournal.com

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1. Introduction

Thanks to continuing progress in childhood cancertreatment, survival rates in children are on the increase.It is estimated that �80% survive their disease [1–3].Unfortunately, enhanced effectiveness of these treat-

* Corresponding author Tel: �32 2 764 95 01; fax: �32 2 7649 18.

nE-mail address: christine.wyns@uclouvain.be (C. Wyns).

093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.oi:10.1016/j.theriogenology.2011.10.015

ments may be accompanied by an increase in theirtoxicity, especially with respect to fertility. Becauseof their young age, sperm cryopreservation is not an op-tion in these patients. Cryopreservation of their immaturetesticular tissue (ITT) with a view to restoring their fertil-ity after autotransplantation therefore emerged as a prom-ising and ethically acceptable approach [4].

Indeed, in vivo survival, proliferation and initiationf differentiation of frozen-thawed human spermatogo-

ia (SG) were demonstrated after orthotopic xenograft-

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1009J. Poels et al. / Theriogenology 77 (2012) 1008–1013

ing in a mouse model [5]. However, rapid loss of ahighly significant number of SG was observed, withrecovery rates of 14.5% after 3 wks [6] and just 3.7%fter 6 mo of xenografting [5]. Although hypoxia due tovascular grafting may have been the cause of this loss,he freezing technique might also have played a part.

While controlled slow-freezing is the most widelymplemented technique, vitrification is an innovativetrategy preventing ice crystal formation by use of highoncentrations of cryoprotectant and ultrafast coolingelocity, which could minimize cellular damage. Prom-sing results were recently obtained with ITT in micefter 3 d of organotypic culture [7] and in piglets after

xenotransplantation [8,9].In humans, vitrification of ITT has never been eval-

uated in vivo. However, in vitro evaluation showedsurvival of SG, with maintenance of proliferative ac-tivity in tissue after vitrification and organotypic culturefor 10 d [10]. There have so far been no reports on thesurvival or functionality of vitrified and transplantedITT in primates.

2. Materials and methods

We performed heterotopic and orthotopic xenograft-ing in a nude mouse model previously established toassess the developmental competence of cryopreservedITT [5]. Fresh tissue grafts (n � 24, 3 mice) andvitrified grafts (n � 24, 3 mice) were compared. Thevitrification protocol recently described by Abrishami,et al [9] was applied, albeit slightly modified.

2.1. Vitrification and xenografting

Briefly, testicular tissue was pretreated with anequilibration solution (5 ml) consisting of 7.5% (v/v)ethylene glycol (EG, Sigma Aldrich, Bornem, Bel-gium), 7.5% (v/v) dimethylsulfoxide (DMSO, SigmaAldrich, Bornem, Belgium), 0.25 M sucrose and 25

g/ml human serum albumin (HSA) in Leibovitz L-15L-15, Sigma Aldrich, Bornem, Belgium) for 10 min-tes at 4 °C. It was then transferred to a vitrificationolution (5 ml) consisting of 15% e.g., 15% DMSO,.5 M sucrose and 25 mg/ml HSA in L-15 for 5inutes at 4 °C.The testicular tissue was then placed on a piece of

auze to remove the surrounding vitrification me-ium, transferred to open cryostraws (CryoBioSys-em, l’Aigle-France, France), and plunged into liquiditrogen (LN2). The straws were inserted into pre-ooled cryotubes (Simport, Canada), sealed and

tored for 24 h in LN2. K

For warming, the cryotubes were removed from theN2 and the straws were quickly immersed in a 35 °Carming solution containing sucrose (1 M) in L-15 with

25 mg/ml HSA. The testicular fragments were thenserially transferred to three baths of warming solutionswith decreasing sucrose concentrations (0.5, 0.25, and0 M) for 5 minutes each.

Grafts of different sizes (scrotal: 4 mm3; back skin:mm3, 9 mm3 and 16 mm3) were implanted in both

roups to test the influence of fragment size on theitrification and grafting process. After 3 wks, the miceere euthanized by an overdose of anesthesia admin-

stered by intraperitoneal injection, and the fresh anditrified grafts were recovered to be directly fixed inaraformaldehyde 4%. This time point (3 wks) washosen to analyze the grafts after the initial period ofschemia resulting from the inevitable delay that occursefore revascularization is established [5]. All experi-ents in this study were approved by the Animal Ethicseview Board of the Catholic University of Louvain.

The grafts were embedded in paraffin and cut into 5�m-thick serial sections for histologic and immunohis-tochemical analysis. Two sections every 50 �m werestained with hematoxylin-eosin (HE) for evaluation bylight microscopy. Digital images were captured with aMirax Midi digital camera (Zeiss, Germany). Seminif-erous tubule (ST) integrity was evaluated. STs wereconsidered intact when good adhesion of cells to thebasement membrane, good cell cohesion, and no scle-rosis were observed. Subsequent sections were used forimmunohistochemistry (IHC).

