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Apoptosis Is Physiologically Restricted to a Specialized Cytoplasmic Compartment in Rat Spermatids

^a Department of Cell Biology, School of Medicine, Valladolid University, 47005 Valladolid, Spain

	ABSTRACT

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Cytoplasmic caudal tags of maturing spermatids condense and are detached from the spermatidal cells just before the spermatids are released as spermatozoa. The detached cytoplasmic masses are termed "residual bodies." Features of residual bodies seem to be compatible with those of apoptosis and, just as occurs with apoptotic bodies, residual bodies are phagocytosed by Sertoli cells. Since in vitro studies have demonstrated that nucleus and cytoplasm apoptosis events can be independent phenomena, we reasoned that apoptosis pathways might be restricted to the caudal tag of the maturing spermatids in order to originate residual bodies. Consistent with this idea, here we showed that annexin V specifically bound the membranes of isolated residual bodies and that expression levels of caspase-1, c-jun, p53, and p21 were specifically increased in these cytoplasmic compartments. Electron microscopy of cytoplasmic lobes and residual bodies confirmed that their ultrastructural features were those of apoptosis. These data indicate that the mechanism responsible for the formation of residual bodies is similar to that for apoptotic bodies; and the study presents evidence, for the first time, that apoptotic signaling molecules can be restricted to a cytoplasmic compartment and proceed in the presence of a healthy nucleus.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Spermatogenesis in mammals is a precisely controlled and cyclical timed process comprising mitotic proliferation of spermatogonia, meiotic divisions of spermatocytes, and the maturation and differentiation of haploid spermatids. In the rat, the spermatogenic cycle involves 14 (I–XIV) stages, each composed of a characteristic cellular association of different progenies of germ cells at a certain step of development [1]. Spermatid differentiation or spermiogenesis takes place through 19 steps, ending at stage VIII of the spermatogenic cycle, at which early step 8 and mature step 19 spermatids coexist. Just before step 19 spermatids pass down the seminiferous tubules as free spermatozoa, they each cast off a small mass of cytoplasm from their caudal tags to which Regaud [2] gave the term "residual body." At stage VIII, residual bodies coalesce along the tubule lumen beneath step 19 spermatid nuclei but, after spermiation, they are engulfed by Sertoli cells. The presence of residual bodies, distributed through the seminiferous epithelium, is a major event characterizing the next stage of the cycle, i.e., stage IX, at which they appear as highly basophilic bodies of different sizes within the epithelium. Thus, as occurs with programmed cell death taking place during embryo development, the generation of residual bodies during spermatogenesis is predictable in both place and time. In addition, it is well known that residual bodies display a dense concentration of organelles [3–5] and that they are phagocytosed by the Sertoli cells [3], which also phagocytose apoptotic germ cells [6–9]. Basophilia, high condensation of organelles, and phagocytosis by Sertoli cells are all consistent with the characteristic features of apoptotic bodies [10].

Since recent in vitro studies have demonstrated that apoptotic events occurring in the nucleus and in the cytoplasm can be independent phenomena [11–13], we reasoned that in some particular cases, apoptotic pathways might be restricted to a specialized cytoplasmic compartment. Thus, apoptosis might be the mechanism responsible for the formation of residual bodies from the highly specialized region constituting the caudal cytoplasm of maturing spermatids.

Consistent with this idea, in this study we provide evidence that 1) the membranes of isolated residual bodies are specifically labeled with annexin V, a Ca²⁺-dependent phospholipid-binding protein with high affinity for phosphatidylserine, exposed on the surface of apoptotic cells [14]; 2) ICE (caspase-1) antibody specifically stains residual bodies, indicating an accumulation of this protease, which is involved in some forms of apoptosis [15–17]; 3) the expression level of some proteins involved in apoptosis regulation, such as c-jun, p53, and p21 [18–21], gradually increases in the caudal cytoplasmic compartment of maturing spermatids and is maximal within residual bodies. Moreover, electron microscopy confirmed that the ultrastructural features of these bodies are compatible with those of apoptotic bodies. All these data indicate that a specialized region of cytoplasm, the caudal tags of the maturing spermatids, can undergo apoptosis and form apoptotic bodies, the so-called residual bodies, while the cell remains healthy and releases as a free spermatozoon.

