Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print November 22, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.054924
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
76/2/279    most recent
biolreprod.106.054924v1
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rasoulpour, R. J.
Right arrow Articles by Boekelheide, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rasoulpour, R. J.
Right arrow Articles by Boekelheide, K.
Agricola
Right arrow Articles by Rasoulpour, R. J.
Right arrow Articles by Boekelheide, K.
BIOLOGY OF REPRODUCTION 76, 279–285 (2007)
DOI: 10.1095/biolreprod.106.054924
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

NF-kappaB Activation Elicited by Ionizing Radiation Is Proapoptotic in Testis1

Reza J. Rasoulpour , and Kim Boekelheide 2 

Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02903

ABSTRACT

The transcription factor NF-kappaB modulates apoptotic machinery following activation by the IkappaB kinase (IKK) complex. Inhibiting activity of one of the catalytic subunits of the IKK complex, IKKbeta (also known as IKBKB and IKK2) severely inhibits NF-kappaB nuclear translocation in response to most stimuli, including ionizing radiation. Doubly floxed IkkbetaF/F mice (control) were compared to haplo-insufficient IkkbetaF/delta mice (NF-kappaB knockdown) to examine the in vivo apoptotic role of NF-kappaB in the testis. Although IkkbetaF/F control adult mice had spermatid head counts and testis and body weights similar to IkkbetaF/delta mice, cellular stress in the form of ionizing radiation elicited a differential phenotype. Lower body exposure to 5 Gy of ionizing radiation induced a greater NF-kappaB activation in IkkbetaF/F than in IkkbetaF/delta mice. In addition, exposure to ionizing radiation resulted in fewer apoptotic germ cells 3, 6, and 12 h after injury in NF-kappaB knockdown mice than in controls, concomitant with the reduced cleavage of caspases 3 and 9 at 3 h. There was also a reduction in total germ cells lost after radiation with NF-kappaB inhibition. Correspondingly, real-time RT-PCR showed a significant reduction in Cdnk1a (also known as p21) and Fasl expression 3 and 6 h, respectively, after irradiation in IkkbetaF/delta compared to control testes. These data indicate that, despite acting in an antiapoptotic manner in many tissue types, NF-kappaB is proapoptotic in modulating the germ cell response to ionizing radiation.

apoptosis, ionizing radiation, NF-kappaB, spermatogenesis, toxicology

INTRODUCTION

The mammalian testis is a site of extensive proliferation, differentiation, and apoptosis. To achieve optimal sperm output, proliferation must be balanced with germ cell carrying capacity. Thus, culling of mitotic spermatogonia is essential during spermatogenesis to prevent overwhelming the somatic Sertoli cells (for a review, see Holdcraft and Braun [1]). The balance of proliferation and apoptosis that controls germ cell output makes the testis an excellent model to study apoptotic machinery. A pivotal regulator of apoptotic genes is the transcription factor NF-kappaB.

To assess the NF-kappaB activity profile during testicular development, an Nfkb1 (p105/p50) promoter/LacZ reporter mouse was utilized [2]. A detailed examination of NF-kappaB expression during testis development demonstrated a peak of NF-kappaB activity in germ cells undergoing meiosis at Postnatal Day 18, which persisted throughout adulthood. In adult mouse testis, NF-kappaB activity was stage-specific and strongly localized to pachytene spermatocytes [2]. These mouse data correlated with immunolocalization studies in rat [3, 4] and human [5] testes.

Further investigation of Sertoli cells revealed a number of putative testis genes under the control of NF-kappaB [6–8]. In vitro studies of NF-kappaB regulation of the cAMP response element binding protein in Sertoli cells provided strong evidence highlighting a basic biological role for NF-kappaB in the testis [6]. This signaling pathway may be activated through binding of androgen to the Sertoli cell androgen receptor, suggesting that NF-kappaB regulates, in part, androgen response in these somatic cells [8]. In addition, NF-kappaB acting in Sertoli cells can lead to increased androgen receptor expression.

A role for NF-kappaB regulation of germ cell apoptosis in the testis was first suggested by studies of cultured human seminiferous tubules. Upon addition of the anti-inflammatory drug sulfasalazine, NF-kappaB activity was decreased concomitantly with germ cell apoptosis. These correlative results suggested a proapoptotic role for NF-kappaB [5]. In contrast, in vitro experiments with rat seminiferous tubule segments incubated in the presence of tumor necrosis factor (TNF) (also known as TNF{alpha}), a TNF inhibitor (infliximab) or an NF-kappaB inhibitor (SN50) suggested an antiapoptotic activity of NF-kappaB [9]. In the presence of TNF, there were fewer TUNEL-positive germ cells and less caspase 3 activity. Addition of infliximab or SN50 produced opposite results, suggesting that TNF-induced germ cell survival was, at least partly, mediated by NF-kappaB. In addition, an antiapoptotic NF-kappaB-regulated gene, Bcl2l1 (also known as Bcl-XL), was expressed at increased levels after TNF treatment [9].

Recent in vivo studies have shown NF-kappaB activation in the testis after ischemic injury [10]. Ischemia/reperfusion activates NF-kappaB through an oxidative stress pathway. In the testis, 2 h of reperfusion after torsion-induced ischemia leads to IkappaB-alpha phosphorylation and degradation, NF-kappaB activation, and alteration of a number of NF-kappaB-regulated genes [10].

