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Regular Article |
a Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901
b Department of Pathology and Medicine, Harvard Medical School, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Boston, Massachusetts 02115
c Department of Pathology, University of Iowa, Iowa City, Iowa 52241
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, sperm maturation, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bax is a multidomain proapoptotic member of the Bcl-2 family, and deficiency of Bax results in decreased cell death in neurons [8, 9] and female germ cells [10]. However, in the male germ line, Bax deficiency results in increased apoptosis and testicular atrophy [11]. Bax deficiency resulted in a large accumulation of premeiotic germ cells in mature animals and a near complete absence of spermatocytes and mature sperm [11]. These findings demonstrate that Bax is required for normal maturation of spermatocytes but did not determine the molecular basis for this requirement. These findings could represent a novel function for Bax in male germ cell maturation or may represent a requirement for Bax-dependent apoptosis during male germ cell development. In the present study, we further characterize Bax-deficient mice to examine the basis for the block in maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used antibodies against murine c-Kit-PE (01905B; Pharminigen, San Diego, CA) and the control anti-Rat IgG2a-PE (11025A; Pharminigen). Type I collagenase (C0130) was from Sigma Chemical Company (St. Louis, MO). Trypsin and fetal bovine serum (FBS) were from Gibco/BRL (Rockville, MD). Ham F12 and L-15 media were from ICN Biotech (Irvine, CA).
Animals and Tissue Preparation
The mice were generated and genotyped as previously described [11, 12]. Testicular weights were determined one of two ways. For morphometric studies, male Bax-deficient (-/-) and wild-type littermates (+/+) were anesthetized, weighed, and perfused via the left ventricle with a blunt 18-gauge needle. The animal was perfused with lactated Ringers with heparin (10 U/ml) until the red blood cells were largely removed from the liver and then the perfusion medium was switched to 0.05 M sodium cacodylate (pH 7.4) with 5% glutaraldehyde (vol/vol) (16500; Electron Microscopy Sciences, Fort Washington, PA) until adequately fixed based on rigidity and tissue color (
10 min). The testes were then removed and weighed before further submersion fixation in the same buffer. For other experiments (cell culture, etc.) the testes were dissected and weighed before fixation. Testicular weights did not appear to change dramatically with fixation (compare Table 1 and Fig. 1). Animal studies were all performed with approval of the institutional animal care and use committee.
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The testes were processed for light and electron microscopic examination as previously described [13]. Most animals were of the C57/B6 background, but some were C3H or hybrid B6/C3H. In all cases littermates were used as controls whenever possible. No differences were observed in the phenotype of Bax-deficient mice on the different genetic backgrounds. Control and knockout littermates were used in most age groups. The day of birth was considered Day 1.
Testes were cut into thin slabs perpendicular to their long axis and then diced such that the length and width of the slab was approximately 1 mm and the thickness approximately 0.05 mm. Subsequently, tissues were washed three times with buffer (one wash was overnight) and postfixed for 1 h in a mixture of osmium and ferrocyanide [13]. Alcohol dehydration and infiltration with propylene oxide were conducted using standard procedures, and the tissue was embedded in epoxy resin. Semithin sections were obtained using an ultramicrotome. Sections were first examined qualitatively, and the microtome was calibrated to make subsequent sections for morphometric examination (described below).
Germ Cell Isolation, Staining, Culture, and Viability Measurement
Germ cells from control and Bax-deficient mice were prepared using a modified procedure from Nagao et al. [14]. Briefly, the testes were dissected and the seminiferous tubules were removed and placed in a conical tube in PBS. The tubules were allowed to settle and excess PBS removed before digestion with 0.25% (wt/vol) collagenase for 20–30 min at 32°C. The tubules were washed with PBS and then further digested with 0.25% (wt/vol) trypsin for 15–20 min at 32°C. FBS was added to a final concentration of 10% (vol/vol), the cells were filtered through cheesecloth, and centrifuged for 5 min at 1000 x g in a tabletop centrifuge. Cells were counted and cultured at 750 000 cells/ml in media containing Pen-Strep, 15 mM HEPES in a 1:1 mixture of F12 and L-15 media. The amount of FBS was varied between 0.1% and 10%. After 45 h in culture, cells were harvested and viability was determined by cells that were negative for both annexin V-fluorescein isothiocyanate (FITC) (Trevigen, Inc., Gaithersburg, MD) and propidium iodide as analyzed on a FACScan flow cytometer. Annexin V-FITC/PI staining was performed following the manufacturer's protocol.
For c-kit staining, cells were prepared as described above and then stained for 25 min at 4°C with either c-kit-PE (1 µl/million cells) or control anti-immunoglobulin G2A PE. Because many dead cells took up both c-kit and control antibodies, dead cells were excluded from the analysis by staining them with annexin V-FITC. After running the cells on a FACScan flow cytometer, the percentage of c-kit positive-annexin V-FITC negative cells was determined. Using this strategy, less than 1% of cells were positive for the rat immunoglobulin G-PE antibody (data not shown).
TdT TUNEL Labeling of Immature Testes
Bax-deficient and control mice were killed at Postnatal Day 7 and the testes were fixed in formalin. After paraffin embedding, 7-micrometer sections were cut and placed on slides. Apoptotic cells were labeled by the TdT TUNEL assay following the manufacturer's directions (4810-45-K; Trevigen Inc.). TUNEL staining was quantified by counting the number of apoptotic cells in 15 random fields at 200x magnification. The mean ± SD were plotted and compared using the Student two-sided t-test (assuming unequal variances).
Determination of the Integrity of the Sertoli Cell Barrier
Perfusion was conducted as described above with the exception that 10% (wt/vol) dextrose was added to the perfusion solution to increase its tonicity and to provoke cell shrinkage within the basal compartment [1]. Tissues were subsequently prepared in a similar manner to that described above, except the postfixation washes were conducted with 10% (wt/vol) sucrose added to the buffer. Integrity of the Sertoli cell barrier was determined by the absence of cell shrinkage, which existed only in the basal compartment of the testis. Tissue shrinkage was judged by both light and electron microscopic evaluation of the testis. Electron microscopy was also used to examine the normality of the region of tight junctions formed by Sertoli cells within the epithelium.
Morphometry
Preliminary examination of tubules from animals of 21 days of age revealed an overproduction of germ cells in Bax-deficient animals compared with wild-type animals. Morphometry was used to determine both the time of onset and the cell types that were responsible for the overproduction of germ cells. Five Bax-null mutants and five wild-type animals were used at 9, 12, and 15 days of age for morphometric determinations. and three were used at Postnatal Day 5.
The volume of individual germ cell nuclei was determined by serially sectioning them at a known section thickness (0.92 µm). The cell types targeted for sectioning were type A spermatogonia (all types), intermediate type spermatogonia, and Sertoli cells. Five cells from each animal were sectioned completely, and all nuclei from these cell types were drawn with a camera lucida. The area of each sectioned nucleus was measured by a manual digitizer and all areas were summed and multiplied by the section thickness. The mean volume of the nucleus of each cell type was determined for Bax-deficient and wild-type animals.
Point counting methods were used to determine the relative volumes of the nuclei of the aforementioned cell types within the seminiferous epithelium of Bax-deficient and wild-type animals. Ten tubules (16 for P9) were selected by lottery from those tubules of the appropriate stage and a 441-point lattice grid was placed over the tubules at 1000x magnification. The percentage occupancy of specific nuclei in seminiferous tubules was determined by dividing the points over the nucleus of interest by the total points over the tubule. The percentage occupancy of the tubules within the testis were determined by point counting at 400x magnification. The total volume of seminiferous tubules was determined by multiplying the volume density of seminiferous tubules by the testis weight. The total volume of nuclei within the testis was determined by multiplying the seminiferous tubule volume by the percentage occupancy of specific cell type nuclei. The number of cells of specific types was determined by dividing the total volume of the nucleus of a particular cell by the mean nuclear volume of the same cell, determined as described above. There were no significant differences in nuclear size in either of the groups studied, with one exception (out of nine determinations) (Table 3). At Postnatal Day 9, the wild-type Sertoli cell nucleus was significantly larger. There was no apparent biological reason for this difference, and we therefore believe this result is spurious. Thus, the nuclear size used to determine cell number was a mean of both Bax-deficient and wild-type animals.
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The relative volume of degenerating cells was determined during the point counting described above. Degenerating cells were expressed as percentage volume density of the seminiferous tubule volume in both Bax-deficient and wild-type animals. In a second method for quantification of degenerating cells, profiles of degenerating cells were counted and expressed per tubule.
Statistics
After analysis of variance, individual Student t-tests were performed to determine significant differences between Bax-deficient and wild-type animals. The cutoff for significance was predetermined to be the 95% probability level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously reported that Bax deficiency results in testicular atrophy and hypocellular tubules in adult animals [11]. To examine the time course of atrophy, testicular weights at various ages in Bax-deficient and control animals were determined. Bax deficiency did not significantly affect testicular weights in very young animals, but resulted in increased weights between 3 and 5 wk of age (Fig. 1, Table 1). Thus, testicular atrophy in Bax-deficient mice is preceded by increased testicular weight between 20 and 40 days of age. This raises the possibility that decreased cell death in Bax-deficient testis accounts for the increased size. To test this, TUNEL staining was performed on P7 testes from control and Bax-deficient animals. Quantification of the TUNEL labeling demonstrates that Bax-deficient animals show a significant reduction in the number of apoptotic cells at this age (Fig. 2). This would be the expected result if the normal function of Bax was to promote apoptosis, as has been shown for oocytes [10]. The specificity of the TdT TUNEL assay for apoptosis has been questioned [15, 16]. Therefore, cell death was also quantified in Bax-deficient and control mice using morphologic criteria. The number of degenerating cells in immature Bax-deficient mice at 5, 9, 12, and 15 days of age was significantly reduced (Table 2). This was true whether calculated by quantifying the number or volume of degenerating cells. Therefore, two independent methods demonstrate reduced cell death in vivo in immature Bax-deficient testis.
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In Vitro Survival Studies
The resistance of Bax-deficient testicular germ cells to apoptosis was further examined by isolation and in vitro culture. The extent of cell death following withdrawal of FCS was determined by annexin V staining in combination with propidium iodide staining. Control cells were sensitive to serum withdrawal because concentrations of FCS below 5% (vol/vol) were associated with increased cell death (Fig. 3). In contrast, Bax-deficient germ cells did not show increased death following 2 days of serum withdrawal. Thus in vitro, Bax is required for the increased cell death of male germ cells following serum withdrawal.
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Quantitation of Germ Cell Numbers by Morphometry
To determine which cell type is protected from apoptosis in Bax-deficient mice, high resolution sections of testis at various ages were weighed and prepared for morphometry (Table 1). Because the heterozygous mice are fertile and phenotypically identical to wild-type animals, these groups were combined for this analysis. Testicular weights were not significantly different between these groups in mice between 5 and 12 days of age. At 9 days of age, abnormal germ cells appearing like gonocytes or large type A spermatogonia were an occasional feature of Bax-deficiency, but not control seminiferous tubules (Fig. 4, a and b). These were centrally located and were not seen at later periods (12 and 15 days) in this study. In Bax-deficient animals at P12 and P15, the number of type A, intermediate type, and type B spermatogonia appeared to be increased compared to wild-type animals (Fig. 4, c–f). This apparent increase was confirmed by quantitative morphometry. Germ cell nuclear size was not significantly different between Bax-deficient and control cells (Table 3). However, both the relative volume of germ cells (Fig. 5) and the total number of type A spermatogonia and intermediate spermatogonia (Table 4) were increased in Bax-deficient mice at P12, P15, or both. The ratio of germ cells to Sertoli cells was, importantly, also increased (Table 4). These differences increased with age between 9 and 15 days, and were significantly different by 15 days of age. The c-kit tyrosine receptor kinase is expressed on spermatogonia and is critical for normal spermatogenesis [17]. Upon examination of c-kit levels in Bax-deficient mice between 3 and 5 wk of age, we found a marked increase in the percentage of c-kit positive cells supporting an increase of immature germ cells in Bax-deficient mice (Fig. 6).
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At 21 days of age there was a pronounced increase in the number of intermediate type A, type B, and preleptotene spermatocytes in Bax-deficient mice (Fig. 7, a and b; Fig. 8). By 25 days of age, massive death of germ cells was observed in Bax-deficient animals compared with wild-type animals (Fig. 7, c and d; Fig. 9). Electron microscopy indicates the degenerating cells had various morphological features, but do not have all the hallmarks of apoptosis, with chromatin condensation lacking in several of the degenerating cells (Fig. 9) [18]. Thus, the death that occurs late in development of Bax-deficient mice may occur by a nonapoptotic pathway [4, 5]. Thus, the massive cell death that leads to the eventual cellular hypoplasia seen in the testis of Bax-deficient mice may not be apoptotic based on morphological criteria.
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Examination of the Sertoli Cell Barrier
The marked increase in cells that normally reside within the basal compartment of the testis prompted us to examine the location and integrity of the Sertoli cell barrier in Bax-deficient mice. Disruption of the Sertoli cell barrier could result in the block in maturation observed in Bax-deficient mice. Tight junctions normally form within one to two cell layers of the basement membrane and separate spermatogonia and immature spermatocytes from the more mature cells. Tight junctions in Bax-deficient mice were assessed by electron microscopic examination of Bax animals following hypertonic perfusion and fixation. Intact tight junctions protect the luminal cells from shrinkage following hypertonic perfusion. The Sertoli cell barrier was intact in Bax-deficient mice as evidenced by extensive shrinkage of cells in the basal compartment (and large intercellular spaces) and an absence of shrinkage of cells in the adluminal compartment (Fig. 7, c and d; Fig. 8). However, in contrast to tubules from control mice, the tight junction often formed four to five cell layers (mostly preleptotene spermatocytes) from the basement membrane. Nonetheless, the region of contact of Sertoli cells forming the Sertoli cell barrier was normal in appearance (Fig. 8). Thus, the Sertoli-Sertoli tight junctions are preserved by both morphological and functional criteria (resistance to hypertonic fixation). We conclude that the defect in spermatocyte maturation is not due to complete disruption of the Sertoli cell barrier.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Which Bcl-2 family member normally protects from Bax mediated germ cell death? Bcl-2-deficient mice have normal spermatogenesis and mice double-deficient in Bax and Bcl-2 did not alter the abnormal testicular phenotype in Bax-deficient mice [27]. However, animals with reduced expression of Bcl-x demonstrate Bax-dependent death of primordial germ cells [28]. This suggests that the unopposed activity of Bcl-x may account for the decreased germ cell death in Bax-deficient mice. Mice completely deficient in Bcl-x are not viable, so spermatogenesis in Bcl-x and Bax double-deficient mice cannot been examined [29].
Does the defect in cell death account for the complete absence of germ cell maturation and infertility in Bax-deficient mice? Sertoli cells have the capability of supporting a relatively fixed number of germ cells in any particular species [30]. Normal spermatogenesis in the presence of an increased ratio of germ cells to Sertoli cells has not been described to date. In fact, the only means shown to increase germ cell number and maintain normal spermatogenesis is to increase the number of Sertoli cells [31, 32]. The excess germ cells present in Bax-deficient animals is substantial and may overwhelm the Sertoli cell's ability to support such a massive increase. For example, in Bax-deficient animals there are four or more layers of preleptotene spermatocytes, whereas normally, there is only one incomplete layer. If all of these cells in Bax animals successfully completed meiosis, then there would be an overwhelmingly large number of spermatids. The Sertoli cells would have to increase their support for germ cells over threefold to fourfold according to germ cell:Sertoli cell ratios presented for P15 animals (Table 4). The decreased cell death results in changes in the Sertoli cell barrier. The barrier remains intact but is located farther from the basement membrane. Whether this altered location is sufficient to impair normal spermatogenesis has not been directly addressed. However, the overexpression of Bcl-2 or Bcl-x in germ cells results in a phenotype very similar to Bax-deficient mice [6, 7]. These findings argue against a unique and perhaps novel function of Bax in spermatogenesis and suggest that preventing the proapoptotic activity of Bax (either by genetic deletion or expression of Bcl-2 or Bcl-x) is sufficient to disrupt normal spermatogenesis.
The data suggest that Bax promotes cell death in the cell divisions of type A spermatogonia that lead to the production of intermediate type spermatogonia. The small increases in type A spermatogonia and large increases in intermediate type spermatogonia are consistent with this hypothesis. A3 and A4 spermatogonia (the progeny of A2 and A3 spermatogonia, respectively) compose only a portion of the total type A spermatogonial population at any one stage [33]. It is difficult to record a significant increase in the total population of type A spermatogonia, given that the various type A spermatogonial cell types were not designated and separated out. This designation requires normal maturation within the tubule and is therefore not possible in Bax-deficient animals.
Previous studies suggest that spermatogonial numbers are governed by a process of density-dependent regulation [34]. In this model, spermatogonia undergo apoptosis when the number of cells exceeds the number that can be supported by Sertoli cells. The factor or factors responsible for supporting germ cell survival is not known, but many candidates exist [35]. Whatever the signal, our data strongly support a model in which decreased trophic support result in the activation of a Bax-dependent cell death pathway.
One interesting finding in the present study is that the Bax-deficient testis is not overwhelmed with very young germ cells (late spermatogonia and early spermatocytes). One recent study found that Bax levels went up in the rodent testes between 10 and 30 days of age, and this correlated with increased cell death at this time [36]. It is only after the young spermatocytes attempt to go through further development to more advanced spermatocytes in animals of about 20–30 days of age that massive cell degeneration occurs in Bax-deficient testes. Therefore, the ability of germ cells to undergo a Bax-independent mechanism for cell death may depend on the level of maturation of the cell. These results suggest that distinct mechanisms of cell death occur within the normal testis. Cells within the basal compartment are killed by a Bax-dependent mechanism that more closely resembles apoptosis. Cell death of more mature spermatocytes in the luminal compartment may occur via a Bax-independent pathway and does not involve classic apoptotic morphology.
In summary, Bax-deficient animals lack the ability to promote the cell death that is known to occur in the most mature of the type A (A2 and A3) spermatogonia. This death is initiated early in development at or just before Day 5 of spermatogenesis in the mouse. Lack of Bax then results in the overproduction of spermatogonia, hyperplasia of the seminiferous tubule, and a block in normal maturation of spermatocytes. This maturational block then results in extensive Bax-independent germ cell death that eventually leads to massive testicular atrophy present in adult Bax-deficient mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 We acknowledge National Institutes of Health grants HD 35494 to L.D.R. and R01 CA50293 to S.J.K., a Latin American Fellowship to H.C.-G., and the Howard Hughes Medical Institute grant RRP 76292-550301 to C.M.K. C.M.K. is a Charles E. Culpeper Medical Scholar and the work was supported by the Rockefeller Brothers Fund. 
2 Correspondence. FAX: 319 335 6555; c-knudson{at}uiowa.edu
3 Current address: Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil 31270-901
Accepted: November 1, 2001.
Received: July 19, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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