1 Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France, 2 MAR&Gen, Molecular Assisted Reproduction and Genetics, calle Gracia 36, 18002 Granada, Spain, 3 American Hospital of Istanbul, Istanbul, Turkey, 4 University of Granada, Granada, Spain and 5 European Hospital, Rome, Italy
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Abstract |
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Key words: FSH/in-vitro spermatogenesis/maturation arrest/spermatid/spermatocyte
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Introduction |
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The recourse to in-vitro culture in an attempt to improve morphological and functional maturity of testicular germ cells has been encouraged by the pioneering work of Ian Craft's group (Craft et al., 1995; Zhu et al., 1997
) who demonstrated, for the first time, that the fertilizing ability of testicular spermatozoa can be improved by additional in-vitro maturation. The recent observation that round spermatids, and even primary spermatocytes, from some patients with maturation arrest can develop in vitro up to late elongated spermatids that can be used with success for assisted reproduction (Tesarik et al., 1999a
, b
) indicates that, under appropriate culture conditions (Tesarik et al., 1998a
, b
), a subpopulation of germ cells that are blocked in vivo at meiotic and postmeiotic stages of spermatogenesis can overcome the block and undergo further differentiation. However, germ cells from some men with in-vivo maturation arrest remain blocked under the same culture conditions that allow germ cells from other men with the same diagnosis to differentiate (Tesarik et al., 1999a
). No clinical or laboratory feature according to which the success or failure of germ cell in-vitro culture can be predicted is currently available.
In this study, the ability of germ cells from patients with maturation arrest to resume spermatogenesis in vitro was evaluated in relation to the stage at which spermatogenesis was arrested in vivo and to the serum FSH concentration.
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Materials and methods |
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Samples of testicular tissue were obtained by open testicular biopsy from multiple sites of both testes. Pieces of tissue were plunged immediately in modified Earle's balanced salt solution (ME) and minced with the use of two sterile microscope slides followed by repeated vigorous aspiration into a tuberculin syringe. ME was prepared by supplementing Earle's balanced salt solution (Sigma, St. Louis, MO, USA) with penicillin G (75 mg/l), pyruvic acid (11 mg/l), human serum albumin (10 mg/ml), and HEPES (5 ml of 1 mol/l HEPES solution added to 995 ml of medium). All the additives were from Sigma except for human serum albumin which was from Irvine Scientific (Irvine, CA, USA). All tissue samples from the same patient were mixed together, and large fibrotic pieces not containing seminiferous tubules were removed with forceps.
In the first series of cases (n = 8), each testicular biopsy sample was mechanically disintegrated, homogenized and divided into three parts. The first part was fixed immediately for the cytological and histological analysis (see below). The second part was cultured for 24 h at 30°C in ME medium supplemented with 25 mIU/ml recombinant human FSH (Puregon; Organon, Oss, The Netherlands). The choice of the culture temperature was based on preliminary experiments with testicular biopsy samples from men with non-obstructive azoospermia (n = 5), showing a significantly better germ cell survival at 30°C as compared with 34°C or 37°C, and on the previously published data on in-vitro differentiation of germ cells from men with normal spermatogenesis (Tesarik et al., 1998a, b
). The cell concentration in the culture tubes was approximately 1x106/ml. The third part was cultured under the same conditions but for the omission of FSH in culture medium to serve as a control. Each of the three parts was finally further divided into two equal specimens. One of them was treated for 1 h, at 37°C, with collagenase I (1000 units/ml) and elastase (10 units/ml) (Sigma), washed in phosphate-buffered saline (PBS), smeared on slides, fixed with 5% glutaraldehyde in 0.05 mol/l cacodylate buffer (pH 7.4) and analysed by immunocytochemistry with 4D4 monoclonal antibody against the germline marker proacrosin (Tesarik et al., 1998a
). Individual stages of germ cell development, including postzygotene primary spermatocytes, secondary spermatocytes, and normal (Sa, Sb1, Sb2, Sc and Sd) and abnormal (Saf, Sbp and Scp) spermatid forms were distinguished by using the previously described criteria (Tesarik et al., 1998a
, b
). The other specimen was fixed with Bouin's solution without any enzymatic treatment, embedded in paraffin, serially sectioned and stained with haematoxylin and eosin for histological evaluation. Primary and secondary spermatocytes and round (Sa) spermatids were distinguished according to the standard anatomopathological criteria based on the cell size, nuclear staining and the cell position within the seminiferous tubule. For microscopic evaluation, preparations were blinded so that the person performing the evaluation was not aware of the nature of individual preparations.
In the second series of cases (n = 25), the control incubations without FSH and the examinations of fresh and cultured tissues in histological preparations were omitted. All the other procedures, including tissue homogenization, in-vitro culture and preparation of smears for cytological analyses were the same as in the first series. In addition to the immunocytochemical visualization of the germline marker proacrosin (see above), at least two smear preparations from each patient and from each treatment group were processed, respectively, for Papanicolaou staining (World Health Organization, 1992), two-colour fluorescence in-situ hybridization (FISH) with probes for chromosomes 15 and 16 (D15Z1 and D16Z1 respectively) and one-colour FISH (probe D15Z1) combined with proacrosin immunocytochemistry (Tesarik et al., 1998b
). The use of FISH facilitated the distinction of meiotic stages of spermatogenesis, particularly those of secondary spermatocytes whose size and immunostaining pattern with anti-proacrosin monoclonal antibodies is similar to that of round spermatids.