We have also recently shown NF-kappaB activation in the testis in response to injury. We demonstrated that NF-kappaB is activated in rat testes in response to the toxicant mono-(2-ethylhexyl) phthalate (MEHP) [11]. Induction of NF-kappaB by MEHP correlated with nuclear translocation of NF-kappaB, enhanced immunolocalization, and a temporal reduction in apoptotic germ cells. Expanding on this earlier study, we sought to discover the biological roles of NF-kappaB in spermatogenesis. Classical NF-kappaB signaling transduction occurs through activation of the IkappaB kinase (IKK) complex, and subsequent IKK phosphorylation of the inhibitors of NF-kappaB (IkappaBs), to allow NF-kappaB nuclear translocation (for excellent reviews, see Karin [12, 13]). Since there are numerous NF-kappaB subunits and nearly as many inhibitors (IkappaBs), we sought to globally inhibit NF-kappaB activation through disruption of the IKK complex [12]. Inhibition of one of the catalytic subunits of the IKK complex, namely IKKß (also known as IKBKB), results in severe inhibition of the canonical NF-kappaB activation [14]. Homozygous deletion of the Ikkß gene (also known as Ikbkb) results in an embryo lethal phenotype similar to p65 –/– mice [14, 15]. To this end, the well-characterized Ikkß floxed mouse (IkkßF/F) was utilized [16]. For our purposes, we mated this well-documented mouse to a male germ cell-specific Cre recombinase-expressing mouse to create heterozygous IkkßF/{Delta} mice [17].

In the present study, we show that although control (IkkßF/F) and NF-kappaB knockdown (IkkßF/{Delta}) mice have no appreciable difference in testicular phenotype under basal conditions, the stress of {gamma}-radiation unmasks a proapoptotic phenotype. Taken together, these data suggest a proapoptotic role for NF-kappaB in the regulation of germ cell apoptosis in the testis.

MATERIALS AND METHODS

Animals

Mice were maintained on a 12L:12D cycle and housed in 30%–70% humidity and 70 ± 2°F temperature-controlled rooms with access to Purina Rodent Chow 5001 and water ad libitum. All procedures involving animals were performed in accordance with the guidelines of Brown University's Institutional Animal Care and Use Committee in compliance with National Institutes of Health guidelines [13, 18]. Sycp1-Cre mice were purchased from the Jackson Laboratory (Bar Harbor, ME) under strain name B6;D2-Tg(Sycp1-cre)4Min/J and stock number 003466. These Sycp1-Cre mice were created and have been characterized in detail [19–22]. The Ikbkbtm2Mka/Ikbkbtm2Mka (hereafter referred to as IkkßF/F) mice were kindly provided by the laboratory of Michael Karin (University of California at San Diego). These IkkßF/F mice have LoxP sites in the introns flanking exon 3 of the Ikkß gene, such that with expression of a Cre recombinase, exon 3 is excised ({Delta}), and the Ikkß is not translated into protein. Characterization of the IkkßF/F mice has been performed [16]. In addition, the IkkßF/F mice have been used in similar experiments to evaluate the effects of NF-kappaB inhibition in different cell and tissue types [23]. Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich.

Genotyping

Tail snips (4–6 mm long) were performed upon weaning and incubated with proteinase K and ATL buffer (DNeasy Kit) overnight at 55°C. DNA extraction and purification was performed with the DNeasy kit (Qiagen). DNA yield ranged from 20 to 80 ng/µl, and 7.5 µl was used for the subsequent 50-µl PCR reaction. Primers used were as follows: IKKß-lox 5'-GTCATTTCCACAGCCCTGTGA-3' and 5'-CCTTGTCCTATAGAAGCACAAC-3'; IKKß-{Delta} 5'-TAGTCCAACTGGCAGCGAATAC-3' and 5'-GCCTAGGTAAGATGGCTGTCT-3'; and Cre 5'-TGATGGACATGTTCAGGGATC-3' and 5'-CAGCCACCAGCTTGCATGA-3'.

Irradiation

Male mice, 8–10 wk old, were exposed to 5.0 Gy of lower body {gamma}-radiation with a Cs137 irradiator at a rate of 6.85 Gy/min. For total apoptosis measurements (see Fig. 3), mice were exposed to 0.5 Gy of {gamma}-radiation, and spermatid head counts were performed after 14 days (for the meiotic spermatocyte population) or 29 days (for the mitotic spermatogonial population). Mice were restrained in polystyrene chambers with upper body lead shielding.


Figure 76030303
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3. Quantification of spermatogonia and spermatocytes after radiation by spermatid head counts. Control (black bar) and NF-kappaB knockdown (gray bar) mice were exposed to 0.5 Gy of lower body ionizing radiation, and spermatogonia or spermatocytes were allowed to age into elongate spermatid heads for quantification (29 and 14 days, respectively). There is a preferential loss of mitotic versus meiotic germ cells after ionizing radiation in both IkkßF/F and IkkßF/{Delta} mice. NF-kappaB knockdown mice (IkkßF/{Delta}) have more spermatogonia than the control (IkkßF/F) after radiation (n > 3; *P < 0.05).

Spermatid Head Count

Freshly isolated testes were detunicated, weighed, and homogenized in 1.5 ml of SMT (saline-merthiolate-thimerosol) buffer as previously described [24]. Samples were blinded, and spermatid heads were counted on a hemacytometer four independent times to ensure consistency.

