1 Karolinska Institute, Division of Obstetrics and Gynaecology, Department of Clinical Science, 2 Clinical Research Centre, Karolinska University Hospital, Huddinge, SE 141 86 Stockholm, Sweden and 3 Department of Reproductive Systems Cryobiology, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of the Ukraine, 61 015 Kharkov, Ukraine
4 To whom correspondence should be addressed. Email: victoria.keros{at}klinvet.ki.se
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Abstract |
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Key words: cryopreservation/culture/electron microscopy/spermatogonia/testicular tissue
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Introduction |
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The standard procedure for fertility protection in male cancer patients is cryopreservation of ejaculated spermatozoa, which is well established for adults and sexually mature boys (Muller et al., 2000; Lass et al., 2001
). For pre-pubertal boys who are not yet able to produce ejaculates, and who will lose spermatogenic cells as a result of chemotherapy or radiotherapy, cryopreservation of testicular tissue is an option for fertility preservation (Bahadur et al., 2000
; Hovatta, 2003
). This technique could also be valuable in older boys and young adults who, after retransplantation of the tissue, might achieve spermatozoa production and hence avoid the need for assisted reproduction in later life (Hovatta, 2001
). Azoospermic men who undergo testicular biopsy for diagnostic or treatment purposes also benefit from the cryopreservation of testicular tissue (Tuuri et al., 1999
).
Most studies on testicular tissue cryopreservation have been aimed at evaluation of the condition of testicular spermatozoa for future use in ICSI treatment of infertility (Hovatta et al., 1996a; Allan and Cotman, 1997
; Oates et al., 1997
; Tuuri et al., 1999
).
We tried to find the optimal cryopreservation protocol for testicular tissue, particularly for spermatogonia, which would also save cellcell contacts between different testicular cells. The structural integrity of the tissue after freezing, storage in liquid nitrogen and thawing was examined by the methods of light and transmission electron microscopy. The percentage of spermatogonia detached from the basement membrane, the neighbouring Sertoli cells and spermatocytes was counted to reveal the cryodamage of the seminiferous tubules. The spermatogonia were recognized using immunohistochemical staining for MAGE-A4 antibody against the so-called cancer/testis MAGE family antigen (Takahashi et al., 1995; Yakirevich et al., 2003
).
Leydig cells also suffer as a result of chemotherapy. Interstitial cells of the testes are not as sensitive as the germinal epithelial cells to the cytotoxic treatment. However, increased concentration of LH; reduction in testosterone concentration; symptoms of hypogonadism such as fatigue, sexual dysfunction and altered mood; reduced bone mineral density as consequences of Leydig cells dysfunction have been described (Siimes et al., 1992). Hence, we also studied the function of the Leydig cells after cryopreservation by measuring testosterone production in culture.
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Materials and methods |
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Light and electron microscopy
Before cryopreservation, testicular tissue from each subject was divided into eight pieces (12 mm3), one of which was fixed for routine histological examination and one for transmission electron microscopy (TEM). These samples were used as controls for light and electron microscopy.
The morphology was evaluated by analysing 47 sections from fresh control samples (n=16), 49 sections after cryopreservation using DMSO, 40 sections after PrOH and 16 sections using glycerol. A total of 34 samples was used for these frozen sections. A total of 1312 seminiferous tubules was analysed, of which 402 were controls, 410 were frozen using DMSO, 390 using PrOH and 110 using glycerol. This basic evaluation was done by light microscopy because it enabled us to count a large number of tubules. The TEM samples were used as qualitative controls.
Two independent experienced observers estimated and scored the morphology of the sections and the electron micrographs. The codes of the object glasses and the photos were blinded from the observers. A particular counting system on each glass was used to make sure that the two observers counted every fifth but different sections on each glass.
Light microscopy
Samples for light microscopy were fixed in Bouin's solution for 610 h at 4°C and then dehydrated in 70% ethanol and embedded in paraffin (Paraplast; Sherwood Medical, USA). Four to six (4 µm) sections from each testicular tissue sample were cut at intervals of 20 µm and stained with haematoxylineosin. Histological examinations were performed using a conventional light microscope (Nikon Eclipse E 400). Serial digital images were recorded by using a digital camera (Nikon, Coolpix950) at magnifications of x100, x200 and x400. Evaluation was performed under the microscope.
Histological features of the frozenthawed testicular tissue samples were compared with those of samples of control tissue, which were fixed immediately after testicular biopsy.
