Hum. Reprod. Advance Access originally published online on July 28, 2008
Human Reproduction 2008 23(11):2402-2414; doi:10.1093/humrep/den272
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Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue
1 Gynecology Research Unit, Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium 2 Department of Urology, Université Catholique de Louvain 1200, Brussels, Belgium
3 Correspondence address. Tel: +32-2-764-95-01; Fax: +32-2-764-95-07; E-mail: donnez{at}gyne.ucl.ac.be
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Abstract |
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BACKGROUND: Preservation of the male germ line in prepubertal boys undergoing gonadotoxic treatment is a crucial consideration in terms of their future quality of life. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival, proliferation and differentiation capacity of spermatogonia after cryopreservation and long-term transplantation of immature testicular tissue pieces.
METHODS: Single pieces of testicular tissue (2–8 mm3) from prepubertal boys (7–14 years) were cryopreserved, thawed and transplanted into the scrotum of mice for 6 months. Upon removal, histological, immunohistochemical and ultrastructural analyses and testicular sperm extraction (TESE) were used to evaluate the tissue.
RESULTS: Histology showed 55 ± 42% of tubules to be intact. MAGE-A4 immunostaining showed mean spermatogonial recovery to be 3.7 ± 5.5%, with 35% of these cells expressing Ki67, evidencing proliferation in tissue from boys <14 years of age. No apoptosis was found, as demonstrated by the absence of active caspase-3 and TUNEL staining. Numerous premeiotic spermatocytes, a few spermatocytes at the pachytene stage and spermatid-like cells were observed. No immunostaining was observed for lactate dehydrogenase-C, ACE or proacrosin, indicating that the spermatid-like structures observed by histology did not express the meiotic and post-meiotic markers characteristic of normal spermatids. No ultrastructural alterations of the tissue were encountered.
CONCLUSIONS: The present study demonstrates that spermatogonia are able to survive and proliferate after cryopreservation and long-term transplantation. Complete regeneration of normal spermatogenesis was not observed since, beyond the pachytene stage, no adequate characterization of germ cells was obtained. Further studies are thus required to investigate the differentiation potential of cryopreserved germ cells.
Key words: cryopreservation/spermatogonia/testicular tissue/xenografting
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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Preservation of the male germ line in prepubertal boys undergoing gonadotoxic treatments is a crucial consideration in terms of their future quality of life. Indeed, with cure rates of childhood cancers now as high as 80% (Aslam et al., 2000
), it is estimated that, by 2010, 1 in 250 young adults (aged 20–29 years) will be affected. This number is even higher if we take into account certain benign diseases, such as drepanocytosis or thalassemia major, which are cured during childhood with highly gonadotoxic regimens (Vermylen, 2003; Lucarelli and Gaziev, 2007).
Cryopreservation of immature testicular tissue has recently emerged as a promising new approach to long-term tissue storage with a view to preserving fertility in patients undergoing such gonadotoxic therapy (Keros et al., 2007).
Although previous studies in non-human primates show that spermatogonia are highly cryosensitive (Jahnukainen et al., 2007), an orthotopic xenotransplantation model in nude mice recently demonstrated a 14.5% spermatogonial cell survival rate in frozen-thawed immature human cryptorchid testicular tissue transplants after 3 weeks (Wyns et al., 2007). Long-term evaluation is nevertheless required to evaluate their long-term survival as well as their functional capacity in terms of proliferation and differentiation after cryopreservation.
Successful xenografting of cryopreserved testicular tissue has been reported in various non-primate species (Honaramooz et al., 2002; Schlatt et al., 2002; Shinohara et al., 2002) and non-human primates (Orwig and Schlatt, 2005).
However, the differentiation capacity of frozen spermatogonia shows conflicting results, as in xenografts of immature rhesus monkey testicular tissue, a blockade was encountered at the spermatocyte stage (Jahnukainen et al., 2007). No data are available in humans as yet.
Therefore, to further characterize the potential of the remaining spermatogonial population after freezing and xenografting, we examined its functional characteristics at 6 months after transplantation using our previously described orthotopic xenografting model (Wyns et al., 2007).
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Materials and Methods |
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Study design
This study was designed to evaluate the capacity of frozen-thawed and grafted spermatogonial cells to survive, proliferate and differentiate. Morphological criteria were used to identify the type of germ cell. The biological characteristics of spermatogonial cells were analyzed after freezing and thawing and orthotopic xenografting for 6 months.
Morphological evaluation included histological and immunohistochemical analysis by light microscopy (LM) and ultrastructural assessment.
Biological determination of spermatogonial cells was based on their long-term survival ability, proliferation capacity and their ability to yield differentiating germ cells. This was investigated by histological examinations of whole tissue mounts or cell suspensions after testicular sperm extraction (TESE) procedures, by immunohistological analysis using LM and by ultrastructural studies in our nude mouse xenografting model.