TUNEL Assays

TUNEL assays were performed with the DeadEND Apoptosis Assay kit (cat no. G3250; Promega) per the manufacturer's instructions, with propidium iodide as a counterstain as previously described [11]. Testes were flash frozen by liquid nitrogen in OCT embedding medium (cat no. 4583; Tissuetek). Testis cross sections (7 µm) were dried onto polylysine-coated slides and postfixed in –20°C methanol for 3 min. For quantification, slides were blinded, and all tubules in two different sections of testis per slide (two slides per testis) were counted for the absence or presence of 1–3 or >3 TUNEL-positive germ cells. For every animal, at least 400 tubules were scored for TUNEL positivity. Fluorescent microscopic images were visualized on a Zeiss Axiovert 35 microscope connected to a Spot RT camera (Diagnostic Instruments Inc., Sterling Heights, MI). Images were downloaded into Photoshop 7.0 imaging software (Adobe Systems Inc., San Jose, CA). All final figures were assembled by Canvas 8.0 software (Deneba Systems Inc., Miami, FL).

Western Blotting

Testes from 8- to 10-wk-old irradiated and control mice were detunicated, weighed, and homogenized in 3 vol of RIPA buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) with fresh protease inhibitor cocktail at 1:100 (cat no. P8340; Sigma) by 10 strokes in a Dounce homogenizer. The homogenate was centrifuged at 14 000 x g for 10 min at 4°C to remove particulate matter, and the supernatant was transferred to a fresh tube. Protein assays were performed with the Bradford-based Bio-Rad Dc (detergent compatible) kit (cat no. 500-0116; Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions in 1:20 and 1:40 duplicates. For Western blotting, 40 µg of testis protein extract was separated by 15% SDS-PAGE and subsequently transferred to Immobilon-P nitrocellulose membranes. Membranes were blocked for 1 h in 5% nonfat dry milk suspended in PBS + 0.1% Tween. Primary antibodies from the Apoptosis Antibody Sampler Kit (cat no. 9930; Cell Signaling, Beverly, MA) were diluted 1:250 in blocking solution and incubated with the membranes overnight at 4°C. The {alpha}-rabbit secondary antibody was detected by enhanced chemiluminescence according to the manufacturer's instructions (Amersham Pharmacia Biotech). Experiments were performed with three different mice per time point at least twice.

EMSA Experiments

Electrophoretic mobility shift assays (EMSAs) and subsequent supershifts were performed as previously described in detail [11, 25]. Briefly, whole detunicated testes were homogenized with 10 strokes by a Dounce homogenizer in lysis buffer with detergent. After 4°C centrifugation (10 min, 14 000 x g), the supernatant (cytoplasmic extract) was removed, and the pellet was washed. The nuclei were then incubated on ice for 40 min in high salt buffer; centrifugation was repeated, and the nuclear protein in the resulting supernatant was collected.

Real-Time RT-PCR

RNA was isolated from detunicated whole testes by TriReagent (cat. no. T9424; Sigma) according to the manufacturer's protocol. RNA underwent DNase digestion and then RT to cDNA by the iScript kit (cat. no. 170-8890; Bio-Rad). Real-time RT-PCR was performed on a Bio-Rad iCyclerIQ thermocycler with SYBR Green (cat. no. 170-8885; Bio-Rad). PCR primer efficiencies were calculated on the basis of standard curves, and quantitation was performed by the Pfaffl method with Hprt1 as the housekeeping gene [26]. Primers used were as follows: Hprt1 5'-CAGGCCAGACTTTGTTGGAT-3' and 5'-TTGCGCTCATCTTAGGCTTT-3'; Fas 5'-TGCACCCTGACCCAGAATAC-3' and 5'-GCCAGGAGAATCGCAGTAGAA-3'; Fasl 5'-GCAAATAGCCAACCCCAGTACAC-3' and 5'-GCCACCTTTCTTATACTTCACTCCAG-3'; Cdnk1a 5'-CAATGGCTGATCCTTTCTCAGTGTT-3' and 5'-CCAGGATGTTACAGAAACAGGGATGT-3'; and Bcl2l1 5'-CCGCTGTGTCTCTGGGTCTC-3' and 5'-GGTTCTGGTCCTTGTCTCATTATCC-3'.

Densitometry

Western autoradiograms were scanned, and band intensity was quantified by ImageJ software (http://rsb.info.nih.gov/ij/).

Statistics

The mean and SEM were calculated for each data point and represented as mean ± SEM. One-way ANOVA pairwise, followed by the Bonferroni correction and/or the Student t-test, was used for all statistical analyses with significance at P < 0.05.

RESULTS

Baseline Evaluation of IkkßF/{Delta} NF-kappaB Knockdown Mice

The IkkßF/{Delta} (NF-kappaB knockdown) mice were created through mating of the IkkßF/F and synaptonemal complex protein-1 Cre recombinase mice [17]. The IkkßF/F mice have LoxP sites within the flanking introns of exon 3 and are the control mice for these experiments. Recombination of exon 3 ({Delta}) results in a null Ikkß (also known as Ikbkb) genotype such that IkkßF/{Delta} mice are haplo-insufficient. NF-kappaB knockdown mice (IkkßF/{Delta}) were compared to control (IkkßF/F) mice under nonirradiated conditions. Body and testis weights were not significantly different between the two genotypes (Table 1). Similarly, a particularly sensitive spermatogenesis output endpoint, the measure of the number of compacted elongate spermatid heads per gram of testis tissue per day, showed no difference between genotypes (Table 1). Histopathological evaluation of testis sections from these mice was also indistinguishable (data not shown). These data indicated that male mice with these different Ikkß genotypes had a similar baseline spermatogenesis.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		Table 1. Differences between genotypes for body and testis weights, and number of spermatid heads per gram of testis tissue per day.

