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Endocrinology Vol. 147, No. 9 4213-4221
Copyright © 2006 by The Endocrine Society

Akt1 Suppresses Radiation-Induced Germ Cell Apoptosis in Vivo

Teresa Rasoulpour, Kathryn DiPalma, Branko Kolvek and Mary Hixon

Department of Pathology and Laboratory Medicine (T.R., B.K., M.H.), Brown University, Providence, Rhode Island 02912; and Providence College (K.D.), Providence, Rhode Island 02918

Address all correspondence and requests for reprints to: Mary Hixon, Ph.D., GE505, Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912. E-mail: Mary_Hixon{at}Brown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Radiation exposure is a well-characterized germ cell injury model leading to cell cycle arrest or apoptosis. The serine-threonine kinase, Akt1, has been implicated in inhibiting cell death induced by different stimuli including growth factor withdrawal, cell cycle discordance, DNA damage, and loss of cell adhesion. However, the in vivo relevance of this prosurvival pathway has not been explored in the testis. To evaluate a protective role for Akt1 in the testis in vivo, we examined the incidence of apoptosis in Akt1-deficient mice after radiation-induced germ cell injury. We found that Akt kinase activity increases in the testes of wild-type mice after ionizing radiation, and that loss of Akt1 results in an earlier onset of germ cell apoptosis and enhanced sensitivity of mitotic spermatogonia to ionizing radiation. At both the mRNA and protein level, neither Akt2 nor Akt3 expression were induced in the absence of Akt1. These data demonstrate an important survival function governed by Akt1 and, to a lesser extent, Akt2 in the survival of germ cells after radiation-induced testicular injury. In addition, the results point to a role for Fas ligand in the regulation of this response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPERMATOGENESIS REQUIRES signaling pathways to regulate a precise balance between cell survival, proliferation, and differentiation. The overall growth of the testis is based on the survival and proliferation of both germ and Sertoli cells. Elimination of germ cells via apoptosis occurs under normal physiologic conditions and is heightened after testicular injury (1, 2). Germ cell apoptosis during testicular development in the mouse has two peaks corresponding to the time of migration of primordial germ cells into the gonads and the beginning of the first round of spermatogenesis (3), which occurs approximately 10–13 d after birth (4, 5). Importantly, the number of germ cells is determined by the supportive capacity of the Sertoli cells (6). Thus, the ratio of the different types of germ cells to Sertoli cells remains relatively constant in mammalian spermatogenesis, and control of this ratio is instrumental during differentiation (4). Given the significance of normal apoptotic signaling in testicular homeostasis, it is important to identify the signaling networks that modulate apoptosis after toxicant-induced testicular injury as they may be representative of the normal homeostasis that occurs in the testis.

The testis is one of the most radiosensitive tissues of the body (7, 8), with very low doses of radiation causing significant impairment of function. Radiation exposure of the testis primarily targets actively dividing germ cells without causing significant injury to Sertoli cells (9, 10). Testicular injury induced by irradiation can result in a cell cycle block, followed by DNA repair, or by apoptosis if the damage is severe (11). Ionizing radiation efficiently induces apoptosis of germ cells, with the actively dividing spermatogonia being the most susceptible, followed by spermatocytes, stem cell spermatogonia, and the highly resistant spermatids (9, 10, 12).

There are three major isoforms of Akt/protein kinase B (PKB) that have been found in mammalian cells; these are termed Akt1/PKB{alpha}, Akt2/PKBß, and Akt3/PKB{gamma}, which are encoded by three separate genes with more than 85% amino acid sequence identity. Despite a high degree of sequence homology, Akt1 and Akt2 appear to differ in their cellular functions with regard to cell survival and metabolism (13, 14, 15). Mice deficient for Akt1 are more sensitive to genotoxic stress (13). In contrast, Akt2 is important in insulin signaling, and male mice deficient for Akt2 display a severe diabetic phenotype (15). The distribution of Akt3 mRNA is more limited than that of either Akt1 or Akt2, with highest expression in the brain and testes (16), and Akt3-deficient mice exhibit a smaller brain size (17). In tumor cells, activation of the phosphatidylinositol 3 kinase (PI3K) kinase/Akt1 pathway has been shown to reduce apoptosis, thereby enhancing tumor cell survival (reviewed in Ref. 18). Inhibition of the PI3 kinase/Akt1 pathway in vitro leads to enhanced susceptibility to cell death by both radiation and chemotherapeutic agents (reviewed in Ref. 19). However, the extent to which the different Akt isoforms contribute to germ cell survival in vivo after toxicant-induced testicular injury has not been explored.

Induction of Fas ligand (FasL) is considered an important pathway of transcription-dependent apoptosis (20). In the testis, the Fas signaling system plays an important role in regulating germ cell apoptosis after toxicant-induced injury (21, 22, 23). Although controversial (24), the Fas receptor has been localized to testicular germ cells; whereas, FasL expression has been localized to the Sertoli cells (reviewed in Refs. 1 and 2 ; see also Refs. 21, 22, 23). After {gamma}-radiation exposure, FasR expression increases dramatically in the rodent testis (21, 22, 23). In contrast, FasL expression remains relatively constant (21, 22, 23). In certain tumor cell lines, inhibition of the PI3 kinase/Akt1 pathway contributes to the up-regulation of FasL resulting in apoptosis (25, 26, 27). Alternatively, IGF1-induced phosphorylation of a forkhead family transcription factor, FKHRL1, by Akt1 prevents forkhead mediated induction of FasL expression consequently inhibiting apoptosis (28, 29).

Based on this information, we tested the hypothesis that Akt1 plays a protective role in the mouse testis after radiation-induced germ cell injury. We found that a threshold of Akt activity is necessary to suppress germ cell apoptosis in the testis. Loss of this Akt1-mediated protective effect results in both an earlier onset of germ cell apoptosis and enhanced sensitivity of mitotic spermatogonia to ionizing radiation-induced injury. In addition, our data provide the first in vivo evidence that both the Akt1 and Akt2 isoforms promote the survival of germ cells after radiation-induced testicular injury. These data point to a role for FasL induction in the modulation of the testicular injury response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Akt1–/– mice were obtained from the laboratory of Dr. Morris Birnbaum (University of Pennsylvania, Philadelphia, PA) and have been backcrossed a minimum of five times into a C57BL/6 background. C57BL/6 mice of the same strain and age were used as control mice. Eight-week-old wild-type mice were purchased from Charles River (Wilmington, MA). Mice were given water and standard lab chow ad libitum. Animals were allowed to acclimatize for at least 1 wk before experiments. The animal room climate was kept at a constant temperature (23.3 ± 2 C) at 30–70% humidity with an alternating 12-h light, 12-h dark cycle. All procedures involving animals were performed in accordance with the guidelines of the institutional animal care and use committee of Brown University in compliance with the guidelines established by the National Institutes of Health.

Primers
For genotyping by PCR, the following primers were used in a single reaction: 853, 5'-GTGGATGTGGAATGTGTGCGAG-3'; 854, 5'-GCTCAGTCAGTGAGGCCAGACC-3'; 855, 5'-CACCCCACAAGCTCTTCTTCCA-3'. The PCR were run with an initial denaturing step of 94 C for 5 min, 39 cycles of 94 C for 30 sec, 63 C for 30 sec, 72 C for 45 sec, followed by a final extension at 72 C for 5 min. PCR genotyping of progeny, the wild-type and targeted bands are 310 and 194 bp, respectively.

Radiation exposure
Adult 8-wk-old Akt1 wild-type, heterozygous, and homozygous C57BL6 mice were used for experiments. Mice were given half-body irradiation at a single dose of 5 Gy with a delivery rate of 6.84 Gy/min using a 137Cesium source (J. L. Sheperd, Mark I Irradiator, Glendale, CA). Mice were restrained in polystyrene chambers, and the upper body was shielded using lead. Control and irradiated mice were killed by carbon dioxide asphyxiation at 0, 1, 3, and 6 h after ionizing radiation, and the testes were excised. Testes were used for in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, protein, or RNA work as indicated.

Assay of Akt1 kinase activity
Mouse testes were lysed in ice-cold lysis buffer (Cell Signaling Technology, Beverly, MA). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). A total of 500 µg of protein from the lysate samples was incubated with gentle rocking at 4 C overnight with immobilized anti-Akt antibody cross-linked to agarose hydrazide beads. After Akt1 was selectively immunoprecipitated from the testes homogenates, the immunoprecipitated products were washed twice in lysis buffer and twice in kinase assay buffer (25 mM Tris, pH 7.5; 10 mM MgCl2; 5 mM ß-glycerolphosphate; 0.1 mM sodium orthovanadate; and 2 mM dithiothreitol), and the samples were resuspended in 25 µl of kinase assay buffer containing 200 µM ATP and 1 µg GSK3{alpha} fusion protein (Cell Signaling Technology). The kinase reaction was allowed to proceed at 30 C for 30 min and was stopped by the addition of 3x SDS sample buffer. Reaction products were resolved by 15% SDS-PAGE followed by Western blotting with an anti-phospho-GSK3{alpha}/ß antibody according to the manufacturer’s specifications.

TUNEL staining and quantitation
For cryosections, unfixed testes were submerged in OCT embedding medium (Sekura Finetek, Inc., Torrance, CA) and snap frozen by immersion in liquid nitrogen. Sections were then cut to 7 µm thickness and mounted on poly-L-lysine-coated glass slides (VWR Scientific, West Chester, PA). Germ cell apoptosis was detected in sections of fresh-frozen testis by the TUNEL labeling method using the ApopTag kit (Chemicon, Temecula, CA). Tissue was counterstained with methyl green. Testis sections were viewed using a Nikon E800 microscope (Melville, NY) using differential interference contrast microscopy. The images were captured with a Kodak DC120 digital camera equipped with a MDS120 adapter (Eastman Kodak Co., Rochester, NY) and processed using Adobe Photoshop 6.0 software (Adobe, San Jose, CA). TUNEL-positive germ cells were quantitated in each tissue section by counting the number of TUNEL-positive cells in each essentially round seminiferous tubule. For each testis section, approximately 100–200 tubules were counted from at least three different mice. The incidence of apoptosis was then categorized into either of three groups, defined as none, one to three, or more than three TUNEL-positive germ cells per seminiferous tubule cross-section. In the control mouse testis, the percentage of seminiferous tubules with more than three TUNEL-positive cells is less than 10%, so that an increase in apoptosis is easily determined using this data presentation. The data, calculated as a percentage of the total, are expressed as the mean ± SEM.

Spermatid head counts
Testes obtained 29 d after irradiation were homogenized separately, and sperm heads were counted on a hemocytometer using previously described methods (30). The counts from the two testes of each of at least three animals were averaged for statistical analysis.

