Germ cell apoptosis in men with complete and incomplete spermiogenesis failure

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1 Molecular Human Reproduction vol.4 no.8 pp , 1998 Germ cell apoptosis in men with complete and incomplete spermiogenesis failure Jan Tesarik 1,4, Ermanno Greco 2, Paul Cohen-Bacrie 1 and Carmen Mendoza 3 1 Laboratoire d Eylau, 55 Rue Saint-Didier, Paris, France, 2 Centre of Reproductive Medicine, European Hospital, Via Portuense 700, Rome, Italy, and 3 Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Campus Universitario Fuentenueva, Granada, Spain 4 To whom correspondence should be addressed at the Laboratoire d Eylau, 55 Rue Saint-Didier, Paris, France Germ cell apoptosis was evaluated in 11 men suffering from nonobstructive azoospermia and enrolled in a spermatid conception programme. In six of these patients, round spermatids (Sa stage) were the most advanced spermatogenic cells recovered from testicular biopsy samples. This condition is referred to as complete spermiogenesis failure. In the remaining five men, a few late elongated spermatids (Sd stage) were unexpectedly found in the testicular biopsy samples on the day of treatment. This condition is referred to as incomplete spermiogenesis failure. Germ cell apoptosis in both groups of patients was examined by analysing cell smears prepared from mechanically disintegrated testicular tissues using terminal deoxyribonucleotidyl transferase-mediated dutp nick-end labelling (TUNEL), which detects apoptosis-specific DNA fragmentation, and annexin-v binding, detecting apoptosis-related translocation of plasma membrane phosphatidylserine to the membrane s outer surface. Both methods were combined, in double-fluorescence labelling preparations, with immunocytochemical detection of proacrosin, a specific germline marker. Patients with complete spermiogenesis failure had significantly higher frequencies of primary spermatocytes and round spermatids carrying the apoptosis-specific DNA damage in comparison with patients with incomplete spermiogenesis failure. Surprisingly, apoptosis-related phosphatidylserine externalization occurs rarely until the advanced stages of spermiogenesis. Since externalized phosphatidylserine is expected to be involved in the recognition of apoptotic cells by phagocytes, apoptotic spermatocytes and round spermatids may not be removed easily by phagocytosis. The high frequency of DNA damage in round spermatids from patients with complete spermiogenesis failure explains the low success rates of spermatid conception in these cases. The evaluation of apoptosis can help predict success rates of spermatid conception. Key words: apoptosis/germ cells/spermatid conception/spermiogenesis failure/tunel Introduction The first reports on human pregnancies and births after fertilization of oocytes with round (Tesarik et al., 1995, 1996) and elongated (Fishel et al., 1995, 1996) spermatids were followed by a series of papers describing round spermatid injection (ROSI) and elongated spermatid injection (ELSI) treatment cycles with highly variable results (Mansour et al., 1996; Amer et al., 1997; Antinori et al., 1997; Araki et al., 1997; Vanderzwalmen et al., 1997; Yamanaka et al., 1997). At the same time, the original indication, which was nonobstructive azoospermia with previous detection of mature spermatozoa in the ejaculate and with the unexpected sperm absence on the day of treatment (Tesarik et al., 1995), was extended to cases in which sperm production could never be detected in the patient s history; these situations are collectively termed complete spermiogenesis failure (Amer et al., 1997). Recently, two independent studies (Amer et al., 1997; Vanderzwalmen et al., 1997) have shown that ROSI success rates are considerably lower in cases of complete spermiogenesis failure as compared to incomplete spermiogenesis failure, where a few late elongated spermatids or spermatozoa can also be detected in testicular biopsy samples, or to azoospermia alternating with severe oligozoospermia. The causes of complete spermiogenesis failure are unknown. However, similar testicular pathologies are known from animal models (reviewed in Tesarik et al., 1998). These include the withdrawal of some developmentally important ligands, such as testosterone (Troiano et al., 1994; O Donnell et al., 1996) or vitamin A (Eskild and Hansson, 1994), mutations of the receptors with which these ligands and their metabolites can act, such as the retinoic acid receptor α (Akmal et al., 1997) or the retinoid X receptor β (Kastner et al., 1996), alterations of molecules involved in signal transduction pathways downstream of receptors, such as camp-responsive element modulator (CREM) protein (Blendy et al, 1996; Nantel et al., 1996), or mutations of components of cell repair systems, such as the HR6B ubiquitin-conjugating DNA repair enzyme (Roest et al., 1996). Such conditions are often associated with germ cell apoptosis (Sassone-Corsi, 1997). Recently, reduced expression of CREM was also detected in patients with predominant round spermatid maturation arrest in comparison with men with normal spermatogenesis or with mixed testicular atrophy (Weinbauer et al., 1998), and increased apoptosis of European Society for Human Reproduction and Embryology 757

