Human Reproduction vol.15 no.8 pp.1713–1716, 2000
In-vitro differentiation of germ cells from frozen testicular biopsy specimens
Jan Tesarik1,2,6, Carmen Mendoza2,3, Reno Anniballo4 and Ermanno Greco5 1Laboratoire d’Eylau, 55 rue Saint-Didier, 75116 Paris, France, 2MAR&Gen, Molecular Assisted Reproduction and Genetics, 3Department of Biochemistry and Molecular Biology, University of Granada Faculty of Sciences, Granada, Spain, 4Andrology Centre ‘John MacLeod’, Naples and 5Centre for Reproductive Medicine,
European Hospital, Rome, Italy 6To
whom correspondence should be addressed at: Laboratoire d’Eylau, 55 rue Saint-Didier, 75116 Paris, France. E-mail:
[email protected]
In some men with germ cell maturation arrest, spermatogenesis can be resumed during in-vitro culture of testicular biopsy samples. In this study, we examined whether similar differentiation events can be induced in cultured germ cells from cryopreserved testicular biopsy specimens. Fresh and cryopreserved aliquots of the same testicular biopsy samples were cultured in medium supplemented with FSH and testosterone. After 24 and 48 h of culture, the progression of spermatogenesis and the percentage of Sertoli cells with DNA damage, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), were evaluated. Spermatogenesis progressed in a similar way in fresh and cryopreserved aliquots over the first 24 h of culture. However, in contrast to fresh aliquots, no additional progress of spermatogenesis was detected between the 24 and 48 h time points. The percentage of TUNEL-positive Sertoli cells in fresh aliquots showed only a moderate increase after 24 h of culture, whereas most Sertoli cells from cryopreserved aliquots became TUNELpositive during the same culture period. These data show that limited progression of spermatogenesis can be achieved by culturing cryopreserved testicular biopsy specimens for 24 h, but no additional benefit can be expected from prolonging the culture beyond this time point. Key words: cryopreservation/in-vitro spermatogenesis/spermatid/spermatocyte/testicular biopsy
Introduction We have shown recently that human germ cells can undergo unusually rapid trans-meiotic and post-meiotic differentiation when cultured in vitro in media supplemented with high concentrations of FSH and testosterone (Tesarik et al., 1998a,b). When applied to patients with spermatogenesis arrest, this method was at the origin of the first birth after fertilization with elongated spermatids obtained by in-vitro differentiation of primary spermatocytes from a patient with maturation arrest © European Society of Human Reproduction and Embryology
(Tesarik et al., 1999). Situations may arise, however, in which couples included in a testicular sperm extraction and intracytoplasmic sperm injection (ICSI) programme are confronted with an unexpected lack of spermatozoa and late elongated spermatids on the day of oocyte recovery, while no decision about the eventual use of in-vitro matured germ cells for assisted reproduction has yet been taken. In such cases, immediate cryostorage of testicular biopsy samples would make it possible to make an attempt at in-vitro maturation of germ cells contained in these samples at a later date. This study was undertaken to evaluate the in-vitro developmental potential of germ cells from frozen testicular biopsy specimens. The in-vitro development of germ cells from the frozen specimens was compared with that of germ cells originating from the same testicular biopsy samples but subjected to in-vitro culture immediately after recovery. In addition to germ cell differentiation, the intactness of Sertoli cell DNA before and after in-vitro culture of fresh and frozen testicular specimens was also examined. Materials and methods Patients This study involved nine patients with non-obstructive azoospermia due to maturation arrest and five patients with obstructive azoospermia. All participants had normal karyotypes. A search for Y-chromosome microdeletions was performed in two of the nine patients with nonobstructive azoospermia and gave a negative result. Testicular tissue sampling and preparation Testicular tissue was sampled by open testicular biopsy under local anaesthesia. Pieces of tissue were put in Gamete-100 culture medium (Scandinavian IVF, Gothenburg, Sweden) and disintegrated by stretching between sterile microscope slides followed by repeated aspirations into a tuberculin syringe. This preparation was known to release some single germ cells from the seminiferous tubules, but most cells remained embedded in aggregates consisting of different stages of germ cells as well as Sertoli cells (Tesarik et al., 1998b). Both single and aggregated testicular cells were pelleted together by gentle centrifugation (200 g; 10 min), and the resulting pellets were used in further experiments. Testicular tissue cryopreservation After homogenization, testicular cells in Gamete-100 medium were mixed with the SpermFreeze (Scandinavian IVF) sperm-freezing solution (containing glycerol, 0.4% human serum albumin and HEPES) in a 10:7 ratio, equilibrated for 10 min at laboratory temperature and aspirated into French straws for sperm freezing (Cryo Bio System, l’Aigle, France). Sealed straws were rapidly frozen by plunging into liquid nitrogen after previous chilling in liquid nitrogen vapour. For thawing, straws were removed from liquid
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Figure 1. (A) Human round spermatid nucleus (counterstained with 4,6-diamidino-2-phenylindole) processed for two-colour FISH with probes for chromosomes 15 (D15Z1, clear blue spot) and 16 (D16Z1, pink spot). A single spot for each of the two chromosomes evaluated is visible. (B) Human primary spermatocyte nucleus (counterstained with 4,6-diamidino-2-phenylindole) processed by a combined method associating one-colour FISH with a chromosome-16 probe (D16Z1, pink spots) and immunocytochemistry with a germline marker antibody (4D4 monoclonal antibody against human proacrosin, clear blue area corresponding to a developing proacrosomal granule). Two FISH spots, each corresponding to a pair of closely attached chromatids, are apparent in this preparation. Only occasionally can each of the four chromatids present in the primary spermatocyte nucleus be distinguished as an individual spot. Scale bar ⫽ 5 µm.
human recombinant FSH (Puregon, Organon, Oss, The Netherlands) and with 1 µmol/l testosterone (water-soluble, Sigma, St Louis, MO, USA) as described (Tesarik et al., 1998b). The choice of the culture temperature was based on preliminary experiments in which cultures at 30°C resulted in an increased proportion of living germ cells as compared to 34 or 37°C. Control incubations without the addition of hormones were not performed in this study because of the limited quantity of testicular tissue that could be used in these experiments and because previous studies had shown clearly an improvement of both meiotic and postmeiotic in-vitro differentiation of human germ cells in the presence of FSH and testosterone (Tesarik et al., 1998a,b).
Figure 2. Cluster of human Sertoli cells processed for detection of DNA fragmentation by using TUNEL. TUNEL-positive nucleus (arrow), containing damaged DNA, shows intense green fluorescence and can be easily distinguished from TUNEL-negative nuclei (N) of healthy cells that remain unlabelled. Scale bar ⫽ 10 µm. nitrogen and warmed rapidly in a water bath at 20°C. The homogenized tissue was expelled from the straws into Gamete-100 medium, washed three times in the same medium and used in individual experiments. In-vitro culture Fresh and frozen testicular biopsy specimens were cultured in the dark at 30°C in Gamete-100 medium supplemented with 50 IU/l
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Identification of germ cells Parts of fresh and frozen–thawed testicular biopsy specimens were taken apart both before and after in-vitro culture. These samples were used for germ cell identification only. Unlike the rest of the specimens, they were placed in Gamete-100 medium supplemented with collagenase I (1000 IU/ml) and elastase (10 IU/ml) (both purchased from Sigma) and incubated at 37°C as described (Tesarik et al., 1998a) to achieve disintegration of cell clusters to single cells. The cell suspensions were then pelleted by centrifugation, smeared onto microscope slides and left to air-dry. At least eight smears were prepared for each experimental treatment group (fresh cells before culture, fresh cells after culture, cryopreserved cells before culture, and cryopreserved cells after culture) with cells from each patient. The smears were fixed with 5% glutaraldehyde in 0.05 mol/l cacodylate buffer (pH 7.4) and stored at –40°C for up to 3 weeks. For each type of cell treatment, two smears were subsequently processed for fluorescent in-situ hybridization (FISH) with markers for chromosomes 15 and 16 (Figure 1A), two were subjected to immunocytochemical detection of the germline marker proacrosin by using 4D4 monoclonal antibody (Escalier et al., 1991), and two were treated by a combination of both (Figure 1B). Details of these methods were described previously (Mendoza et al., 1996; Tesarik et al., 1998b). Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole. The distinction of individual developmental stages of germ cells was
Frozen germ cell culture
Table I. In-vitro differentiation of germ cells from fresh testicular biopsy specimens from nine patients with non-obstructive azoospermia
Table II. In-vitro differentiation of germ cells from frozen testicular biopsy specimens from nine patients with non-obstructive azoospermia
Patient Most advanced stage of spermatogenesis after different periods of no. culture
Patient Most advanced stage of spermatogenesis after different periods of culture no.a
0h
24 h
48 h
1 2 3 4 5
Sa spermatid Sa spermatid Primary spermatocyte Primary spermatocyte Primary spermatocyte
Sc spermatid Sc spermatid Sa spermatid Secondary spermatocyte Primary spermatocyte
6 7
Primary spermatocyte Primary spermatocyte
Secondary spermatocyte Primary spermatocyte
8 9
Primary spermatocyte Primary spermatocyte
Secondary spermatocyte Primary spermatocyte
Sd spermatid Sc spermatid Sc spermatid Sa spermatid Primary spermatocyte Sc spermatid Primary spermatocyte Sa spermatid Primary spermatocyte
Sa ⫽ round; Sc ⫽ elongating; Sd ⫽ elongated.
based on differences in their cell and nuclear size, on the pattern of their immunoreactivity with the anti-proacrosin antibody and on the determination of ploidy by FISH (Tesarik et al., 1998a,b).
0h
24 h
48 h
1 2 3 4
Sa spermatid Sa spermatid Primary spermatocyte Primary spermatocyte
Sc spermatid Sb spermatid Sa spermatid Secondary spermatocyte
5
Primary spermatocyte
Primary spermatocyte
6
Primary spermatocyte
Secondary spermatocyte
7
Primary spermatocyte
Primary spermatocyte
8
Primary spermatocyte
Secondary spermatocyte
9
Primary spermatocyte
Primary spermatocyte
Sc spermatid Sb spermatid Sa spermatid Secondary spermatocyte Primary spermatocyte Secondary spermatocyte Primary spermatocyte Secondary spermatocyte Primary spermatocyte
Sb ⫽ elongating; See Table I for other definitions. aThe same identification numbers indicate the same patients as in Table I.
Evaluation of Sertoli cell DNA damage DNA fragmentation in Sertoli cell nuclei (Figure 2) was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) using the in-situ Cell Death Detection KitFluorescence (Boehringer Mannheim, Mannheim, Germany) as described (Tesarik et al., 1998b).
Table III. Proportion of TUNEL-positive Sertoli cell nuclei before and after in-vitro culture of fresh and frozen testicular biopsy specimens from nine patients with non-obstructive azoospermia
Statistics Percentages of TUNEL-positive Sertoli cell nuclei in different types of preparation were compared by χ2 and Kruskal–Wallis tests.
Fresh (n ⫽ 9) Frozen (n ⫽ 9)
Results The examination of the nine original testicular biopsy samples from the patients with non-obstructive azoospermia (before their use in different experimental protocols) showed spermatogenesis arrest at the round spermatid stage in two cases and at the primary spermatocyte stage in seven cases. In fresh aliquots, spermatogenesis was resumed in vitro in both patients with the in-vivo arrest at the round spermatid stage and in four of the seven patients with the in-vivo arrest at the primary spermatocyte stage, and it progressed to different meiotic and postmeiotic stages in each individual case (Table I). In all patients whose fresh testicular biopsy aliquots showed in-vitro progression, spermatogenesis was also resumed during culture of frozen aliquots. However, progression of spermatogenesis was only detectable during the first 24 h of culture of frozen aliquots, and no additional maturation occurred during further incubation up to 48 h (Table II). Thus, the final stage of spermatogenesis achieved by the end of culture of the frozen aliquots (Table II) was less advanced as compared to that achieved in fresh aliquots (Table I). The percentage of primary spermatocytes that underwent meiosis in vitro and that of round spermatids that showed signs of in-vitro elongation was always very low and never exceeded 1% of the total germ cell
Table IV. Proportion of TUNEL-positive Sertoli cell nuclei before and after in-vitro culture of fresh and frozen testicular biopsy specimens from nine patients with obstructive azoospermia
Specimen
Specimen
Fresh (n ⫽ 5) Frozen (n ⫽ 5)
% TUNEL-positive nuclei (mean ⫾ SEM) Before culture
After culture
13 ⫾ 2 14 ⫾ 3
26 ⫾ 4 72 ⫾ 10
% TUNEL-positive nuclei (mean ⫾ SEM) Before culture
After culture
10 ⫾ 2 12 ⫾ 3
22 ⫾ 2 55 ⫾ 11
population. Thus, a quantitative comparison among individual samples was not possible. Because the beneficial effect of the high concentrations of FSH and testosterone in culture media on the in-vitro differentiation of human male germ cells is likely to be mediated by intact Sertoli cells present in the cultured samples (Tesarik et al., 1998b), the poor differentiation potential of germ cells from the frozen aliquots over incubation periods exceeding 24 h may have been due to a shorter survival of Sertoli cells from the frozen aliquots. To test this hypothesis, we compared the percentage of TUNEL-positive Sertoli cells in fresh and frozen testicular biopsy aliquots before and after culture (Table III). In spite of a slight increase in the percentage 1715
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of TUNEL-positive Sertoli cells during in-vitro culture of fresh aliquots (P ⬍ 0.01), the increase in the percentage of TUNELpositive Sertoli cells was much more pronounced after culture of their frozen counterparts (Table III). Similar findings were obtained with testicular biopsy specimens from the five patients with obstructive azoospermia (Table IV). Discussion The present results of in-vitro culture of fresh testicular biopsy specimens confirm the previous observation that germ cells from some, but not all, men with spontaneous in-vivo maturation arrest resume spermatogenesis during incubation with high concentrations of FSH and testosterone (Tesarik et al., 1999). As compared with the above study, the concentration of FSH used in the present study was higher (50 versus 25 IU/l). Although the FSH concentrations of 25 and 50 IU/l were equally effective in supporting in-vitro spermatogenesis in testicular biopsy samples from men with normal testicular function (Tesarik et al., 1998b), higher concentrations of this hormone may be needed for samples from men with spermatogenesis disorders, in whom FSH receptors may be partly desensitized by elevated concentrations of the endogenously produced hormone. The optimal FSH concentration for germ cell in-vitro culture in these cases and the possible relationship between this concentration and serum FSH concentrations in each patient remain to be determined. As to the frozen testicular biopsy aliquots, in-vitro resumption of spermatogenesis was detected in all those patients in whom spermatogenesis progressed during the culture of the fresh testicular biopsy counterparts. However, the final stage achieved at the end of the 48 h culture period was less advanced because germ cell differentiation, after the initial progression, ceased between the 24 and 48 h time points. This difference is likely to be related to the relatively poor survival of frozen–thawed Sertoli cells in culture as compared to the culture of fresh testicular biopsy specimens. Although germ cells may possess FSH receptors and be responsive to direct FSH action (Baccetti et al., 1998), the functional significance of these putative FSH receptors is not known, and FSH effects on germ cells are thus likely to be largely mediated by Sertoli cells (Griswold et al., 1993). Recent studies on in-vitro differentiation of human germ cells have also confirmed the dependence of the effects of FSH on meiosis and spermiogenesis on the presence of intact Sertoli cells (Tesarik et al., 1998a,b). However, the freezing and thawing protocol used in this study was developed for germ cells, not for Sertoli cells. The results of this study show that this protocol does not ensure adequate protection of Sertoli cells during the freezing and thawing procedures. Moreover, the cryopreservation procedure may also have produced some functional defects in germ cells through a direct action, without mediation of Sertoli cells. These effects may not have been manifest immediately after thawing but may have impaired the meiotic and postmeiotic differentiation of germ cells during the extended postthaw incubation period. Alternative protocols have also been suggested for cryopreservation of human male germ cells (Yamamoto et al., 1999). It remains to be determined whether 1716
the developmental potential of germ cells and the viability of Sertoli cells could be better preserved by using alternative freezing and thawing protocols as compared to that used in the present study. In practical terms, the present data suggest that IVF centres that have frozen testicular biopsy samples lacking spermatozoa with the use of one of the standard sperm cryopreservation methods may still hope to achieve a limited degree of germ cell differentiation by culturing thawed samples for 24 h. On the other hand, there is no additional benefit from prolonging the culture of such samples beyond 24 h. For future germ cell cryopreservation policies, it is strongly recommended to perform in-vitro culture for 48 h with fresh samples and to cryopreserve the cultured samples with the use of a standard sperm-freezing protocol. Our preliminary data (J.Tesarik, unpublished data) suggest excellent cryosurvival of cultured germ cells which, however, should be used for assisted reproduction immediately after thawing, without additional post-thaw culture. Reports on an ongoing pregnancy (Antinori et al., 1997) and birth (Gianaroli et al., 1999) after fertilization with cryopreserved spermatids show that the reproductive capacity of spermatids is not destroyed by the freezing and thawing procedures when these sperm precursor cells are cryopreserved shortly after recovery from the testis. Further study is needed to determine whether the same applies to spermatids resulting from in-vitro maturation of earlier germ cell stages. References Antinori, S., Versaci, C., Dani, G. et al. (1997) Successful fertilization and pregnancy after injection of frozen–thawed round spermatids into human oocytes. Hum. Reprod., 12, 554–556. Baccetti, B., Collodel, G., Costantino-Ceccarini, E. et al. (1998) Localization of human follicle-stimulating hormone in the testis. FASEB J., 12, 1045–1054. Escalier, D., Gallo, J.-M., Albert, M. et al. (1991) Human acrosome biogenesis: immunodetection of proacrosin in primary spermatocytes and of its partitioning pattern during meiosis. Development, 113, 779–788. Gianaroli, L., Selman, H.A., Magli, M.C. et al. (1999) Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue. Fertil. Steril., 72, 539–541. Griswold, M.D. (1993) Action of FSH on mammalian Sertoli cells. In Russel, L.D. and Griswold, M.D. (eds), The Sertoli Cell. Cache River Press, Clearwater, pp. 493–508. Mendoza, C., Benkhalifa, M., Cohen-Bacri, P. et al. (1996) Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridization for evaluation of human ejaculated germ cells. Zygote, 4, 279–283. Tesarik, J., Greco, E., Rienzi L. et al. (1998a) Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum. Reprod., 13, 2772–2781. Tesarik, J., Guido, M., Mendoza, C. et al. (1998b) Human spermatogenesis in vitro: respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis, and Sertoli cell apoptosis. J. Clin. Endocrinol. Metab., 83, 4467–4473. ¨ zcan, C. et al. (1999) Restoration of fertility by inTesarik, J., Bahceci, M., O vitro spermatogenesis. Lancet, 353, 555–556. Yamamoto, Y., Sofikitis, N. and Miyagawa, I. (1999) Ooplasmic injections of rabbit round spermatid nuclei or intact round spermatids from fresh, cryopreserved and cryostored samples. Hum. Reprod., 14, 1506–1515. Received on October 25, 1999; accepted on April 7, 2000