Donor testicular tissue
Immature testicular tissue was obtained from five boys aged between 7 and 14 (7, 12, 12, 14 and 14) years. All of them were undergoing testicular biopsy as a fertility preservation measure. This option is proposed to prepubertal patients and their parents before initiating any therapy carrying a high risk of permanent infertility, such as high-dose chemotherapy prior to stem cell transplantation (Thomson et al., 2002). Testicular volume was below 4 cc in two boys (7 and 12 years of age), and 6 cc, 
8 cc and 10 cc in the others, aged 12, 14 and 14 years, respectively. Tanner stages were I or II, except for the 14-year-old boys, who were both Tanner Stage III. Only one testis was biopsied in each child and <5% of the testicular volume was removed. No complications occurred during or after tissue retrieval.
In all cases, the majority of the biopsied testicular tissue was cut into pieces (2–4 mm3), cryopreserved and stored in the cryobank for later clinical use. A small part of the available tissue (approximately one-fourth) was used for this study after obtaining informed consent from the parents, and child, where applicable.
The fragments, measuring 2–8 mm3, were immediately transferred to Falcon tubes containing Hank's balanced saline solution (HBSS) at 4°C and placed on ice.
Each biopsy of immature tissue was divided into two pieces in a Petri dish containing HBSS and placed on ice. The smallest piece was immediately fixed in 1 ml buffered formol for reference histology and immunohistochemistry (n = 5). The remaining piece was cryopreserved within 10 min of recovery. There were four pieces of cryopreserved tissue available for grafting from each of the 14-year-old donors, three pieces from the 7-year-old donor, and just one piece each from the 12-year-old donors.
All experiments in this study were approved by the Ethical Review Board of the Catholic University of Louvain.
Freezing media and protocol
Testicular tissue samples were frozen as previously described (Wyns et al., 2007). Dimethylsulfoxide (DMSO) 0.7 mol/l was supplemented with sucrose 0.1 mol/l and human serum albumin (HSA) 10 mg/ml as cryoprotectants. Tissue pieces were placed in 1 ml freezing media at 4°C in a 2 ml cryovial. Using a programmable freezer (Minicool 40 PC Air Liquide, Marne-la-Vallee, France), the vials were maintained at 0°C for 9 min, cooled at a rate of 0.5°C/min to –8°C and then held for 5 min before seeding manually at –8°C. After holding for a further 15 min at –8°C, a cooling rate of 0.5°C/min was used from –8°C to –40°C before final dehydration for 10 min at –40°C. After cooling at 7°C/min to –80°C, the vials were transferred to liquid nitrogen (–196°C).
Thawing
After retrieval from storage (2–12 months), the cryopreserved tissue was kept for 2 min at room temperature, thawed in a water bath at 37°C for 2 min, and then washed three times in a reversed sucrose concentration gradient solution (0.1, 0.05 and 0 M sucrose) for 5 min per bath, using HBSS medium on ice.
Recipient animals and procedures for xenografting
Xenografting was performed as previously described (Wyns et al., 2007). Briefly, 13 6-week-old nude mice (NMRI nu/nu, Janvier Laboratories, Le Genest-St-Isle, France) with a deficient T lymphocyte system were used. The mice were first castrated and, during the same surgery, one piece of donor tissue (1–6 mm3) was grafted into the scrotum without suture. The whole procedure was performed through a median scrotal incision and the grafts were placed in the peritoneal bursa, after open incision of the cremaster, close to the castration wound. The peritoneal bursa was then closed with 7/0 Prolene (Johnson and Johnson Intl, Belgium).
Analysis of recipient mice
Recipient mice were euthanized 6 months after grafting by cervical dislocation and all the grafts were recovered.
The grafts were dissected and fixed in Bouin's solution for histology and immunohistochemistry (n = 7; at least one piece of tissue from each donor), in glutaraldehyde for transmission electron microscopy (TEM) (n = 3; one piece of tissue from three different donors), or placed in sperm preparation medium (SPM, MediCult, Denmark) before mechanical dissociation for TESE (n = 3; one piece of tissue from three different donors).
Histological evaluation
After fixation, fresh control tissue samples and grafts were routinely embedded in paraffin and cut into 5 µm-thick serial sections. Consecutive serial sections were used for histological and immunohistochemical evaluation.
One section every 50 µm was stained with hematoxylin–eosin for evaluation by LM, and digital images were captured with a digital camera (Leica DFC 320, Zeiss, Germany). Subsequent sections were used for immunohistochemistry, as described below.
Seminiferous tubule integrity was recorded. Tubules were considered intact when no sclerosis, good adhesion of cells to the basement membrane and good cell cohesion were observed. When seminiferous tubules were found to be intact, they were classified according to the most advanced type of germ cell found on hematoxylin–eosin sections, according to Clermont (1963).
All data were blindly examined and compared with the reference histology (before freezing and grafting).
Immunohistochemical evaluation
Consecutive 5 µm serial sections were used for immunohistological evaluation, respectively, for MAGE-A4 (mouse anti-human monoclonal antibody purified from hybridoma 57B, kindly provided by Giulio Spagnoli MD, University of Basel, Switzerland) as a spermatogonial marker (Yakirevich et al., 2003); Ki67 (mouse anti-human monoclonal antibody clone Mb1; DAKO ref M7240; directed against the nuclear Ki67 antigen of cells not in G0 of the cell cycle) as a proliferation marker; caspase-3 (rabbit anti-human polyclonal antibody directed against a peptide from the p18 fragment of human active caspase-3; Promega G7481) as an apoptotic marker; LDH-C (lactate dehydrogenase-C rabbit anti-human monoclonal antibody; Abcam: ab52747) as a meiotic marker (Patrizio et al., 2000); ACE (mouse monoclonal CD143 anti-human antibody; Serotec ref: MCA2056) as a post-meiotic marker (Pauls et al., 2003); 4D4 anti-proacrosin monoclonal antibody that labels a sperm-specific protein in meiotic and post-meiotic human germ cells (kindly provided by Denise Escalier, Reproductive Biology and Development Laboratory, France) (Escalier et al., 1992); and 3β-HSD (3 beta-hydroxysteroid dehydrogenase rabbit anti-human polyclonal antibody; SantaCruz ref sc-28206) as a marker of functionally active Leydig cells (Dupont et al., 1991; Gaskell et al., 2004).
