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DNA Replication and Germ Cell Apoptosis During Spermatogenesis in the Cat

From the Department of Cell Biology, School of Medicine, Valladolid University, Spain.

Correspondence to: Josefa Blanco Rodríguez, MD, PhD, Departamento de Biología Celular, Facultad de Medicina, Ramón y Cajal, 7, 47005 Valladolid, Spain (e-mail: jblanco{at}med.uva.es ).

Received for publication August 27, 2001; accepted for publication January 8, 2002.

	Abstract

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Stages at which DNA synthesis and germ cell death take place have recently been found to be equivalent in rabbits and rats. Preservation of the timing of these processes in different orders of mammals indicates that this timing may be crucial for testis cell biology. Since there is no previous study on either germ cell proliferation or apoptosis in upper mammals, an analysis of DNA replication (by bromodeoxyuridine labeling) and of the location of apoptotic germ cells (by the TUNEL assay) has been performed in 3 young adult cats. Our observations indicated that in this animal, spermatogonial DNA synthesis occurs at stages V (at which point the first generation of replicating spermatogonia appears, together with replicating preleptotene spermatocytes), early VII, VIII, and early I, II, and IV. Apoptosis of both spermatogonia and spermatocytes was located mostly at stages early I, early VI, early VII, and VIII. Interestingly, DNA synthesis and germ cell death were found to occur at the same stages of the spermatogenic cycle (that is, to occur at the same specific stages of development) as those reported for the rabbit and small rodents.

     Key words: Spermatogonia, spermatocytes, spermatogenic stages, DNA synthesis, programmed cell death

Previous studies of DNA synthesis leading to cell proliferation were carried out in the rat (Clermont, 1962) and in the mouse (Monesi, 1962) by ³H incorporation, while the occurrence of germ cell apoptosis at specific stages has been well established in the rat (reviewed in Blanco-Rodríguez, 1998). More recently, we have shown that DNA synthesis and apoptosis in the rabbit occur at stages showing cellular associations with stages at which these processes take place in other animals previously studied (Blanco-Rodríguez, in press).

Preservation of the cellular associations between stages at which these events take place in members of 2 different orders of mammals (that is, Rodentia and Lagomorpha) suggests that establishing such specific cellular associations could play an important role in seminiferous epithelium biology. Nevertheless, no previous studies on germ cell proliferation or apoptosis in upper mammals exist. In this study, I analyze the occurrence of DNA synthesis (by immunostaining of bromodeoxyuridine incorporated in vivo to the new DNA strand) as well as the appearance of germ cell apoptosis (by the TUNEL assay) in the cat, a member of an upper order of mammals, the Carnivora. The results demonstrate that DNA replication occurred at stages V (at which point the first generation of replicating spermatogonia appears scattered among replicating preleptotene spermatocytes), early VII, VIII, and early I, II, and IV; on the other hand, germ cell apoptosis was detected mostly at stages VIII (coinciding with the meiotic divisions of spermatocytes) and early VII (coinciding with zygotene and ending pachytene), as well as, to a lesser extent, stages early I and early VI. Since the cellular composition of these stages is equivalent to those at which both processes take place in mice, rats, and rabbits, we hypothesize that these specific cellular associations could play an important role in seminiferous epithelium cell biology.

	Materials and Methods

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Animals and Tissue Preparation

Three 1-year-old (young adult) male cats were maintained under conventional, controlled conditions. After anesthesia with an intraperitoneal injection of sodium pentobarbital (70 mg/kg body weight), the testes were prepared by making an incision from the proximal to the distal pole, and pieces of parenchyma were immersion fixed with 10% formaldehyde in 0.1 M phosphate buffer, pH 7.4. The immersion-fixed testes were sliced transversely into approximately 3-mm-thick slabs and processed for paraffin embedding. Sections (5 µm thick) across the seminiferous tubules were mounted in aminopropyltriethoxy silane—coated slides. Paraffin sections were deparaffinized, hydrated, and used for the subsequent detection of DNA synthesis and apoptosis.

Bromodeoxyuridine DNA Labeling

One hour prior to sacrifice, the animals were intraperitoneally injected with 100 mg/kg bromodeoxyuridine (Sigma Chemical Co, St Louis, Mo). Hydrated sections were further processed for immunocytochemical staining with an avidin-biotin complex (ABC)-based method, using the Santa Cruz immunoperoxidase staining kits (ABC reagents, Santa Cruz Biotechnology Inc, Santa Cruz, Calif), as recommended. In brief, the endogenous peroxidase was quenched by a 15-minute incubation in 2% hydrogen peroxide in phosphate-buffered saline (PBS). Bromodeoxyuridine exposure was carried out by incubation for 20 minutes in 0.1% trypsin (Sigma), in 0.1% CaCl₂ pH 7.8 at 37°C, and by DNA denaturation in 2 N HCl for 1 hour at 37°C prior to the antibody reaction. Antibromodeoxyuridine antibody (Dakopatts, Glostrup, Denmark) was used at a concentration of 10.5 µg/mL. Negative controls were processed in an identical manner except that the primary antibody was substituted by PBS. After incubating the sections with a biotinylated secondary antibody, biotin was detected using an avidin-biotinylated horseradish peroxidase detection reagent.