NF-kappaB Activation after Ionizing Radiation

To assess nuclear NF-kappaB activity in the testis before and after injury, EMSAs were performed (Fig. 1). Whole testis nuclear protein from both IkkßF/F and IkkßF/{Delta} nonirradiated animals had minimal active NF-kappaB. One hour after {gamma}-irradiation, there was a large increase in NF-kappaB band intensity in IkkßF/F mice testes, indicating nuclear translocation of active NF-kappaB. In contrast, the IkkßF/{Delta} NF-kappaB knockdown mice at 1 h had reduced nuclear NF-kappaB, indicating an inhibited NF-kappaB response (Fig. 1). The EMSA of TCFAP2 (also known as AP2) was used as a loading control. Supershift experiments of pooled IkkßF/F protein (1 h) indicated the presence of p65, p50, and c-Rel components of gel-shifted bands. Given the heterogeneous cell population collected with whole testis nuclear extracts, mixed NF-kappaB subunit complexes were expected and observed.


Figure 76030301
View larger version (70K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1. Electrophoretic mobility shift assays (EMSAs) of IkkßF/F and IkkßF/{Delta} testis nuclear protein before and 1 h after radiation (A) and corresponding antibody supershift (B). Faint NF-kappaB-shifted bands appear in control IkkßF/F and IkkßF/{Delta} lanes (every lane is a separate animal), while there is strong intensity in IkkßF/F testes 1 h after 5 Gy of ionizing radiation (IR). Shifted bands for IkkßF/{Delta} testes are less intense than their IkkßF/F counterparts. Pooled 1-h IkkßF/F protein incubated with specific cold oligonucleotide competitor (SC) displaces all three complexes, while mutated cold oligonucleotide competition (NC) has no effect. Nuclear protein was equally loaded on the basis of protein assay, and the TCFAP2A (AP2) EMSA is shown as DNA binding loading control. Specific and mutated cold control AP2 oligos were used for SC and NC AP2 lanes. B) Supershift of pooled 1-h IkkßF/F protein with p65, p50, or c-Rel antibodies reduced the intensity of the bands compared to no antibody (–) (n = 3).

IkkßF/{Delta} Testes Are More Resistant to Ionizing Radiation Injury

Although the baseline phenotype of IkkßF/{Delta} mice was not significantly different from IkkßF/F mouse testes, injury showed a phenotype. Lower body exposure of 5 Gy of {gamma}-radiation causes germ cell apoptosis, which was quantified by TUNEL analysis. Figure 2 presents a comparison of IkkßF/F and IkkßF/{Delta} mice for the percentage of seminiferous tubules with >3 TUNEL-positive germ cells. Control mice, IkkßF/F, have a baseline TUNEL staining similar to IkkßF/{Delta} testes; however, upon injury, the percentage of TUNEL-positive germ cells was significantly increased in control IkkßF/F mice compared to NF-kappaB inhibited IkkßF/{Delta} mice at 3, 6, and12 h. These data suggest either a delayed onset or a reduced amount of apoptosis in the IkkßF/{Delta} (NF-kappaB knockdown) mice.


Figure 76030302
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2. Percentage of seminferous tubules with >3 TUNEL-positive germ cells in IkkßF/F (black bar) and IkkßF/{Delta} (gray bar) testes untreated (control) or 3, 6, and 12 h after 5 Gy of {gamma}-irradiation. Untreated NF-kappaB knockdown mice (IkkßF/{Delta}) show a nonsignificant trend toward fewer TUNEL-positive germ cells than their control (IkkßF/F) counterparts. Upon exposure to ionizing radiation, IkkßF/{Delta} mice had statistically fewer seminiferous tubules with >3 TUNEL-positive germ cells than did IkkßF/F mice at 3, 6, and 12 h (n > 3 mice; *P < 0.05).

To address the question of delayed onset, or reduced total apoptosis, mice were exposed to 0.5 Gy of ionizing radiation, and spermatid head counts were performed 14 days (for spermatocytes) or 29 days (for spermatogonia) after exposure (Fig. 3). This highly sensitive endpoint for total spermatocyte or spermatogonia germ cell loss showed a preferential death of mitotic versus meiotic germ cells in both genotypes. Corresponding with TUNEL data, there were more IkkßF/{Delta} than IkkßF/F spermatogonia, indicating a reduction in diploid germ cell apoptosis with NF-kappaB inhibition. Contrastingly, there was no significant difference between the meiotic spermatocyte Ikkß control and inhibited genotype germ cell populations, suggesting that the eventual extent of spermatocyte apoptosis in IkkßF/F and IkkßF/{Delta} mice was similar. These data with the TUNEL assay time course indicate that inhibition of NF-kappaB causes both a delayed onset of and reduction in total apoptosis after {gamma}-irradiation.

Caspase Activation

To corroborate these TUNEL results that indicate delayed onset of apoptosis with inhibition of NF-kappaB, caspase cleavage was determined. Caspases 3 and 9 (Fig. 4) cleavage was significantly increased in IkkßF/F compared to IkkßF/{Delta} whole testis protein 3 h after exposure to ionizing radiation. In addition, cleaved caspase 3 was significantly higher in IkkßF/F than in IkkßF/{Delta} untreated mice. Levels of full-length caspases were consistent throughout the genotypes and time course following exposure.


Figure 76030304
View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4. Western blots of caspases and cleaved caspases 3 and 9 before and 3 h after 5 Gy of {gamma}-radiation for IkkßF/F and IkkßF/{Delta} mice testes. A) Full-length caspases 3 (C3) and 9 (C9) are unchanged after ionizing radiation (IR), while cleaved caspases 3 (cC3) and 9 (cC9) increase significantly in IkkßF/F testes. Unlike IkkßF/F, IkkßF/{Delta} cleaved caspases do not change after IR. B) Densitometry of cleaved caspases relative to ACTB, also known as beta-actin (ßA), with IkkßF/F control set at 1.0, shows statistically different cleaved caspase 3 in control and IR IkkßF/{Delta} mice. Cleaved caspase 9 increases after IR in IkkßF/F, but not IkkßF/{Delta}, mice at 3 h (n = 3, *P < 0.05).

RNA Expression Profiling during the Injury Time Course

Real-time RT-PCR on IkkßF/F and IkkßF/{Delta} testes for control and 1, 3, and 6 h after ionizing radiation was performed with primers for genes known to be regulated by NF-kappaB and/or modulated during radiation (Fig. 5). The expression of Fas (Fas receptor) increased maximally to 3.6- and 3.8-fold (IkkßF/F and IkkßF/{Delta}, respectively) of nonirradiated by 3 h after radiation, as observed previously during this injury [27]. Interestingly, there was no difference in Fas expression between IkkßF/F and IkkßF/{Delta} mice, as both increased significantly at 3 and 6 h after {gamma}-radiation (Fig. 5). Fas ligand (FasL) RNA increased in a nonsignificant trend 1 h after exposure for both IkkßF/F and IkkßF/{Delta} mice (compared to their nonirradiated counterparts). In IkkßF/F mice, Fasl expression increased significantly by 6 h after injury. In contrast, IkkßF/{Delta} mice had significantly less Fasl RNA at 3 and 6 h than their nonirradiated counterparts. In addition, the rise of Fasl expression (2-fold increase) in IkkßF/F mice at 6 h was strikingly absent from IkkßF/{Delta} mice at this time point, as Fasl decreased further to one third of the nonirradiated mice. Expression of Cdnk1a (also known as p21) increased in both genotypes 1 and 3 h after radiation; however, the increase was significantly higher at 3 h for IkkßF/F mice than for their IkkßF/{Delta} counterparts (2.8- versus 1.8-fold). In addition, Cdnk1a levels 6 h after injury remained significantly elevated in IkkßF/F mice, while IkkßF/{Delta} mice returned to basal levels. The antiapoptotic gene Bcl2l1 (also known as Bcl-XL) decreased in IkkßF/F mice by 6 h, dropping to 0.58-fold of nonirradiated, while this gene was significantly decreased in IkkßF/{Delta} mice at 1, 3, and 6 h compared to IkkßF/{Delta} nonirradiated. Real-time RT-PCR of these samples for Trp53 (p53), Bcl2, Birc2 (also known as cIAP-1), and Birc5 (also known as survivin) showed no significant difference in RNA levels during the injury time course or between genotypes (data not shown).


Figure 76030305
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5. Real-time RT-PCR of Fas, Fasl, Cdnk1a, and Bcl2l1 (top to bottom) for IkkßF/F (black bar) and IkkßF/{Delta} (gray bar) mice testes after 5 Gy of {gamma}-irradiation. Fas RNA expression increases significantly in IkkßF/F and IkkßF/{Delta} testes 3 and 6 h after radiation. Fasl is significantly increased in IkkßF/F but is decreased in IkkßF/{Delta} mice testes at 6 h. The expression of Cdnk1a in the testis is increased after ionizing radiation (IR) in IkkßF/F (1, 3, and 6 h) and IkkßF/{Delta} (1 and 3 h) compared to controls. Interestingly, IkkßF/F and IkkßF/{Delta} RNA levels for Cdnk1a are different at 3 and 6 h. Bcl2l1 RNA levels decreased in IkkßF/F (6 h) and IkkßF/{Delta} (1, 3, and 6 h) testes. Note earlier decrease in Bcl2l1 expression for IkkßF/{Delta} testes. (*Indicates significantly different from control; +indicates significantly different from IkkßF/F, P < 0.05.)

DISCUSSION

The IkkßF/{Delta} (NF-kappaB knockdown) mice showed no discernable basal testicular phenotypic differences compared to the IkkßF/F (control) mice. We, and others, have recently shown that NF-kappaB is activated in the rodent testis following injury, such as toxicant exposure, ischemia reperfusion, and cytokine stimulus [9–11]. Despite the recent efforts in this field, the biological role of activated NF-kappaB in the testis remains an enigma. To unmask a phenotype, we challenged the IkkßF/{Delta} mice with {gamma}-irradiation. These NF-kappaB knockdown mice are an invaluable tool for addressing questions of apoptosis regulation during spermatogenesis vis-à-vis NF-kappaB.

The lack of a basal testicular phenotype in our NF-kappaB knockdown (IkkßF/{Delta}) mice was surprising. Testis weights, body weights, and sperm output (as determined by spermatid head count) were quite similar between the genotypes. In addition, there was no reduction of fertility or fecundity in our NF-kappaB knockdown mice (data not shown). We hypothesized that despite the haplo-insufficient status of IkkßF/{Delta} mice, enough NF-kappaB activity remained to maintain normal spermatogenesis. Similarly, the initial p50 –/– mouse reported in 1995 had no basal phenotype [28]. Despite the lack of major developmental abnormalities in the p50 –/– mouse, pathogen exposure detected an interesting phenotype. The p50 –/– mice were far more sensitive to Streptococcus pneumoniae infection and died within 24 h from overwhelming sepsis. In contrast to the enhanced susceptibility to S. pneumoniae infection, p50 –/– mice had greater resistance to murine encephalomyocarditis (EMC) infection. Although 75% of control mice died within 1 wk of EMC infection, all p50 –/– mice survived [28]. This dual role of p50 in immune response to challenge and previous studies in our laboratory [11] urged us to challenge the IkkßF/{Delta} mice to unmask a potential latent phenotype.

