help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-1338
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shetty, G.
Right arrow Articles by Weng, C. C. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shetty, G.
Right arrow Articles by Weng, C. C. Y.
Endocrinology Vol. 149, No. 6 2773-2781
Copyright © 2008 by The Endocrine Society

p53-Dependent Apoptosis in the Inhibition of Spermatogonial Differentiation in Juvenile Spermatogonial Depletion (Utp14bjsd) Mice

Gunapala Shetty, Shan H. Shao and Connie C. Y. Weng

Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Address all correspondence and requests for reprints to: Gunapala Shetty, Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030. E-mail: sgunapal{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In adult male mice homozygous for the juvenile spermatogonial depletion (Utp14bjsd) mutation in the Utp14b gene, type A spermatogonia proliferate, but in the presence of testosterone and at scrotal temperatures, these spermatogonia undergo apoptosis just before differentiation. In an attempt to delineate this apoptotic pathway in jsd mice and specifically address the roles of p53- and Fas ligand (FasL) /Fas receptor-mediated apoptosis, we produced jsd mice deficient in p53, Fas, or FasL. Already at the age of 5 wk, less degeneration of spermatogenesis was observed in p53-null-jsd mice than jsd single mutants, and in 8- or 12-wk-old mice, the percentage of seminiferous tubules showing differentiated germ cells [tubule differentiation index (TDI)] was 26–29% in the p53-null-jsd mice, compared with 2–4% in jsd mutants with normal p53. The TDI in jsd mice heterozygous for p53 showed an intermediate TDI of 8–13%. The increase in the differentiated tubules in double-mutant and p53 heterozygous jsd mice was mostly attributable to intermediate and type B spermatogonia; few spermatocytes were present. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining showed that most of these differentiated spermatogonia still underwent apoptosis, thereby blocking further continuation of spermatogenesis. In contrast, the percentage of tubules that were differentiated was not significantly altered in either adult Fas null-jsd mice or adult FasL defective gld-jsd double mutant mice as compared with jsd single mutants. Furthermore, caspase-9, but not caspase-8 was immunochemically localized in the adult jsd mice spermatogonia undergoing apoptosis. The results show that p53, but not FasL or Fas, is involved in the apoptosis of type A spermatogonia before/during differentiation in jsd mice that involves the intrinsic pathway of apoptosis. However, apoptosis in the later stages must be a p53-independent process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPERMATOGENESIS IS A well-coordinated complex process in which spermatogonia proliferate and differentiate into terminally differentiated spermatozoa. The undifferentiated spermatogonia at the basement membrane of the seminiferous tubule are at the beginning of the spermatogenic lineage and can be subdivided according to their topographical arrangement into A single, A paired, or A aligned (Aal) (1, 2). Most of the Aal cells differentiate into differentiating type A1 spermatogonia, and after a series of five subsequent divisions (A2-B), differentiating spermatogonia divide into spermatocytes, which after the completion of meiosis develop into spermatids. However, many pathological situations or genetic defects block spermatogonial differentiation.

In mice carrying the juvenile spermatogonial depletion mutation (Utp14b jsd, henceforth called jsd), the heterozygous males and homozygous females are fertile, but adult homozygous males are azoospermic and sterile due to a block in spermatogonial differentiation (3). The Utp14b gene is a retrotransposed copy of the X-linked Utp14a gene (4, 5). Whereas Utp14a is ubiquitously expressed but silenced in pachytene spermatocytes, Utp14b is almost exclusively expressed in germ cells, including pachytene, to compensate for Utp14a’s absence (6). Utp14a and -b are mouse homologs of the yeast UTP14 gene, which is necessary for 18S rRNA production and whose deletion is lethal to the yeast (7). The block in spermatogonial differentiation is due to a defect within the spermatogonia (8).

In 8-wk-old jsd mice on some genetic backgrounds, only spermatogonia and Sertoli cells are seen in the seminiferous tubules, whereas on other backgrounds the decline occurs later (9). The block in spermatogonial differentiation can be reversed by suppressing testosterone or raising the testicular temperature (9, 10). Males maintain spermatogenesis for the first few weeks after birth but eventually fail to maintain the differentiation of type A spermatogonia (11). The spermatogonia continuously proliferate but undergo apoptosis instead of differentiation. In this study we attempted to determine whether the block in spermatogonial differentiation could also be alleviated by blocking the apoptotic pathways involved.

Two major pathways, intrinsic and extrinsic, are involved in the process of apoptosis in mammalian cells (12, 13). The intrinsic pathway for apoptosis involves the release of cytochrome c into the cytosoplasm in which it binds to the apoptotic protease-activating factor-1. Once activated by cytochrome c, apoptotic protease-activating factor-1 binds to procaspase-9 via the caspase recruitment domain, resulting in the activation of initiator caspase-9 and subsequent proteolytic activation of executioner caspases-3, -6, and -7. Members of Bcl-2 family of proteins play a major role in governing this mitochondria-dependent apoptotic pathway, with proteins such as Bcl-2-associated X protein (Bax) functioning as an inducer and proteins such as Bcl-2 as suppressors of cell death. In contrast, the extrinsic pathway for apoptosis is initiated by the binding of a ligand such as Fas ligand (FasL) to its receptor. Binding of FasL to Fas induces the trimerization of the Fas receptors, which recruits Fas-associated death domain (FADD). The Fas/FADD complex then binds to an initiator caspase of the extrinsic pathway such as caspase-8 or -10 through interactions between the death effector domain of FADD and these caspase molecules. Caspase-8 or -10 then activates the executioner caspases-3 and -7, resulting in the cellular disassembly.

The tumor suppressor protein p53 is activated by DNA damage- or oncogene-induced signaling pathways and promotes transcripts of a number of genes that are involved in apoptosis, including those encoding death receptors (14, 15) and proapoptotic members of the Bcl-2 family (16, 17, 18). In addition, transcription-independent signaling can also be launched directly by targeting p53 to mitochondria (19). In most cases, p53-induced apoptosis proceeds through mitochondrial release of cytochrome c, which leads to caspase activation (20). p53 is highly expressed in the testis under cellular stress (21) and known to be involved in apoptosis of testicular germ cells (21, 22, 23, 24, 25, 26).

In the present study, we attempted to determine the pathway of apoptosis that is activated in the jsd spermatogonia. The results indicate that in jsd mice, p53, but not FasL/Fas, is involved in the apoptosis of type A spermatogonia before or during their differentiation, but the apoptosis in the later stages must be a p53-independent process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mice were housed in animal facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulations and standards of the U.S. Department of Agriculture and the Department of Health and Human Services, National Institutes of Health (NIH). They were maintained on a 12-h light, 12-h dark cycle and were allowed food and water ad libitum. All the experimental procedures carried out were approved by the Institutional Animal Care and Use Committee.

The male mice mutant for the apoptosis-associated genes, tumor suppressor p53 (Trp53tm1Tyj)(27), Fas receptor (Fas, Tnfrsf6tm1Osa, TNF receptor superfamily, member 6) (28), and FasL [generalized lymphoproliferative disease (gld), Tnfsf6gld, TNF superfamily, member 6] (29) were obtained from Jackson Laboratory (Bar Harbor, ME). Male mice heterozygous for p53-null mutation (p53+/–), on a 129 background, were bred to jsd/jsd females on C3H-B6–129 mixed background (30). The resulting jsd/+,p53+/– littermates were intercrossed to obtain jsd/jsd,p53–/– males. Eventually for more efficient production of the double homozygotes, jsd/+,p53–/– males were crossed to jsd/jsd,p53–/– females. For comparison jsd/jsd,p53+/–, jsd/jsd,p53+/+, and (jsd or +)/+,p53+/+ littermates were used. Similarly, males homozygous for the targeted mutation of Fas (Fas–/–) on a B6–129 background were crossed with jsd/jsd females, and the double heterozygotes produced were intercrossed to obtain jsd/jsd,Fas–/– males and jsd/jsd,Fas+/–, jsd/jsd,Fas+/+, and (jsd or +)/+,Fas+/+ littermates. A similar breeding protocol was followed to obtain jsd/jsd,gld/gld males and jsd/jsd,gld/+, jsd/jsd,+/+, and (jsd or +)/+,+/+ littermates from male mice homozygous for gld mice that were on B6-C3H background. There were no phenotypic differences between jsd/+ and wild-type mice as indicated by the lack of significant differences in testis histology or weights of these mice and the fathering of normal numbers of pups by jsd/+ mice. Hence, mice of both the above genotypes were used as wild type in the present study.

Screening the mice for jsd, p53, Fas, and FasL mutations
Initially jsd mice were genotyped by PCR analysis of DNA using loci D1Mit 415 and D1Mit 181, which encode microsatellite DNA sequences close to the jsd locus (10). Subsequently, after the jsd gene was identified, the genotyping was done using the primers described and testing the PCR product for sensitivity to HphI digestion (4).

The p53-null and gld mutant mice were genotyped by PCR analysis as described earlier (31). The Fas mutants were identified by PCR using the primers designed for the Fas gene (28) and the neomycin with slight modifications. The neomycin primers were 5'-CTG AAT GAA CTG CAG GAC GA-3' and 5'-ATA CTT TCT CGG CAG GAG CA-3'. The PCR was performed in a PTC-100 programmable thermal controller (MJ Research Inc., Waltham, MA) with the conditions for the neomycin being 94 C for 3 min, followed by 35 cycles of 94 C for 20 sec, 55 C for 30 sec, and 72 C for 30 sec. The cycles for Fas were similar except that the selected annealing temperature was 60 C. The constituents of the PCR buffer used were same as described earlier (10).

Experimental design
Spermatogonial differentiation was analyzed at 8 wk of age in jsd mutant mice also carrying the additional mutation of p53-null, Fas-null, or gld. The p53-null mice were also killed at the age of 5 and 12 wk to assess the age dependence of the decline in spermatogenesis and analyze intratesticular testosterone (ITT) levels at wk 12. In all these experiments, single-mutant littermates and wild-type mice served as controls. The effect of heterozygosity of p53 on spermatogonial differentiation in jsd males was also analyzed.

Histological analysis
In all experiments testes were weighed. The left testis was fixed in Bouin’s fluid and embedded in paraffin or methacrylate, and sections were stained in hematoxylin or periodic acid-Schiff-hematoxylin; the right testis was used for ITT measurements. All seminiferous tubules (~100–150) in the testis section were categorized as either differentiating (containing germ cells at the stage of B spermatogonia or beyond) or not, and the tubule differentiation index (TDI) was taken as the percentage of tubules that were differentiating. The numbers of A spermatogonia per 100 Sertoli cells, differentiating spermatogonia [intermediate spermatogonia, B spermatogonia and preleptotenes (IBPs)] per 100 Sertoli cells, apoptotic indices, and mitotic indices were measured as described earlier (32) in methacrylate sections of testes from 8-wk-old mice. The morphological criterion used for apoptosis of mouse spermatogonia was the appearance of dense chromatin distributed in the cell nucleus (33).

Stereological techniques
The optical disector method (34) was used to determine the total number of cells per testis in 25-µm-thick methacrylate sections. All estimates were performed using a x100 objective on a DM LB microscope (Leica, Wetzlar, Germany) with a motorized stage controller and joystick (35). A software package, Stereo Investigator (MicroBrightField, Colchester, VT), was used to superimpose an unbiased counting frame on the video image. Fields were selected by a systematic uniform random sampling scheme as described previously (34).

A counting frame with an area of 3600 µm2 was used to count Sertoli cells, type A spermatogonia, and intermediate spermatogonia/B spermatogonia/preleptotene spermatocytes. Leptotene/zygotene/pachytene spermatocytes were counted only in jsd and p53-null-jsd mice. We counted 40–700 cells of each type in each animal. The average number of sampling sites was 100 per section.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay
The TUNEL staining of the Bouin’s fixed testis sections was performed using the DeadEnd Colorimetric TUNEL system (Promega, Madison, WI) following the manufacturer’s instructions. Slides were counterstained with hematoxylin, and dehydrated in graded alcohol solutions. Control sections containing no recombinant terminal deoxynucleotidyl transferase enzyme were processed in parallel. The number of TUNEL-positive germ cells was counted in 100–150 tubules and the percentage of tubules that were TUNEL positive (showing at least one TUNEL-positive germ cell) or the number of TUNEL-positive cells/tubule was determined.

Immunohistochemical staining for caspases
Bouin’s fixed, paraffin-embedded testicular sections were immunostained for caspase-9 and caspase-8 as described previously (36). Two different rabbit polyclonal antibodies for caspase-9 were used (Cell Signaling Technology, Beverly, MA), both corresponding to residues surrounding the cleavage site of mouse caspase-9. The first one detects endogenous levels of the 37-kDa subunit of mouse caspase-9 only after cleavage at aspartic acid 353. It does not cross-react with full-length caspase-9 or other caspases at endogenous levels. The second one recognizes both full-length and large fragments of mouse caspase-9 after cleavage and has been successfully used to identify apoptotic testicular germ cells in mice (37). These antibodies were used at concentrations of 1:25 and 1:50, respectively. An antihuman active caspase-8 antibody (Novus Biologicals, Littleton, CO) was used (1:2000) that cross-reacts specifically with mouse active caspase-8. Immunoreactivity was detected using biotinylated goat antirabbit IgG secondary antibody followed by avidin-biotinylated horseradish peroxidase complex visualized with diaminobenzidine according to the manufacturer’s instructions (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Slides were counterstained with hematoxylin.

Hormone measurements
The entire right testis was weighed, immediately frozen in liquid nitrogen, and stored at –80 C. The testis was homogenized, debris was removed by centrifugation, and the supernatant was used for the assay of ITT, using the DSL-4000 (Diagnostic Systems Laboratories, Webster TX)-coated tube RIA kit as described earlier (38). The lower limit of detection for testosterone by this assay was 0.041 ng/ml.

Cryptorchid surgery
Normal adult mice from C57BL6/J background were unilaterally cryptorchidized. The adipose tissue of the right caput epididymis was sutured to the inner peritoneal wall that placed the testis at the level of the urinary bladder as described earlier (10). The left testis remained in the scrotum and was used as the control. Mice were killed 2 d later for analysis.

Statistical analysis
The data were represented as the mean ± SEM of the untransformed data for TDI, and testis weight, and as the mean ± SEM calculated from log-transformed data for ITT. The differences between the groups were first analyzed by one-way ANOVA. If the differences were significant (P < 0.05), Dunnett’s post hoc test for multiple comparisons was performed to determine the significance of differences between wild-type or double mutants and the jsd/jsd mutant group. When only two groups were compared, a t test was performed instead to analyze the significance of the difference. A computer-assisted statistics program (SPSS, Inc., Chicago, IL) was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Germ cell apoptosis in developing jsd testis
To determine the timing and types of germ cells that undergo cell death, we analyzed the testicular sections from pubertal and adult jsd mice for apoptosis. At 4–5 wk of age, widespread apoptosis of spermatocytes and spermatogonia (Fig. 1Go, A and B) were observed with 32 ± 5% of the tubules showing TUNEL-positive germ cells. In some cases, even at 5 wk of age, many tubules were depleted of advanced germ cells and showed few spermatocytes, most of which were TUNEL positive, and both healthy and TUNEL-positive spermatogonia (Fig. 1Go, C and D). In the adults, the spermatogonia, the only germ cells that remained, continued to undergo apoptosis instead of differentiation with 14 ± 3 and 13 ± 3% of the tubules being TUNEL-positive tubules at 8 and 12 wk of age, respectively.


Figure 1
View larger version (145K):
[in this window]
[in a new window]

  		FIG. 1. TUNEL-stained testicular sections from 4.5- to 5-wk-old jsd mice showing varying degrees of germ cell loss. A and B, Initiation of apoptosis in spermatocytes and spermatogonia in 4.5-wk-old jsd testis. C and D, Persistence of both apoptotic and healthy type A spermatogonia in tubules in which most of the germ cells are lost, in 5-wk-old jsd testis. B and D are magnified views of outlined portions from A and C, respectively. Note the presence of TUNEL-positive spermatocytes (arrowhead) and spermatogonia (solid arrow) and a normal type A spermatogonium (open arrow). Bars, 50 µm (A and C) and 10 µm (B and D).

 
Spermatogonial differentiation and apoptosis in p53-deficient jsd mice
To determine whether p53 is involved in the apoptosis of germ cells in jsd testis, we produced p53-null-jsd double-mutant mice. Similar to that observed in jsd single mutant mice the testis weights in double mutants at 5, 8, and 12 wk of age were drastically reduced, compared with wild-type mice (Fig. 2Go, A–C). However, the testis weights in the p53-null-jsd mice were significantly higher than jsd single mutants at 5 and 8 wk of age. Although there was an age-related decline in the percentage of tubules with differentiated cells (TDI) in both genotypes, consistent with the above observations, the values for p53-null-jsd mice were higher than the jsd single mutants at all time points (Fig. 2Go, D–F). By and large, only type A spermatogonia were observed in the tubules of adult jsd single mutants (Fig. 3Go, A and B), with less than 4% of the tubules showing B spermatogonia as most advanced differentiated germ cells. In contrast, about 30% of the tubules contained differentiating cells in the adult p53-null-jsd double mutants, and in all these differentiated tubules, B spermatogonia or a few spermatocytes were the latest stages observed (Fig. 3Go, C and D). Thus, at 8 wk of age in p53-null-jsd/jsd testis, of 29 ± 5% of the differentiated tubules, 19 ± 5% of the tubules had B spermatogonia as the most advanced stages and the remaining 10 ± 2% of the tubules showed spermatocytes also.


Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		FIG. 2. Testis weights (A–C) and tubule differentiation indices (D–F) in the jsd single-mutant, p53 heterozygous-jsd, and p53-null-jsd littermate mice at different ages, compared with wild-type and p53-null mice. Significance of differences for testis weights of p53 heterozygous-jsd and p53-null-jsd mice, compared with jsd single mutants (Student’s t test). **, P < 0.01; *, P < 0.05. The testis weights of wild-type and p53-null mice were significantly higher than all three other groups of mutants at each tested age (P < 0.001). Significance of differences for TDI of p53 heterozygous-jsd and p53-null-jsd mice, compared with jsd single mutants (Student’s t test). ***, P < 0.001; *, P < 0.05. Significance of difference for p53-null-jsd mice, compared with p53 heterozygous-jsd (Student’s t test). {dagger}, P < 0.05 (n = 6–11).

 

Figure 3
View larger version (120K):
[in this window]
[in a new window]

  		FIG. 3. Photomicrographs of testis sections from 8-wk-old jsd single mutant (A and B) and p53-null-jsd double-mutant mice (C and D) stained with periodic acid-Schiff-hematoxylin. Low-power micrographs are presented for the two genotypes (A and C) with the magnified views of outlined portions of each (B and D). The tubules from jsd testis show type A spermatogonia (arrowhead) only as the germ cells. The differentiated tubules in p53-null-jsd mice have B spermatogonia (arrow) or spermatocytes (open arrow) as the most advanced stages. Bars, 50 µm (A and C) and 10 µm (B and D).

 
Consistent with the above observations and the known effects of haploinsufficiency for p53 (39, 40), testis weights (Fig. 2Go, A–C) and TDIs (Fig. 2Go, D–F) in p53 heterozygous-jsd (+/p53,jsd/jsd) mice at the different ages studied were intermediate between the jsd single-mutant and p53-null-jsd double-mutant mice.

Furthermore, a quantitative estimation of the germ cells by stereological optical disector techniques and calculation of their numbers relative to the Sertoli cells in 8-wk-old mice showed an increase in the number of A spermatogonia and early differentiated spermatogenic cells in p53-null-jsd mice, compared with the jsd single mutants (Fig. 4Go and Table 1Go). However, the numbers of these cells in p53-null-jsd mice were still below that of the p53-null or wild-type mice, suggesting the occurrence of a p53-independent mechanism for loss of germ cells even at this early stage of spermatogonial differentiation. The continued loss of differentiating germ cells in jsd mutants as well as p53-null-jsd double mutants was indicated by the percentage of early spermatogenic cells showing morphological characteristics of apoptosis, which was 13 ± 4% in the p53-null-jsd double mutants, similar to that of 7 ± 1% in jsd single mutants. The mitotic indices of the spermatogonia were 25 ± 3% in jsd single mutants, compared with a value of 22 ± 6% in p53-null-jsd mice, indicating that the proliferative status of spermatogonia was unchanged after p53 deletion in jsd mice.


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 4. Absolute numbers of type A spermatogonia (A gonia), differentiated early spermatogenic cells (IBPs: intermediate spermatogonia, B spermatogonia, and preleptotene), spermatocytes (Scytes), and Sertoli cells in 8-wk-old p53-null-jsd mice and jsd/jsd, p53-null, and wild-type littermates. Different letters on the bars indicate significant differences between the specific germ cell types of mice from different genotypes (P < 0.05, Student’s t test); same letter on the bars indicates the differences were not significant (n = 4).

 

View this table:
[in this window]
[in a new window]

  		TABLE 1. Numbers (mean ± SEM) of type A spermatogonia and early spermatogenic cells per 100 Sertoli cells in 8-wk-old mice of different genotypes

 
We analyzed the ITT levels in the p53-null-jsd mice to rule out the possibility that low ITT levels in these mice stimulated spermatogonial differentiation, as it does in GnRH antagonist-treated jsd single mutants (41). Although the ITT concentrations of p53 heterozygous-jsd and p53-null-jsd mice were lower than that in jsd single-mutant littermates (Fig. 5Go), this was mainly due to the increased weights of the testis in the former; the concentrations in the p53-null-jsd mice (937 ng/g testis) were still 10-fold higher than the wild-type mice, which rules out that low levels of ITT could be responsible for the stimulation of spermatogonial differentiation in p53-null-jsd mice. The factors that determine the dramatic increase in the ITT concentration in jsd mice, with or without additional mutations, are the greater proportion of the Leydig cells in the testis as the germ cells are lost, apparent decreased clearance rate of the testosterone from testis due to decline in blood flow (42), and elevated LH levels (41).


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 5. ITT levels in 12-wk-old wild-type, p53-null, jsd single-mutant, p53 heterozygous-jsd, and p53-null-jsd mice. Significance of the difference for mice of different genotypes, compared with wild-type mice (Dunnett’s test) is as follows: {dagger}, P < 0.001. Significance of the difference for wild-type and mice of different genotypes, compared with jsd single mutant littermates (Dunnet’s test) is as follows: *, P < 0.001 (n = 4–10).

 
Spermatogonial differentiation in Fas-null-jsd and gld-jsd mice
We further tested jsd mice to determine whether the apoptosis of early spermatogenic cells was mediated by an effector of the extrinsic pathway by testing the role of Fas-mediated pathway of apoptosis in these mice. In 8-wk-old Fas-null, jsd double mutants, the TDI, spermatogonial numbers, numbers of differentiated early spermatogenic cells, and apoptotic indices were similar to those in the jsd single-mutant littermates (Fig. 6Go, A–D), ruling out the involvement of Fas-mediated pathway in the apoptosis of spermatogonia in jsd mice. To further confirm that FasL was not acting on receptors other than Fas, we also produced gld-jsd mice, which have functionally inactive FasL. The double-mutant gld-jsd mice had a TDI of 1 ± 0.5%, compared with a value of 4 ± 1% in jsd single-mutant littermates at 8 wk of age, confirming that the FasL-mediated apoptosis of spermatogonia also did not occur in jsd mice. Although significantly reduced TDI values in the gld-jsd mice raises the question that FasL action might have a role in supporting the differentiation of spermatogonia, such low levels of TDI may not be biologically significant.


Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 6. TDI (A), relative numbers of type A spermatogonia (B), relative numbers of differentiated early spermatogenic cells (intermediate spermatogonia, B spermatogonia, and preleptotene) (C), and the apoptotic indices (D) in 8-wk-old jsd single-mutant mice and Fas-null, jsd double-mutant littermates. The various parameters were not significantly different between the two genotypes (Student’s t test; n = 4–5).

 
Involvement of caspase-9 in the apoptosis of jsd spermatogonia
To determine whether the death of spermatogonia undergoing apoptosis involved an intrinsic pathway, jsd testes were immunostained for caspase-9, the key initiator caspase specific for the intrinsic pathway of apoptosis. Normal mouse testis exposed to increased abdominal temperature by cryptorchid surgery for 2 d were concurrently stained as positive controls because it has been shown that a mitochondria-dependent intrinsic pathway is involved in heat-induced death of male germ cells through caspase-9 activation (37). Cleaved caspase-9 staining was observed in many spermatocytes in the stage X-XII tubules of cryptorchidized wild-type mouse testis (Fig. 7Go, A and B). These spermatocytes should correspond to cells that are initiating and undergoing apoptosis as shown previously in heat-exposed testis (37, 43). In 10-wk-old jsd mice, many spermatogonia were also positive for cleaved caspase-9 (Fig. 7Go, C and D), suggesting the involvement of caspase-9 in the apoptosis of these spermatogonia. In 5-wk-old jsd testis, active caspase-9 was present in both some spermatogonia and a few spermatocytes (data not shown). In the 5-wk-old p53-null-jsd testis in those tubules that almost lost germ cells of advanced stages, few active caspase-9-positive spermatogonia, likely in the differentiated stages, and some active caspase-9-positive spermatocytes were observed, with many normal spermatogonia being present (Fig. 7Go, E and F). This suggests that even p53-independent apoptosis in jsd mice occurs via a caspase-9 pathway at least in some of the differentiated spermatogonia and spermatocytes that undergo apoptosis. The immunostaining with the caspase-9 antibody that recognizes both full-length and cleaved active caspase-9 also displayed a similar pattern of staining in the cryptorchidized, jsd, and p53-null-jsd testis (data not shown) and supported the above observations. Caspase-9-positive germ cells as detected using either of the two antibodies were rarely seen in the normal wild-type testis (data not shown).


Figure 7
View larger version (127K):
[in this window]
[in a new window]

  		FIG. 7. Immunostaining for active caspase-9 in wild-type mouse testis cryptorchidized for 2 d (A and B), 10-wk-old jsd testis (C and D), and 5-wk-old p53-null-jsd testis (E and F). B, D, and F are the magnified views of outlined portions from A, C, and E, respectively. Note the active caspase-9-positive spermatocytes in a stage XII tubule in the cryptorchidized testis (arrowheads) and active caspase-9-positive spermatogonia in the jsd and p53-null-jsd testes (solid arrows) in addition to the presence of normal spermatogonia that are negative for caspase-9 (open arrows) and an active caspase-9 spermatocyte in p53-null jsd testis (arrowhead). Bars, 50 µm (A, C, and E) and 10 µm (B, D, and E).

 
Involvement of caspase-8 in the apoptosis of jsd spermatogonia
The testes from jsd mice were immunostained for active caspase-8 to test the prevalence of extrinsic pathway of apoptosis in the death of spermatogonia during differentiation. Many glandular cells from the mouse gastric mucosa, used as positive control, showed positive staining for the antibody (Fig. 8AGo). It is known that glandular cells of gastric mucosa periodically undergo apoptosis through the extrinsic pathway (44, 45). In 10-wk-old jsd testes that had only spermatogonia, no positive staining was observed (Fig. 8BGo), suggesting that spermatogonial apoptosis does not occur through the extrinsic pathway. Furthermore, even in the 5-wk-old-jsd testes, no positive staining was observed in germ cells up to the stage of preleptotene spermatocytes. However, surprisingly, most of the spermatocytes of later stages and round spermatids in the 5-wk-old jsd testis showed positive staining for active caspase-8 antibody (Fig. 8Go, C and D). A similar staining pattern was observed in 5-wk-old p53-null-jsd testis also (data not shown). Although these results indicate the possible involvement of extrinsic pathway of apoptosis in the death of spermatocytes and round spermatids in jsd testis that occurs at around this age, that has yet to be confirmed. In fact, the weak positive staining that was also observed in the healthy spermatocytes of adult wild-type mice, especially in tubules at stages X-XII (Fig. 8Go, E and F) makes it difficult to conclude with certainty whether the presence of active caspase-8 in spermatocytes and round spermatids in jsd testis is the mechanism by which they undergo apoptosis.


Figure 8
View larger version (149K):
[in this window]
[in a new window]

  		FIG. 8. Immunostaining for active caspase-8 in glandular cells of mouse gastric mucosa (A), 10-wk-old jsd testis (B), 5-wk-old jsd testis (C and D), and adult wild-type mouse testis (E and F). D and F are the magnified views of outlined portions from C and E, respectively. Note the active caspase-8-positive glandular cells of gastric mucosa, absence of active caspase-8-positive staining in the spermatogenic cells up to preleptotene spermatocytes including morphologically apoptotic spermatogonia in 10- and 5-wk-old jsd testis, positive active caspase-8 staining in most of the spermatocytes and round spermatids of 5-wk-old jsd testis, and a weak positive staining in the spermatocytes of a stage XI tubule from adult wild-type mouse testis. Gl, glandular cells; Sg, spermatogonia; ApSg, apoptotic spermatogonium; Pls, preleptotene spermatocytes; Cytes, spermatocytes; rtids, round spermatids. Bars, 20 µm (A), 50 µm (B, C and E), 10 µm (D and F).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results from this study show that p53 is involved in the apoptosis of type A spermatogonia and early differentiating germ cells up the preleptotene spermatocyte stage in jsd mice, but apoptosis in the later stages must be a p53-independent process. The study also rules out a role for the Fas/FasL-mediated pathway in the death of the spermatogonia and spermatocytes in jsd testis. Furthermore, the involvement of caspase-9 and the absence of Fas/FasL-mediated apoptosis suggests that such loss of spermatogonia in jsd mice likely occurs through a cell intrinsic pathway of apoptosis.

Because testosterone is known to be a causative factor for the apoptosis of type A spermatogonia in jsd mice (41) and spermatogonia do not have androgen receptors, one of the possible mechanisms for spermatogonial apoptosis is that testosterone acted on androgen receptor-positive somatic cells, causing the apoptosis of spermatogonia through an extrinsic pathway, such as the Sertoli cells. Sertoli cells are known to express FasL and activate the Fas receptors on the germ cells (46, 47, 48) to target germ cells for death after exposure to cytotoxic agents (31, 46, 49, 50, 51, 52, 53) or heat stress (24); or germ cells with genetic differentiation defects may also be targets of Fas-mediated apoptosis (49). However, the results from this study indicate that Sertoli cells did not mediate the testosterone-dependent apoptosis of spermatogonia, at least by the FasL or Fas pathway.

Although it was difficult to identify the cellular subtype of the TUNEL-positive spermatogonia, in jsd mice immediately after testicular descent at 4 wk of age, it is likely that most of the type A spermatogonia that started undergoing apoptosis along with the later stages were differentiating. Adult jsd mice contain almost exclusively undifferentiated spermatogonia, with few differentiating spermatogonia on the genetic background we used (HB129) (9). In addition, the presence of relatively few apoptotic spermatogonia (0.23 TUNEL-positive spermatogonia/tubule; apoptotic index: 7%) at 8 wk of age suggests that part of these apoptotic spermatogonia were differentiating ones in addition to those that were undergoing apoptosis during Aal to A1 transition (9, 54). The increase in the number of type A spermatogonia in the p53-null, jsd double-mutant mice indicates differentiating A spermatogonia were protected from undergoing apoptosis (21) as discussed later. It is unlikely that such an increase in the spermatogonia in p53-null, jsd double-mutant mice was due to the increased proliferation of the spermatogonia because there was no change in the percentage of spermatogonia undergoing mitosis.

During spermatogenesis, a distinct induction of p53 was found in spermatogonia after irradiation, and in the p53-null testis, the differentiating A2-B spermatogonia were found to be more radio resistant than their wild-type controls, indicating that p53 is essential for removal of lethally irradiated differentiating spermatogonia (21, 26). Furthermore, the numbers of A1 spermatogonia were also increased in the p53-null mice, indicating that the production of differentiating spermatogonia from the undifferentiating ones is enhanced in these mice (21) by the inhibition of apoptosis of these spermatogonia. Surprisingly Sertoli cell numbers were significantly reduced in jsd testis but not p53-null-jsd testis, compared with wild-type mice (Fig. 4Go). The slight reduction in the Sertoli cells in adult jsd testis appears to be a result of frequent sloughing of Sertoli cells, some of which appeared degenerative, to the lumen in these atrophic tubules. The population of the seminiferous epithelium with increased numbers of spermatogonia in the adult p53-null-jsd testis may inhibit the sloughing of the Sertoli cells. Even accounting for increased number of Sertoli cells in these mice, the numbers of type A spermatogonia per Sertoli cell was still increased, indicating that the p53 gene acts within spermatogonia to promote apoptosis of jsd-mutant spermatogonia. The early stages of spermatogonial differentiation are also known to be susceptible to p53-mediated apoptosis under heat stress, but the apoptosis of jsd-mutant spermatocytes and spermatids were independent of p53 (25). The results from our study are consistent with these previous reports on the role of p53-mediated apoptosis of spermatogonia after radiation or heat stress.

p53 is known to act at multiple sites, in both the intrinsic and extrinsic pathway of apoptosis (55). The data in the present study suggest that the induction of apoptosis of jsd spermatogonia at least partially involves p53 and occurs through the intrinsic pathway of apoptosis as evidenced by the absence of a role for FasL/Fas, absence of active caspase-8, and the activation of caspase-9 in these cells. Although FasL/Fas was not involved, another member of the tumor necrosis receptor family, DR5 death receptor gene, is also known to be a target for p53 (53) and was not tested here. Moreover, the possible death receptor activation-induced recruitment of intrinsic/mitochondrial pathway via the cleavage of Bid (56, 57), a BH3-only member of the Bcl-2 family leading to apoptosome (cytochrome c-Apaf1-procaspase-9 complex) activation, does not totally rule out the occurrence of extrinsic death receptor-mediated apoptosis of spermatogonia in jsd mice.

Our data suggest the possible involvement of extrinsic pathway in the apoptosis of spermatocytes and round spermatids of jsd testis. Because p53 is a major regulator of the intrinsic pathway of apoptosis, loss of pachytene spermatocytes and round spermatids via extrinsic pathway but the death of earlier stages of germ cells only though the intrinsic pathway would be consistent with the rescue of germ cells only up to the preleptotene or early spermatocyte stage in p53-null-jsd testis.

The discovery of the mutation responsible for the jsd phenotype suggests that the intrinsic pathway is the most likely pathway causing apoptosis of germ cells. The jsd mutation results in the loss of the Utp14b gene that is involved in 18S RNA processing (7). It has also been shown that 18S rRNA processing is defective in jsd mutant gem cells (Zhao, M., and M. L. Meistrich, unpublished results), and it is reported that the germ cells are defective in the jsd mutants (8, 58). The apoptosis of these germ cells, at least that of spermatogonia, is likely activated through the intrinsic pathway as a cellular stress response, which could be due to the reduced global protein synthesis as a result of defective RNA processing (59, 60, 61).

There is evidence that p53 is involved in response to the abnormal pre-rRNA processing. Functional inactivation of the mouse nucleolar protein block of proliferation 1 (Bop1) that inhibits pre-RNA processing was also reported to induce p53-dependent cell cycle arrest (62, 63). However, the partial rescue of spermatogonial differentiation in p53-null, jsd double-mutant mice is not likely to be determined by enhanced pre-rRNA processing because elimination of p53 does not affect rRNA processing (64).

These results do not support a direct role for testosterone in the apoptosis of spermatogonia in the jsd mouse. Moreover, we also have evidence to suggest that testosterone may act to maintain a scrotal temperature more reduced than the abdominal temperature, which is detrimental for spermatogonial differentiation in jsd mice (9, 10) (Shetty, G., and M. L. Meistrich, unpublished data). The mechanism by which elevated temperature restores spermatogenesis in jsd mice is not known. It may restore protein synthesis, or it is also possible that in jsd mice apoptosis is reduced at elevated temperatures, even with decreased protein synthesis.

Thus, the spermatogonia in jsd mice undergo apoptosis by the intrinsic pathway as the result of a cell-autonomous defect, a pathway that does not involve FasL-Fas system. p53 plays a role in apoptosis of type A and B spermatogonia during differentiation, but apoptosis in the later stages must be independent of p53.


   Acknowledgments
 
Our sincere gratitude goes to Dr. Marvin L. Meistrich for his guidance throughout the study, suggestions during the preparation of the manuscript, and material and monetary assistance. We are thankful to Mr. Kuriakose Abraham for histological preparations, Ms. Jun Ju for technical assistance, and Mr. Walter Pagel for editorial advice.


   Footnotes
 
This work was supported by Research Grant HD 40397 from the National Institutes of Health (NIH)/National Institute of Child Health and Human Development, Cancer Center Support Grant CA 16672 from the NIH, and an institutional research grant from The University of Texas M. D. Anderson Cancer Center.

Disclosure Statement: The authors have no conflicts of interest to declare.

First Published Online March 20, 2008

Abbreviations: Aal, A aligned; FADD, Fas-associated death domain; FasL, Fas ligand; IBP, intermediate spermatogonia, B spermatogonia and preleptotenes; ITT, intratesticular testosterone; TDI, tubule differentiation index; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Received September 27, 2007.

Accepted for publication March 7, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Oakberg EF 1971 Spermatogonial stem-cell renewal in the mouse. Anat Rec 169:515–532[CrossRef][Medline]
  2. de Rooij DG 1998 Stem cells in the testis. Int J Exp Pathol 79:67–80[CrossRef][Medline]
  3. Beamer WG, Cunliffe-Beamer TL, Shultz KL, Langley SH, Roderick TH 1988 Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferations of male mice. Biol Reprod 38:899–908[Abstract]
  4. Rohozinski J, Bishop C 2004 The mouse juvenile spermatogonial depletion (jsd) phenotype is due to a mutation in the X-derived retrogene, mUtp14b. Proc Natl Acad Sci USA 101:11695–11700[Abstract/Free Full Text]
  5. Bradley J, Baltus A, Skaletsky H, Royce-Tolland M, Dewar K, Page DC 2004 An X-to-autosome retrogene is required for spermatogenesis in mice. Nat Genet 36:872–876[CrossRef][Medline]
  6. Zhao M, Rohozinski J, Sharma M, Ju J, Braun RE, Bishop CE, Meistrich ML 2007 Utp14b: a unique retrogene within a gene that has acquired multiple promoters and a specific function in spermatogenesis. Dev Biol 304:848–859[CrossRef][Medline]
  7. Dragon F, Gallagher JE, Compagnone-Post PA, Mitchell BM, Porwancher KA, Wehner KA, Wormsley S, Settlage RE, Shabanowitz J, Osheim Y, Beyer AL, Hunt DF, Baserga SJ 2002 A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417:967–970[CrossRef][Medline]
  8. Boettger-Tong HL, Johnston DS, Russell LD, Griswold MD, Bishop CE 2000 Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis. Biol Reprod 63:1185–1191[Abstract/Free Full Text]
  9. Bolden-Tiller OU, Chiarini-Garcia H, Poirier C, Alves-Feitas D, Weng CC, Shetty G, Meistrich ML 2007 Genetic factors contributing to defective spermatogonial differentiation in juvenile spermatogonial depletion (Utp14b/jsd) mice. Biol Reprod 77:237–246[Abstract/Free Full Text]
  10. Shetty G, Weng CCY 2004 Cryptorchidism rescues spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mice. Endocrinology 145:126–133[Abstract/Free Full Text]
  11. Kojima Y, Kominami K, Dohmae K, Nonomura N, Miki T, Okuyama A, Nishimune Y, Okabe M 1997 Cessation of spermatogenesis in juvenile spermatogonial depletion (jsd/jsd) mice. Int J Urol 4:500–507[Medline]
  12. Hengartner MO 2000 The biochemistry of apoptosis. Nature 407:685–687[CrossRef]
  13. Kumar S, Vaux DL 2002 A Cinderella caspase takes center stage. Science 297:1290–1292[Free Full Text]
  14. Owen-Schaub LB, Zhang W, Cusack JC, Angelo LS, Santee SM, Fujiwara T, Roth JA, Deisseroth AB, Zhang WW, Kruzel E, Radinsky R 1995 Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression. Mol Cell Biol 15:3032–3040[Abstract]
  15. Muller M, Wilder S, Bannasch D, Israeli D, Lehlbach K, Li-Weber M, Friedman SL, Galle PR, Stremmel W, Oren M, Krammer PH 1998 p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. J Exp Med 188:2033–2045[Abstract/Free Full Text]
  16. Miyashita T, Reed JC 1995 Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293–299[CrossRef][Medline]
  17. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T, Tanaka N 2000 Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288:1053–1058[Abstract/Free Full Text]
  18. Nakano K, Vousden KH 2001 PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 7:683–694[CrossRef][Medline]
  19. Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, Moll UM 2003 p53 has a direct apoptogenic role at the mitochondria. Mol Cell 11:577–590[CrossRef][Medline]
  20. Schuler M, Green DR 2001 Mechanisms of p53-dependent apoptosis. Biochem Soc Trans 29:684–688[CrossRef][Medline]
  21. Beumer TL, Roepers-Gajadien HL, Gademan IS, van Buul PP, Gil-Gomez G, Rutgers DH, de Rooij DG 1998 The role of the tumor suppressor p53 in spermatogenesis. Cell Death Differ 5:669–677[CrossRef][Medline]
  22. Ohta Y, Nishikawa A, Fukazawa Y, Urushitani H, Matsuzawa A, Nishina Y, Iguchi T 1996 Apoptosis in adult mouse testis induced by experimental cryptorchidism. Acta Anat 157:195–204[Medline]
  23. Jordan SA, Speed RM, Bernex F, Jackson IJ 1999 Deficiency of Trp53 rescues the male fertility defects of KitW-v mice but has no effect on the survival of melanocytes and mast cells. Dev Biol 215:78–90[CrossRef][Medline]
  24. Yin Y, Stahl BC, Dewolf WC, Morgentaler A 2002 p53 and Fas are sequential mechanisms of testicular germ cell apoptosis. J Androl 23:64–70[Abstract]
  25. Ohta H, Aizawa S, Nishimune Y 2003 Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells. Biol Reprod 68:2249–2254[Abstract/Free Full Text]
  26. Hasegawa M, Wilson G, Russell LD, Meistrich ML 1997 Radiation-induced cell death in the mouse testis: relationship to apoptosis. Radiat Res 147:457–467[Medline]
  27. Jacks T, Remington L, Williams BW, Schmitt EM, Halachmi S, Bronson RT, Weinberg RA 1994 Tumor spectrum analysis in p53-mutant mice. Curr Biol 4:1–7[Medline]
  28. Adachi M, Suematsu S, Kondo T, Ogasawara J, Tanaka T, Yoshida N, Nagata S 1995 Targeted mutation in the Fas gene causes hyperplasia in peripheral lymphoid organs and liver. Nat Genet 11:294–300[CrossRef][Medline]
  29. Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T, Nagata S 1994 Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76:969–976[CrossRef][Medline]
  30. Shetty G, Weng CC, Porter KL, Zhang Z, Pakarinen P, Kumar TR, Meistrich ML 2006 Spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mice with androgen receptor or follicle stimulating hormone mutations. Endocrinology 147:3563–3570[Abstract/Free Full Text]
  31. Embree-Ku M, Venturini D, Boekelheide K 2002 Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis. Biol Reprod 66:1456–1461[Abstract/Free Full Text]
  32. Shuttlesworth GA, de Rooij DG, Huhtaniemi I, Reissmann T, Russell LD, Shetty G, Wilson G, Meistrich ML 2000 Enhancement of A spermatogonial proliferation and differentiation in irradiated rats by GnRH antagonist administration. Endocrinology 141:37–49[Abstract/Free Full Text]
  33. Hasegawa M, Zhang Y, Niibe H, Terry NHA, Meistrich ML 1998 Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in p53-deficient mice. Radiat Res 149:263–270[Medline]
  34. Wreford NG 1995 Theory and practice of stereological techniques applied to the estimation of cell number and nuclear volume in the testis. Microsc Res Tech 32:423–436[CrossRef][Medline]
  35. Shetty G, Weng CC, Meachem SJ, Bolden-Tiller OU, Zhang Z, Pakarinen P, Huhtaniemi I, Meistrich ML 2006 Both testosterone and FSH independently inhibit spermatogonial differentiation in irradiated rats. Endocrinology 147:472–482[Abstract/Free Full Text]
  36. Sinha Hikim AP, Lue Y, Yamamoto CM, Vera Y, Rodrigues S, Yen PH, Soeng K, Wang C, Swerdloff RS 2003 Key apoptotic pathways for heat-induced programmed germ cell death in the testis. Endocrinology 144:3167–3175[Abstract/Free Full Text]
  37. Vera Y, Diaz-Romero M, Rodrigues S, Lue Y, Wang C, Swerdloff RS, Sinha Hikim AP 2004 Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice. Biol Reprod 70:1534–1540[Abstract/Free Full Text]
  38. Shetty G, Wilson G, Huhtaniemi I, Shuttlesworth GA, Reissmann T, Meistrich ML 2000 Gonadotropin-releasing hormone analogs stimulate and testosterone inhibits the recovery of spermatogenesis in irradiated rats. Endocrinology 141:1735–1745[Abstract/Free Full Text]
  39. Venkatachalam S, Tyner SD, Pickering CR, Boley S, Recio L, French JE, Donehower LA 2001 Is p53 haploinsufficient for tumor suppression? Implications for the p53+/– mouse model in carcinogenicity testing. Toxicol Pathol 29(Suppl):147–154
  40. Ranaldi R, Palma S, Tanzarella C, Lascialfari A, Cinelli S, Pacchierotti F 2007 Effect of p53 haploinsufficiency on melphalan-induced genotoxic effects in mouse bone marrow and peripheral blood. Mutat Res 615:57–65[Medline]
  41. Shetty G, Wilson G, Huhtaniemi I, Boettger-Tong H, Meistrich ML 2001 Testosterone inhibits spermatogonial differentiation in juvenile spermatogonial depletion mice. Endocrinology 142:2789–2795[Abstract/Free Full Text]
  42. Wang J, Galil KAA, Setchell BP 1983 Changes in testicular blood flow and testosterone production during aspermatogenesis after irradiation. J Endocrinol 98:35–46[Abstract/Free Full Text]
  43. Lue YH, Hikim AP, Swerdloff RS, Im P, Taing KS, Bui T, Leung A, Wang C 1999 Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity. Endocrinology 140:1709–1717[Abstract/Free Full Text]
  44. van Grieken NC, Meijer GA, zur Hausen A, Meuwissen SG, Baak JP, Kuipers EJ 2003 Increased apoptosis in gastric mucosa adjacent to intestinal metaplasia. J Clin Pathol 56:358–361[Abstract/Free Full Text]
  45. Vollmers HP, Dammrich J, Hensel F, Ribbert H, Meyer-Bahlburg A, Ufken-Gaul T, von Korff M, Muller-Hermelink HK 1997 Differential expression of apoptosis receptors on diffuse and intestinal type stomach carcinoma. Cancer 79:433–440[CrossRef][Medline]
  46. Lee J, Richburg JH, Younkin SC, Boekelheide K 1997 The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 138:2081–2088[Abstract/Free Full Text]
  47. Suda T, Takahashi T, Golstein P, Nagata S 1993 Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75:1169–1178[CrossRef][Medline]
  48. French LE, Hahne M, Viard I, Radlgruber G, Zanone R, Becker K, Muller C, Tschopp J 1996 Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover. J Cell Biol 133:335–343[Abstract/Free Full Text]
  49. Sakata S, Sakamaki S, Watanabe K, Nakamura N, Toyokuni S, Nishimune Y, Mori C, Yonehara S 2003 Involvement of death receptor Fas in germ cell degeneration in gonads of kit-deficient W-v/W-v mutant mice. Cell Death Differ 10:676–686[CrossRef][Medline]
  50. Richburg JH, Nanez A, Williams LR, Embree ME, Boekelheide K 2000 Sensitivity of testicular germ cells to toxicant-induced apoptosis in gld mice that express a nonfunctional form of Fas ligand. Endocrinology 141:787–793[Abstract/Free Full Text]
  51. Lee J, Richburg JH, Shipp EB, Meistrich ML, Boekelheide K 1999 The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis. Endocrinology 140:852–858[Abstract/Free Full Text]
  52. Taylor MF, de Boer-Brouwer M, Woolveridge I, Teerds KJ, Morris ID 1999 Leydig cell apoptosis after the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process. Endocrinology 140:3797–3804[Abstract/Free Full Text]
  53. Chandrasekaran Y, Richburg JH 2005 The p53 protein influences the sensitivity of testicular germ cells to mono-(2-ethylhexyl) phthalate-induced apoptosis by increasing the membrane levels of Fas and DR5 and decreasing the intracellular amount of c-FLIP. Biol Reprod 72:206–213[Abstract/Free Full Text]
  54. de Rooij DG, Okabe M, Nishimune Y 1999 Arrest of spermatogonial differentiation in jsd/jsd, Sl17H/Sl17H, and cryptorchid mice. Biol Reprod 61:842–847[Abstract/Free Full Text]
  55. Haupt S, Berger M, Goldberg Z, Haupt Y 2003 Apoptosis—the p53 network. J Cell Sci 116:4077–4085[Abstract/Free Full Text]
  56. Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B, Roth KA, Korsmeyer SJ 1999 Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400:886–891[CrossRef][Medline]
  57. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X 1998 Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94:481–490[CrossRef][Medline]
  58. Ohta H, Yomogida K, Tadokoro Y, Tohda A, Dohmae K, Nishimune Y 2001 Defect in germ cells, not in supporting cells, is the cause of male infertility in the jsd mutant mouse: proliferation of spermatogonial stem cells without differentiation. Int J Androl 24:15–23[CrossRef][Medline]
  59. Meijerman I, Blom WM, de Bont HJ, Mulder GJ, Nagelkerke JF 1999 Induction of apoptosis and changes in nuclear G-actin are mediated by different pathways: the effect of inhibitors of protein and RNA synthesis in isolated rat hepatocytes. Toxicol Appl Pharmacol 156:46–55[CrossRef][Medline]
  60. Silverman RH 2003 Implications for RNase L in prostate cancer biology. Biochemistry 42:1805–1812[CrossRef][Medline]
  61. Shimazu T, Degenhardt K, Nur EKA, Zhang J, Yoshida T, Zhang Y, Mathew R, White E, Inouye M 2007 NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition. Genes Dev 21:929–941[Abstract/Free Full Text]
  62. Strezoska Z, Pestov DG, Lau LF 2000 Bop1 is a mouse WD40 repeat nucleolar protein involved in 28S and 5. 8S RRNA processing and 60S ribosome biogenesis. Mol Cell Biol 20:5516–5528[Abstract/Free Full Text]
  63. Strezoska Z, Pestov DG, Lau LF 2002 Functional inactivation of the mouse nucleolar protein Bop1 inhibits multiple steps in pre-rRNA processing and blocks cell cycle progression. J Biol Chem 277:29617–29625[Abstract/Free Full Text]
  64. Sugimoto M, Kuo ML, Roussel MF, Sherr CJ 2003 Nucleolar Arf tumor suppressor inhibits ribosomal RNA processing. Mol Cell 11:415–424[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shetty, G.
Right arrow Articles by Weng, C. C. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shetty, G.
Right arrow Articles by Weng, C. C. Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals