Hum. Reprod. Advance Access originally published online on December 13, 2006
Human Reproduction 2007 22(4):1060-1067; doi:10.1093/humrep/del471
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Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts
1 Department of Cell Biology and Physiology, Center for Research in Reproductive Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA 2 Pediatric Endocrinology Unit, Department of Woman and Child Health, Karolinska Institute and University Hospital, Stockholm, Sweden 3 Department of Pediatrics, University of Turku, Turku, Finland
4 To whom correspondence should be addressed at: Department of Pediatrics, University of Turku, FIN-20520 Turku, Finland. E-mail: kirsi.jahnukainen{at}ki.se
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Abstract |
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BACKGROUND: Successful cryopreservation of gonadal tissue is an important factor in guaranteeing the fertility preservation via germ cell or testicular tissue transplantation. The aim of this study was to evaluate the effects of cooling and cryopreservation on spermatogonial stem cell survival and function of immature non-human primate testicular tissue xenografted to nude mice.
METHODS: Group 1 (control group) received subcutaneous grafts of fresh immature rhesus monkey testes. The treatment groups received grafts after 24 h cooling in ice-cold medium (Group 2), after 24 h of cryopreservation without cryoprotectant (Group 3), with ethylene glycol (Group 4: 1.4 M) or with dimethylsulphoxide (DMSO) (group 5: 1.4 M; group 6: 0.7 M), using cooling rates of 0.5°C/min. The graft number, weight and histology were examined 3–5 months later.
RESULTS: After xenografting, grafts from fresh and cooled tissue showed good survival and spermatogenic induction to spermatocytes. Cryopreservation in 1.4 M DMSO also allowed grafts to initiate spermatogenesis. In contrast, 0.7 M DMSO and ethylene glycol showed inferior protection.
CONCLUSIONS: Our observations suggest that cryopreservation of immature primate testis is a feasible approach to maintain spermatogonial stem cells and may serve as a promising tool for fertility preservation of prepubertal boys. The possibility to delay the transplantation of cooled samples suggests an option for clinical centralization of testicular tissue cryopreservation.
Key words: cryopreservation/prepuberty/primate/testis/xenograft
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Introduction |
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Storage of gametes and gonadal tissue is a key requirement for current and future approaches to secure fertility in tumor survivors (Jahnukainen et al., 2006b). Single cell preparations and fragments of testes from adult men and young boys have been cryopreserved using propanediol, glycerol, ethylene glycol or dimethylsulphoxide (DMSO) (Brook et al., 2001
; Keros et al., 2005; Kvist et al., 2005). None of these previous studies has assessed the potential for spermatogenic recovery or the number and functionality of spermatogonial stem cells after cryostorage.
Grafting immature rhesus monkey tissue into immunodeficient mice has recently been introduced as a novel strategy to evaluate spermatogonial stem cell potential in primate testicular tissue (Honaramooz et al., 2004; Jahnukainen et al., 2006a). The recipient serves as a living incubator for the testicular fragments and provides a milieu that allows pubertal changes to occur in the grafted testicular tissue. Xenografting of testicular tissue from immature rhesus monkeys resulted in acceleration of testicular maturation, production of round spermatids 5 months and fertilization-competent sperm 7 months after grafting (Honaramooz et al., 2004). Cryopreservation of functional spermatogonial stem cells from mouse and rabbit has been reported. After heterotopic transplantation, complete spermatogenesis was observed in cryopreserved grafts and live offspring was generated by microinjection of sperm recovered from these grafts (Kanatsu-Shinohara et al., 2003).
Here, we used the xenografting approach to evaluate the effects of short-term storage and cryopreservation of immature monkey testes. Endpoints were survival and degree of spermatogenic progression in xenografts. We compared immediate grafting of fresh tissue with a 24-h cooling procedure or a 24-h cryostorage. DMSO and ethylene glycol were used as cryoprotectants.
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Materials and methods |
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Animals and graft preparation
Testicular tissue was obtained from four immature male rhesus monkeys (18, 18, 21 and 21 months of age). In this species, the onset of puberty, as reflected by the initiation of nocturnal testosterone secretion, occurs at approximately 30 months of age (Plant, 1985
). Monkey testes were decapsulated and cut into 50–80 fragments. The fragments of tissue (
0.5–1.0 mm3) were dissected and maintained in ice-cold sterile Leibovitz-L15 medium (Gibco, Paisley) with 10% fetal calf serum (FCS) until immediate grafting, delayed grafting (24–26 h later) or until cryopreserved (see below). The tissue pieces from all donors were used for immediate and delayed grafting. Tissue pieces from the two first donors were used for cryopreservation with ethylene glycol, 1.4 M DMSO and with no cryoprotectant and from the two latter donors for cryopreservation with 0.7 and 1.4 M DMSO.
Cryopreservation
Two different cryoprotective agents ethylene glycol (1.4 M) and DMSO (0.7 and 1.4 M) were used. These cryoprotective agents were added drop by drop to the cell culture medium (containing 10% FCS) containing the tissue samples until the final concentrations were reached. During this procedure, up to 200 tissue samples were suspended in 40 ml of the ice-cold medium, to which the cryoprotectants were slowly added over a time of 5 min under constant manual agitation. The vial containing medium and tissue samples was then incubated on ice for an additional 15 min and shaken periodically to allow for complete cryoprotectant diffusion into the tissue samples. Forty to fifty grafts were then transferred to one 1.8-ml cryovials (Nunc) containing a final volume of 1.5 ml of cryopreservation medium. One group of grafts was cryopreserved without cryoprotective agents.
For freezing, the grafts were first equilibrated on ice for 40 min and thereafter placed in –20°C standard freezer for 60 min. To monitor the temperature during the cooling and freezing period, one vial containing the respective freezing medium was fitted with a thermocouple (wires of 0.5 mm diameter). Typical runs with 0.7 and 1.4 M DMSO are shown in Figure 1. The seeding of the samples occurred after 50 min (0.7 M DMSO) or 75 min (1.4 M DMSO), when a temperature of around –13°C was reached. The cryotubes were then plunged into liquid nitrogen and stored for 24 h. The grafts were thawed in 37°C water bath for 2 min and the grafts were washed in fresh ice cold medium.
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Xenografting
Five- to seven-week-old immunodeficient intact male nude mice (Crl:Nu/Nu-nuBR, Charles River Laboratories, Wilmington, MA) were used as recipients. Testicular fragments of monkey tissue were placed under the dorsal skin on either side of the dorsal midline by using cancer implant G13 needle (Popper Precision Instruments, Lincoln, RI). Mice were maintained in groups of 5–6 per cage, with food and water available ad libitum.
Two separate experiments were done. In the first experiment, the effect of cryopreservation with and without 1.4 M ethylene glycol or DMSO on graft survival was compared to that after cooling into +4°C and immediate grafting. Altogether four testicular grafts in each seven recipients per group were injected, except in the group with immediate grafting where eight testicular grafts in each eight recipients were injected. Three recipients were lost for infection complications before cessation of experiment 3 months after xenografting. In the second experiment, the effect of cryopreservation with DMSO using doses 0.7 and 1.4 M on graft survival was compared to that after cooling of grafts or immediate grafting. In each group 4 testicular grafts were injected in six recipients and the experiment was terminated 5 months post-transplantation.
At the respective time points mice were anaesthetized, weighed and blood was collected by cardiac puncture. The seminal vesicles were dissected and weighed, the back skin was removed and photographed and the number of visible grafts was recorded. The grafts were dissected from the skin and fixed in Bouin's solution. All animal experiments were approved by and performed under the guidance of the Animal Care and Use Committee at the University of Pittsburgh, School of Medicine.