	MATERIALS AND METHODS

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Separation and Morphological Characterization of Residual Bodies

Ten adult male Wistar rats (about 3 mo old), weighing 250–300 g and housed under conventional, controlled standard conditions, were used in this part of the study. The animals were anesthetized with sodium pentobarbital, and the testis contents were gently expressed through an incision in the tunica albuginea. Approximately 2.5 g of testis tissues, suspended in 25 ml of 0.01 M PBS (pH 7.2) containing 0.1% glucose and 3 mM lactate (PBSGL), was incubated with 100 U/ml collagenase H (Boehringer Mannheim GmbH, Mannheim, Germany) for 20 min at 33°C in a shaker water bath (150 counts/min). To isolate the dispersed seminiferous tubules, they were allowed to sediment for 3–4 min and the supernatant was decanted. This process was repeated three times to ensure removal of the dissociated interstitial tissue and blood cells. The seminiferous tubules were then incubated in 25 ml PBSGL containing 0.25 mg/ml trypsin (Sigma Chemical Co., St. Louis, MO) for 20 min (33°C). The cells were dispersed by gently pipetting. The suspension was diluted to approximately 40 ml with PBSGL and was filtered through a 30-mm nylon mesh. The cells were pelleted by centrifugation (800 x g, 10 min), washed (2 times), and resuspended in 40 ml PBSGL. The cell suspension was pelleted at 200 x g for 3 min. Supernatants were collected and, after gently pipetting, pelleted again at 200 x g for 3 min (2 times). The resulting suspensions containing an enriched fraction of residual bodies (F1), as well as the pellets, containing most of the cells (F2), were retained. Slides were prepared, and cell populations were determined microscopically after staining with cresyl violet. Cellular viability was determined by trypan blue exclusion.

Annexin-V Assays

Annexin-V-biotin (Boehringer Mannheim GmbH) was used as recommended to detect the presence of phosphatidylserine in the outer layer of the plasma membrane of isolated residual bodies. In brief, fraction F1 was pelleted by centrifugation (800 x g, 10 min), and pelleted cells from F1 and F2 were resuspended in 20 ml PBS. Then 1 ml F1, 1 ml F2, and 0.5 ml F1 + 0.5 ml F2 were separately resuspended in annexin-V-biotin labeling solution and incubated for 30 min at room temperature. After washing in incubation buffer, the cells were incubated for 30 min in avidin and biotinylated horseradish peroxidase (ABC reagents; Santa Cruz Biotechnology, Santa Cruz, CA). Annexin-V-biotin binding was revealed after subsequent detection of the enzyme activity using diaminobenzidine as substrate for 10 min at room temperature.

Tissue Preparation

Five additional adult male Wistar rats were used in this part of the study. After the animals were anesthetized, the tunica albuginea of the right testis was incised from the proximal to the distal pole, and the parenchyma was immersion fixed with 10% formaldehyde in 0.1 M phosphate buffer, pH 7.4. The thoracic aorta was then cannulated and the vasculature flushed with buffered sodium chloride before perfusion fixation of the left testis with 2% glutaraldehyde and 2% paraformaldehyde in 0.05 M phosphate buffer, pH 7.4.

Immunohistochemistry

The immersion-fixed right testes were sliced transversely into approximately 3-mm-thick slabs and processed for paraffin embedding. Consecutive orthogonal sections (5 µm thick) across the seminiferous tubules were mounted in polylysine-coated slides. The first section of each set of sections was stained with periodic acid-Schiff and used to determine tubule stages. The remaining sections were further processed for immunocytochemical staining with an ABC-based method, using the Santa Cruz immunoperoxidase staining kits (ABC reagents; Santa Cruz Biotechnology) as recommended. The endogenous peroxidase was quenched by 15-min incubation in 2% hydrogen peroxide in PBS. Unmasking of the epitope was carried out by boiling deparaffinized rehydrated sections for 10 min (2 times, 5 min each) in 10 mM citrate buffer, pH 6.0, using a microwave oven at 500 W power output. To optimize p53 detection, sections were subsequently incubated for 5 min with 0.5 mg/ml trypsin (Sigma) in 0.05% CaCl₂, pH 7.8, at 37°C prior to the antibody reaction. Primary antibodies were as follows: anti-p53 monoclonal antibody (1:10) directed to residues 212–217 of human p53 and 206–211 of mouse p53 (Calbiochem, La Jolla, CA); anti-c-Jun polyclonal antibody (1:150), epitope corresponding to amino acids 91–105 of mouse c-Jun; anti-p21 polyclonal antibody (1:400) directed to amino acids 125–143 of mouse p21; and anti-ICE p20 polyclonal antibody (1:500) directed to the carboxy terminus of the p20 subunit (SC-045-G, SC-471-G, SC-1218, respectively; Santa Cruz Biotechnology). Negative controls were processed in an identical manner except that the primary antibody was replaced by PBS.