Cross-sections were deparaffinized and rehydrated. Endogenous peroxidase activity was then blocked by incubating the samples for 30 min in 0.3% H2O2 (for MAGE-A4, LDH-C, ACE, Ki67 and caspase-3); in 3% H2O2 (for 3β-HSD) and in 7.5% H2O2 (for 4D4) at room temperature. Sections were placed in citrate buffer at 98°C for 75 min (for MAGE-A4, ACE, Ki67 and caspase-3). Non-specific antibody binding was subsequently blocked by incubation of samples in 10% non-immune goat serum and 1% bovine serum albumin (BSA) for 30 min at room temperature, except for 4D4, where 5% BSA was applied for 20 min at room temperature. The primary antibody (diluted to 1:500 for MAGE-A4, 1:100 for Ki67, 1:200 for caspase-3, 1:10 000 for LDH-C, 1:20 for ACE, 1:2 for proacrosin and 1:100 for 3β-HSD) was added to the samples and incubated at 4°C overnight (for MAGE-A4, Ki67, ACE and 3β-HSD), at 4°C for 1 h (for LDH-C) or at room temperature for 1 h (for caspase-3) or 45 min (for 4D4).
Secondary anti-mouse (for MAGE-A4, Ki67 and ACE) or anti-rabbit (for caspase-3, LDH-C and 3β-HSD) antibody (EnVision+System-Labeled Polymer-HRP; DAKO K4001, K4003) was added and incubated at room temperature for 1 h (MAGE-A4, Ki67, caspase-3, ACE and 3β-HSD) or 30 min (LDH-C). A three-step immunoperoxidase technique using a secondary rabbit anti-mouse biotinylated antibody (DAKO E0464) diluted to 1:100 (incubated at room temperature for 30 min) and an avidin-HRP system (DAKO P0397) was applied for the 4D4 antibody. Diaminobenzidine (DAB) (DAKO K 3468) was used as a chromogen for HRP-conjugated secondary antibodies. Samples were incubated for 10 min (MAGE-A4, Ki67, caspase-3, ACE, 4D4 and 3β-HSD) or 2 min (LDH-C) at room temperature.
Nuclei were counterstained with hematoxylin.
Assessment of spermatogonial cell number and proliferation
The number of spermatogonia was evaluated after MAGE-A4 immunodetection.
As only one serial section every 50 µm was stained for MAGE-A4; double counting of the same spermatogonia was avoided. The mean number of positive cells per intact tubule and the mean percentage of positive tubules were recorded and compared with fresh tissue. Recovery of spermatogonia after freeze-thawing and grafting for 6 months was thus calculated. The proportion of MAGE-A4-positive cells showing proliferative activity by Ki67 immunostaining was recorded.
Assessment of germ cell differentiation
In addition to the morphological aspects of the tubules observed by histology, a more accurate evaluation of germ cell differentiation was performed by the detection of immunohistological markers specific to the germ cell type: MAGE-A4 as a spermatogonial marker, LDH-C as a meiotic marker, proacrosin as a meiotic and post-meiotic marker and ACE as a post-meiotic marker.
The most advanced type of germ cell found was recorded and the number of positive tubules for each given germ cell marker was calculated and expressed as a percentage of the total number of intact tubules observed.
Assessment of germ cell apoptosis
In addition to the immunohistochemical detection of active caspase-3, apoptosis was analyzed by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method to detect DNA fragmentation. Sections were dewaxed, rehydrated and washed in deionized water. The slides were then pretreated with 20 µg/ml of proteinase K solution (Roche Applied Science 745723) in 10 mM Tris–HCl (pH 7.5) for 30 min at 37°C in a humidified chamber.
DNA strand breaks occurring during the apoptotic process were detected using the In Situ Cell Death Detection Kit, TMR Red (Roche Applied Science 2156792), a TUNEL assay. After washing with PBS, slides were incubated with a TUNEL reaction mixture: 50 µl enzyme solution (terminal deoxynucleotidyl transferase) and 450 µl label solution (nucleotide mixture in reaction buffer) for 60 min at 37°C in a humidified chamber protected from light, followed by rinsing in PBS. Slides were covered with Vectashield Mounting Medium with 4',6-diamino-2-phenylindole (DAPI) (Vector Laboratories). This special formulation is intended to preserve fluorescence during prolonged storage and, at the same time, counterstain DNA by means of DAPI. Human tonsil tissue was used as a positive control, and negative control sections were incubated with label solution without enzyme solution. Slides were coverslipped and sealed around the perimeter with nail polish, stored at 4°C and protected from light until examination.
TUNEL-stained and DAPI-counterstained slides were examined under an inverted fluorescence microscope (Leica; Van Hopplynus Instruments). Red fluorescence was visualized in TUNEL-positive cells using an excitation wavelength in the 520–560 nm range and by observing the emitted light at a wavelength between 570 and 620 nm. DAPI reached excitation at 360 nm, and emitted at 460 nm when bound to DNA, producing blue fluorescence in all nuclei.
TESE procedure
Testicular tissue was placed in a Petri dish containing HEPES-buffered Earle's medium supplemented with HSA (SPM, MediCult, Jyllinge, Denmark). Wet preparations of testicular tissue were shredded roughly using two microscopic glass slides in a Petri dish (model 353003; Becton-Dickinson, Le Pont De Claix, France) on the warmed stage of a stereomicroscope at x40 magnification (Verheyen et al., 1995). During this procedure, the seminiferous tubules were unravelled and broken. The tissue was then further minced with two fine forceps (Lawton, Tuttlingen, Germany) in a Petri dish, until tissue fragments of <1 mm3 in size or free tubule pieces of a few millimeters in length were obtained. The shredded tissue was then placed in a Falcon tube (model 352058; Becton-Dickinson; Meylan Cedex; France) containing 3 ml of SPM. After washing and concentration of the suspension, the preparation was checked under an inverted microscope (x400 magnification) for the presence of spermatozoa, according to Clermont (1963).
Ultrastructural assessment
Electron microscopy was used to evaluate tissue and cell integrity. Samples used for ultrastructural analysis were obtained from the two 14-year-old boys and the 7-year-old boy. For corresponding fresh and frozen-grafted samples, four randomly taken consecutive sections of 500 Å, each containing at least 10 seminiferous tubular sections were analyzed. After glutaraldehyde fixation at 4°C, fresh and grafted tissue was rinsed in cacodylate buffer, post-fixed with 1% osmium tetroxide (Agar Scientific, Essex, UK) and rinsed again in cacodylate buffer (pH 7.4; 0.1 M). The samples were dehydrated through ascending series of ethanol, immersed in propylene oxide overnight for solvent substitution and embedded in Epon 812 (Fluka 45345). Specimens were then sectioned with a Reichert-Jung Ultracut E ultramicrotome. Semithin sections (1 µm thick) were stained with toluidine blue (Sigma, St Louis, MO, USA) and examined by LM with a Zeiss Axioskop microscope (Zeiss, Munich, Germany). Ultrathin sections (500 Å) were cut with a diamond knife, mounted on copper grids and contrasted with saturated uranyl acetate followed by lead citrate. They were examined and photographed using Zeiss EM109 and Zeiss EM10 electron microscopes at 80 kV.
Statistical analysis
Analyses were performed using the SPSS 11.5 program. All data are presented as mean ± SD.
Statistical significance of the difference between variables was analyzed using the Mann–Whitney U-test. P-value of <0.05 was considered statistically significant.
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Histological evaluation of seminiferous tubule integrity and spermatogenesis
The histological appearance of donor testicular tissue on hematoxylin–eosin sections was characterized by complete immaturity in three patients (aged 7, 12 and 12 years, respectively) and immature seminiferous tubules with focal spermatogenesis in two patients (both 14 years of age).
The immature tissue was composed of seminiferous cords with Sertoli cells, spermatogonial cells and interstitial tissue containing Leydig cells (Fig. 1).
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In fresh and frozen-grafted tissue, an average of 105.0 ± 91.0 and 1704.4 ± 1453.5 seminiferous tubules, respectively, were examined to assess tubular integrity at a final magnification of x200. After freeze-thawing and grafting for 6 months, 55 ± 42% of tubules were considered intact, compared with 100% in fresh tissue (P = 0.009).
The most extensive tubular damage (78.2% of tubules) was observed in the two oldest boys, in whom focal spermatogenesis was seen at the time of biopsy (Fig. 1E and F).
The evaluation of germ cell differentiation is shown in Fig. 1. An average of 188 ± 195 (range: 56–517) intact or 241.6 ± 160.8 total seminiferous tubules was examined in frozen-grafted tissue to assess germ cell differentiation at a final magnification of x400. Spermatogonia in close contact with the basement membrane, characterized by their typical white cytoplasmic halo around the nucleus, were easily identified in all the grafts. Some of these cells were in mitosis, as shown in Fig. 1B. In all donors, premeiotic spermatocytes were observed, whereas spermatocytes in prophase of the first meiotic division, recognized by their characteristic large nucleus and very thick, short chromatin filaments (Clermont, 1972), were only occasionally seen in a 12-year-old donor (Fig. 1D).
In 50% (49.7 ± 2.5) of intact tubules or 36% (35.8 ± 19.8) of total tubules from all donors (including those which were completely immature at the time of freezing), germ cells with the morphological aspect of spermatids were found on hematoxylin–eosin-stained sections after freeze-thawing and 6 months' grafting. Germ cells considered to be spermatids were localized in the adluminal compartment of seminiferous tubules (Fig. 1D). As described by Clermont, they were small in size, with condensed chromatin in their nucleus and looked pear-shaped or paddle-shaped, with a thinner anterior half appearing less stained than the caudal half. Interestingly, spermatids were slightly smaller in size in frozen-grafted tissue than in control tissue, respectively, 3.8 ± 1.4 and 4.6 ± 1.2 µm (P = 0.045).