Histology and statistical analysis
The grafts were fixed for 18–24 h, weighed, transferred for storage into 70% ethanol and embedded in Technovit (Kulzer, Germany) for sectioning at 2 µm. Tissue sections were stained with periodic acid-Schiff's reagent (PAS)/Gill's hematoxylin and examined with oil immersion under the light microscope. Graft survival was defined by light microscopic observation of seminiferous tubules with Sertoli cells in the retrieved graft. All cross-sections of seminiferous tubules were analyzed from smaller grafts. If the grafts were large, 50 tubular cross-sections were selected for analysis by random systematic sampling. Each cross-section was scored for the presence of germ cell types (spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, round and/or elongating spermatids) or a complete absence of germ cells (Sertoli cell-only (SCO)). The identification of type A dark (Ad) and A pale (Ap) spermatogonia followed the scheme of Clermont and Leblond (1959). Tubules with spermatogonia which were located in the centre of seminiferous tubules and were detached from the basement membrane were scored as a separate category named adluminal spermatogonia. The light microscopic determinations were conducted by one observer (K.J.).
Data are presented as mean ± SEM. The Mann–Whitney U-test was employed for single statistical comparison of independent groups of samples and the Kruskall–Wallis analysis with Dunn's post-hoc test for multiple comparisons of independent groups of samples. A P-value of <0.05 was considered to indicate a statistically significant difference.
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Results |
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Graft survival
Three months after fresh tissue xenografting 51% (33/64) of the grafts were recovered. A 24-h delay in grafting of fresh and cooled tissue fragments did not have any effect on graft survival (52% recovery, 14/27). Cryopreservation decreased graft survival. None of the grafts frozen without cryoprotective agent survived. DMSO was superior to ethylene glycol as a cryoprotectant. While 30% (7/23) of the grafts survived after cryopreservation with 1.4 M DMSO, only 7% (2/27) were recovered after cryopreservation with ethylene glycol. There was no difference in the weights of dissected testicular grafts (Table I).
Five months after fresh tissue xenografting, 73% (35/48) of immediately implanted grafts and 79% (38/48) of grafts which were implanted with a 24-h delay were recovered. The grafts which were xenografted after cryopreservation with 1.4 M DMSO showed very similar survival 73% (35/48). Xenograft survival was severely reduced when 0.7 M DMSO was used as cryoprotective agent (13%; 6/48). The graft weight after delayed xenografting was significantly higher compared to both cryopreserved groups and the immediately grafted group (Table I).
Histological evaluation of spermatogenic recovery
At the time of grafting, the testicular tissue from all donor monkeys consisted of seminiferous cords with spermatogonia as the most advanced germ cell type (Figure 2a). Histological morphology of grafts cryopreserved with 0.7 or 1.4 M DMSO (Figure 2b) was examined after thawing. No morphological changes compared to fresh or cooled grafts could be detected.
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Three months after grafting 1% of the seminiferous tubules contained B spermatogonia or spermatocytes after immediate or delayed xenografting (Table I). No difference in relative numbers of tubules with A-spermatogonia or tubules with Sertoli cell only were seen between these groups (Table I). In cryopreserved grafts, no B-spermatogonia or more advanced differentiating germ cells were detected (Table I). Cryopreservation with 1.4 M DMSO caused a decrease in the number of seminiferous tubules containing Ad-spermatogonia but this difference did not reach statistical significance (Table I). The grafts cryopreserved with ethylene glycol (Figure 2d) showed comparable histological morphology to the grafts cryopreserved with DMSO. Low number of these grafts prevented a profound morphological analysis (Table I).
Five months after grafting B spermatogonia were recorded in fresh (immediate: 8%; delayed: 12%) and cryopreserved (1.4 M DMSO, 2%) grafts (Figure 2h, Table I). These grafts also contained spermatocytes up to the pachytene stage. Cryopreservation decreased the number of seminiferous tubules with Ad and Ap spermatogonia and increased relative number of seminiferous tubules with SCO (Table I). Complete absence of germ cells (SCO) was determined in six surviving grafts after cryopreservation with 0.7 M DMSO (Figure 2g).
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Analysis of the most advanced germ cell type at three months after xenografting revealed the presence of at least B-spermatogonia after immediate (15%; B spermatogonia present in 5 out of 33 grafts) and delayed xenografting (21%; B spermatogonia present in 21 out of 14 grafts), (Figure 3). None of the cryopreserved grafts showed initiation of spermatogenesis (Figure 3). Ad and Ap and adluminal spermatogonia were detected in all cryopreserved grafts. In contrast, five months after xenografting spermatogenic induction was more widespread with B-spermatogonia or more advanced germ cells in 63% (22/35) of grafts after immediate grafting, in 79% (30/38) after delayed grafting and in 31% (11/35) after cryopreservation with 1.4 M DMSO (Figure 3). The most advanced germ cell types in each of these groups were pachytene spermatocytes, showing that five months after grafting, monkey spermatogenesis had not yet reached the post-meiotic stage.
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In all recovered xenografts a morphologically normal interstitium with blood vessels, macrophages, peritubular and Leydig cells was observed. Cryopreservation did not affect the light microscopic morphology of Sertoli cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Our results demonstrate that the spermatogonial stem cells in xenografted testicular tissue from immature non-human primates survive cryopreservation and retain their capacity to initiate spermatogenesis when exposed to gonadotrophins of an adult mouse host. This observation suggests that the cryopreservation of immature primate testicular tissue is a feasible approach to maintain the stem cell potential of spermatogonia and may serve as a promising method for fertility preservation of prepubertal boys (Jahnukainen et al., 2006a
,b).
The survival rate of 73% we present here for cryopreserved testicular tissue after 5 months of cryopreservation was obtained using an un-controlled freezing program and using cryopreservation media with 10% FCS and 1.4 M DMSO. According to earlier studies, an even further enhanced graft survival can be expected if a controlled cooling rate, adjustments of the cryoprotectant concentrations and sucrose substitution were to be employed (Izadyar et al., 2002; Frederickx et al., 2004). Our present results suggest that the cooling rate may not be that critical since the significant survival of testicular grafts was obtained here even without a control of the freezing rate. This observation is in line with a previous report stating that a high yield of living cells after thawing was obtained when bovine A-spermatogonia were cryopreserved with similar freezing methods (Izadyar et al., 2002). However, we cannot rule out that a more precisely controlled freezing procedure would result in an even better survival. Since our conditions were maintained constant for each procedure, we are confident that the comparison of the various procedures is valid. Our results encourage the application of testicular cryopreservation even under circumstances and in laboratories where no computer-assisted freezing equipment is available, for example in less developed countries.
The type of cryoprotective agent employed seems to be much more critical for graft survival. No viable grafts were recovered when grafts were cryopreserved without cryoprotective agent. There was also a clear difference between cryoprotective agents. Ethylene glycol showed an inferior protection for immature primate testicular tissue than DMSO. This observation is in accordance with previous reports that DMSO with its low molecular weight and high tissue penetration provides superior cryoprotection especially in solid tissues (Keros et al., 2005). DMSO also provides superior cryoprotection in cell suspensions (Frederickx et al., 2004). The DMSO concentration in cryopreservation media had a significant effect on both graft survival and on the survival of the spermatogonial stem cells. Graft survival five months after xenografting decreased from 73% to 13% when the concentration of DMSO in the medium was lowered from 1.4 to 0.7 M. Histological evaluation revealed that none of the grafts cryopreserved with 0.7 M DMSO contained germ cells. This observation suggests that the concentration of the cryoprotective agent was especially critical for the survival of spermatogonial stem cells and that the stem cells are more sensitive to cryopreservation related stress than the somatic components of testicular tissue. It is also important to notice that there was no difference in histological morphology of thawed grafts at the time of xenografting. Pre-grafting morphology did not reveal the later observed difference in cryoprotective effect of different doses of DMSO. Xenografting the testicular tissue into immunodeficient hosts was the only way to detect differences in spermatogonial stem cell potential. Our observations confirm earlier studies stating that histological detection of spermatogonia in cryopreserved tissues does not necessarily imply that those (cryopreserved) spermatogonial stem cells are actually functional (Frederickx et al., 2004).
Cryopreservation delayed the initiation of spermatogenesis in the grafted tissue. Early focal differentiation of spermatogonia and a progression of spermatogenesis up to the level of spermatocytes 3 months after xenotransplantation were detected only in non-cryopreserved grafts. There was also a significantly reduced number of seminiferous tubules with A-spermatogonia in the cryopreserved grafts when compared to non-cryopreserved grafts. In addition, no increase in weight of cryopreserved grafts was detected between three and five months after xenografting. The relative number of seminiferous tubules with SCO morphology increased concurrently. Our observations indicate that cryopreservation either affects the number of surviving A-spermatogonia or their capacity to colonize the seminiferous tubules. The observed poor increase in cryopreserved graft weights further suggest that pubertal increase in number of differentiating germ cells is delayed in xenografts. Since cryopreservation, as performed here, had a negative impact on graft development, future studies should focus on comparing cryopreservation media and procedures in order to optimize the outcome.
Importantly, 24 h of cooling to +4°C followed by delayed grafting did not affect the survival of primate testicular xenografts or the capacity of spermatogonial stem cells to colonize or initiate spermatogenesis. The cooled grafts showed a fast increase in graft weight between 3 and 5 months after xenografting and were even significantly heavier than the immediately grafted control samples. The grafts after 24 h cooling also showed the highest rate of graft survival and relative number of seminiferous tubules with initiated spermatogenesis. Observations suggest that using our tissue processing procedure, the immature primate testicular tissue does not suffer significantly from ischemia and that the 24 h of cooling of the tissue before xenografting appears to even improve the performance of the testicular grafts. Ischemia resistance of immature testicular tissue seems to be comparable to that reported on ovarian cortical tissue. Survival of primordial follicles have not shown to be affected by 4-h delay in transplantation when ovarian tissue was transported on ice to another institution (Schmidt et al., 2003). Our present observations suggest that for cooled immature primate testicular tissue, transplantation may be delayed up to 24 h without loss of spermatogonial stem cell potential.
The present findings from immature primate testis are important for a putative clinical practice to cryobank prepubertal testicular tissue before sterilizing cancer treatment or other gonadotoxic therapy. Morphology and hormonal function of testicular tissue from young prepubertal boys is very similar to immature non-human primate testis. Immature human testis most probably starts pubertal maturation when either autotransplanted into cured patients or xenotransplanted into a mouse host. The possibility to transport cooled samples without the loss of spermatogonial stem cell potential is significant for the clinical applicability of this technique. The transportation of cooled samples would allow the centralization of tissue storage. Each clinical unit treating cancer patients would not need to establish cryopreservation routines. Flexibility in timing of cryopreservation would help to meet the special time constringency demands which the onset of cancer treatment is causing for fertility laboratories. Cryopreservation could be done during office hours. Our present observations obtained from non-human primate testicular tissue need to be confirmed with studies using prepubertal human testicular tissue.
We conclude that ectopic xenografting is a simple and powerful approach for the study of spermatogonial stem cell potential in immature primate testes. Presently the effect of delayed grafting and cryopreservation on spermatogonial stem cell potential was evaluated. Significant differences in graft survival and spermatogonial stem cell potential were detected in cryopreserved tissue using different cryoprotective agents and different agent doses. Cryopreservation with 1.4 M DMSO was shown to successfully preserve immature primate testicular tissue. Spermatogonial stem cells were able to initiate spermatogenesis even when xenografting of cooled tissue was delayed by 24 h.
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Acknowledgements |
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Grant support: Start up funds of Pittsburgh University and NIH-grants 1RO1 01050617; 2U54 HD008610, The Swedish Barncancerfonden, the Finnish Cancer Society, the Helsingin Sanomat Centennial Foundation, the Finnish Pediatric Research Foundation and the Nona and Kullervo Väre Foundation.
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References |
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Submitted on October 4, 2006; resubmitted on October 28, 2006; accepted on November 15, 2006.
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