2.2. Immunochemistry

The presence of SG was evaluated by immunostain-ing with MAGE-A4 mouse antihuman monoclonal an-tibody purified from hybridoma 57B, kindly providedby Giulio Spagnoli, M.D. (University of Basel, Swit-zerland) [11].

Proliferative activity was assessed by detection ofKi-67 mouse antihuman monoclonal antibody (DAKOM7240), directed against the nuclear Ki-67 antigen ofcells not in G0 of the cell cycle.

Immunostaining was performed with three beta-hy-droxysteroid dehydrogenase (3�-HSD) rabbit antihu-

an polyclonal antibody (Santacruz ref sc-28,206) as aarker of functionally active Leydig cells (LCs) [12].For positive and negative controls, mature testicular

issue with normal spermatogenesis, tonsil tissue anduman fetal testicular tissue were used for MAGE-A4,

i-67 and 3�-HSD staining, respectively.

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1010 J. Poels et al. / Theriogenology 77 (2012) 1008–1013

Sections mounted on Superfrost plus slides (Menzel-Glazer, Braunschweig) were deparaffinized and rehy-drated. Endogenous peroxidase activity was blockedby incubating the sections with 0.3% H2O2 (for

AGE-A4 and Ki-67) or 3% H2O2 (for 3�-HSD) for

Fig. 1. Histologic appeareance of fresh (A, C, E, G and I) and vitrifiedmonkey grafted for 3 wks to nude mice: hematoxylin-eosin (HE)-stai(C: back skin 9 mm3, D: back skin 16 mm3), Ki-67 (E: back skin 9 m9 mm3). Double immunostaining with MAGE-A4 and Ki-67 (I andspermatogonia (pink coloration) and red arrows show non-proliferatinserved as positive and negative controls for MAGE A4, E’ and E’’ (fand G’ and G’’ (human fetal testicular tissue) served as positive an0 �m.

30 minutes at room temperature (RT).

After washing under deionized water for 5 minutes,sections were placed in citrate buffer for 75 minutes at98°C (for MAGE-A4 and Ki-67), followed by washingin tris-buffered saline (TBS) 0.05 M and 20% TritonX-100 (Sigma Aldrich, Bornem, Belgium), before in-

F, H and J) immature testicular tissue (ITT) from a 4 year old rhesusand B: scrotal site); immunohistochemical staining with MAGE-A4back skin 16 mm3) and 3�-HSD (G: back skin 9 mm3, H: back skin

skin 9 mm3); black arrows show proliferating (brown coloration)atogonia (pink coloration). C’And C’’ (fresh human testicular tissue)man tonsil tissue) served as positive and negative controls for Ki67,tive controls for 3�-HSD. Original magnification � 400; scale bar

(B, D,ning (Am3, F:J: back

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cubation with 10% normal goat serum (NGS) and 1%

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1011J. Poels et al. / Theriogenology 77 (2012) 1008–1013

bovine serum albumin (BSA) to block non-specificbinding sites for 30 minutes (for MAGE-A4 and Ki-67)or 45 minutes (for 3�-HSD) at RT. Primary antibodydiluted to 1/500 for MAGE-A4, 1/150 for Ki-67 and/100 for 3�-HSD) was added to the sections andncubated overnight at 4 °C in a humidified chamber.

The following day, the slides were washed in TBS.05 M and 20% Triton X-100 three times for 2 minutesach and secondary antimouse antibody (EnVision�ystem-labeled polymer horseradish peroxidase (HRP);AKO K4001) was added and incubated for 60 min-tes at RT, followed by washing in TBS 0.05 M and

20% Triton X-100 three times for 2 minutes each.Sections were incubated with diaminobenzidine

(DAKO K3468) as a chromogen for 10 minutes at RTand nuclei were counterstained with HE after washingunder tap water for 3 minutes. Finally, the Superfrostslides were dehydrated and mounted.

For double immunostaining, Ki-67 sections (notconterstained with HE) were washed under acidifiedwater (HCl 0.1 M) for 60 minutes, followed by distilled

ater for 5 minutes and then TBS 0.05 M and 20%Triton X-100 three times for 2 minutes each. Non-specific antibody binding was blocked by incubation of

Fig. 1

samples in 10% NGS and 1% BSA for 30 minutes at a

RT. MAGE-A4 antibody was added to the samples andincubated at 4 °C overnight in a humidified chamber.