In Situ DNA 3' End Labeling of Apoptotic Cells

Labeling of DNA fragmentation was performed using the Oncor ApopTag nonradioactive detection kit (Oncor Inc, Gaithersburg, Md). After hydration, testis sections were treated with 20 µg/mL proteinase K (Boehringer Mannheim, Mannheim, Germany) for 15 minutes at room temperature. DNA 3' end labeling with digoxigenin-deoxyuridine triphosphate (dig-dUTP) was performed by incubation at 37°C in a humidified chamber for 1 hour. The reaction mixture, containing terminal transferase reaction buffer, dig-dUTP, and terminal deoxynucleotidyl transferase (TdT), was used following the suppliers' guidelines. After incubation, DNA strand breaks were revealed with antidigoxigenin antibody conjugated to peroxidase at room temperature for 30 minutes and the subsequent detection of enzyme activity using diamino benzidine as substrate, as recommended. Negative controls were processed in an identical manner except that the TdT enzyme was substituted by the same volume of distilled water. The absence of the enzyme abolished dig-dUTP incorporation to the 3' end of DNA fragments and the apoptotic cell staining.

Image Analysis and Quantification

After bromodeoxyuridine immunodetection or DNA 3' end labeling, sections were counterstained in periodic acid-Schiff (PAS)/cresyl violet for accurate stage classification. Tubule staging was undertaken following the criteria proposed by Böhme and Pier (1986). Nevertheless, for purposes of comparison with other species, we have subdivided some of the stages described by these authors into 2 substages. Thus, I have considered stage Ia (early I) when round spermatids have just appeared, but intermediate spermatogonia can not be observed, and stage Ib (late I) when these type of spermatogonia are present. In the same way, early stage VI has been designated VIa, and late stage VI (when spermatid elongation is clearly apparent), VIb. Stage VII has also been subdivided into early (VIIa, when pachytene spermatocytes are still present) and late (VIIb, when the oldest generation of spermatocytes are in diplotene).

Images were captured using a computer-assisted (Spot, RT Color, Diagnostic Instruments Inc, Sterling Heights, Mich) charge-coupled device (1520 by 1080 pixels) on a Zeiss Axiophot light microscope at 630x magnification, using a 63x (1.4 numerical aperture) planapochromatic oil immersion objective (Carl Zeiss Inc, Jena, Germany). In addition to cross-sections, overlapping longitudinal sections were further analyzed to follow the whole spermatogenic wave, in order to ascertain that we had analyzed every spermatogenic stage. Since mitosis occurs immediately after DNA replication, metaphase spermatogonia were also localized in these longitudinal sections to ensure that each type of DNA replicating spermatogonia was recorded. Digital images were processed using Adobe Photoshop 4.0 (Adobe Systems Inc, San Jose, Calif).

Apoptotic cells observed in circular or near-circular TUNEL-stained tubule sections were identified according to their morphological features and positions. All the labeled cells were recorded except for dying elongated spermatids. For each animal, 10 nonconsecutive sections were chosen at random from each stage of the spermatogenic cycle. A total of 30 tubules from each stage were studied. Counts were expressed as a ratio of counted cells to Sertoli cell nucleolus.

	Results

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Bromodeoxyuridine DNA Labeling