RNA analysis
Total RNA was isolated from testes of control, 1-, 3-, 6-, and 12-h Akt1 wild-type, heterozygous, and homozygous mice. These testes were detunicated, weighed, and homogenized in TriReagent (Sigma Aldrich, St. Louis, MO) and further RNA isolation was preformed according to the TriReagent manufacturer’s protocols.

Quantitative RT-PCR
Total RNA (1 µg) was DNase-I (Invitrogen, Carlsbad, CA) treated and reverse-transcribed using iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s protocols, and the cDNA templates were amplified with each of the primer pairs (see Table 1Go) in independent sets of PCR using iQ SYBR Green Supermix (Bio-Rad) on an iCycler iQ Multicolor Real-time PCR Detection System (Bio-Rad). Mouse-specific primers were designed using Molecular Beacon Design 4.0 Software (Bio-Rad). The concentration of Mg2+ and the linear range of amplification of cDNAs with each primer pair first were optimized, and cDNAs subsequently were tested. Each sample was run in triplicate, and mRNA levels were analyzed relative to hypoxanthine phosphoribosyltransferase, a housekeeping gene that was not altered in response to ionizing radiation. Log2-transformed relative expression ratios were calculated as described using the equation set forth by Pfaffl (31), in which efficiencies for both the gene of interest and the calibrator hypoxanthine phosphoribosyltransferase were used.


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  		TABLE 1. Primer sequences for real-time RT-PCR analysis

 
Western blotting
Testes from control, 1-, 3-, 6-, and 12-h wild-type and Akt1-deficient mice were detunicated, weighed, and homogenized in three volumes of ice-cold RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail (P2714; Sigma) by 10 strokes in a Dounce homogenizer. Samples were incubated on ice for 30 min. The homogenate was then centrifuged at 13,500 x g for 10 min at 4 C. For Western blotting, 50 µg of testis supernatant was separated by 10% SDS-PAGE unless otherwise specified and transferred to Immobilon-P membrane. Blocking solution (20 mM Tris, pH 7.4; 137 mM NaCl; 10% nonfat dry milk) was added to the membranes for 60 min. Primary antibodies were diluted in blocking solution and added to the membranes at 4 C overnight. After washing three times with 20 mM Tris (pH 7.4), 137 mM NaCl, and 0.1% Tween 20 (TBS-Tween), horseradish peroxidase-coupled secondary antibody diluted in blocking solution was incubated with the membranes for 1 h at room temperature. Membranes were washed three times with PBS-Tween (0.1%), and secondary antibody was detected by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham Pharmacia Biotech). The antibodies used were anti-phospho-Akt (Ser473), anti-Akt1, anti-Akt2, anti-Akt3 from Cell Signaling Technology; Fas and FasL from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); and ß-actin from Sigma. All antibodies were used at a dilution of 1:1000; except for FasL, which was used at a dilution of 1:100, and ß-actin (1:2000). Akt kinase activity was measured with Akt Kinase Assay Kit from Cell Signaling Technology.

Statistical analysis
The Student’s t test or one-way ANOVA with Bonferonni post hoc analysis were performed using Sigma Stat software (SPSS, Chicago, IL). A P value < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of the Akt signaling pathway by ionizing radiation
To determine whether Akt activation functions in germ cell apoptosis, Akt phosphorylation status was examined by Western analyses in the testes of wild-type mice after exposure to ionizing radiation at 0, 1, 3, and 6 h (Fig. 1AGo). The level of Akt phosphorylation of the Ser473 phosphorylation site was markedly higher in the testes of radiated mice at 1, 3, and 6 h (Fig. 1AGo), indicating that Akt signaling is activated in the testis under conditions of radiation-induced germ cell injury. To confirm that the increase in phosphorylated Akt was indeed activation of the Akt signaling pathway after ionizing radiation exposure, Akt kinase activity was assessed by an in vitro Akt kinase assay using a known Akt substrate, GSK3. Ionizing radiation (5 Gy) significantly elevated Akt kinase activity approximately 2-fold relative to control lysates at 1, 3, and 6 h (Fig. 1BGo). These results indicate that the Akt signaling pathway is induced after exposure to ionizing radiation in the testis.


Figure 1
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  		FIG. 1. Activation of the Akt kinase afterionizing radiation. Time course of Akt1 in wild-type mice exposed to 5 Gy ionizing radiation. A, Representative Western blots from three testis samples for each time point are provided. Shown are phosphorylated Ser473 Akt and GSK3{alpha}ß protein levels. B, A graphical representation of densitometric analysis in the mice exposed to 5 Gy ionizing radiation. The intensities of phosphorylated Ser473 Akt and Ser21/9 GSK3{alpha}ß were normalized to that of ß-actin. The values represent the mean ± SEM. The asterisk denotes a significant difference relative to control (P < 0.05).

 
Early onset of germ cell apoptosis in Akt1-deficient mice after radiation-induced injury
Akt1 is reported to play a protective role in reducing the radiosensitivity of certain tumor cells (reviewed in Ref. 32). Therefore, to determine the potential consequences of decreased Akt kinase activity in vivo in the testis, we examined the level of germ cell apoptosis in wild-type, Akt1 heterozygous, and Akt1-deficient testes after exposure to 5 Gy of radiation. Animals were killed at 0, 1, 3, 6, and 12 h after lower body exposure to ionizing radiation to evaluate TUNEL-labeled cells as depicted in Fig. 2Go, A–C. Apoptosis, as assessed by counting seminiferous tubules with more than three apoptotic germ cells per cross section, was increased at 6 h in Akt1-deficient mouse seminiferous tubules (P < 0.03) (Fig. 2Go, C and D) when compared with wild-type (Fig. 2AGo) and Akt1 heterozygous mouse seminiferous tubules (Fig. 2BGo). Similar to previously published findings in normal rodent testis (31), the number of tubules with greater than three apoptotic germ cells was increased in wild-type and Akt1 heterozygous seminiferous tubules at 12 h (Fig. 2DGo), but to a lesser extent than in Akt1-deficient testes.


Figure 2
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  		FIG. 2. Akt suppresses germ cell apoptosis in mouse seminiferous tubules. Apoptosis as detected by TUNEL in mouse seminiferous tubules after 5 Gy of ionizing radiation at 6 h. A, Akt1 wild-type seminiferous tubules; B, Akt1 heterozygous seminiferous tubules; and C, Akt1-deficient seminiferous tubules. Note the numerous TUNEL-positive germ cells (brown stain) D, Bar graph representing time course of radiation-induced germ cell apoptosis in Akt1 wild-type (black bar), Akt1 heterozygous (gray bar), and Akt1-deficient mouse seminfierous tubules (striped bar). A minimum of three mice per genotype per timepoint were analyzed. Statistical analyses were conducted using one-way ANOVA (P < 0.05); error bars, SEM.

 
Enhanced sensitivity of mitotic spermatogonia to ionizing radiation
To more rigorously address the issue of earlier onset of apoptosis vs. increased apoptosis or "sensitivity" of a specific germ cell population, spermatid head counts were evaluated 29 d after 0.5 Gy of ionizing radiation (Fig. 3Go). This experiment examines the ability of A1 through B spermatogonia to survive and differentiate into late spermatids (41). As predicted, after exposure of control mice to 0.5 Gy radiation (Fig. 3Go), spermatid head counts were reduced by 49% in Akt1 wild-type (Fig. 3Go, 0.5 Gy, black bar), by 47% in Akt1 heterozygous (Fig. 3Go, 0.5 Gy, gray bar), and by 66% in Akt1-deficient testes (Fig. 3Go, 0.5 Gy, striped bar), relative to their respective nonirradiated counterparts (Fig. 3Go, controls). Comparison of spermatid head count number in the irradiated groups of mice indicated a significant reduction in the number of spermatid heads (33%) of Akt1-deficient mice (Fig. 3Go, 0.5 Gy, striped bar) relative to irradiated wild-type mice (Fig. 3Go, 0.5 Gy, black bar). These data indicate that not only was there an earlier onset of apoptosis in mice deficient for Akt1, but also an enhanced sensitivity of the mitotic spermatogonia to ionizing radiation.


Figure 3
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  		FIG. 3. Akt1 protects mitotic spermatogonia from ionizing radiation. Response of Akt1 wild-type, Akt1 heterozygous, and Akt1-deficient mice to 0.5 Gy of ionizing radiation. For all experiments, a minimum of three mice per time point per genotype were analyzed. Statistical analyses were conducted using one-way ANOVA (P < 0.05); error bars, SEM. Asterisk (*) indicates significance compared with wild-type control. Cross (+) indicates significance from wild-type at a specific time point.

 
Delayed induction of Fas expression after ionizing radiation in Akt1-deficient mice
It is recognized that testicular germ cell apoptosis after ionizing radiation is dependent, at least in part, on a functional Fas system. Radiation directly targets germ cells leading to a rapid induction of Fas and a delayed induction of FasL (33). Therefore, we examined expression of Fas and FasL after exposure to ionizing radiation. Figure 4AGo demonstrates an approximate 2-fold increase of Fas expression in the testes of wild-type mice at 1 h after exposure, with expression remaining elevated at 3 and 6 h and returning to control levels by 12 h after irradiation. In contrast, Fas expression was not elevated at 1 h in Akt1 heterozygous (Fig. 4AGo, gray bars) or Akt1-deficient mouse testes (Fig. 4AGo, striped bars); however, by 3 h, Akt1 heterozygous and Akt1-deficient mice exhibited a 2- to 3-fold increase in Fas expression, which remained elevated at 6 h and then returned to control levels by 12 h (Fig. 4AGo, gray bars).


Figure 4
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  		FIG. 4. Delayed induction of Fas and increased FasL expression after ionizing radiation in Akt1-deficient mice. A, A time course of relative expression of Fas mRNA after exposure of Akt1 wild-type, Akt1 heterozygous, and Akt1-deficient mice to lower body ionizing radiation (5 Gy). B, A time course of relative expression of FasL mRNA after exposure of Akt1 wild-type (black bar), Akt1 heterozygous (gray bar), and Akt1-deficient (striped bar) mice to lower body ionizing radiation (5 Gy). For all experiments, a minimum of three mice per time point per genotype were analyzed. Statistical analyses were conducted using one-way ANOVA (P < 0.05); error bars, SEM. Asterisk (*) indicates significance compared with wild-type control. Cross (+) indicates significant difference from wild-type at a specific time point.

 
Akt1-deficiency results in inducible expression of FasL after radiation
Others have shown that inhibition of the PI3 kinase/Akt1 signaling pathway leads to induction of FasL and apoptosis (34, 35, 36). Although there was a trend for decreased expression of FasL in wild-type testes throughout the time course examined (P = 0.07) (Fig. 4BGo; black bars), it was not found to be statistically significant. In contrast to wild-type testes, FasL was unchanged in both Akt1 heterozygous (Fig. 4BGo, gray bars) and Akt1-deficient mouse testes (Fig. 4BGo, striped bars) after exposure to ionizing radiation. Due to the trend of decreased expression of FasL in wild-type mouse testes (Fig. 4BGo, black bars) after ionizing radiation, FasL expression was found to be significantly elevated in the Akt1 heterozygous (Fig. 4BGo, gray bars) and Akt1-deficient mouse testes (Fig. 4BGo, striped bars) at 3 h (P < 0.05). At 12 h, only the Akt1-deficient mice (Fig. 4BGo, striped bars) exhibited significantly higher levels of FasL compared with wild-type mice at the 12-h time point. These data suggest that the lack of a decrease in FasL expression contributes to the enhanced radiosensitivity exhibited by Akt1-deficient mice.

No change in Fas and FasL protein levels after ionizing radiation in Akt1-deficient mice
We next examined the levels of Fas and FasL protein of testicular homogenates of wild-type and Akt1-deficient animals (Fig. 5AGo). After ionizing radiation, no significant increases in Fas or FasL protein were observed relative to controls. The Fas and FasL time points are normalized with those of ß-actin (Fig. 5BGo).


Figure 5
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  		FIG. 5. Western blot analysis of Fas and FasL of testicular homogenates exposed to ionizing radiation. A, Representative blots from three testis homogenates for each time point of wild-type and Akt1-deficient mice after exposure to 5 Gy ionizing radiation are provided. B, A graphical quantitation of Fas and FasL levels in wild-type and Akt1-deficient mice after 5 Gy ionizing radiation. The intensities of Fas and FasL were normalized to that of ß-actin. Values represent the mean ± SEM.

 
Induction of Akt1 and Akt2 mRNA expression after ionizing radiation in wild-type mice
Although the Akt gene family is implicated in a survival response to genotoxic injury in multiple cell types, it is not known to what degree there is functional overlap between these family members in the testis. Thus, we examined the level of Akt1, Akt2, and Akt3 mRNA levels after ionizing radiation in wild-type, Akt1 heterozygous, and Akt1-deficient mice. In response to ionizing radiation, wild-type animals exhibited an approximate 2-fold increase in Akt1 mRNA expression, peaking at 1 h, and returning to control levels by 3 h (Fig. 6AGo, black bars). In contrast to wild-type mice, Akt1 heterozygous mice (Fig. 6AGo, gray bars) exhibited no induction in Akt1 mRNA after ionizing radiation at 1 h. Akt1-deficient animals (Fig. 6AGo, striped bars) exhibited a 2-fold decrease in Akt1 expression relative to Akt1-deficient control animals (Fig. 6AGo, striped bars). These data suggest that Akt1 plays a role in the transcriptional response to radiation-induced germ cell injury.


Figure 6
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  		FIG. 6. Induction of Akt1 and Akt2 mRNA expression after ionizing radiation in wild-type mice. A, A time course of relative expression of Akt1 mRNA after exposure of Akt1 wild-type, Akt1 heterozygous, and Akt1-deficient mice to lower body ionizing radiation (5 Gy). B, A time course of relative expression of Akt2 mRNA after exposure of Akt1 wild-type, Akt1 heterozygous, and Akt1-deficient mice to lower body ionizing radiation (5 Gy). C, A time course of relative expression of Akt3 mRNA after exposure of Akt1 wild-type, Akt1 heterozygous, and Akt1-deficient mice to lower body ionizing radiation (5 Gy). For all experiments, a minimum of three mice per time point per genotype were analyzed. Statistical analyses were conducted using one-way ANOVA (P < 0.05); error bars, SEM. Asterisk (*) indicates significance compared with wild-type control. Cross (+) indicates significant difference from wild-type at a specific time point.

 
Similar to the induction observed for Akt1 expression after ionizing radiation in wild-type animals, Akt2 mRNA levels (Fig. 6BGo, black bars) were also increased 1.8-fold in response to ionizing radiation. In contrast to wild-type animals, no induction of Akt2 was observed in the Akt1 heterozygous (Fig. 6BGo, gray bars) and Akt1-deficient animals (Fig. 6BGo, striped bars) after exposure to ionizing radiation. Although there was a trend for elevated Akt3 expression in wild-type mice (Fig. 6CGo, black bars) after ionizing radiation, it was not significant. Again, our data suggest that Akt1 and Akt2 play a role in the transcriptional response to germ cell injury. Moreover, lack of an induction of either Akt2 or Akt3 in the Akt1-deficient mouse testes after ionizing radiation indicates that these isoforms likely do not compensate for lack of Akt1; and, consequently contributes to the enhanced radiosensitivity exhibited by the Akt1-deficient mice.

Neither Akt2 nor Akt3 protein compensate for Akt1 after ionizing radiation
Western blot analysis of Akt1 (Fig. 7AGo) in the testis homogenates of wild-type and Akt1-deficient testes after exposure to 5 Gy ionizing radiation confirmed not only the loss of Akt1 protein in Akt1-deficient testes (Fig. 7AGo); but also, that Akt1 protein levels were not significantly elevated after ionizing radiation in wild-type mice. Despite a trend toward increased Akt2 and Akt3 protein levels in the Akt1-deficient mice exposed to ionizing radiation, Akt2 and Akt3 protein did not differ significantly from wild-type mice exposed to ionizing radiation (Fig. 7BGo).


Figure 7
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  		FIG. 7. Western blot analysis of Akt1, Akt2, and Akt3 of testicular homogenates exposed to ionizing radiation. A, Representative blots from three testis homogenates for each time point of wild-type and Akt1-deficient mice after exposure to 5 Gy ionizing radiation are provided. B, A graphical quantitation of Akt2 and Akt3 protein levels in wild-type and Akt1-deficient mice after 5 Gy ionizing radiation. The intensities of Akt1, Akt2, and Akt3 were normalized to that of ß-actin. Values represent the mean ± SEM. Asterisk (*) indicates significance compared with wild-type control (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Over the past decade, numerous studies have demonstrated a critical role for the Akt family of proteins in cell survival, growth, and differentiation (34). Research is now focused on understanding how each Akt isoform contributes to the survival of various cell types and organs after exposure to both endogenous and exogenous stimuli. Recently, Akt1 down-regulation was shown to sensitize tumor cells to chemotherapeutic agents (19). Correspondingly, overexpression of Akt1 in human lung cancer cells has been shown to delay the onset of apoptosis after treatment with cisplatin or mitoxantrone (35). In addition, inhibition of either Akt1 or Akt2 using small interfering RNA technology has resulted in enhanced radiosensitivity of some tumor cell lines (36).

To better understand the in vivo biology of Akt1 in response to radiation-induced testis germ cell injury, we exposed wild-type, Akt1 heterozygous, and Akt1-deficient mice to 5 Gy ionizing radiation. Our results have identified an important role for Akt1 in protecting testicular function after exposure to ionizing radiation. Germ cell apoptosis was increased 2-fold in Akt1-deficient mouse testes compared with irradiated wild-type animals 6 h after exposure. In addition, we determined that the mitotic spermatogonial germ cell population as a whole in Akt1-deficient mice is more sensitive to radiation as exemplified by a 33% decrease in the ultimate survival of these cells relative to their wild-type irradiated counterparts. Taken together, the earlier onset and increased sensitivity of Akt1-deficient mice to ionizing radiation provides strong evidence that the Akt1 signaling pathway plays a direct role in protecting against germ cell apoptosis after radiation-induced germ cell injury.

Accumulating evidence suggests that the PI3 kinase/Akt signaling pathway is a major contributor to radioresistance (18, 19). Activation of Akt by ionizing radiation occurred within 1 h after ionizing radiation exposure. This activated Akt increased phosphorylation of the downstream substrate GSK3 within a similar time frame, indicating a role for Akt activation in the cytoprotective response of germ cells to ionizing radiation-induced germ cell injury.

Subsequent research on Akt1 signaling has focused on the phosphorylation and subsequent activation or inactivation of Akt1-target genes associated with cell survival, cell growth, and apoptosis in vitro. Therefore, we set out to determine how Akt1-dependent gene expression is altered in the testes of Akt1-deficient mice. The Fas/FasL system has fundamental roles in testicular germ cell apoptosis (37). Radiation exposure in the rat testis leads to an up-regulation of Fas, but not FasL, gene expression (23, 24, 25). Moreover, exposure of Fas receptor mutant mice (lpr(cg)) to 5 Gy ionizing radiation resulted in a significant reduction of germ cell apoptosis compared with that of wild-type mice (22). Surprisingly, we observed an initial delay in the ability of ionizing radiation to increase Fas mRNA levels in both Akt1-heterozygous and Akt1-deficient mice compared with irradiated wild-type controls.

Previous work failed to demonstrate a role for FasL in radiation-induced germ cell apoptosis (21, 22, 23). FasL does not increase in the rat testis after exposure to {gamma}-irradiation (21, 22, 23), and gld mice, which have a mutant FasL are as sensitive as wild-type mice to {gamma}-radiation (38). However, our results support a role for FasL as a mediator of enhanced germ cell apoptosis after ionizing radiation in the Akt1-deficient mice. We found that the timing of germ cell apoptosis in the Akt1-deficient mice coincided with increased FasL expression relative to irradiated wild-type mice at 3 and 12 h after irradiation. After ionizing radiation, there was a rapid up-regulation of Fas mRNA in wild-type animals and FasL down-regulation. In contrast, Akt1-deficient testes display an initial delay in Fas transcriptional activation but elevated FasL expression relative to wild-type animals resulting in enhanced germ cell sensitivity and an earlier onset of germ cell apoptosis. In agreement with our observations, inhibition of the PI3 kinase/Akt1 signaling pathway in certain tumor cell types has been reported to increase expression of FasL and subsequent apoptosis in vitro (25, 26, 27). Furthermore, overexpression of Akt has been shown to delay the onset of p53-mediated, transcriptional dependent apoptosis (39).

The delay in Fas transcription in Akt1-deficient mice prompted us to examine expression of the other Akt isoforms, Akt2 and Akt3, to determine whether these genes could possibly compensate for the loss of Akt1 after injury. In wild-type mice, both Akt1 and Akt2 expression was radiation inducible. No induction of Akt1, Akt2, or Akt3 was observed in Akt1-deficient testes after exposure to ionizing radiation; in fact, we observed significant decreases in Akt1 transcription in Akt1-deficient mice after ionizing radiation exposure.

Recently, dosage-dependent effects of Akt1 and Akt3 on the thymus, skin, and cardiovascular and nervous system have been reported (40). Intriguingly, we found that Akt1 heterozygous mice exhibit a transcriptional response similar to the Akt1-deficient mice after germ cell injury; despite the fact that the level of germ cell apoptosis was not significantly altered. Although the Akt1 heterozygous mouse response to injury was similar to that of Akt1-deficient mice, it was not as extensive. This trend suggests that a certain threshold level of Akt1 activity may be necessary for overall testicular homeostasis. At the RNA level, our data indicate that Akt2 and Akt3 do not compensate for Akt1 function in the testis after exposure to ionizing radiation. Similarly, we did not observe any significant changes at the protein level in the Akt family isoforms after exposure to ionizing radiation.

In summary, although the precise signaling pathways and cell types associated with radiation-inducible germ cell death remain to be elucidated, it is clear from this work that the Akt1-signaling pathway plays a major role in the protection of germ cells after injury. In an Akt1-deficient testis, Akt2 and Akt3 cannot compensate for Akt1, and hence this survival pathway is overwhelmed and germ cells are more susceptible to apoptosis. Intriguingly, previous work has shown that insulin-like growth factor 1 mediated Akt activation postpones the onset of UV B-induced apoptosis by promoting the removal of cyclobutane thymine dimers (41). We propose a model in which the cell survival genes Akt1 and Akt2 are activated to promote germ cell survival by repair of damaged germ cells in a normal testicular stress response to radiation-induced germ cell injury.


   Acknowledgments
 
We thank Dr. Morris Birnbaum for providing two breeding pair of Akt1 knockout mice and Bob Monk for much useful advice. We thank the Boekelheide and Freiman laboratories for critical reading of the manuscript.


   Footnotes
 
This work was supported by a Biomedical Research Infrastructure Network/IDeA Network of Biomedical Research Excellence Grant No. P20 RR016457 from the National Center for Research Resources/National Institutes of Health (to M.H.).

T.R., K.D., B.K., and M.H. have nothing to declare.

First Published Online June 8, 2006

Abbreviations: FasL, Fas ligand; PI3K, phosphatidylinositol 3 kinase; PKB, protein kinase B; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling.

Received February 9, 2006.

Accepted for publication May 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Richburg, JH 2000 The relevance of spontaneous- and chemically-induced alterations in testicular germ cell apoptosis to toxicology. Toxicol Lett 112–113:79–86
  2. Richburg JH, Johnson KJ, Schoenfeld HA, Meistrich ML, Dix DJ 2002 Defining the cellular and molecular mechanisms of toxicant action in the testis. Toxicol Lett 135:167–183[CrossRef][Medline]
  3. Wang RA, Nakane PK, Koji T 1998 Autonomous cell death of mouse male germ cells during fetal and postnatal period. Biol Reprod 58:1250–1256[Abstract/Free Full Text]
  4. Rodriguez I, Ody C, Araki K, Garcia I, Vassalli P 1997 An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J 16:2262–2270[CrossRef][Medline]
  5. Bartke A 1995 Apoptosis of male germ cells, a generalized or a cell type specific phenomenon? Endocrinology 136:3–4[CrossRef][Medline]
  6. de Franca LR, Ghosh S, Ye SJ, Russell LD 1993 Surface and surface-to volume relationships of the Sertoli cell during the cycle of the seminiferous epithelium in the rat. Biol Reprod 49:1215–1228[Abstract]
  7. Hughes GC 1962 Radiosensitivity of male germ cells in neonatal rats. Int J Rad Biol 4:511–519[Medline]
  8. Moreno SG, Dutrillaux B, Coffigny H 2001 High sensitivity of rat foetal germ cells to low dose-rate irradiation. Int J Radiat Biol 77:529–538[CrossRef][Medline]
  9. 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]
  10. 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]
  11. Fornace Jr AJ 1992 Mammalian genes induced by radiation; activation of genes associated with growth control. Annu Rev Genet 26:507–526[Medline]
  12. Meistrich ML, Hunter NR, Suzuki N, Trostle PK, Withers HR 1978 Gradual regeneration of mouse testicular stem cells after exposure to ionizing radiation. Radiat Res 74:349–362[Medline]
  13. Chen WS, Xu PZ, Gottlob K, Chen ML, Sokol K, Shiyanova T, Roninson I, Weng W, Suzuki R, Tobe K, Kadowaki T, Hay N 2001 Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene. Genes Dev 15:2203–2208[Abstract/Free Full Text]
  14. Cho H, Thorvaldsen JL, Chu Q, Feng F, Birnbaum MJ 2001 Akt1/PKB{alpha} is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem 276:38349–38352[Abstract/Free Full Text]
  15. Garofalo RS, Orena SJ, Rafidi K, Torchia AJ, Stock JL, Hildebrandt AL, Coskran T, Black SC, Brees DJ, Wicks JR, McNeish JD, Coleman KG 2003 Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKBß. J Clin Invest 112:197–208[CrossRef][Medline]
  16. Yang ZZ, Tschopp O, Hemmings-Mieszczak M, Feng J, Brodbeck D, Perentes E, Hemmings BA 2003 Protein kinase B{alpha}/Akt1 regulates placental development and fetal growth. J Biol Chem 278:32124–32131[Abstract/Free Full Text]
  17. Easton RM, Cho H, Roovers K, Shineman DW, Mizrahi M, Forman MS, Lee VM, Szabolcs M, de Jong R, Oltersdorf T, Ludwig T, Efstratiadis A, Birnbaum MJ 2005 Role for Akt3/protein kinase B{gamma} in attainment of normal brain size. Mol Cell Biol 25:1869–1878[Abstract/Free Full Text]
  18. Testa JR, Tsichlis PN 2005 AKT signaling in normal and malignant cells. Oncogene 24:7391–7393[CrossRef][Medline]
  19. Cheng JQ, Lindsley CW, Cheng GZ, Yang H, Nicosia SV 2005 The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 24:7482–7492[CrossRef][Medline]
  20. Sharma K, Wang RX, Zhang LY, Yin DL, Luo XY, Solomon JC, Jiang RF, Markos K, Davidson W, Scott DW, Shi YF 2000 Death the Fas way: regulation and pathophysiology of CD95 and its ligand. Pharmacol Ther 88:333–347[CrossRef][Medline]
  21. Boekelheide K, Lee J, Shipp EB, Richburg JH, Li G 1998 Expression of Fas system-related genes in the testis during development and after toxicant exposure. Toxicol Lett 102–103:503–508
  22. 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]
  23. 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]
  24. D’Alessio A, Riccioli A, Lauretti P, Padula F, Muciaccia B, De Cesaris P, Filippini A, Nagata S, Ziparo E 2001 Testicular FasL is expressed by sperm cells. Proc Natl Acad Sci USA 98:3316–3321[Abstract/Free Full Text]
  25. Ciechomska I, Pyrzynska B, Kazmierczak P, Kaminska B 2003 Inhibition of Akt kinase signalling and activation of Forkhead are indispensable for upregulation of FasL expression in apoptosis of glioma cells. Oncogene 22:7617–7627[CrossRef][Medline]
  26. Arimoto-Ishida E, Ohmichi M, Mabuchi S, Takahashi T, Ohshima C, Hayakawa J, Kimura A, Takahashi K, Nishio Y, Sakata M, Kurachi H, Tasaka K, Murata Y 2004 Inhibition of phosphorylation of a forkhead transcription factor sensitizes human ovarian cancer cells to cisplatin. Endocrinology 145:2014–2022[Abstract/Free Full Text]
  27. Osaki M, Kase S, Adachi K, Takeda A, Hashimoto K, Ito H 2004 Inhibition of the PI3K-Akt signaling pathway enhances the sensitivity of Fas-mediated apoptosis in human gastric carcinoma cell line, MKN-45. J Cancer Res Clin Oncol 130:8–14[CrossRef][Medline]
  28. Zheng WH, Kar S, Quirion R 2002 Insulin-like growth factor-1-induced phosphorylation of transcription factor FKHRL1 is mediated by phosphatidylinositol 3-kinase/Akt kinase and role of this pathway in insulin-like growth factor-1-induced survival of cultured hippocampal neurons. Mol Pharmacol 62:225–233[Abstract/Free Full Text]
  29. Schwab TS, Madison BB, Grauman AR, Feldman EL 2005 Insulin-like growth factor-I induces the phosphorylation and nuclear exclusion of forkhead transcription factors in human neuroblastoma cells. Apoptosis 10:831–840[CrossRef][Medline]
  30. Meistrich ML, van Beek MEAB 1993 Spermatogonial stem cells: assessing their survival and ability to produce differentiated cells. In: Chapin RE, Heindel J, eds. Methods in toxicology. Vol 3A. New York: Academic Press; 106–123
  31. Pfaffl MW 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
  32. Zhan M, Han ZC 2004 Phosphatidylinositide 3-kinase/AKT in radiation responses. Histol Histopathol 19:915–923[Medline]
  33. 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]
  34. Bellacosa A, Testa JR, Moore R, Larue L 2004 A portrait of AKT kinases: human cancer and animal models depict a family with strong individualities. Cancer Biol Ther 3:268–275[Medline]
  35. Hovelmann S, Beckers TL, Schmidt M 2004 Molecular alterations in apoptotic pathways after PKB/Akt-mediated chemoresistance in NCI H460 cells. Br J Cancer 90:2370–2377[Medline]
  36. Kim IA, Bae SS, Fernandes A, Wu J, Muschel RJ, McKenna WG, Birnbaum MJ, Bernhard EJ 2005 Selective inhibition of Ras, phosphoinositide 3 kinase, and Akt isoforms increases the radiosensitivity of human carcinoma cell lines. Cancer Res 65:7902–7910[Abstract/Free Full Text]
  37. Boekelheide K, Fleming SL, Johnson KJ, Patel SR, Schoenfeld HA 2000 Role of Sertoli cells in injury-associated testicular germ cell apoptosis. Proc Soc Exp Biol Med 225:105–115[Abstract/Free Full Text]
  38. 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
  39. Sabbatini P, and McCormick F 1999 Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis. J Biol Chem 274:24263–24269[Abstract/Free Full Text]
  40. Yang ZZ, Tschopp O, Di-Poi N, Bruder E, Baudry A, Dummler B, Wahli W, Hemmings BA 2005 Dosage-dependent effects of Akt1/protein kinase B{alpha} (PKB{alpha}) and Akt3/PKB{gamma} on thymus, skin, and cardiovascular and nervous system development in mice. Mol Cell Biol 25:10407–10418[Abstract/Free Full Text]
  41. Decraene D, Agostinis P, Bouillon R, Degreef H, Garmyn M 2002 Insulin-like growth factor-1-mediated AKT activation postpones the onset of ultraviolet B-induced apoptosis, providing more time for cyclobutane thymine dimer removal in primary human keratinocytes. J Biol Chem 277:32587–32595[Abstract/Free Full Text]



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