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BOR - Papers in Press, published online ahead of print January 22, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.014779
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BIOLOGY OF REPRODUCTION 68, 2249–2254 (2003)
DOI: 10.1095/biolreprod.102.014779
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Functional Analysis of the p53 Gene in Apoptosis Induced by Heat Stress or Loss of Stem Cell Factor Signaling in Mouse Male Germ Cells

Hiroshi Ohta4, Shinichi Aizawa3,5, and Yoshitake Nishimune2,4

Department of Science for Laboratory Animal Experimentation,4 Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan Department of Morphogenesis,5 Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).

apoptosis, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During normal spermatogenesis, apoptosis is common and is believed to play an important role in controlling germ cell numbers and eliminating defective germ cells to produce functional spermatozoa [1–4]. The apoptosis that occurs during spermatogenesis is a highly complex process that involves genes for various factors, such as the Bcl-2 family, Fas, Fas ligand, and p53 [1–4]. Germ cell apoptosis can also be induced by various pathological conditions such as heat stress [5], exposure to ionizing radiation [6, 7] or toxic substances [8], hormonal depletion [9], and loss of stem cell factor (SCF) signaling [10, 11]. Thus, numerous genes and environmental conditions are associated with male germ cell apoptosis, and the specific molecular mechanisms that govern germ cell apoptosis under different apoptotic conditions have not yet been characterized.

The tumor suppressor protein p53 is highly expressed in the testis [12] and is known to be involved in apoptosis [13, 14]. The presence of p53 mRNA and protein in primary spermatocytes [15, 16] suggests that p53 plays a role at the meiotic prophase. The expression of p53 in irradiated testes is enhanced not only in spermatocytes [16], but also in spermatogonia [17]. These results suggest that p53 is important in the regulation of the apoptotic process in spermatocytes and spermatogonia, and that p53 might be associated with male infertility. In male infertility models, experimental cryptorchidism [18] and c-kit/SCF mutation (Sl/Sld) [19] are known to cause the loss of differentiated germ cells in the testes. Although the causes of germ cell loss in these testes are different, heat stress in experimental cryptorchid testes [18] and the loss of c-kit receptor stimulation by mutation in its ligand (SCF in Sl/Sld testes [19]), both testes show similar morphology (i.e., only undifferentiated spermatogonia together with Sertoli cells were observed in the seminiferous tubules [18, 20]). Several studies have indicated that p53-dependent apoptosis occurs in the testes of these mice [21, 22]. However, the types of germ cells that are most affected and the contribution of p53-dependent pathways to apoptosis in these two conditions remain to be elucidated.

In the present study, we attempted to define the role of p53 in male infertility models using a germ cell transplantation technique that has been established by injecting donor germ cells into seminiferous tubules of recipient mice [23, 24]. In our previous paper, we transplanted the "green germ cells" of enhanced green fluorescence protein (eGFP) transgenic mice and demonstrated proliferation and differentiation of the colonized spermatogonial stem cells [25]. This technique is useful for the analysis of the functions of spermatogonial stem cells per se [26], and the testicular microenvironment of supporting somatic cells [27, 28].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

Male WBB6F1 Sl/Sld and C57BL/6 mice were purchased from the Shizuoka Laboratory Animal Center (Hamamatsu, Japan). The p53-deficient mice, which carry a germ-line disruption in the p53 gene, were constructed by gene targeting in TT2 embryonic stem cells that originated from an F1 C57BL/6 x CBA embryo [29] and back-crossed into the C57BL/6 strain over eight generations in our facility. To introduce the eGFP gene into the p53-deficient mouse line, male p53(-/-) mice were mated with eGFP transgenic [C57BL/6TgN(acro/act-EGFP)OsbN01] [25] female mice. The derived mice, which were heterozygous for p53 and hemizygous for the eGFP transgene, were mated with each other to obtain p53(-/-), p53(+/-), and p53(+/+) mice that all expressed eGFP. The presence of eGFP was examined under excitation light, and the knockout allele of p53 was verified by polymerase chain reaction as described previously [29]. Busulfan was injected i.p. at a dose of 40 mg/kg into the C57BL/6 male wild-type recipients to destroy almost all of the endogenous germ cells. The busulfan-treated males were used as recipients 4 wk after injection. All animal experiments conformed to the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Research Institute for Microbial Diseases, Osaka University).

Transplantation of Germ Cells Into Seminiferous Tubules

Donor cells for transplantation were collected from 7- to 10-day-old testes using the two-step digestion procedure described previously [23, 25], and subsequently transplanted into the seminiferous tubules via the efferent ductules [30]. The donor cell concentrations were adjusted to 1 x 108 cells/ml, and approximately 10 µl was transplanted for busulfan-treated testes and 5 µl was transplanted for Sl/Sld mutant testes. The recipient testes were analyzed 3 mo after transplantation. To investigate the potential association between p53-dependent apoptosis and cryptorchidism, the busulfan-treated recipient testes were surgically manipulated 4 wk after transplantation to induce experimental cryptorchidism as described previously [18], and the testes were analyzed 2 mo after the cryptorchid operation.

Histological Analysis and Evaluation of Transplanted Germ Cell Differentiation

Recipient mice were killed by cervical dislocation 3 mo after transplantation. The testes were fixed in 4% paraformaldehyde at 4°C overnight, and embedded in glycol methacrylate (Technovit 8100; Kulzer, Wehrheim, Germany). Histological 5-µm-thick serial sections of the testes were prepared. Randomly chosen sections, each separated by more than 100 µm, were observed with a fluorescence microscope. All of the cross sections of the seminiferous tubules were differentially counted as being "gonia" (containing only spermatogonia), "gonia-cytes" (containing differentiated germ cells up to spermatocytes from spermatogonia), or "gonia-tids" (containing differentiated germ cells up to spermatids from spermatogonia). The sections that emitted green fluorescence under excitation light were photographed, stained with hematoxylin, and observed with a photomicroscope.

Detection of Apoptotic Cells

TUNEL staining was performed in order to detect apoptotic germ cells. The testes were fixed in 4% paraformaldehyde and embedded in methyl methacrylate (MMA) resin [31]. The sections (5 µm thick) were collected on Superfrost microslide glasses with Aminopropylsilane (APS) coating (Matsunami Glass Ind., Ltd, Osaka, Japan). The MMA resin was removed using xylene at 37°C for 20 min, and the sections were stained using an in situ cell death detection kit (POD; Boehringer-Mannheim, Mannheim, Germany), according to the manufacturer's instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histological Analysis and TUNEL Staining of Cross Sections of Sl/Sld and Cryptorchid Testes

Histological analyses and TUNEL staining were performed to examine the cause of germ cell loss in Sl/Sld mutant and cryptorchid testes. The Sl/Sld mutant and cryptorchid testes were 20%–25% smaller than the wild-type testes (Fig. 1A). Only undifferentiated type A spermatogonia and Sertoli cells were observed in the seminiferous tubules of the Sl/Sld mutant and cryptorchid testes (Fig. 1, B and C) [20]. TUNEL-positive germ cells were frequently observed in the seminiferous tubules of Sl/Sld mutant and cryptorchid testes (Fig. 1, E and F), whereas they were rarely seen in wild-type testes (Fig. 1D), which suggests that germ cell apoptosis was stimulated in Sl/Sld and cryptorchid testes. Therefore, although the cause of germ cell loss remains to be clarified, the Sl/Sld and cryptorchid testes showed similar morphologies and patterns of germ cell apoptosis.



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  		FIG. 1. Histological analysis and TUNEL staining of testicular cross sections from normal adult, Sl/Sld mutant, and experimental cryptorchid mice. A) Comparison of testicular sizes in normal adult (left), Sl/Sld (middle), and cryptorchid (right) mice. Testicular cross sections of Sl/Sld mutant (B) and experimental cryptorchid (C) mice. TUNEL staining of testicular cross sections of normal adult (D), Sl/Sld mutant (E), and experimental cryptorchid (F) mice. Inset in (D) shows a higher magnification view. The arrows in (D–F) indicate TUNEL-positive cells. Bars = 1 mm (A), 100 µm (B–D), and 50 µm (E, F)

Stem Cell Activity of p53-Deficient Spermatogonia on Transplantation

To investigate the stem cell activity of p53-deficient spermatogonia, p53(-/-) germ cells were labeled with eGFP and transplanted into busulfan-treated wild-type testes. The donor germ cells settled in the seminiferous tubules of the recipient testis, and initiated normal spermatogenesis (Fig. 2, A and B). These results were similar to those of our previous transplantation experiments with wild-type eGFP-labeled spermatogonia [25]. Histological evaluation showed that the differentiation of transplanted cells in busulfan-treated testes occurred at the same frequency when donor cells were used from p53(-/-) or control, wild-type p53(+/+) germ cells (Table 1). These results indicate that p53 deficiency does not affect the establishment of spermatogonial stem cells or the differentiation of transplanted germ cells in seminiferous tubules.



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  		FIG. 2. Transplantation of p53-deficient germ cells that express eGFP into the seminiferous tubules of busulfan-treated wild-type or Sl/Sld mutant mice. Transplantation of eGFP-labeled p53(-/-) germ cells into busulfan-treated wild-type testes (A, B) or Sl/Sld mutant testes (C, D). Cross sections of recipient testes 3 mo after transplantation. Fluorescence microscopic photographs (A, C) and photomicroscopic photographs (B and D, respectively) of the same sections stained with hematoxylin. Bars = 100 µm (A, B) and 50 µm (C, D)


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  		TABLE 1. Histological analysis of eGFP-labeled p53(+/+), p53(+/-), and p53(-/-) germ cells transplanted into busulfan-treated, cryptorchid, or S1/S1d mouse testes.*

Apoptosis of Differentiated Germ Cells in the c-kit/SCF Mutant is p53-Independent

Undifferentiated spermatogonia in the seminiferous tubules of the Sl/Sld mutant mouse proliferated, but did not differentiate into the more advanced cellular stages, indicating that important roles of stem cell factor (SCF) produced by Sertoli cells were represented in the differentiation or maintenance of differentiated spermatogonia [26]. To investigate whether germ cell apoptosis in Sl/Sld mutant testes was p53-dependent, we transplanted p53(-/-) germ cells into the seminiferous tubules of Sl/Sld mutant mice. Three months after transplantation, the settlement of donor germ cells was observed in recipient seminiferous tubules, although differentiated germ cells, such as differentiated spermatogonia, spermatocytes, and spermatids, were completely absent (Fig. 2, C and D). Thus, all of the transplanted p53(-/-) germ cells in the Sl/Sld mutant testes were undifferentiated type A spermatogonia and were negative for the c-kit receptor (data not shown). These results clearly demonstrate that germ cell loss in Sl/Sld mutant testis is not due to p53-dependent apoptosis.

Involvement of p53-Dependent Apoptosis in Germ Cell Loss Associated with Experimental Cryptorchidism

To further examine the association between p53-dependent apoptosis and spermatogenesis, transplanted eGFP-labeled p53(+/+), p53(+/-), and p53(-/-) spermatogonia were examined under cryptorchid conditions. Transplanted p53(+/+) germ cells did not differentiate in the seminiferous tubules of cryptorchid testes (Fig. 3, A and B, Table 1). On the other hand, donor germ cell differentiation was observed when p53(+/-) or p53(-/-) germ cells were transplanted into the seminiferous tubules of cryptorchid testes (Fig. 3, C–F). Semiquantitative analyses of these histological observations indicated that the degree of differentiation was higher following the transplantation of homozygous p53(-/-) donor cells than with the heterozygous p53(+/-) cells, which suggests that the p53 expression level is important for the accomplishment of heat stress-induced germ cell apoptosis (Table 1). These results indicate that germ cell loss as a result of heat stress is the result of p53-dependent apoptosis.



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  		FIG. 3. Transplantation of eGFP-labeled p53(+/+), p53(+/-), and p53(-/-) germ cells into the seminiferous tubules of cryptorchid testes. Almost all of the transplanted germ cells were undifferentiated spermatogonia 3 mo after the transplantation of eGFP-labeled p53(+/+) germ cells into cryptorchid testes (A, B). C–F) Numerous spermatocytes (arrowheads) and differentiated transplanted germ cells are observed 3 mo after the transplantation of eGFP-labeled p53(+/-) (C, D) or p53(-/-) (E, F) germ cells into cryptorchid testes. Fluorescence-microscopic photographs (A, C, E) and photomicroscopic photographs of the same sections stained with hematoxylin (B, D, F, respectively). Bars = 50 µm (A–F)

Although the differentiation of transplanted p53(-/-) and p53(+/-) germ cells was observed in the recipient cryptorchid testes, almost all of the differentiation was stopped at the stage of meiotic prophase (i.e., haploid-genesis still remained to be impaired by cryptorchidism even if p53 was inactivated) (Table 1, Fig. 3, C–F). These results were confirmed by the cryptorchid manipulation of p53(-/-) mice. Numerous spermatocytes and TUNEL-positive apoptotic cells were observed in the testicular cross sections of p53(-/-) cryptorchid mice (Fig. 4, A and B). In addition, the number of pycnotic nuclei in p53(-/-) cryptorchid testis increased 7-fold compared with the p53(-/-) testis in normal conditions [97 pycnotic nuclei in 192 tubular cross sections of p53(-/-) cryptorchid testis, 13 pycnotic nuclei in 175 tubular cross sections of p53(-/-) testis in normal conditions]. These results suggest that different apoptotic pathways are involved in heat-induced germ cell apoptosis; apoptosis during the spermatogonia and spermatocyte stages is p53-dependent, whereas apoptosis during spermiogenesis is p53-independent.



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  		FIG. 4. TUNEL staining of testicular cross sections of a p53(-/-) mouse 3 mo after the experimental cryptorchid operation. Bars = 100 µm (A) and 50 µm (B)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study reveals that p53-dependent germ cell apoptosis occurs in cryptorchid testes (Fig. 3, Table 1), and that the main cause of germ cell impairment is heat stress. On the other hand, apoptosis caused by the loss of SCF was independent of p53 (Fig. 2, Table 1). Although male germ cell apoptosis induced by heat stress was partially p53-dependent (Fig. 3, Table 1), the existence of other apoptosis pathways was also suggested, because germ cell apoptosis still occurred during the later stages of germ cell differentiation, such as spermiogenesis, in the cryptorchid testes of p53(-/-) mice (Fig. 4). Thus, several apoptosis pathways appear to function in male germ cell differentiation under different stress conditions.

It was previously shown that spermatogenesis in Wv/Wv (c-kit receptor-deficient) mice could be rescued by loss of the p53 gene [22]. This indicates that germ cell apoptosis due to defective c-kit receptor signal transduction is mediated by a p53-dependent pathway. In the present study, we investigated whether spermatogenic cell differentiation could be enhanced by p53 deficiency in Sl/Sld mutant (deficient in the ligand for the c-kit receptor) testes. Our results clearly demonstrate that the loss of p53 gene function could not promote or rescue spermatogenesis in Sl/Sld mutant testis (Fig. 2, Table 1), which indicates that germ cell deficiency in the c-kit/SCF mutant is not dependent on a p53-mediated apoptotic pathway. The discrepancies in these two findings may be attributed to two major factors. First, differences in the genetic backgrounds of the mouse strains used in these two studies may account for the different results. In our study, the genetic background of the p53(-/-) mouse was C57BL/6, whereas a mixed background of 129 and CBA was used in the previous report [22]. It is generally accepted that genetic background is important in considering mutant mouse phenotypes, such as testicular germ cell differentiation. Second, differences in the experimental systems used in the two studies may explain the different outcomes. In the previous report, the mice that carried mutations in both the Wv/Wv and p53 genes were generated by mating. In contrast, we used a transplantation technique to reconstitute spermatogenesis from a single spermatogonial stem cell. Thus, differences in the mouse strains used and different approaches to the reconstitution of spermatogenesis and normal development may have produced the divergent results. In any case, if p53-dependent apoptosis occurs in Sl/Sld mutant testis, we should be able to observe a pattern of differentiation for the transplanted p53-deficient germ cells in seminiferous tubules that resembles the results of the transplantation study involving heat-stress induced apoptosis (Fig. 3).

Recently, it was proposed that phosphatidylinositol 3'-kinase (PI 3-kinase) functioned downstream of the c-kit receptor in male germ cell differentiation [32, 33]. Activated PI 3-kinase mediates c-kit/SCF signaling to a wide array of downstream targets, including the protein kinases PDK1, Akt, and PKC{delta}, and the small GTPase Rac1 [34–36]. Several targets of the PI 3-kinase/Akt signaling pathway have recently been identified, and the targets associated with this regulatory cascade may be directly involved in the promotion of cell survival [37]. These substrates include two components of the intrinsic cell death machinery, Bad and caspase 9, transcription factors of the forkhead family, which is believed to regulate the death signal, Fas ligand, and a kinase, IKK, that regulates the NF-{kappa}B transcription factor [37–39]. These components of cell death machinery differ from the components of the p53 pathway, and may be associated with germ cell apoptosis in c-kit/SCF mutants.

Mechanism of Heat Stress-Induced Apoptosis During Spermatogenesis

We demonstrated that germ cell apoptosis that was induced by exposure to heat stress was partially p53-dependent (Fig. 3, Table 1). In the C57BL/6 genetic background, male germ cell differentiation in experimental cryptorchidism is inhibited at the step of differentiation from undifferentiated type A spermatogonia to differentiated spermatogonia [20]. In contrast, we could observe transplanted p53(-/-) germ cell differentiation in the cryptorchid condition, although the differentiation was stopped in meiotic prophase (Fig. 3, Table 1). Haploid germ cells derived from p53(-/-) donor cells were entirely absent in the cryptorchid testes (Fig. 3, Table 1). We observed many differentiated spermatogonia and meiotic prophase cells in addition to TUNEL-positive germ cells in cryptorchid testis of p53(-/-) mice (Fig. 4). Therefore, it is likely that the role of p53 in heat stress is to control germ cell apoptosis in cells that differentiate from type A spermatogonia to spermatocytes, but not in haploid cells or in cells undergoing meiotic division from spermatocytes. This suggests that the apoptotic mechanisms of spermatocytes and haploid spermatids are quite different. Consistent with our results, Yin et al. [21] indicated the existence of a p53-dependent or p53-independent pathway in germ cell apoptosis that was induced by heat stress, although it was unclear what type of germ cells were sensitive to p53-dependent apoptosis. In this study, we indicate that heat-induced apoptosis in differentiated spermatogonia and spermatocytes (but not at the spermatid stage) is p53-dependent (Fig. 3, Table 1). Although Yin et al. [40] showed that p53 and Fas were involved in testicular germ cell apoptosis induced by heat stress, it was not enough to prevent germ cell apoptosis in a cryptorchid testis, even in a double mutant for p53 and Fas. This suggests that cell type-specific and other apoptotic systems control germ cell apoptosis that is induced by heat stress.


   FOOTNOTES
 
1 H.O. is the recipient of a Research Fellowship from the Japan Society for the Promotion of Science (2000–2003). Back

2 Correspondence: Yoshitake Nishimune, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp Back

3 Current address: Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami Cho, Chuou-Ku, Kobe, Hyougo 650-0047, Japan Back

Received: 17 December 2002.

First decision: 5 January 2003.

Accepted: 21 January 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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