One hour after injection, bromodeoxyuridine immunolabeling appeared within the nuclei of early spermatocytes and spermatogonia. The youngest labeled spermatocytes were preleptotene spermatocytes, showing mature spermatozoa close to spermiation, located at stage V (Figure 1a), whereas the oldest spermatocytes showing bromodeoxyuridine incorporation were leptotene spermatocytes appearing just after sperm release to the tubule lumen (early stage VI). A few labeled spermatogonia nuclei were also observed to be scattered among spermatocyte nuclei at these stages (Figure 1a). Bromodeoxyuridine immunostaining also labeled the nuclei of spermatogonia appearing at 5 additional stages: early VII (VIIa), showing young spermatocytes at the zygotene phase and old spermatocytes ending pachytene (Figure 1b); VIII, coinciding with the meiotic divisions (Figure 1c); early I (Ia), showing recently appeared spermatids starting differentiation (Figure 1d); II, showing round spermatids with acrosomes starting to spread over the nuclei (Figure 1e); and IV, showing round spermatids with acrosomes covering one third of their nuclei (Figure 1f). Spermatogonia underwent mitosis immediately after the completion of DNA synthesis, as revealed by the presence of metaphase images close to or even mixed with labeled nuclei, which could be especially well observed in longitudinal tubule sections (data not shown). Analysis of these longitudinal sections allowed us to ascertain that, in the cat, as in other species, spermatogonia underwent 6 consecutive mitotic divisions. Since the last generation of spermatogonia (B spermatogonia) appeared to be undergoing mitosis in order to originate preleptotene spermatocytes at stage IV, we assumed that the few labeled spermatogonia that could be observed, together with DNA replicating preleptotene spermatocytes, constituted the first spermatogonium generation.

View larger version (84K): [in this window] [in a new window]   Figure 1. Bromodeoxyuridine DNA labeling, periodic acid-Schiff (PAS)/cresol violet counterstaining. (a) Stage V tubule. Arrows point to nuclei of the first generation of dividing spermatogonia. Several labeled nuclei of preleptotene spermatocytes also appear (arrowheads). (b through f) Labeled nuclei correspond to the second (stage VIIa) (b), third (stage VIII) (c), fourth (stage la) (d), fifth (stage II) (e), and sixth (stage IV) (f) generations of spermatogonia, respectively. Bar = 10 µm.

In Situ DNA 3' End Labeling and Quantification of Apoptotic Germ Cells

As occurs in other species, each germ cell type can be found dying at each of the developmental stages. Nevertheless, most apoptotic germ cells appeared at specific stages (Figures 2a and 3). These stages were stage Ia (Figures 2b and 3a), VIa (Figures 2c and 3a), VIIa (Figures 2d and 3a), and VIII (Figures 2a and e and 3a). Most apoptotic cells were observed at stage VIII, which showed about 40% of the dying cells, followed by early stage VII (VIIa) and, to a lesser extent, early stages I (Ia) and VI (VIa) (Figure 3). In contrast, germ cell deaths were very scarce at other stages (Figures 2a and 3). This finding indicates that apoptotic germ cells were observed mainly when the second (stage VIIa) and the third (stage VIII) generations of spermatogonia underwent mitotic divisions, and, to a lesser extent, coinciding with stages at which the fourth (stage Ia) and the first generations of spermatogonia divide (stage VIa). Apoptotic cells at these stages were mainly spermatogonia and pachytene spermatocytes at stages Ia (Figures 2b and 3a) and VIa (Figure 2c); spermatogonia and zygotene spermatocytes at stage VIIa (Figures 2d and 3c); and metaphase I spermatocytes, spermatogonia, and pachytene spermatocytes at stage VIII (Figures 2e and 3d). Nevertheless, each germ cell type is prone to die at these stages (Figures 2d and 3b through d). Our results are summarized in Figure 4.

View larger version (160K): [in this window] [in a new window]   Figure 2. Periodic acid-Schiff (PAS)/cresol violet counterstained in situ DNA 3' end labeled apoptotic germ cells. (a) Low power magnification photomicrograph showing the characteristic pattern of apoptosis in cat testis sections. Arrowhead points to a apoptotic spermatogonia, and arrows point to metaphase I spermatocytes. Bar = 100 µm. (b through d) High magnification. Bar = 20 µm. (b) Stage la tubule section. Arrow points to a metaphase spermatogonia, and arrowhead points to an apoptotic pachytene spermatocyte. (c) Stage VIa. Arrowhead points to an apoptotic spermatogonia. (d) Stage VIIa. Arrowhead points to an apoptotic spermatogonia, arrow points to a pachytene spermatocyte, and small arrows point to zygotene spermatocytes. (e) Stage VIII tubule showing 2 apoptotic cells (very likely a metaphase I spermatocyte [arrow] and a pachytene spermatocyte [arrowhead]).

View larger version (24K): [in this window] [in a new window]   Figure 3. Spontaneous germ cell apoptosis in adult cat. TUNEL-stained germ cells were identified, recorded at every stage, and expressed as a ratio to Sertoli cell nucleoli. Values are the mean plus or minus SEM. (a) Number of apoptotic germ cells at every stage. (b) Analysis of apoptotic germ cell types appearing at stage la. (c) Apoptotic germ cells at stage VIIa. (d) Apoptotic germ cells at stage VIII. G indicates spermatogonia; Z, zygotene spermatocytes; P, pachytene spermatocytes; M, metaphase I spermatocytes; SS, secondary spermatocytes; and S, round spermatids.

View larger version (51K): [in this window] [in a new window]   Figure 4. Stages of the spermatogenic cycle of the cat. indicates germ cell death; s, DNA synthesis; and m, mitotic division.

	Discussion

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Typical cellular associations of germ cells at various developmental steps characterize the different stages of the spermatogenic cycle in mammals (Leblond and Clermont, 1952; Oakberg, 1956; Swierstra and Foote, 1963; Ibach et al, 1976; Böhme and Pier, 1986; Johnson et al, 1990; Komatsu et al, 1996). Data on spermatogonial proliferation in small rodents have led to the hypothesis that the architectural arrangement of these stages or spermatogenic waves might be determined by the sequential synchronization of mitoses of committed spermatogonia (Chaturvedi and Johnson, 1993; Johnson, 1994; de Rooij and Grootegoed, 1998; França et al, 1998). In fact, current knowledge of cell proliferation control at cell cycle checkpoints is consistent with such a hypothesis (Giancotti, 1997; Kelly and Brown, 2000). In addition to its precise organization and the high rates of cell proliferation, apoptosis is also a main feature of the seminiferous epithelium (reviewed in Blanco-Rodríguez, 1998). Therefore, it is reasonable to assume that more detailed knowledge about the occurrence of events subject to or related to checkpoints during spermatogenesis, such as DNA replication or germ cell apoptosis (Basu and Haldar, 1998; Guo and Hay, 1999; Murakami and Nurse, 1999), is important for our understanding of seminiferous epithelium biology. Nevertheless, studies on these events have thus far been restricted to small rodents.

Spermatogenic stages of the cat and their corresponding cellular associations have been described by Böhme and Pier (1986). I have followed the criteria proposed by these authors, except that, for comparative purposes, I have subdivided stages I, VI, and VII in early (a) and late (b), depending on the absence (Ia) or presence (Ib) of intermediate spermatogonia; the absence (VIa) or presence (VIb) of young spermatid nuclei clearly elongated; and the presence (VIIa) or absence (VIIb) of ending pachytene spermatocytes. However, there is no previous study of either DNA replication or germ cell apoptosis in this animal.

Data in this article demonstrate that in the cat, as in other mammals, the onset of DNA synthesis in the first generation of spermatogonia occurs at the same time as in preleptotene spermatocytes, when spermatozoa are about to be shed into the lumen (that is, at late stage V). Then, spermatogonia enter a series of 5 additional mitotic divisions. Bromodeoxyuridine incorporation as a consequence of DNA replication preceding these divisions was detected at stages VIIa, VIII, Ia, II, and IV. These observations are consistent with previous data on DNA synthesis in mammals such as the rat (Clermont, 1962), the mouse (Monesi, 1962), and the rabbit (Blanco-Rodríguez, 2002). That is, DNA synthesis in the cat started at stages showing cellular associations that are equivalent to those stages at which DNA synthesis was known to occur in these animals.

Germ cell apoptosis has not previously been analyzed in the cat. Data presented here indicate that in the cat, as in the rat (see Blanco-Rodríguez, 1998, for a review) and in the rabbit (Blanco-Rodríguez, in press), germ cell death primarily occurred at specific stages, which mainly coincided with those at which the second, the third, and the fourth generations of spermatogonia underwent mitotic divisions. The apoptotic germ cell types found most frequently at these stages were also spermatogonia, as well as zygotene and metaphase I spermatocytes. In addition, a considerable number of dying germ cells appeared at early stage VI (VIa) in the cat. Interestingly, at this stage, the first generation of differentiating spermatogonia underwent mitotic division.

In conclusion, these results demonstrate that DNA synthesis during spermatogenesis in the cat—a member of a different order of mammals than previously studied, the Carnivora—occurs at the same stages as those at which it occurs in the rat and in the rabbit. Likewise, in the cat, as in other species analyzed, spontaneous germ cell apoptosis generally appeared at the same stages (that is, when differentiating spermatogonia underwent mitotic divisions), indicating that germ cell death in the seminiferous epithelium is related to spermatogonial cell cycle checkpoints. Preservation of the cellular associations of stages at which these processes take place in members of 3 different orders of mammals (that is, Rodentia [rats], Lagomorpha [rabbits], and Carnivora [cats]) suggests that this timing could play a crucial role in spermatogenesis cell biology.

	Acknowledgments

 
The author wishes to thank M. J. Díeguez for her excellent technical assistance.

	Footnotes

 
This work has been supported by grant PM 97-0107 from the Spanish Ministry of Science and Technology (DGSIC).

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