2 J.Tesarik et al. testicular cells has been demonstrated in patients with abnormal spermatogenesis (Lin et al., 1997). However, the identification of specific cell types undergoing apoptosis was not possible in the latter study (Lin et al., 1997). It can thus be postulated that the low efficacy of ROSI in cases of complete spermiogenesis failure (Amer et al., 1997; Vanderzwalmen et al., 1997) is due to the activation of apoptosis-promoting mechanisms similar to those operating in the experimental models of spermiogenesis arrest. To test this hypothesis, we undertook this study in which the frequency of apoptotic germ cells was evaluated in a group of patients with complete spermiogenesis failure and compared with another group of azoospermic patients in whom spermiogenesis failure was incomplete. These evaluations were made with the use of two kinds of complementary methodological approaches for the assessment of apoptosis, detecting the presence of apoptosis-specific DNA damage and the typical translocation of plasma membrane phospholipids, respectively. Each of these methods was coupled with the use of a specific germline marker to distinguish specific stages of germ cell development from non-germ testicular cells. Materials and methods Patients Germ cells used in this study were obtained from six men suffering from complete spermiogenesis failure and from five men with incomplete spermiogenesis failure. All these patients were azoospermic and were undergoing an assisted reproduction attempt during which open testicular biopsy was performed for sperm or spermatid recovery. All of them had a normal karyotype. Late elongated spermatids (Sd) and spermatozoa were found in the five men with incomplete spermiogenesis failure in whom ICSI was subsequently performed. In the six patients with complete spermiogenesis failure, only round spermatids (Sa) were recovered and used for ROSI. Serum follicle stimulating hormone (FSH) concentrations (mean SEM) in the incomplete and the complete spermiogenesis failure group were and IU/l respectively (not significant). Serum testosterone concentrations in the two groups were and nmol/l respectively (not significant). The terminology used for individual stages of spermatid development is that introduced by de Kretser and Kerr (1988) and recommended by Tesarik (1997) for the use in therapeutic ROSI and ELSI cycles. After the ROSI or ICSI procedure, half of the remaining testicular biopsy sample was frozen for eventual future assisted reproduction attempts. The other half was used for the examinations described in this study. These examinations were performed with the patient s informed consent and made part of the complex diagnostic setup to be used for the prediction of the chances of success of eventual future therapeutic attempts. Recovery and preparation of testicular cells Testicular tissue was sampled by open testicular biopsy from multiple sites of both testes. Tissue samples were put in Gamete-100 medium (Scandinavian IVF Science AB, Gothenburg, Sweden). Within 5 min after biopsy, they were disintegrated by stretching between two microscope slides followed by repeated aspiration with a 1 ml sterile plastic syringe. After the removal of large tissue pieces that could not be disintegrated mechanically, testicular cell suspensions were pelleted by centrifugation (500 g, 10 min), resuspended in 10 ml of protein-free phosphate-buffered saline (PBS) and pelleted again. 758 Aliquots (2 µl) were then smeared onto microscope slides, fixed with 5% glutaraldehyde in 0.05 M cacodylate buffer (ph 7.4) and kept at 40 C for a maximum of 10 days until further processed for the detection of DNA fragmentation. The rest of the testicular cell pellets, prepared as above, was used immediately for the evaluation of plasma membrane phosphatidylserine externalization. Details of these methods are given below. Detection of DNA fragmentation To detect the presence of apoptosis-specific DNA strand breaks, fixed smears of testicular cells (see above) were processed for terminal deoxynucleotidyltransferase-mediated dutp nick-end labelling (TUNEL) using a Cell Death Detection Kit (Boehringer, Mannheim, Germany) containing fluorescein isothiocyanate (FITC)- labelled dutp. This method adds the FITC-labelled nucleotides to the exposed ends of multiple DNA fragments resulting from the apoptosis-induced internucleosomal DNA breakage; this generates an in-situ fluorescent signal in the respective nuclei. Preliminary experiments were carried out according to the manufacturer s instructions for use, including supravital staining of cell suspensions with propidium iodide to distinguish between apoptotic and necrotic cells. These preliminary experiments showed that, under the conditions of this study, many dead cells (staining positive with propidium iodide) did not generate a detectable TUNEL fluorescent signal (necrotic cells), whereas most cells with the TUNEL signal were able to exclude the supravital dye (apoptotic cells). This observation was in agreement with a previous report on experimental germ cell apoptosis and necrosis in the rat (Sinha Hikim et al., 1997) and probably reflected the size difference between DNA fragments resulting from apoptosis and those resulting from necrosis (see Discussion). In fact, the apoptosis-related internucleosomal DNA fragmentation yields much smaller fragments as compared with nonspecific DNA fragmentation in necrotic cells, leading to a much higher availability of DNA sites available for the end-labelling. The supravital staining was thus left out in further experiments and was replaced with immunocytochemical detection of a germline marker (see below). Control incubations were carried out with incomplete reaction mixture in which terminal deoxyribonucleotidyltransferase was not included. Detection of plasma membrane phosphatidylserine externalization The apoptosis-associated translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane was assessed with the use of FITC-labelled annexin V, a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine. This examination was performed with native testicular cell suspensions using the Annexin-V-FLUOS Staining Kit (Boehringer Mannheim) and the procedure recommended by the manufacturer. Part of the testicular cell suspension from each patient was stained with incomplete staining solution in which propidium iodide (cell viability stain) was left out. These preparations were subsequently used in double-fluorescence experiments including a specific germline marker (see below). After incubation with the reaction mixture, testicular cells were smeared onto microscope slides and either evaluated immediately in a fluorescence microscope or fixed with 4% depolymerized paraformaldehyde and stored at 40 C for further processing. Identification of germ cells in fluorochrome-labelled testicular cell preparations To distinguish between germ and non-germ cells, testicular cell preparations, previously processed by one of the above methods for

3 Germ cell apoptosis the detection of apoptosis (excluding the viability assessment stain), were counterstained with 4D4 monoclonal antibody against human proacrosin (BioMérieux, Marcy l Etoile, France). This antibody recognizes proacrosin located within the Golgi apparatus and in the developing acrosomal granules as early as the primary spermatocyte at pachytene of the first meiotic division (Escalier et al., 1991; Mendoza et al., 1996). After the completion of the apoptotic cell staining procedure, smears were incubated, sequentially, with 4D4 antbody and with tetramethylrhodamine isothiocyanate (TRITC)- labelled anti-mouse immunoglobulin G (IgG; Sigma, St Louis, MO, USA) as described (Mendoza et al., 1996). Control slides were incubated with the secondary antibody only. Amongst 4D4-positive cells, primary spermatocytes, secondary spermatocytes and round spermatids were distinguished according to their respective nuclear size (Mendoza et al., 1996). Statistical analysis When possible, 100 germ cells at each developmental stage studied were analysed for each patient. However, the numbers of cells available were sometimes lower for some stages and some patients (see Results). The frequency of germ cells showing apoptotic changes in patients with complete and incomplete spermiogenesis failure was compared by analysis of variance and χ 2 test using StatView II (Abacus Concepts, Berkeley, CA, USA) statistical package. Results DNA fragmentation Germ cells presenting apoptotic DNA fragmentation were clearly recognizable because of their bright FITC-fluorescence signal in their nuclei and because of their TRITC-fluorescence signal in developing acrosomal structures (Figures 1 and 2). For quantitative evaluation, spermatogonia were not included because of the unavailability of a reliable cytological marker to distinguish those cells from other cell types. For the same reason, the category of primary spermatocytes only involves spermatocytes from the pachytene stage onwards, since earlier primary spermatocytes cannot be distinguished with the use of the 4D4 monoclonal antibody. Secondary spermatocytes were not included because of the very low numbers of these cells in testicular biopsy samples. For the same reason, Sb and Sc elongating spermatids were grouped together. Accordingly, in samples from patients with incomplete spermiogenesis failure, the frequency of germ cells with apoptotic DNA damage was evaluated in four stage-categories, namely postzygotene primary spermatocytes, Sa round spermatids, Sb Sc elongating spermatids and Sd elongated spermatids (Table I). The latter stage is morphologically identical with spermatozoa at this level of observation. The same staging was also applied to samples from patients with complete spermiogenesis failure where, however, elongating (Sb, Sc) and elongated (Sd) spermatids were lacking, in agreement with the definition of this condition (Table II). When comparing the frequency of germ cells carrying apoptotic DNA damage in the two stage-categories that are present in both groups of patients (Tables I and II), it is apparent that apoptosis was more frequent both in primary spermatocytes (P 0.01) and Sa round spermatids (P 0.01) recovered from patients with complete spermiogenesis failure than in those with incomplete spermiogenesis failure. Figure 1. Paired fluorescence micrographs showing an apoptotic round spermatid (Sa). (A) TUNEL reaction resulting in a diffuse fluorescent signal in the spermatid nucleus (N). (B) Immunofluorescence with the germline marker 4D4 antibody giving a positive signal in the developing acrosomal granule (arrow). Bar 5 µm. Plasma membrane phosphatidylserine externalization Even though phosphatidylserine externalization was occasionally present in round testicular cells, these cells were only rarely identified as germline cells. In fact, the frequency of annexin-v-positive primary spermatocytes and Sa round spermatids was very low both in samples from patients with incomplete spermiogenesis failure (Table III) and in those with complete spermiogenesis failure (Table IV). In contrast, the frequency of annexin-v-positive cells was higher in Sb Sc elongating spermatids and, particularly, in Sd elongated spermatids (Figure 3) in this group of patients (Table III). Discussion Apoptosis is a genetically programmed and physiological mode of cell death, which is important for normal tissue differentiation and remodelling. This is also true for the male germline as demonstrated by the development of a highly abnormal pattern of adult spermatogenesis and sterility in transgenic mice expressing high levels of the anti-apoptotic BclxL or Bcl2 proteins (Rodriguez et al., 1997). In pathological situations, apoptosis serves to remove those germ cells whose further differentiation has been arrested because of an intrinsic deficiency, such as the absence of CREM, a transcriptional 759

4 J.Tesarik et al. Table II. Occurrence of apoptosis-specific DNA fragmentation amongst germ cells from patients with complete spermiogenesis failure a Fraction (%) of germ cells with DNA fragmentation a Patient no. Primary spermatocytes Sa spermatids 1 96/100 (96) 60/64 (94) 2 85/100 (85) 59/74 (80) 3 98/100 (98) 46/52 (88) 4 48/100 (48) 27/57 (47) 5 87/100 (87) 39/63 (62) 6 92/100 (92) 42/55 (76) a In agreement with the definition of complete spermiogenesis failure, Sb Sd (elongating and elongated) spermatids were absent in the samples. b Where available, 100 cells were evaluated for each patient and each stage. Sa round spermatids. Table III. Occurrence of apoptosis-specific phosphatidylserine externalization amongst germ cells from patients with incomplete spermiogenesis failure Fraction (%) of germ cells with phosphatidylserine externalization a Figure 2. Paired fluorescence micrographs showing two elongating spermatids (Sc), one of which is apoptotic and the other is healthy. (A) TUNEL reaction resulting in an intense fluorescent signal in the nucleus of the apoptotic spermatid (large arrow), whereas the healthy spermatid (small arrow) is not labelled. (B) Immunofluorescence with the germline marker 4D4 antibody giving a positive signal in the acrosomal granule of both spermatids (arrowheads). Bar 10 µm. Table I. Occurrence of apoptosis-specific DNA fragmentation amongst germ cells from patients with incomplete spermiogenesis failure Primary Sa Sb Sc Sd Patient no. spermatocytes spermatids spermatids spermatids 1 2/100 (2) 1/48 (2) 4/40 (10) 42/78 (54) 2 1/100 (1) 0/29 (0) 5/32 (16) 18/36 (50) 3 0/100 (0) 1/43 (2) 4/29 (14) 14/29 (48) 4 5/100 (5) 2/24 (8) 1/33 (3) 13/45 (29) 5 2/100 (2) 0/36 (0) 1/28 (4) 12/60 (20) a Where available, 100 cells were evaluated for each patient and each stage. Sa round spermatids; Sb elongating spermatids; Sc elongating spermatids; Sd late elongated spermatids. Fraction (%) of germ cells with DNA fragmentation a Primary Sa Sb Sc Sd Patient no. spermatocytes spermatids spermatids spermatids 1 33/100 (33) 10/100 (10) 3/28 (11) 12/84 (14) 2 26/100 (26) 21/100 (21) 5/25 (20) 2/33 (6) 3 49/100 (49) 22/100 (22) 3/13 (23) 15/45 (33) 4 15/100 (15) 18/100 (18) 5/37 (14) 8/41 (20) 5 22/100 (22) 14/100 (14) 3/20 (15) 5/53 (9) a Where available, 100 cells were evaluated for each patient and each stage. Sa round spermatids; Sb elongating spermatids; Sc elongating spermatids; Sd late elongated spermatids. regulator responsive to the camp signalling pathway (Sassone- Corsi, 1997), or because of the lack appropriate external stimuli, such as post-hypophysectomy gonadotrophin deficiency, the suppression of pituitary gonadotrophins by gonadotrophinreleasing hormone (GnRH) agonist treatment or immunoneutralization of FSH (Tapanainen et al., 1993; Shetty et al., 760 Table IV. Occurrence of apoptosis-specific phosphatidylserine externalization amongst germ cells from patients with complete spermiogenesis failure a Patient no. Fraction (%) of germ cells with phosphatidylserine externalization b Primary spermatocytes Sa spermatids 1 3/100 (3) 0/16 (0) 2 4/100 (4) 0/23 (0) 3 0/100 (0) 0/17 (0) 4 1/100 (1) 3/32 (9) 5 2/100 (2) 0/19 (0) 6 3/100 (3) 1/15 (7) a In agreement with the definition of complete spermiogenesis failure, Sb-Sd spermatids were absent in the samples. b Where available, 100 cells were evaluated for each patient and each stage. Sa round spermatids; Sb elongating spermatids; Sc elongating spermatids; Sd late elongated spermatids.

5 Germ cell apoptosis Figure 3. Paired fluorescence micrographs showing a round spermatid (Sa) and an elongated spermatid (Sd). (A) Annexin-V binding reveals phosphatidylserine externalization in the Sd elongated spermatid (large arrow) but not in the Sa round spermatid (small arrow). (B) Immunofluorescence with the germline marker 4D4 antibody giving a positive signal in the developing acrosomal granule of the Sa round spermatid (arrowhead) and a more diffuse reaction in the Sd elongated spermatid, possibly resulting from apoptotic damage to the acrosome leading to proacrosin leakage. Bar 10 µm. 1996; Sinha Hikim et al., 1997). By analogy, the removal of arrested germ cells in human spermatogenic arrest can also be expected to employ apoptosis. Apoptosis involves the stimulation of a complex cell signalling cascade, which ultimately leads to the activation of interleukin 1β-converting enzyme-like, aspartate-specific cystein proteases (caspases), one of which (caspase 3) activates an apoptosis-specific DNase (CAD) responsible for the characteristic internucleosomal chromatin cleavage observed in apoptotic cell nuclei (Enari et al., 1998; Sakahira et al., 1998). Terminal deoxynucleotidyltransferase-mediated addition of labelled dutp to free 3 -ends of the resulting DNA fragments in the TUNEL reaction can reveal this kind of DNA damage. The specificity of CAD, which is activated exclusively in apoptosis (Enari et al., 1998; Sakahira et al., 1998), explains why TUNEL positivity is rarely seen in necrotic cells in which DNA fragments, resulting from the action of non-specific DNases, are larger and less numerous, thus exposing less free DNA 3 -ends available for the reaction. In fact, detectable TUNEL positivity was not associated either with necrotic human germ cells from patients suffering from nonobstructive azoospermia (this study) or with necrotic rat germ cells recovered from animals exposed to cadmium, a known germ cell toxicant (Sinha Hikim et al., 1997). Our results thus confirm the working hypothesis that apoptosis is involved in the removal of arrested germ cells from the testis of patients with spermatogenic disorders. The degree of spermatocyte and spermatid DNA fragmentation in the group of patients with incomplete spermiogenesis failure (this study) appears higher as compared to men with normal spermatogenesis (Sinha Hikim et al., 1998). In addition to DNA fragmentation, apoptotic cells also undergo a rearrangement of plasma membrane lipids, leading to translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer, probably as a result of disintegration of plasma membrane cytoskeleton that, in healthy cells, stabilizes membrane structure by connecting plasma membrane components to the cellular interior (reviewed in van Engeland et al., 1997). It was suggested recently that this plasma membrane modification may serve to mark apoptotic cells for subsequent recognition and removal by the phagocytotic machinery (van Engeland et al., 1997). Here we show that phosphatidylserine externalization is a rare phenomenon in human primary spermatocytes and round spermatids, even when these cells come from patients in whom the TUNEL assay shows high degrees of apoptosis at these stages of spermatogenesis. By contrast, frequencies of cells showing phosphatidylserine externalization increased with spermatid elongation in patients with incomplete spermiogenesis failure, reaching a maximum in late elongated spermatids. If phosphatidylserine externalization is instrumental in phagocytosis of apoptotic cells, the absence of this phenomenon in primary spermatocytes and round spermatids is likely to compromise the phagocytotic removal of apoptotic germ cells at these stages. This can partly explain the frequent finding of spermatocytes and round spermatids in the ejaculate of patients suffering from nonobstructive azoospermia (Mendoza and Tesarik, 1996; Mendoza et al., 1996). In fact, a very small testicular output of spermatozoa may not be sufficient for the spermatozoa to appear in the ejaculate because of phagocytosis during epididymal transit, whereas the same small output of spermatids and spermatocytes, less readily recognized by phagocytes, may allow some of these cells to reach the ejaculate. These findings are important for the eventual use of spermatids and, in a longer perspective, of spermatocytes for assisted reproduction. The present results show that apoptotic DNA damage is more frequent in spermatids and spermatocytes from patients with complete spermiogenesis failure as compared to patients in whom spermiogenesis failure is incomplete. Moreover, spermatids carrying this DNA damage cannot be distinguished from healthy spermatids by any of the available viability tests. The risk of injecting an apoptotic germ cell into the spouse s oocyte is thus high in these cases. There is no reason, a priori, why the apoptotic DNA damage should be responsible for fertilization failure. However, it is hardly compatible with ongoing embryonic development. This is in agreement with the poor embryo implantation rate after transfer of embryos originating from oocytes fertilized with round spermatids from patients with complete spermiogenesis failure (Amer et al., 1997). On the other hand, the activation of caspases, which is required for the activation of the apoptosis-specific DNase (see above), can also lead to proteolytic degradation of oocyte-activating factor(s) in the spermatid cytoplasm, either directly or indirectly via the activation of 761

6 J.Tesarik et al. another protease. This may be behind the poor ability of spermatids from patients with complete spermiogenesis failure to activate human oocytes (Tesarik et al., 1998), in comparison with the performance of round spermatids recovered from men with normal spermatogenesis (Sousa et al., 1996). Poor fertilization rates with the use of spermatids from patients with complete spermiogenesis failure have, indeed, been reported (Vanderzwalmen et al., 1997). In conclusion, apoptosis is a frequent phenomenon in germ cells from patients with spermatogenic disorders, especially in those suffering from complete spermiogenesis failure. Because a reliable clinical and histopathological diagnosis of complete spermiogenesis failure is not available, mainly because of the possible persistence of small foci of complete spermatogenesis that may not be sampled during a diagnostic biopsy and whose output may not be sufficient for spermatozoa to reach the ejaculate, a direct evaluation of germ cell apoptosis offers an easier and more straightforward approach. One of the methods described in this study, combining a standard TUNEL assay with immunodetection of a specific germline marker, has the advantage of being applicable both to cell smears prepared from disintegrated testicular tissue and to ejaculated cells. The latter might provide a preferrable screening method with which preliminary information can be obtained without the need for an invasive sampling technique. References Akmal, K.M., Dufour, J.M. and Kim, K.H. 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