The integrity and the structural changes of the fresh control and frozenthawed sections were evaluated semiquantitatively. Leydig cells, stroma and lamina propria were evaluated separately. The normal structure was scored as 2, small changes as 1 and severe damage as 0. Overall, tissues with >50% damage were scored 0 and evaluated as bad morphology. The tissue, which scored 2 and 1 was evaluated as good. The total number of all sections judged as good (scored 2 or 1) was divided by the number of sections investigated in the sample. This gave the percentage of sections with good morphology in each sample. The mean value between the two independent observers was used in the calculations.
(i) When the integrity of the seminiferous tubules was appraised as undamaged (judged 2) or impaired (judged 1), the structure was scored as good. The tubules were scored as 0 when two or more of the following changes in structure were observed: disruption of cellcell connections in the seminiferous tubules; pyknotic nuclei in the spermatogonia; detachment of spermatogonia from the basement membrane.
(ii) The Leydig cells were evaluated on the basis of nuclear morphology, and the integrity of the cell membrane.
(iii) The stroma was scored on the basis of the disruption and/or disintegration. Also the disruption and/or division into layers of the lamina propria were scored, and the swelling or shrinkage of the basement membrane was evaluated.
Transmission electron microscopy
For TEM the tissue samples were fixed in 2% glutaraldehyde and 0.5% paraformaldehyde buffered with 0.1 mol/l sodium cacodylate containing 0.1 mol/l sucrose and 3 mmol/l calcium chloride, pH 7.4. The fixed samples were stored at 4°C and then postfixed in 2% osmium tetroxide, dehydrated in an ascending series of alcohol into acetone and embedded in LX-112 epoxy resin (Ladd Research Industries Inc., USA). Thin sections (0.5 µm) were placed on glass slides and stained with toluidine blue for LM examination. Ultrathin sections (5070 nm) were cut from selected samples and treated with 2% uranyl acetate and lead citrate. Ultrastructural examinations were performed using Leo 906 equipment (Leo, Germany) at 80 kV. Micrographs were taken at different magnifications between x2784 and x7750.
To distinguish different testicular cell types, the following ultrastructural morphological characteristics were used. Spermatogonia are located on the basal lamina of the seminiferous tubules. Type A spermatogonia were identified as cells with an ovoid nucleus with the nucleoli close to the nuclear membrane. The electron-dense cytoplasm contained a small Golgi apparatus, few mitochondria and many free ribosomes. Type B spermatogonia were identified by having a more rounded nucleus and heavily stained chromatin masses attached to the nuclear membrane or to the nucleoli, located at the centre of the nucleus. Sertoli cells were recognized by their location on the basal laminae of the tubuli, by their extension to the lumen of the tubule, and by their large deeply indented nucleus with a homogeneous nucleoplasm and a prominent nucleolus. The cytoplasm contained oval mitochondria, a small Golgi apparatus, an agranular endoplasmic reticulum, lipid droplets, and primary and secondary lysosomes. The Leydig cells were found in the stroma between the tubules. The cytoplasm of the Leydig cells contained a high number of mitochondria, an agranular endoplasmic reticulum, varying numbers of lipid droplets and occasionally some protein crystals.
Immunochistochemical staining and evaluation of the detachment of spermatogonia
Detection of the spermatogonia was carried out using monoclonal mouse anti-human MAGE-A4 antibody (purified from hybridoma 57B), which was kindly provided by Dr Giulio C.Spagnoli (University of Basel, Switzerland).
All the stages were performed at room temperature. After deparaffinization and rehydration in descending concentrations of alcohol, formalin-fixed 4 µm sections were treated with 3% H2O2 in methanol for 10 min to block the non-specific peroxidase. After incubation with 10% horse serum in phosphate-buffered saline (PBS), the samples were placed into a humidified chamber and incubated with the primary anti-MAGE antibody at a dilution of 1:50 for 1 h. Mouse IgG1 at 1:250 concentration was used as a negative control. After double washing in 0.01% Tween 20 in PBS the samples were treated by secondary antibody (horse anti-mouse Ig, Vector Laboratories Inc., USA). Following 30 min incubation with avidinbiotin complex (Vector Laboratories) and treating with fresh diaminobenzidine tetrahydrochloride solution (Vector Laboratories) the samples were counterstained with haematoxylin.
Dark and pale type A and type B spermatogonia, located on the tubular basement membrane, showed strong expression of MAGE-A4 proteins in the nuclei and cytoplasm. They were counted by two independent observers. No other spermatogenic cells were counted. Early primary spermatocytes were clearly distinguished from spermatogonia because of a weaker cytoplasmic staining and unstained nuclei. No MAGE-A4-staining was observed in spermatids, Sertoli or Leydig cells, which were also distinguished by their morphology. We did not count spermatogonia from tubular cross-sections, which were only partially visible.
Testicular tissue obtained from 12 patients was examined. Three samples from each patient were evaluated: control tissue, fixed immediately after operation, and tissue cryopreserved using protocols with DMSO and PrOH as cryoprotectants (DMSO and PrOH groups respectively). The tissue which had been frozen using the sperm-freezing protocol with glycerol as a cryoprotectant was excluded from these analyses because of the very severe damage of the basal compartment of the seminiferous tubules. The total number of spermatogonia in the samples, and also the total number and percentage of spermatogonia detached from basal membrane and neighbouring Sertoli cells and spermatocytes were counted within the investigated groups. The mean value of total number of spermatogonia in each group between the two independent observers was used in the evaluation.
Tissue culture
Cultures were carried out in a humidified incubator (Galaxy, UK) at 33°C, in 5% CO2 in air for up to 12 days. The medium was first changed on the 4th day of culture. The concentration of testosterone measured in the culture medium collected at that time reflects both intracellular testosterone released from cryodamaged cells and newly synthesized by undamaged Leydig cells. The second change of the culture medium was performed on day 12. It shows only the concentration of testosterone released by viable Leydig cells.
The tissue pieces were cultured individually within Matrigel Matrix (Becton Dickinson, Sweden) in 0.5 ml of minimum essential medium (Life Technologies, UK) supplemented with fetal calf serum (FCS; 15%) (Life Technologies) and antibiotic/antimycotic solution (Life Technologies). 8-Bromo-cGMP (Sigma, USA) was added to minimize apoptosis (Hreinsson et al., 2002
). All culture medium from the outside of the inserts was removed on the fourth and 12th day and replaced with 0.3 ml of fresh pre-equilibrated medium. Removed media from the samples were placed in individual 0.5 ml Eppendorf tubes, labelled and stored at 20°C for later testosterone assay.
Twelve samples obtained from four subjects were studied in this part of the work (Table III). Testicular tissue samples from three men were divided into three groups; nine pieces in total. One piece from every sample was placed directly in culture and used as control. The other two pieces were frozen using two different protocols, one with DMSO and one with PrOH, and cultured after thawing (Table III).
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Testosterone measurement
Concentrations of testosterone in the culture media were determined by direct radioimmunoassay using Coat-a-Count® Testosterone commercial kits (Diagnostic Products Corp., USA). There were no differences in matrix between culture medium and the calibrators supplied in the kit. The samples were assayed in duplicate according to the manufacturer's instructions. The detection limit and the within- and between-assay coefficients of variation were 0.1 nmol/l, 6% and 10% respectively.
Statistical analysis
All data are presented as mean ±SD. Statistical differences between the numbers of detached cells in the different investigated groups were analysed using the MannWhitney U-test in Statistica software (StatSoft Inc., USA). P<0.05 was considered as statistically significant.
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Results |
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In these experiments, 70 ±6% of the tubules were judged to be good after freezing with DMSO as cryoprotectant (Figure 2m, n). No major differences versus controls were observed and the remaining 30%, considered poor, reflected the same types of changes as in the controls. In tissue biopsies subjected to PrOH as cryoprotectant, only 37±3% were judged to be good (Figure 2i, j). Here, lower quality in terms of tissue morphology was observed mostly in the basal compartment, characterized by detachment of spermatogonia from the basement membrane, rupture of cellular connections, pyknotic nuclei and vacuolization. No changes were observed in the cell cytoplasm when magnifications of x100 and x200 were used. However, at x400 we observed a fuzzy structure of the cell cytoplasm in cases of cryopreservation with PrOH.
Spermatogenic cells had undamaged morphology after using PrOH (41%, 24%) or DMSO (73%, 57%) in the cases of severely impaired spermatogenesis (n=2). When all the stages of spermatogenesis were studied, cryopreservation with DMSO as cryoprotectant always gave better results.
The lamina propria was influenced in 40% of the tubules and showed swelling in 23% after cryopreservation with PrOH. No evident changes were observed when DMSO had been used (Figure 2).
Transmission electron microscopy
The effect of different freezingthawing procedures was studied at a higher resolution using TEM. The integrity of the plasma membrane, the shape and density of the mitochondria, cytoplasm and chromatin of the nuclei, vacuolization, condition of the basement membrane, cellcell connections and attachment of spermatogonia to the basal membrane further contributed to evaluation of the different treatments.
Fresh tissue, fixed after biopsy without cryopreservation and used as control, displayed a well-preserved ultrastructure (Figure 2c, d) and most of the cells displayed a morphology judged to be good. In the control samples of testicular tissue, the outline of the cell membrane of the spermatogonia in most cases was clear and spermatogonial nuclei were characterized by disperse chromatin. The electron-dense cytoplasm had a homogeneous structure with normal endoplasmic reticulum. The mitochondria were darker than the surrounding cytoplasm and had a homogeneous intermatrix with undamaged cristae, indicating an intact structure. At the same time, some changes, mostly unexpected vacuolization, were observed in the non-frozen samples, but probably most of these were the result of handling of the biopsies, since this material was subjected to fixation by immersion. The nature of the subject's condition also had an influence. These samples were used as a starting point when evaluating cryo-damage in the tissue samples after freezing and thawing.
It was impossible to distinguish spermatogonial cell membranes after cryopreservation using glycerol as a cryoprotectant. Detachment of spermatogonia from the basement membrane was observed. Destroyed dark and pale mitochondria were swollen and characterized by dilated cristae. The nuclei of the spermatogonia were dense, pyknotic and dark (Figure 2g, h).
After cryopreservation using PrOH, most of the Sertoli cells displayed a necrotic state. Structural changes of spermatogonia were characterized by a fuzzy outline of the plasma membrane, an increased number of vacuoles in the cytoplasm and a heterogeneously shaped matrix in the mitochondria (Figure 2k, l). The cellcell connections were broken and the chromatin was fragmented and dense compared with the controls (Figure 2k, c).
After cryopreservation using DMSO as a cryoprotectant, the spermatogonia were more mildly influenced and showed a morphology similar to that in the control material, with well-preserved and well-attached cells and a homogeneously stained cytoplasm (Figure 2o, p). At higher magnification, non-lethal changes were observed, such as increased vacuolization (Figure 2o, c) and some changes in the morphology of the mitochondria (Figure 2p, d). However, these changes were slight. The attachment of spermatogonia to the basement membrane of the seminiferous tubuli was intact, as in control tissue (Figure 2p, d).
The morphology of the lamina propria, stromal compartment and, in particular, the Leydig cells after cryopreservation using either DMSO (80%) or PrOH (50%) appeared well-preserved without evidence of major changes compared with the control tissue. The Sertoli cells had better morphology after cryopreservation with DMSO than after PrOH or glycerol treatment.
The spermatogenic cells of the latest stages, spermatocytes, spermatids and spermatozoa, maintained better ultrastructural morphology than the more basally located structures, even when PrOH or glycerol were used as cryoprotectants.
Immunochistochemical staining and evaluation of the detached spermatogonia
We analysed 73 sections of the testicular tissue from the three investigated groups. The total number of cross-sections of the seminiferous tubules was 695. Positively stained spermatogonia were seen in 540 seminiferous tubules of the freshly prepared tissue and that cryopreserved by the protocols using DMSO and PrOH (numbers of seminiferous tubules 195, 207 and 138 respectively); 4752.5396.04±305.09 (total number/mean±SD) MAGE-A4-stained spermatogonia were observed in the control tissue, 2073/172.75±183.94 in tissue frozenthawed using DMSO protocol and 1291.5/107.63±104.58 in tissue frozen thawed using PrOH protocol (Table IV; Figure 3).
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The number of MAGE-A4-positive spermatogonia detached from the neighbouring Sertoli cells and early spermatocytes was significantly higher in the PrOH group (33.2%), as compared with the control (2.5%) and DMSO (4.6%) groups (429/35.75±35.44, 119/9.92±12.02 and 95/7.92±9.96. respectively).
Testosterone production in vitro
Leydig cell activity, measured as concentration of testosterone in tissue culture media, is shown in Figure 4. Statistical analysis was not possible owing to the limited number of observations.
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When DMSO-preserved tissue and cell suspension from the same patients were compared, testosterone concentrations were considerably higher in the cultures with DMSO-preserved tissue (Figure 4).
The concentration of testosterone was dependent on the condition of the tissue, fresh or cryopreserved, ischaemia time and the freezing protocol. It was also correlated to the numbers of Leydig cells present in the tissue pieces, and the concentration of testosterone in the subject's serum.
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Discussion |
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Spermatogonia obtained from various animals can survive when extracted from disaggregated tissue and cryopreserved as a cell suspension (Avarbock et al., 1996; Izadyar et al., 2002
; Kanatsu-Shinohara et al., 2003
). Similar studies have been performed using human testicular tissue from patients with obstructive azoospermia or maturation arrest, or testicular tissue obtained from pre-pubertal boys before cancer treatment, when they were disaggregated and cryopreserved as a cell suspension (Brook et al., 2001
).
The survival rate has been found to be higher in murine than in human specimens, when testicular cells were cryopreserved (Brook et al., 2001). An explanation may be that human tissue is much more fibrous when compared with that of rodents. As a consequence, the procedure of tissue disaggregation in human material is more extensive. Hence, aggressive enzymatic treatment is likely to influence the viability of the cells. Extensive handling, directed to mechanical disruption of the cellcell connections, increases cell hypoxia, which damages the cells. The negative influence of these treatments can be avoided when testicular cells are cryopreserved within a piece of tissue.
For germ cell transplantation to be successful, sufficient numbers of stem cells have to be injected into the testes. It has been estimated that 104 germ cells isolated from an adult rodent testis contain as few as two stem cells (Meistrich and van Beek, 1993). Only 510% of stem cells can colonize the seminiferous tubule after spermatogonial transplantation (Ogawa et al., 2000
). Probably, preservation of testicular tissue as pieces could prevent the loss of spermatogonia that occurs during the process of tissue disintegration. It is obvious that proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially the supporting Sertoli cells (Russell et al., 1990
; Schlatt et al., 1997
). Availability and function of the Leydig cells is also an important factor for successful spermatogonial maturation. Both Leydig and Sertoli cells have endocrine and paracrine functions that influence spermatogenesis. Both of these cell types are involved, for instance, in the feedback regulation of release of FSH and LH, which are necessary for proper function of gonadal cells.
Transplantation of isolated testicular cells from calves, humans and other primates into the testes of recipient mice has not resulted in complete spermatogenesis (Nagano et al., 2001, 2002
; Izadyar et al., 2002
). However, fresh and frozenthawed mouse and rabbit testicular pieces (Shinohara et al., 2002
) and fragments of pig testis (Honaramooz et al., 2002
) showed restored spermatogenesis after transplantation into immunodeficient mice. It was hypothesized that if pieces of whole testicular tissue can maintain their function better than an enzymatically prepared cell suspension, pieces of frozenthawed testicular tissue could be reimplanted inside the tunica albuginea of the testis and possibly result in testicular spermatozoa which could be used for ICSI. The Leydig cells of these individuals might also begin to produce testosterone (Hovatta, 2003
).
Shinohara et al. (2002) transplanted frozenthawed (DMSO/Dulbecco's modified Eagle's medium/FCS) pieces of mouse and rabbit testicular tissue to mice. Live offspring were obtained after microinjection with sperm that developed in the transplanted testicular pieces (Shinohara et al., 2002
). Isografts and xenografts of immature and adult testicular tissue from several animal species became functionally active as spermatogenic cells in the mouse (Schlatt et al., 2002
). The cryoprotectant medium was composed of DMSO with sucrose and human serum albumin.
Cryopreservation of tissue requires better permeation of the cryoprotectant compared with cell suspension (Nugent et al., 1997). Different cell types may need different freezing protocols for optimal survival (Leibo and Mazur, 1971
).
In the present study, the protocol developed for cryopreservation of semen with egg yolk medium containing glycerol was tested to see if it could be used in these small-sized tissue samples. Glycerol is a widely used cryoprotectant for cryopreservation of spermatozoa and suspensions of testicular cells. Nogueira et al. (1999) showed by light microscopy and TEM that cryopreservation of human testicular spermatozoa within tissue, using TESTyolk medium with glycerol and a slow-freezing protocol, caused damage to the membranes and to the acrosome similar to that seen in ejaculated spermatozoa. These morphological observations suggested that late-stage spermatids and spermatozoa were more resistant to the cryopreservation procedure than other germ cells. Similar results were obtained in rat testicular biopsy specimens stored in different cryopreservation media, containing glycerol and/or DMSO at moderate (625%) concentrations (Jezek et al., 2002
). In both studies, the parts of the seminiferous tubules most affected by freezingthawing were the basal tubular compartment and the spermatocytes. Detachment of spermatogonia and Sertoli cells from the basement membrane, nuclear damage, increased vacuolization of the cytoplasm, and swelling and shrinkage of the cells, were frequently observed. In the present study we observed similar changes when human testicular tissue fragments were cryopreserved by the method designed for sperm, using glycerol as a cryoprotectant.
A cryopreservation method optimized for human ovarian tissue using PrOH and sucrose (Hovatta et al., 1996b; Hreinsson et al., 2003
) was found not to be optimal for cryopreservation of testicular tissue.
In TEM, cryo-injury of the Sertoli cells was often observed, also destruction of the spermatogonial cytoplasmic membrane and contents, even when intact morphology of the tissue was observed in light microscopy.
The most suitable cryoprotectant for tissue cryopreservation might be DMSO, owing to its low molecular weight and penetration ability. However, the toxicity of this substance restricts its application in clinical practice. The concentrations of DMSO that are usually used for both cell suspension and tissue cryopreservation are 1020% (Donahoe et al., 1977; Gosden, 1992; Hovatta et al., 1996b
; Jezek et al., 2002
; Shinohara et al., 2002
; Schlatt et al., 2002
). In our previous studies, the concentration of DMSO was decreased to 5% in the cryoprotectant medium. This was successfully used for cryopreservation of human fetal testes (Keros, 1999
; Keros et al., 2001
). Good integrity of the tissue and functional activity of the Leydig cells were confirmed by the results obtained in light microscopy and tissue culture experiments.
In the present study, the best morphology of the basal compartment was obtained when the cryoprotectant medium contained 5% DMSO and the protocol was modified for adult testis tissue. Increased vacuolization was seen, but it was not different from that observed in the fresh control tissue. This probably resulted from the many steps required during the preparation of the samples for TEM. The structure of testicular tissue obtained from infertile men is characterized by an increased number of apoptotic cells (Francavilla et al., 2002). Chromatin condensation in the nuclear periphery, with remaining large empty nuclear areas, dilated or disrupted mitochondria and loss of contact with Sertoli cells are commonly observed changes in instances of apoptotic degeneration of type A spermatogonia. Later apoptotic changes consist of diffuse cytoplasmic vacuolization associated with cell shrinkage, resulting in the formation of an empty pericellular space, as visualized in light microscopy (Francavilla et al., 2002
).
The total number of spermatogonia detached from the basal membrane and neighbouring cells could be effectively evaluated by using immunostaining with MAGE-A4 antibody against the so-called antigen from MAGE family. MAGE-A4 is known as cancer/testis tumour-associated antigen, which is expressed in the tumour cells from several different histological origins. In normal, healthy human tissue this protein is expressed in the placenta and testes (Juretic et al., 2003). Its ability to give strong staining of spermatogonia in the testes proved valuable in our study.
No significant differences were observed in the quantity of the spermatogonia detached from the basal membrane and neighbouring cells, when samples treated with DMSO were compared with fresh control tissue. This confirms the better post-thaw viability of larger-sized cells, such as Sertoli and primary spermatocytes, within testicular tissue when compared with the protocol, in which PrOH was used as cryoprotectant.
In the present study, 80% of the Leydig cells were well preserved and showed normal morphology when DMSO was used. They were more resistant to cryo-damage than spermatogenic cells. In the protocol using PrOH, 50% of the Leydig cells also appeared well-preserved, without evidence of major changes. In agreement with the results of our previous studies on in vitro culture of frozenthawed fetal human testicular tissue (Keros, 1999; Keros et al., 2001
), hormonal activity of the interstitial cells after cryopreservation could be confirmed in culture of the adult testicular tissue samples after thawing.
All the samples showed hormonal activity during culture. Because decreased testosterone production has been reported among men who have undergone leukaemia treatment in childhood (Siimes et al., 1992), using a protocol such as ours, which maintains normal Leydig cell function, is very important.
The concentration of DMSO in our freezing protocol was relatively low, 5%. Even though DMSO is known to be a toxic substance, the viability of the tissue appeared to be better than that obtained by using the other programmes. Five per cent concentration of DMSO is also widely used for cryopreservation of human haematopoietic stem cells for future clinical use. It is also continuously and successfully used for cryopreservation of day 3 human embryos. Regarding the function of germ cells, our data suggest that the best morphology at the electron microscopic level was obtained after cryopreservation of testicular tissue using the protocol with DMSO. Hence, we can recommend this programme for clinical use.
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Acknowledgements |
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Submitted on June 4, 2004; resubmitted on December 1, 2004; accepted on January 20, 2005.