The following day, the slides were washed in TBS0.05 M and 20% Triton X-100 three times for 2 minutesach and secondary antimouse antibody (EnVision�ystem-labeled polymer HRP; DAKO K4001) wasdded and incubated for 60 minutes at RT, followed byashing in TBS 0.05 M and 20% Triton X-100 three

imes for 2 minutes each.Sections were incubated with 3-amino-9-ethylcarba-

ole (AEC; DAKO K3464) as a chromogen for 10inutes at RT and nuclei were counterstained with HE

fter washing under tap water for 3 minutes. Finally,he Superfrost slides were dehydrated and mounted.

. Results

Histologic analysis of fresh and vitrified graftshowed well preserved histologic characteristics of SGnd Sertoli cells (SCs), as well as ST integrity (Fig. 1),ith homogeneous distribution of intact STs after graft-

ng to the scrotum and under the back skin, regardlessf graft size. Cell density was evaluated in 10 roundTs, whose perpendicular diameter ratio was between 1

nued)

nd 1.5. The total number of nuclei (SC and SG) per

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1012 J. Poels et al. / Theriogenology 77 (2012) 1008–1013

round tubule and surface area of round tubules wererecorded. ST cellular density of fresh ungrafted tissuecontrol was 8.35 � 10�3 � 1.55 � 10�3 cells/�m2.Cell density of fresh grafts was 4.04 � 10�3 � 0.34 �10�3 cells/�m2 for orthotopic grafts and 4.14 � 10�3 �0.35 � 10�3 cells/�m2, 4.22 � 10�3 � 0.36 � 10�3

cells/�m2 and 4.19 � 10�3 � 0.23 � 10�3 cells/�m2

for 4 mm3, 9 mm3 and 16 mm3 heterotopic grafts,espectively. For vitrified tissue grafts, cell density was.98 � 10�3 � 0.87 � 10�3 cells/�m2 for orthotopic

grafts and 3.84 � 10�3 � 0.34 � 10�3 cells/�m2, 4.08 �10�3 � 0.72 � 10�3 cells/�m2 and 4.35 � 10�3 �.70 � 10�3 cells/�m2 for 4 mm3, 9 mm3 and 16 mm3

heterotopic grafts, respectively.After vitrification and grafting for 3 wks, mainte-

nance of SG with proliferative activity was evidencedby MAGE-A4 and Ki-67 immunostaining on serialsections (Fig. 1). Preservation of LCs was demonstratedby immunostaining of 3�-HSD in all grafts, regardlessf size (Fig. 1). Proof of their functionality was con-rmed by testosterone impregnation of mice based oneminal vesicle weight, with 46.8 � 4 mg, 46.5 � 1.9g, 133 � 23 mg and 27 � 4 mg in fresh grafted tissue,

vitrified grafted tissue, intact mice and castrated mice,respectively.

4. Discussion

The structural integrity of all vitrified tissues wascomparable to that of fresh tissue, and no histologicdamage was observed by light microscopy. Preserva-tion of SCs with proliferative activity and functionalLCs was achieved after vitrification and avasculartransplantation for 3 wks.

Interestingly, a trend toward a decrease in cell den-sity of fresh and vitrified grafts was noted. Althoughthis observation needs to be further confirmed, it maybe linked to transplantation rather than vitrification.

Appropriate graft size is a major concern for vitri-fication, since tissue damage increases with the size offragments due to longer penetration time, leading tooverexposure of surface cells to cryoprotectant concen-trations and slower cooling rates, related to largeramounts of tissue and greater vapor coats around frag-ments when plunged into LN2 [13].

Homogeneous distribution of ST integrity wasachieved in fragments up to 16 mm3. Since vitrificationof small tissue pieces is time-consuming, establishingmaximum tissue size allowing adequate vitrification

may be important for laboratories.

In conclusion, this pilot study demonstrates preser-vation of tissue integrity and maintenance of prolifer-ating SG and functional LCs after vitrification andtransplantation of non-human primate ITT. These dataare in line with our previous report on the in vitropotential of vitrified ITT in primates [10]. Althoughfurther studies are needed to assess the functionality oftissue with the capacity to complete spermatogenesis,ITT vitrification looks to be promising new strategywith a view to clinical banking.

Acknowledgments

This study was supported by a grant from the FondsNational de la Recherche Scientifique de Belgique(grant Télévie N° 7. 4.572.09.F). The authors are grate-ul to Mira Hryniuk, B.A., for reviewing the Englishanguage of the manuscript.The authors thank the lab-ratory of morphology of the Institute of Experimentalesearch (IREC), in particular Prof. Marie-Christineany, for access to laboratory facilities (premises,orphology laboratory material).

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