Immunohistochemical evaluation of spermatogonial recovery, spermatogonial proliferation and germ cell differentiation
In fresh and frozen-grafted tissue, an average of 94.4 ± 82.5 (range: 22–207) and 335.7 ± 305.9 (range: 176–780) intact seminiferous tubules, respectively, were examined to assess spermatogonial recovery (Fig. 2B and C). As indicated by the presence of MAGE-A4-positive cells in frozen-grafted tissue, spermatogonial cells were present after cryopreservation and 6 months' xenografting, confirming their long-term survival ability. Our data nevertheless reveal a loss of spermatogonia after freezing and grafting. The number of tubules positive for MAGE-A4 was significantly decreased in frozen-grafted tissue (3.1 ± 2.6% of intact tubules) compared with fresh tissue (53.8 ± 22.8% of intact tubules; P = 0.004).
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In addition, the number of spermatogonia decreased from 3.9 ± 3.2 per tubule in fresh tissue to 0.09 ± 0.1 per tubule in frozen-grafted tissue (P = 0.004). Spermatogonial recovery (number of spermatogonia per tubule in frozen-grafted tissue/number of spermatogonia per tubule in fresh tissue x 100) was calculated for each donor (mean = 3.7 ± 5.5%) (Table I).
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An average of 66 ± 65.8 (range: 9–176) intact seminiferous tubules were examined in frozen-grafted tissue to assess spermatogonial cell proliferation at a final magnification of x400.
Ki67 immunostaining, directed against the nuclear Ki67 antigen of cells not in G0 of the cell cycle, was observed in 34.64 ± 11.5% of MAGE-A4-positive cells from donors under 14 years of age (Fig. 3), demonstrating that spermatogonia are able to proliferate after freezing and long-term grafting in this age group. Interestingly, no proliferating spermatogonia were observed in the two 14-year-old boys, but the number of remaining spermatogonia and intact seminiferous tubules examined was low (9 and 22, respectively). Percentages of Ki67-labeled spermatogonia are given for each donor in Table II.
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The immunohistological evaluation of germ cell differentiation before and after cryopreservation and grafting is shown in Fig. 2D–L. LDH-C protein was used as a marker for meiotic germ cells, proacrosin, as a marker for meiotic and post-meiotic germ cells, and ACE protein, as a marker for post-meiotic germ cells. In fresh and frozen-grafted tissue, an average of 111 ± 98 and 143.3 ± 171.3 (range: 80–500); 113.8 ± 100.8 and 79.6 ± 60.7 (range: 80–220); and 111.6 ± 97.6 and 139.7 ± 172.2 (range: 71–499) intact seminiferous tubules were examined for LDH-C, proacrosin and ACE, respectively, to assess germ cell differentiation.
Besides the average numbers of examined seminiferous tubules, the actual numbers for each donor are given in Table III.
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In fresh tissue, LDH-C-positive tubules were observed in, respectively, 5.5% and 0.4% of total tubules in the two 14-year-old patients, whereas ACE-positive tubules were recorded in 0.4% of total tubules in only one of the 14-year-old donors.
In contrast, no positive tubules were found for LDH-C, ACE or proacrosin after cryopreservation and grafting, indicating that the spermatid-like structures observed by histology did not express the meiotic and post-meiotic markers characteristic of normal spermatids (Fig. 2E, F, H, I, K and L).
Assessment of germ cell apoptosis
An average of 70.4 ± 69.6 intact or 86.6 ± 56.0 total seminiferous tubules were examined in frozen-grafted tissue to assess germ cell apoptosis by immunostaining for active caspase-3. As shown in Fig. 4A, no apoptotic germ cells were observed.
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An average of 73 ± 71.4 intact or 91.8 ± 55.1 total seminiferous tubules were examined in frozen-grafted tissue to assess germ cell apoptosis by TUNEL. No DNA fragmentation was observed (Fig. 4B).
Assessment of the interstitial compartment
Identification of interstitial Leydig cells after 6 months' orthotopic xenografting of cryopreserved testicular tissue by 3β-HSD immunostaining, used as a marker of functionally active Leydig cells (Dupont et al., 1991; Gaskell et al., 2004), is shown in Fig. 5. No quantitative evaluation of the number of positive cells was performed.
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Ultrastructural evaluation
In the three grafts analyzed by TEM, qualitative analysis showed a well-preserved ultrastructure of intact seminiferous tubules with regard to Sertoli cell and spermatogonial cell adhesion to the basement membrane, as well as to cell-to-cell contacts after freeze-thawing and grafting (Fig. 6).
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Spermatogonial cells were characterized by good integrity of the nucleus in terms of chromatin dispersion, nucleolus and nuclear membrane in both fresh control and frozen-grafted tissue. The freeze-thawing/grafting procedure did not cause damage to cell membranes or the integrity of the cytoplasm or its components, including mitochondria and endoplasmic reticulum. Normal mitosis was even observed in some spermatogonial cells (Fig. 6D).
Primary spermatocytes were observed. They were considered to be at the preleptotene stage, as they presented with some features common to spermatogonia, but were displaced away from the tubular wall toward the lumen and separated from neighboring cells by expansions of Sertoli cells. They showed a spherical nucleus with a centrally located nucleolus and pale-stained and granulated chromatin. In their cytoplasm, mitochondria were often observed together in pairs separated by granular intermitochondrial material (Fig. 6E).
Sertoli cells displayed normal-looking mitochondria, lipid droplets, dense bodies, Golgi apparatus and structurally intact cell-to-cell junctional complexes (Fig. 6B).
The stroma exhibited a normal morphology of the layers of the lamina propria of the seminiferous tubules and undamaged Leydig cells. Their ultrastructural characteristics, as described by de Kretser and Kerr (1994), showed an inner mantle of peripheral heterochromatin associated with the nuclear envelope, at times forming small dense clumps of electron-dense material at irregularly spaced intervals along the inner aspect of the nuclear membrane. Their cytoplasm contains variable amounts of smooth endoplasmic reticulum and Golgi membranes, correlated with steroid synthesis (Goldblatt and Gunning, 1985). Variable amounts of intracellular lipid inclusions were also seen before and after freeze-thawing and grafting. After transplantation, we observed an increase in smooth endoplasmic reticulum involved in testosterone biosynthesis (Tamaoki, 1973). Mitochondria also appeared normal (Fig. 6F and G).
TESE results
In order to improve detection of germ cell differentiation into spermatozoa, a TESE procedure was performed in three different donors, in whom enough tissue was available. Examination of the microdroplets was characterized by the presence of Sertoli cells and round germinal cells at a final magnification of x400. No spermatozoa with a normal morphology were found after freeze-thawing and grafting for 6 months. However, in one of the 14-year-old patients, we found three spermatozoon-like cells after the 6 month grafting period (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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As prepubertal boys do not yet produce spermatozoa, they cannot benefit from cryopreservation of ejaculated sperm, a procedure routinely used for fertility preservation in adults undergoing gonadotoxic therapies.
Cryopreservation of immature human testicular tissue has recently emerged as a promising new technique for future fertility restoration, as it allows survival and proliferation of frozen spermatogonia (Kvist et al., 2006; Keros et al., 2007; Wyns et al., 2007).
However, since normal and continuous spermatogenesis throughout life in males depends on the strict control of spermatogonial self-renewal, and because the concentration of stem cells in this cell population is extremely low (Tegelenbosch and de Rooij, 1993), it was important to demonstrate survival of at least some spermatogonial stem cells (SGSCs) after cryopreservation and grafting. The morphological characteristics of spermatogonia have already been well described in humans (Clermont, 1966; Meistrich and van Beek, 1993; de Rooij and Russell, 2000), but no unique phenotypic marker has been identified to distinguish SGSCs in spermatogonia.
Long-term survival in a host environment has been reported to be a characteristic of stem cells in studies on hematopoietic as well as germ stem cells (Harrisson, 1980; Lord, 1997; Weissmann, 2000; Nagano et al., 2002). Definitive identification of human SGSCs would therefore require demonstration of the capacity for self-renewal and the ability for complete regeneration of spermatogenesis by means of bioassays. Xenografting, used as a bioassay to demonstrate stem cell activity in terms of colonization capacity of stem cell niches, is not optimal, since the niche of the stem cell is from the donor and not the host.
However, xenografting appears to be a useful approach to study spermatogonial cell survival and differentiation ability after cryopreservation, as previously demonstrated in non-human primates (Jahnukainen et al., 2007).
After xenografting of cryopreserved immature testicular tissue pieces, we observed a long-term spermatogonial survival rate of 3.7 ± 5.5%, with increasing loss over time, if we take into account the recovery rate of 14.5% observed in our grafts at 3 weeks (Wyns et al., 2007). A decrease in remaining spermatogonia numbers, intensifying over time, was also reported when non-frozen whole fragments of adult human testicular tissue were grafted to nude mice (Geens et al., 2006). The maturity of the tissue was proposed as a reason for this cell loss as, in rodent testicular tissue xenografts, the success of grafts appears to be linked to the pubertal state of the donor (Schlatt et al., 2002). Our data appear to corroborate these observations, since we observed a very low recovery rate in our two 14-year-old patients, where focal spermatogenesis was already present in fresh tissue. This raises the question of just how good the survival rate is in tissue from boys of this age.
We analyzed the proliferative and differentiation capacity of our spermatogonial population. Ki67-positive staining, detected in 35% of spermatogonial nuclei from boys <14 years of age, suggests that they maintained the ability to continuously proliferate after freezing and 6 months’ grafting. This was further substantiated by the observation of mitotic spermatogonia in grafts by histology and electron microscopy, evidencing their capacity to produce daughter cells. Although it is highly likely that some of these mitotic cells were premeiotic differentiating cells, it is possible that some of them were stem cells. Interestingly, no proliferating spermatogonia were observed in the two oldest boys, but this needs to be confirmed, since it may have been due to the low number of intact tubules examined in this age group. Another potential explanation for the lack of proliferation may be related to the regulation of the stem cell niche, as it appears to be age-dependent (Hess et al., 2006). Evidence from the literature suggests that SGSC proliferation and differentiation change as the testes develop from perinatal to pubertal age (Chen et al., 2005; Naughton et al., 2006). Indeed, knockout studies appear to show that GDNF, a Sertoli cell factor, is sufficient to maintain SGSCs in the perinatal period. In contrast, ERM, expressed in Sertoli cells from the onset of puberty, is essential for stem cell maintenance after the first wave of spermatogenesis in pubertal and adult testes. This differential regulation might therefore account for the observed differences in spermatogonial proliferation in the two patients in whom small spermatogenic foci were already present at the time of grafting. Produced daughter cells of the SGSCs should, in turn, be able to differentiate into spermatozoa when they are in an appropriate environment, as previously suggested (Ogawa et al., 2000; Shinohara et al., 2000).
As experiments involving human subjects are limited by ethical constraints, xenogenic transplantation, using experimental animal models, has been used to provide an adequate environment to study the different stages of germ cell differentiation. Attempts have been made to transplant human germ cell suspensions into the testes of immunodeficient mice, but unfortunately without success in the case of fresh cell suspensions (Reiss et al., 2000), and with limited success in the case of cryopreserved cells (Nagano et al., 2002).
Transplantation of whole tissue fragments, where human stem cell niches and supporting cells are preserved, should therefore provide a more satisfactory environment to support stem cell differentiation than the model of isolated cell transplantation, which requires human cells to colonize mouse niches and could itself be responsible for the lack of cell differentiation observed by Nagano et al. (2002).
In non-human primates, contradictory results have been obtained on the differentiation capacity of spermatogonia in transplanted tissue. Indeed, after xenografting of immature testicular tissue pieces, maturation arrest was observed at the spermatocyte stage in rhesus monkeys (Jahnukainen et al., 2007) and at the spermatogonial level in marmosets (Schlatt et al., 2002; Wistuba et al., 2004), whereas complete sperm differentiation was seen in macaques, albeit in only 4% of seminiferous tubules (Honaramooz et al., 2004). Species-specific characteristics of the seminiferous tubules were suggested as the cause of this discrepancy between results (Luetjens et al., 2005).
In humans, xenografting of testicular tissue pieces has so far met with limited success, and no differentiation capacity of spermatogonia has been demonstrated (Geens et al., 2006; Schlatt et al., 2006). Apart from the species-specific characteristics of the seminiferous tubules, the actual source of the grafted tissue, i.e. adult subjects with spermatogenic activity (Geens et al., 2006; Schlatt et al., 2006) and adult azoospermic or hormone-treated transsexual patients (Schlatt et al., 2006), may also have been to blame.
We used normal immature human testicular tissue from untreated patients (at the time of biopsy), in whom there was no evidence of any risk of future infertility and in whom we could expect spermatogonial differentiation. Histological evaluation of long-term grafts revealed well preserved spermatogonia and numerous primary spermatocytes, some of them entering meiosis, and a spermatid-like state. Electron microscopy showed most of the observed primary spermatocytes to be preleptotene spermatocytes. Unfortunately, in our study, immunohistochemical and electron microscopy observations failed to confirm the presence of spermatids, suggesting a blockade in the differentiation process, regardless of the source of the testicular tissue.
In order to improve the detection and observation of differentiated germ cells in grafts, we also performed a TESE procedure. No normal spermatozoa were retrieved. However, in a graft from one of the 14-year-old patients, we did observe three germ cells resembling spermatozoa, albeit with an abnormal shape. It is highly unlikely that these spermatozoa were residual from spermatogenic foci present in fresh tissue and still recognizable as sperm-shaped cells after 6 months, as they would probably not have survived after such a long time period, and would possibly have been phagocytosed by Sertoli cells, but this cannot be totally excluded.
Since spermatid- and spermatozoon-like cells were observed in our grafts, the possibility of abnormal differentiation of germ cells in a cellular type morphologically and functionally different from normally differentiated germ cells should be considered. The absence of three distinct differentiation markers that should be present in germ cells at or beyond the mid-pachytene stage, namely ACE, LDH-C and proacrosin, clearly proves that no normally differentiated cells were formed in the grafts.
It may be postulated that a blockade in the differentiation process (in the case of the majority of germ cells) or abnormal differentiation (observed in a few germ cells) could be the result of modifications in paracrine interactions due to different tissue architecture between donor and host or a different environment in terms of hormone requirements.
A number of hypotheses may be suggested to explain our observations. First of all, defective biosynthesis of testosterone could be to blame, as it appears to be important for germ cell survival and differentiation (Erkkila et al., 1997). However, we do not believe that altered steroid secretion by grafts could have been responsible for maturation arrest or impairment, based on our qualitative Leydig cell immunohistochemical and ultrastructural data. Indeed, after freeze-thawing and grafting, we observed positive immunostaining for 3β-HSD, an enzyme essential for the biosynthesis of testosterone in human testes, catalyzing the conversion of 
-5-steroids, pregnenolone or dehydroepiandrosterone to -4-steroids, progesterone or androstenedione, respectively (Dupont et al., 1991; Paine A, Contemporary Endocrinology). In addition, we found well-preserved integrity of Leydig cells by electron microscopy, with a well-developed smooth endoplasmic reticulum, which was previously described as the site of conversion of pregnenolone to testosterone in response to LH stimulation (Tamaoki, 1973; Dym and Raj, 1977).
Second, since demonstration of the preservation of the functionality of frozen spermatogonial cells requires use of a transplantation model (Frederickx et al., 2004), we cannot rule out, in the light of our study, the possibility that transplantation itself causes differential expression of proteins involved in normal germ cell differentiation. Indeed, in bovine xenotransplantation experiments, testicular grafts showed decreased FSH receptor protein expression compared with age-matched testicular tissue, although no difference was observed in the cellular location of FSH-R (Schmidt et al., 2007).
Finally, altered interactions between the murine hormonal environment and grafted human testicular tissue could also be responsible for the absent or abnormal differentiation observed in our study. Therefore, since species-specific differences in LH and/or FSH could also be involved in inadequate germ cell maturation, it may be appropriate to use human gonadotrophin supplementation in this orthotopic transplantation model to further investigate the potential of cryopreserved immature testicular tissue.
In order to identify which phenomenon/phenomena could be involved in the increasing loss of spermatogonia over time, we studied apoptosis to elucidate the poor spermatogonial recovery observed. Active caspase-3 immunostaining and TUNEL led us to the conclusion that no apoptotic events were ongoing in grafts at 6 months. This suggests that further spermatogonial loss is unlikely and, since one-fifth of MAGE-A4-positive cells showed proliferative activity, that it is possible that stem cells might, if still present, preferentially opt for the self-renewal pathway. This type of stem cell behavior was previously observed in testes in some instances, such as after germ cell depletion by cytotoxic agents or irradiation (Van Keulen and de Rooij, 1975; van Beek et al., 1990). However, the absence of apoptosis in our grafts at 6 months does not exclude spermatogonial depletion due to apoptosis at earlier time points. The mechanisms determining which pathway stem cells choose are the subject of numerous investigations, and further studies are required to clarify the reasons for this spermatogonial loss, as well as establish the actual presence of stem cells, after freezing and long-term grafting.
Regarding the possibility that the freezing procedure could itself account for the spermatogonial loss, we already showed that the applied protocol did not appear to be the cause. Indeed, spermatogonial cell number was not found to be different between fresh and frozen-thawed cryptorchid human testicular tissue in an earlier study (P = 0.267) (Wyns et al., 2007). Nevertheless, potential optimization of the cryopreservation method warrants consideration since it was previously suggested, in non-human primates, that higher concentrations of DMSO (1.4 M) could improve the differentiation capacity of testicular stem cells (Jahnukainen et al., 2007). When our cryopreservation protocol was set up, we chose to add sucrose (a membrane-non-permeating cryoprotectant) since it was reported to improve the cryopreservation outcome in different cell populations and embryos (Van den Abbeel et al., 1994), as well as in SGSCs (Izadyar et al., 2002). This, therefore, allowed us to decrease the concentration of DMSO, which has been shown to induce severe toxic reactions when used for clinical purposes (Martino et al., 1996; Zambelli et al., 1998). Both the type of cryoprotectant and the cooling rate curves were shown to be important for the viability of testicular cells, and the endocrine and partial exocrine functions of frozen immature testicular tissue, when different freezing protocols were recently tested in mice (Milazzo et al., 2008). Unfortunately, the lack of availability of normal human immature testicular tissue constitutes a serious limiting factor for comparative studies on SGSC functional potential under different cryopreservation conditions.
In conclusion, the present study demonstrates that spermatogonia are able to survive cryopreservation and transplantation and shows, for the first time, that the few remaining spermatogonia found in long-term orthotopic xenografts of immature human testicular tissue from boys <14 years of age are still able to proliferate, suggesting that some SGSCs could be present in this cell population. This may, therefore, open up potential new avenues for future fertility restoration. However, although these cells were shown to enter the differentiation pathway, complete regeneration of normal spermatogenesis was not observed, and spermatogenic arrest at the pachytene spermatocyte stage was suggested, since we could not prove the presence of normal, newly formed spermatids in grafts. Human gonadotrophin supplementation could therefore be proposed to improve the differentiation environment of spermatogonial cells in this xenografting model. Since only a few remaining spermatogonia without signs of proliferation were observed in the 14-year-old boys, the question arises as to whether cryopreservation with a view to fertility restoration is appropriate in peripubertal boys. Further studies are clearly required to investigate the self-renewal and differentiation potential of cryopreserved germ cells in pre- and peripubertal boys.
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The present study was supported by grants from the FNRS (Fonds National de la Recherche Scientifique), Télévie (Grant No. 7.4619.05), Fondation St Luc, Belgian Federation Against Cancer, Baron Albert Frère and Count Philippe de Spoelberch.
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The authors are grateful to Dr Giulio Spagnoli for supplying the MAGE-A4 antibody and Dr Denise Escalier for supplying the proacrosin antibody. The authors thank Dolores Gonzalez for her help with immunohistochemical procedures and Mira Hryniuk, B.A., for reviewing the English language of the manuscript. We also thank the Department of Anatomopathology of the UCL (Prof Rahier) for their help with the TEM procedure, in particular, Alberte Lefèvre and Stéphane Lagasse.
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Submitted on January 16, 2008; resubmitted on June 10, 2008; accepted on June 17, 2008.
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