Electron Microscopy

The fixed left testes were removed and sliced transversely into approximately 1-mm-thick slabs. Slabs were then cut into small blocks (1 mm³) and placed in the same fixative for an additional 20–24 h. Tissue blocks were washed overnight in 0.1 M phosphate buffer, pH 7.4, and postfixed with 2% osmium tetroxide in the same buffer for 2 h. The tissues were then dehydrated in ascending concentrations of acetone, infiltrated with propylene oxide, and embedded in Spurr (Polysciences Inc., Northampton, UK). Orthogonal sections (1 mm thick) across the seminiferous tubules were stained with 1% toluidine blue-1% sodium borate solution and examined at the light microscope at x630 magnification using a 63x (1.4 N.A.) planapochromatic oil immersion objective. Tubule staging was carried out according to the staging criteria proposed by Hess [22] for plastic semithin sections. Selected tissue blocks containing seminiferous tubules at stages VI–IX were trimmed, and thin sections (silver to gold interference colors) were cut on an LKB (Rockville, MD) ultramicrotome and examined with a Jeol JEM-1200EXII (JEOL, Tokyo, Japan) electron microscope.

	RESULTS

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Annexin-V-Biotin Binding

To assess the occurrence of apoptosis, isolated residual bodies were incubated with annexin-V, a Ca²⁺-dependent phospholipid-binding protein with high affinity for phosphatidylserine. Annexin-V specifically labeled residual bodies contained within either the F1+F2 (Fig. 1a) or the F1 fractions (Fig. 1b), whereas the dispersed cells from either the F1+F2 fractions (Fig. 1a) or the F2 fraction alone (Fig. 1c) remained unlabeled.

View larger version (86K): [in this window] [in a new window]   FIG. 1. Annexin-V-biotin binding. a) Staining of cells from F1+F2 fractions. Arrows point to labeled residual bodies, whereas all the germ cells remained unlabeled (arrowheads). Bar = 10 µm. Higher magnification shows one of the residual bodies. Arrow points to annexin staining. b) Positive staining of residual bodies from the F1 fraction. Bar = 25 µm. c) Unlabeled germ cells from the F2 fraction after incubation with annexin. Bar = 10 µm

ICE p20 Staining

Formation of residual bodies was accompanied by an apparent increase in the immunoexpression of ICE p20 subunit. Therefore, a light staining was present in the caudal region of elongating spermatids (Fig. 2a), whereas at stage VII this staining was readily apparent at the cytoplasmic lobes of mature spermatids (Fig. 2b), and at stages VIII–IX a strong staining was seen in residual bodies (Fig. 2, c and d). The reaction product was found labeling the margins of these cytoplasmic spheres, immediately below the plasma membrane (Fig. 2d). Its location and distribution were similar to those of the numerous vacuoles found surrounding the basophilic central mass (see below). In the negative control sections, staining was not detected (Fig. 2e).

View larger version (87K): [in this window] [in a new window]   FIG. 2. ICE immunolabeling of seminiferous tubule histological sections. a) Low-power photomicrograph. The reaction product labeled the caudal cytoplasm of elongating spermatids in the lumina of seminiferous tubules at several steps of differentiation (arrows). Bar = 30 µm. b) Immunostained cytoplasmic lobes of step 19 spermatids (arrows) delineated the seminiferous tubule lumen at stage VII. Bar = 10 µm. c) Immunolabeled residual bodies (arrows) in a stage VIII seminiferous tubule section. Note that staining increased as cytoplasmic lobes became residual bodies. Bar = 10 µm. d) Residual bodies (arrows) within the seminiferous epithelium at stage IX of the spermatogenic cycle. Note that the strongest immunostaining was found labeling the residual body periphery. Bar = 10 µm. e) Negative control. Arrowheads point to residual bodies at an early stage IX tubule section. Bar = 10 µm

Immunolabeling of c-jun, p53, and p21

Using immunohistochemistry, we also examined the expression level of some oncoproteins that are known to be increased during apoptosis. A positive reaction was observed for c-jun, p53, and p21 antibodies in the cytoplasmic lobes of elongated spermatids. Again, as occurred with ICE p20 antibodies, with advancing spermatid differentiation the immunocytochemical staining shown with these antibodies was more intense. Thus, staining was readily apparent at step 18–19 spermatids of stages VI–VII (Fig. 3, a–c); and at stage VIII an intense immunoreactivity was observed delineating residual bodies along the seminiferous tubule lumen, beneath step 19 spermatid nuclei (Fig. 3, d–f). The strongest reaction was at stage IX, where intensely stained residual bodies appeared distributed throughout the epithelium (Fig. 3, g–i). In all cases the reaction product was located in the central cytoplasm.

View larger version (122K): [in this window] [in a new window]   FIG. 3. Immunocytochemistry for c-jun, p53, and p21. a–c) Stage VII tubule sections, showing immunostaining of cytoplasmic lobes (arrowheads) of step 19 spermatids: c-jun, p53, and p21 (left-right). d–f) Stage VIII tubule sections, showing the positive immunoreaction of residual bodies, lining the tubule lumen (arrowheads): c-jun, p53, and p21 (left-right). g–i) Residual bodies within the epithelium of stage IX tubule section (arrowheads), showing a positive immunoreaction: c-jun, p53, and p21 (left-right). Note that in contrast to anti-ICE antibodies, immunolabeling with anti-c-jun, anti-p53, and anti-p21 antibodies was found located in the central region of residual bodies. Bar = 10 µm

Morphological Analysis of Residual Bodies

The cytoplasm of elongating spermatids lines the tubule lumen from stages XI to VII. In toluidine blue-stained semithin sections, the distal cytoplasm of these spermatids was seen as a region darker than the remainder of the epithelium (Fig. 4a) because of a higher basophilia of these structures. Basophilia gradually increased from stage XI to VII; and at stage VIII, highly basophilic residual bodies appeared distributed along the luminal border (Fig. 4b). After spermiation, at stage IX, residual bodies were enlarged and could be observed as highly basophilic bodies of different sizes throughout the epithelium. They were readily identified because their high basophilia gave them an appearance similar to that of dying cells. Basophilia was greatest at the central region of the body, which showed large vacuoles at the periphery (Fig. 4c). These features distinguished the residual bodies with accuracy from occasional apoptotic germ cells.

View larger version (168K): [in this window] [in a new window]   FIG. 4. Toluidine blue-stained plastic semithin sections of seminiferous tubules. a) Low-power photomicrograph showing the darker staining of the caudal cytoplasm of elongated spermatids, which delineated the tubule lumina (arrowheads). Bar = 30 µm. b) Stage VIII tubule showing highly basophilic residual bodies (arrows) delineating the tubule lumen. Bar = 10 µm. c) Stage IX tubule. Arrows point to residual bodies that were at the top of the epithelium and within the basal compartment, after having been phagocytosed by the Sertoli cell. Note their similarity to apoptotic germ cells. Nevertheless, they showed a highly basophilic irregular mass at the central region and numerous vacuoles at the periphery, allowing them to be distinguished with accuracy. Bar = 10 µm

Ultrastructural study of late spermatids revealed that the increased basophilia of their distal cytoplasm coincided with a high electron density (Fig. 5a). As spermatid differentiation proceeded, the cytoplasmic lobe formed and became progressively more electron dense (Fig. 5b). Due to this gradual condensation, a higher electron density was found in residual bodies when they were distributed along the luminal border (Fig. 5c). Clusters of intact mitochondria were found within this membrane-bound cytoplasmic compartment. Additional observations included an irregular aggregate of tightly packed yet distinct electron-dense particles corresponding to the most highly basophilic central region, previously identified as ribosome particles; numerous dispersed vacuoles containing low-density material, often found indenting the ribosomal mass; clusters of electron-dense droplets with irregular margins of lipid appearance; and a Golgi remnant formed by membrane flattened sacs (Fig. 5, b–d).

View larger version (168K): [in this window] [in a new window]   FIG. 5. Electron microscopy. a) Arrowheads point to the contact between round spermatids (step 6) and the caudal cytoplasm of elongated spermatids (step 18) at a stage VI tubule section. Note the higher condensation of the cytoplasm of mature spermatids. Bar = 2 µm. b) Arrowheads point to cytoplasmic lobes of step 19 spermatids at stage VII showing a high electron density due to the gradual condensation of this cytoplasmic compartment. Bar = 2 µm. c) Residual body at the lumen of a stage VIII seminiferous tubule. e, Highly electron-dense central mass; v, vacuole; l, lipoidal bodies; g, Golgi remnant; m, mitochondria. Bar = 1 µm. d) Magnification showing a detail of lipoidal bodies (l), a vacuole (v), Golgi remnnant (g), and a cluster of intact mitochondria (m). Bar = 200 nm

	DISCUSSION

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During the release of spermatozoa from the seminiferous epithelium, part of the cytoplasm of the mature spermatid is pinched off by a constriction similar to that responsible for separation of the daughter cells in mitotic division [23]. Thus in the rat at stage VIII of the spermatogenic cycle, cytoplasmic stalks extend from the neck of the mature spermatids down to cytoplasmic compartments, which are lodged in the seminiferous epithelium. As the spermatid moves toward the lumen, the stalk breaks, resulting in the release of the spermatozoon. During the next stage, the resulting residual body, ensheathed in the Sertoli cell processes, is drawn deeper and phagocytosed [24].

Basophilia, vacuolation, and a high electron density due to an increased condensation of organelles were reported from classical studies at both the distal cytoplasm of maturing spermatids and at residual bodies [2, 4, 25]. In addition, some ultrastructural aspects of residual bodies have previously been related to a degeneration process [4], whereas phagocytosis of these bodies by the Sertoli cells is also well established [3]. Our morphological study allowed us to confirm these classical data. In addition, we realized that the high basophilia shown by residual bodies is similar to that of apoptotic germ cells, although their morphological features make it possible to distinguish them with accuracy in semithin plastic sections [26, 27]. Furthermore, electron microscopy revealed that clusters of intact mitochondria remain especially well preserved in residual bodies. Basophilia, vacuolation, compaction of cytoplasmic organelles, and the rapid phagocytosis of dead cells have been defined as major features characterizing apoptosis [10, 28–30], a process in which mitochondria play a relevant role [31].

The application of cell-free systems for the study of apoptosis has revealed that essential factors triggering apoptosis are present in the cytoplasm and not in the nucleus [32]. This is consistent with the fact that cytoplasts undergo apoptosis in the absence of nuclei [11, 12] and indicates that nucleus and cytoplasm apoptosis events can be independent phenomena [13].

Taking into account these considerations, we reasoned that apoptosis might be involved in the mechanism responsible for the formation of residual bodies. Our results are indeed consistent with this idea: a high basophilia affects the whole cytoplasmic compartment, and electron microscopy reveals the gradual condensation of organelles. As already appreciated from early studies, mitochondria are numerous [3, 4, 25]. Moreover, these mitochondria always appeared intact, an observation that would correspond with the important role that they play in the control of apoptosis triggering [33–36]. Therefore, all these data confirm that morphological features of residual bodies are compatible with those of apoptotic bodies.

The specific labeling of the membranes of isolated residual bodies with annexin V gives evidence that phosphatidylserine is exposed on the surface of residual bodies. Exposure of this phospholipid is the best-characterized change in the plasma membrane that occurs early after the initiation of programmed cell death [37]. Phagocyte recognition of apoptotic cells and bodies occurs because membrane-bound bodies are tagged with "eat me" signals such as phosphatidylserine [14]. Thus, it is very likely that phosphatidylserine on residual body membranes is exposed to being recognized and subsequently phagocytosed by the Sertoli cells. In this regard it is interesting to note that exposure of phosphatidylserine occurs on the membrane of apoptotic germ cells [9]. In addition, the specifically increased level of ICE, a cysteine protease involved in the execution pathways of apoptosis [15–17], limited to the cytoplasmic lobe and residual bodies, strongly supports the occurrence of a process of apoptosis restricted to this cytoplasmic compartment. It is intriguing that some nuclear oncoproteins such as c-jun, p53, and p21, which are considered apoptosis regulatory proteins [18–21], are also gradually increased in the cytoplasmic lobe of maturing spermatids, reaching their highest levels when they become residual bodies. In contrast to anti-ICE, which stains the periphery of residual bodies, the stronger immunostaining of c-jun, p53, and p21 oncoproteins is found in the central area of the bodies.

As far as we know, the immunocytochemical distribution of ICE in the seminiferous epithelium has not been studied. This is not the case for the nuclear proteins c-jun, p53, and p21 [38–40], but the presence of c-jun or p21 proteins in residual bodies has not previously been reported. Stephan et al. [40] observed a positive immunoreaction of the residual bodies to both anti-p53 and anti-DNase I antibodies. This is an interesting point, since DNase I is involved in nuclear DNA degradation during apoptosis [41, 42]. In this regard, it might be worthwhile to remember that present in the cytoplasmic lobe is a mysterious organelle, the chromatoid body [4, 25, 43]. This cytoplasmic structure exists in several animals from invertebrates to vertebrates [44], indicating that it must have an important function. In the rat, the chromatoid body first appears in pachytene spermatocytes, derived from intranuclear material [45–47], although its composition and function thus far are not clear. Available data indicate that it is mainly composed of ribonucleoproteins [48–50], whereas there are conflicting results concerning the possible occurrence of DNA within this organelle [49, 51]. Therefore, it has been proposed that the chromatoid body directs protein synthesis when the genome of the spermatid is inactive [48, 52]. During spermatid differentiation, the chromatoid body moves from one location to another [48, 53], but it always appears located in close association with the structure whose development characterizes that morphogenetic stage. Therefore, it is tempting to speculate that this organelle might play a key role in directing the development of these structures and in controlling the appearance of an apoptotic process restricted to the cytoplasmic lobe of elongating spermatids.

Another important issue is the function that residual bodies have, if any. Some authors have pointed out that detachment of the residual body not only contributes to diminishing the sperm size but also has a regulatory function [3, 54]. In this respect it might be interesting to take into account that mitotic and meiotic divisions occurring during spermatogenesis do not complete cytokinesis. The persistence of intercellular bridges leads to a large number of spermatids linked at the completion of spermatogenesis [55, 56]; they should be eliminated at spermiation. At the end of spermiogenesis these cytoplasmic contacts are between the residual bodies [4, 23, 24, 56]. That is, the detachment of residual bodies is required to remove intercellular bridges selectively, thus playing an important role in sperm release. Specifically, selective removal of some components not required by the mature spermatozoon might be, in our opinion, the main function of residual bodies. Therefore, it is logical that apoptosis is the mechanism for the disposal of these cytoplasmic masses, since phagocytosis of apoptotic cells and bodies provides a rapid and efficient mechanism to eliminate unnecessary cytoplasmic components [3, 54–56], thus limiting potential damage to neighboring tissues through leakage of intracellular contents. These concepts are consistent with the fact that impairment of this cytoplasmic elimination is linked to impairment of sperm release and causes infertility in a transgenic rat [57]. Alternatively, apoptosis might also be an efficient mechanism to direct informative molecules to being recognized by Sertoli cells, thanks to phosphatidylserine exposure.

The high susceptibility of germ cells to several apoptogenic agents has been evidenced [58]. Therefore, although pathways of apoptosis control in testis are poorly defined, their importance is clearly established [59]. Thus, the occurrence of a programmed death process restricted to a part of the cytoplasmic lobe of maturing spermatids seems to be another important aspect of the key role played by apoptosis control in the highly dynamic but strictly ordered organization of the seminiferous epithelium.

In conclusion, data presented here clearly indicate that a highly specialized region of cytoplasm, the caudal tags of the maturing spermatids, can undergo apoptosis in the presence of a healthy nucleus, forming structures that display the characteristic features of apoptotic bodies, the so-called residual bodies. Meanwhile, the cell is kept healthy and releases as a free spermatozoon.

	ACKNOWLEDGMENTS

 
We would like to thank L. Santiago for excellent technical assistance.

	FOOTNOTES

 
¹ Correspondence: Josefa Blanco Rodríguez, Departamento de Biología Celular, Facultad de Medicina, Ramón y Cajal, 7, 47005 Valladolid, Spain. FAX: 34 983 423022; jblanco{at}oem.es

Accepted: July 28, 1999.

Received: April 13, 1999.

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