Ionizing radiation, such as {gamma}-irradiation, is a potent activator of NF-kappaB in an ATM (ataxia-telangiectasia mutated), receptor-interacting protein-dependent pathway [29–31]. Moreover, this activation of NF-kappaB by ionizing radiation is dependent on a functional IKKß subunit, because HeLa cells containing a mutated IKKß are unable to activate NF-kappaB following {gamma}-radiation [18]. Exposure to {gamma}-irradiation leads to IKKß activation and phosphorylation of IkappaBalpha on serines 32 and 36, while short-wave ultraviolet radiation bypasses the IKK complex.

EMSA experiments demonstrated that 5 Gy of {gamma}-radiation induced NF-kappaB in the IkkßF/F testes within 1 h. The NF-kappaB induction observed in IkkßF/F mice was significantly attenuated in IkkßF/{Delta} testes. Our results show that IKKß-dependent NF-kappaB activity is involved in an overall proapoptotic response to {gamma}-irradiation-induced testicular injury. Fewer seminiferous tubules had >3 TUNEL-positive germ cells in IkkßF/{Delta} mice 3, 6, and 12 h after ionizing radiation. In addition, baseline TUNEL staining showed a nonsignificant trend toward less apoptosis in IkkßF/{Delta} mice testes. These results were surprising, given the antiapoptotic role of NF-kappaB in other systems.

The IkkßF/F mouse was recently used to study the role of NF-kappaB in small intestine crypts in response to 8 Gy of ionizing radiation [23]. In the small intestine crypt epithelium, IKKß-dependent NF-kappaB activation by {gamma}-radiation is protective. Therefore, the proapoptotic effects in our system are intriguing. Similar to the role of p50 –/– in the mouse, in which infection with different pathogens resulted in either protection or sensitization, NF-kappaB appears to have a dual role, depending on tissue type [28]. The testis is a particularly complex organ because of the autocrine, paracrine, and endocrine signaling critical for spermatogenesis. It is possible that NF-kappaB alters Sertoli cell–germ cell communication, manifesting in the observed results.

As a measure of total germ cell apoptosis due to radiation, spermatid head counts were performed on mice exposed to a low dose of {gamma}-radiation. With 0.5 Gy of exposure, the loss of different cell populations can be assessed by waiting the appropriate number of days between exposures and counting to allow the germ cell cohort in question to develop [27]. In this experiment, the meiotic spermatocyte and mitotic spermatogonia cell populations were assayed. Significantly fewer spermatogonia were lost in IkkßF/{Delta} than in IkkßF/F mice, indicating the area under the curve of total cell death following {gamma}-irradiation was attenuated with NF-kappaB inhibition. These data confirmed the delayed onset of apoptosis, as detected by TUNEL, and further strengthened the identification of a proapoptotic role for NF-kappaB in the testis. In contrast to spermatogonia, there was no difference in meiotic spermatocyte germ cell loss between control and NF-kappaB knockdown mice.

Correlating with the observed apoptosis results were elevations of cleaved caspases 3 and 9 in IkkßF/F and IkkßF/{Delta} mice testes 3 h after {gamma}-irradiation. These caspase activation data support a delayed onset of apoptosis, given the relative lack of caspase activation. Also observed was a significant decrease in cleaved caspase 3 in untreated IkkßF/{Delta} mice testes. Inhibition of NF-kappaB has been shown to decrease caspase 3 in vitro, which could, in part, explain the reduction in cleaved caspase 3 in unexposed IkkßF/{Delta} testes that we observed [32].

RNA profiling experiments during the response to injury showed increased Fas and Cdnk1a with decreased Bcl2l1 RNA expression in both genotypes over time. Comparing IkkßF/{Delta} to IkkßF/F testis RNA, there was significantly less Cdnk1a and Bcl2l1 (3 h) in IkkßF/{Delta} than in IkkßF/F mice. The almost 4-fold increase in Fas mRNA after irradiation is a hallmark of this injury [27]. The upregulation of Fas has been attributed to germ cells and precedes the cell death that is seen at later time points. No statistical difference was observed between the IkkßF/F and IkkßF/{Delta} Fas RNA levels before and after radiation, suggesting that although Fas can be regulated by NF-kappaB in other systems, the decreased germ cell apoptosis observed with IkkßF/{Delta} mice is not due to a reduction in Fas transcription.

Fasl, normally expressed on the somatic Sertoli cells, is not significantly different at 1 h, but IkkßF/{Delta} mice have decreased levels at 3 and 6 h postirradiation compared to nonirradiated mice. In addition, the statistically significant increase of Fasl mRNA at 6 h in control IkkßF/F mice is in stark contrast to IkkßF/{Delta} levels at this time point. FAS can activate NF-kappaB, and NF-kappaB has been shown to regulate Fas/FasL transcription as well [32]. This difference at 6 h suggests that NF-kappaB regulates, at least partly, FasL transcription and that the stress-associated induction at 6 h is unable to manifest properly in IkkßF/{Delta} mice. If NF-kappaB-deficient Sertoli cells are unable to mount a strong Fasl mRNA response to stress, the expected result is decreased or delayed FAS/FASL-mediated apoptosis, as observed.

An increase in Cdnk1a RNA is seen throughout the radiation time course in a characteristic bell shape curve peaking at 3 h. With IKKß-dependent NF-kappaB knockdown mice, IkkßF/{Delta}, this increase of Cdnk1a is attenuated. In addition, Cdnk1a mRNA levels for IkkßF/{Delta} mice return to baseline by 6 h, while they remain elevated in IkkßF/F mice. Given the roles of Cdnk1a as a p53-induced cell cycle arrest and apoptosis gene, these data correlate well with the diminished apoptosis observed and support an NF-kappaB-dependent regulation of Cdnk1a transcription after injury. Bcl2l1 can be regulated by NF-kappaB [33]. In IkkßF/{Delta} mice testes, Bcl2l1 mRNA levels were significantly decreased 1, 3, and 6 h after radiation, compared to nonirradiated mice testes. These levels were significantly different from IkkßF/F at the 3-h time point. Although there was a reduction in the antiapoptotic gene Bcl2l1 in IkkßF/{Delta} testes, suggesting a role of NF-kappaB activation in promoting its transcription, the overall result was a decrease in apoptosis in the NF-kappaB knockdown mice.

NF-kappaB promotes cell survival in most tissues and cell types, such as hepatocytes, T cells, and the colon epithelium, although it has been shown to be both pro- and antiapoptotic in the same cell, depending on the stimulus [34]. This may help explain the disparity in recent results regarding NF-kappaB function in the testicular injury response [5, 7, 9, 35]. In the present study, we show that with radiation exposure, IKKß-dependent NF-kappaB activation is proapoptotic on the basis of TUNEL assays, spermatogonial cell counts, caspase cleavage, and altered Cdnk1a and Fasl RNA levels. Since {gamma}-irradiation preferentially targets rapidly dividing cells for apoptosis, these proapoptotic effects of NF-kappaB in the testis may be germ cell-specific. Additional work is needed to identify the testicular cell type specificity resulting in these identified NF-kappaB proapoptotic effects.

ACKNOWLEDGMENTS

The authors would like to thank Teresa Rasoulpour and Mary Hixon for developing and testing the primers used for the real-time RT-PCR used in these experiments. The IkkßF/F mouse was generously provided by Michael Karin at the University of California at San Diego in cooperation with the Bristol-Myers Squibb Pharma Company.

FOOTNOTES

1Supported by P42ES013660 Superfund Basic Research Program (NIEHS). Back

Correspondence: 2Kim Boekelheide, Department of Pathology and Laboratory Medicine, 70 Ship St., Box G-E505, Brown University, Providence, RI 02903. FAX: 401 863 9008; e-mail: kim_boekelheide{at}brown.edu

Received: 22 June 2006.

First decision: 23 July 2006.

Accepted: 31 October 2006.