In the third series of cases (n = 60), FISH was omitted, and the culture medium supplemented with 25 IU/l pure FSH (Serono, Rome, Italy) instead of recombinant FSH. All the other methods were the same in the three series.
Other cases in which late elongated spermatids or spermatozoa were unexpectedly found in fresh samples (n = 7) were excluded from further evaluation.
A blood sample was taken from each patient during the first consultation and serum FSH concentration was determined by immunoenzymology using a commercial kit (FSH-ACS:180; Chiron, Cergy-Pontoise, France; sensitivity threshold, 0.3 IU/l).
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Results |
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In the third series of cases, patients were subdivided into two subgroups according to the stage at which their spermatogenesis was blocked in vivo. From 31 patients with maturation arrest at the primary spermatocyte stage, spermatogenesis was resumed in vitro in seven cases (Table III). As in the second series, FSH concentrations in these patients (15.9 ± 4.1 IU/l) were significantly lower (P < 0.01) as compared with the remaining patients of this group (27.7 ± 4.3 IU/l).
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Discussion |
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Interestingly, early studies reporting in-vitro spermatogenesis in the rat (Parvinen et al., 1983; Toppari and Parvinen, 1985
) also used the culture of intact segments of seminiferous tubules. In contrast, the culture of isolated rat round spermatids on Sertoli cell monolayers did not lead to spermatid elongation even though the spermatidSertoli cell contact was re-established (Cameron and Muffly, 1991
). It is thus possible that the persistence of the initial Sertoli cellgerm cell contacts stimulates the production by Sertoli cells of humoral factors that can positively influence even those germ cells that are outside, but in the proximity of, the intact seminiferous tubule segments.
From the physiological point of view, the rapidity of spermatogenic events under the in-vitro conditions clearly represents an unusual situation. A similar rapid progression has previously been observed in testicular biopsy samples from men with obstructive azoospermia and normal spermatogenesis (Tesarik et al., 1998a, b
). This developmental acceleration may be explained by the action of presumptive hormone or growth factor antagonists that are active in situ but are diluted or inactivated after tissue removal from its natural context. Differences in the synthesis and activity of hormone and growth factor receptors may also exist between the in-vivo and in-vitro systems. For example, it has been shown that multiple promoter elements contribute to the activity of the FSH receptor gene in Sertoli cells, and the requirements for their activation are not the same in vivo and in vitro (Heckert et al., 1998
). Another hypothesis, suggesting that the suppression of a developmental checkpoint controlling the synchronization of individual events occurring during spermiogenesis may be involved in the in-vitro acceleration of germ cell differentiation, has been proposed (Tesarik et al., 1999b
). Further study is needed to elucidate the molecular events underlying these empirical findings.
The observation that immature germ cells whose development is blocked in vivo can eventually resume spermatogenesis in vitro is intriguing, and is complemented by the recent demonstration showing that some of the cultured germ cells can give rise to gametes with a full reproductive capacity (Tesarik et al., 1999a, b
). These observations raise a question about the mechanism of this unexpected improvement. In this study we show that the serum FSH concentration of those patients whose germ cells are capable of resuming spermatogenesis in vitro is significantly lower compared with patients whose germ cells remain blocked. In particular, FSH concentrations exceeding 20 IU/l predict poor prognosis, whereas concentrations between 10 and 20 IU/l are often associated with good in-vitro differentiation potential of germ cells. In-vitro differentiation of germ cells from men with normal spermatogenesis requires the supplementation of culture medium with relatively high (
25 IU/l) concentrations of FSH (Tesarik et al., 1998b
). It is possible that the concentration of FSH used in this study (25 IU/l), though effective in samples from men with normal spermatogenesis in whom serum FSH concentrations are low, is insufficient for men with high serum FSH concentrations which may lead to desensitization of FSH receptors on Sertoli cells. It remains to be determined whether the refractoriness of germ cells from men with elevated serum FSH concentrations can be overcome by increasing the concentration of FSH in culture medium.
It is also possible that, as compared with this study in which testicular biopsy samples were cultured for 24 h only, better results might have been obtained if a longer incubation time had been used, or if the FSH effect had been potentiated by simultaneous addition of testosterone to the culture medium as described previously (Tesarik et al., 1998b). The first human birth after transmeiotic in-vitro differentiation of primary spermatocytes into elongated spermatids has been reported recently after culture of testicular biopsy samples in medium supplemented with both FSH and testosterone for 48 h (Tesarik et al., 1999a
). It is thus possible that improved culture conditions would lead to in-vitro differentiation of germ cells, even in some of those cases in which no progression of spermatogenesis was observed under the conditions used in this study. This possibility is currently being evaluated.
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Notes |
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References |
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Submitted on December 22, 1999; accepted on February 23, 2000.