REFERENCES

  1. Holdcraft RW and Braun RE. Hormonal regulation of spermatogenesis. Int J Androl 2004; 27:335–342[CrossRef][Medline]
  2. Lilienbaum A, Sage J, Memet S, Rassoulzadegan M, Cuzin F, Israel A. NF-kappa B is developmentally regulated during spermatogenesis in mice. Dev Dyn 2000; 219:333–340[CrossRef][Medline]
  3. Sage J, Martin L, Cuzin F, Rassoulzadegan M. cDNA sequence of the murine synaptonemal complex protein 1 (SCP1). Biochim Biophys Acta 1995; 1263:258–260[Medline]
  4. Delfino F and Walker WH. Stage-specific nuclear expression of NF-kappaB in mammalian testis. Mol Endocrinol 1998; 12:1696–1707[Abstract/Free Full Text]
  5. Pentikainen V, Suomalainen L, Erkkila K, Martelin E, Parvinen M, Pentikainen MO, Dunkel L. Nuclear factor-kappa B activation in human testicular apoptosis. Am J Pathol 2002; 160:205–218[Abstract/Free Full Text]
  6. Delfino FJ and Walker WH. NF-kappaB induces cAMP-response element-binding protein gene transcription in Sertoli cells. J Biol Chem 1999; 274:35607–35613[Abstract/Free Full Text]
  7. Delfino F and Walker WH. Hormonal regulation of the NF-kappaB signaling pathway. Mol Cell Endocrinol 1999; 157:1–9[CrossRef][Medline]
  8. Delfino FJ, Boustead JN, Fix C, Walker WH. NF-kappaB and TNF-alpha stimulate androgen receptor expression in Sertoli cells. Mol Cell Endocrinol 2003; 201:1–12[CrossRef][Medline]
  9. Suominen JS, Wang Y, Kaipia A, Toppari J. Tumor necrosis factor-alpha (TNF-alpha) promotes cell survival during spermatogenesis, and this effect can be blocked by infliximab, a TNF-alpha antagonist. Eur J Endocrinol 2004; 151:629–640[Abstract]
  10. Lysiak JJ, Bang HJ, Nguyen QA, Turner TT. Activation of the nuclear factor kappa B pathway following ischemia-reperfusion of the murine testis. J Androl 2005; 26:129–135[Abstract/Free Full Text]
  11. Rasoulpour RJ and Boekelheide K. NF-kappaB is activated in the rat testis following exposure to mono-(2-ethylhexyl) phthalate. Biol Reprod 2005; 72:479–486[Abstract/Free Full Text]
  12. Karin M. How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex. Oncogene 1999; 18:6867–6874[CrossRef][Medline]
  13. Karin M. The beginning of the end: IkappaB kinase (IKK) and NF-kappaB activation. J Biol Chem 1999; 274:27339–27342[Free Full Text]
  14. Li ZW, Chu W, Hu Y, Delhase M, Deerinck T, Ellisman M, Johnson R, Karin M. The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis. J Exp Med 1999; 189:1839–1845[Abstract/Free Full Text]
  15. Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 1995; 376:167–170[CrossRef][Medline]
  16. Li ZW, Omori SA, Labuda T, Karin M, Rickert RC. IKK beta is required for peripheral B cell survival and proliferation. J Immunol 2003; 170:4630–4637[Abstract/Free Full Text]
  17. Rasoulpour RJ and Boekelheide K. The Sycp-1Cre transgenic mouse and male germ cell inhibition of NF-{kappa}B. J Androl 2006; 27:729–733[Abstract/Free Full Text]
  18. Li N and Karin M. Ionizing radiation and short wavelength UV activate NF-kappaB through two distinct mechanisms. Proc Natl Acad Sci U S A 1998; 95:13012–13017[Abstract/Free Full Text]
  19. Vidal F, Sage J, Cuzin F, Rassoulzadegan M. Cre expression in primary spermatocytes: a tool for genetic engineering of the germ line. Mol Reprod Dev 1998; 51:274–280[CrossRef][Medline]
  20. Sage J, Martin L, Meuwissen R, Heyting C, Cuzin F, Rassoulzadegan M. Temporal and spatial control of the Sycp1 gene transcription in the mouse meiosis: regulatory elements active in the male are not sufficient for expression in the female gonad. Mech Dev 1999; 80:29–39[CrossRef][Medline]
  21. Rassoulzadegan M, Magliano M, Cuzin F. Transvection effects involving DNA methylation during meiosis in the mouse. EMBO J 2002; 21:440–450[CrossRef][Medline]
  22. Chung SS, Cuzin F, Rassoulzadegan M, Wolgemuth DJ. Primary spermatocyte-specific Cre recombinase activity in transgenic mice. Transgenic Res 2004; 13:289–294[CrossRef][Medline]
  23. Egan LJ, Eckmann L, Greten FR, Chae S, Li ZW, Myhre GM, Robine S, Karin M, Kagnoff MF. IkappaB-kinase beta-dependent NF-kappaB activation provides radioprotection to the intestinal epithelium. Proc Natl Acad Sci U S A 2004; 101:2452–2457[Abstract/Free Full Text]
  24. Rasoulpour RJ, Schoenfeld HA, Gray DA, Boekelheide K. Expression of a K48R mutant ubiquitin protects mouse testis from cryptorchid injury and aging. Am J Pathol 2003; 163:2595–2603[Abstract/Free Full Text]
  25. Inan MS, Rasoulpour RJ, Yin L, Hubbard AK, Rosenberg DW, Giardina C. The luminal short-chain fatty acid butyrate modulates NF-kappaB activity in a human colonic epithelial cell line. Gastroenterology 2000; 118:724–734[CrossRef][Medline]
  26. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29:e45.[Abstract/Free Full Text]
  27. Embree-Ku M, Venturini D, Boekelheide K. Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis. Biol Reprod 2002; 66:1456–1461[Abstract/Free Full Text]
  28. Sha WC, Liou HC, Tuomanen EI, Baltimore D. Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell 1995; 80:321–330[CrossRef][Medline]
  29. Lee SJ, Dimtchev A, Lavin MF, Dritschilo A, Jung M. A novel ionizing radiation-induced signaling pathway that activates the transcription factor NF-kappaB. Oncogene 1998; 17:1821–1826[CrossRef][Medline]
  30. Piret B, Schoonbroodt S, Piette J. The ATM protein is required for sustained activation of NF-kappaB following DNA damage. Oncogene 1999; 18:2261–2271[CrossRef][Medline]
  31. Li N, Banin S, Ouyang H, Li GC, Courtois G, Shiloh Y, Karin M, Rotman G. ATM is required for IkappaB kinase (IKKk) activation in response to DNA double strand breaks. J Biol Chem 2001; 276:8898–8903[Abstract/Free Full Text]
  32. Peng Y, Gallagher SF, Haines K, Baksh K, Murr MM. Nuclear factor-kappaB mediates Kupffer cell apoptosis through transcriptional activation of Fas/FasL. J Surg Res 2006; 130:58–65[CrossRef][Medline]
  33. Glasgow JN, Qiu J, Rassin D, Grafe M, Wood T, Perez-Pol JR. Transcriptional regulation of the BCL-X gene by NF-kappaB is an element of hypoxic responses in the rat brain. Neurochem Res 2001; 26:647–659[CrossRef][Medline]
  34. Lin B, Williams-Skipp C, Tao Y, Schleicher MS, Cano LL, Duke RC, Scheinman RI. NF-kappaB functions as both a proapoptotic and antiapoptotic regulatory factor within a single cell type. Cell Death Differ 1999; 6:570–582[CrossRef][Medline]
  35. Suomalainen L, Pentikainen V, Dunkel L. Sphingosine-1-phosphate inhibits nuclear factor kappaB activation and germ cell apoptosis in the human testis independently of its receptors. Am J Pathol 2005; 166:773–781[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J AndrolHome page
J. S. Moffit, K. Boekelheide, J. M. Sedivy, and J. Klysik
Mice Lacking Raf Kinase Inhibitor Protein-1 (RKIP-1) Have Altered Sperm Capacitation and Reduced Reproduction Rates With a Normal Response to Testicular Injury
J Androl, November 1, 2007; 28(6): 883 - 890.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
A.-N. Spiess, C. Feig, W. Schulze, F. Chalmel, H. Cappallo-Obermann, M. Primig, and C. Kirchhoff
Cross-platform gene expression signature of human spermatogenic failure reveals inflammatory-like response
Hum. Reprod., November 1, 2007; 22(11): 2936 - 2946.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
76/2/279    most recent
biolreprod.106.054924v1
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rasoulpour, R. J.
Right arrow Articles by Boekelheide, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rasoulpour, R. J.
Right arrow Articles by Boekelheide, K.
Agricola
Right arrow Articles by Rasoulpour, R. J.
Right arrow Articles by Boekelheide, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS