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From the * Department of Urology and the
Department of Cell Biology, University of
Virginia Health System, Charlottesville, Virginia.
| Correspondence to: Dr Jeffrey J. Lysiak, Department of Urology Box 800422, University of Virginia Health System, Charlottesville, VA 22908 (e-mail: jl6n{at}virginia.edu). |
| Received for publication May 26, 2004; accepted for publication September 9, 2004. |
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Abstract |
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B)
is a nuclear transcription factor involved in the control of a number of
cellular processes, and its activation is part of the cellular stress response
to a variety of factors including cytokine stimulation, irradiation, and IR.
The present study investigates NF-B activation after IR of the murine
testis and potential downstream target genes of that activation. Mice were
subjected to a period of testicular ischemia followed by 0-4 hours of
reperfusion. Activation of NF-B was assessed by 1) Western blot
analysis of the NF-B inhibitory protein, IB
; 2)
immunohistochemistry for IB; and 3) TranSignal NF-B
target gene array (107 genes) analysis. Results demonstrate that
IB is phosphorylated on serine 32 reaching a peak by 2 hours
after IR of the testis. A decrease in total IB was also noted at
2 hours after IR, consistent with the rapid degradation of the phosphorylated
protein. Phosphorylation and degradation of IB is indicative of
NF-B activation. Imunnolocalization revealed IB
specifically in Sertoli cells of the murine testis. Results of the TranSignal
target gene array revealed that the expression of 9 genes was consistently
changed 2 hours after IR of the testis, 3 of which increased in expression and
6 of which were down-regulated. Most notably, high-mobility group nucleosomal
binding domain 1 increased in expression while platelet-derived growth factor
B and Wilms tumor homolog decreased. These results suggest that testicular IR
releases the suppression of NF-B by IB in Sertoli cells.
Activation of the NF-B pathway in the testis resulted in an alteration
of expression of potential NF-B target genes, some increased while
others decreased. The specific roles of these genes in the testicular response
to IR remains to be determined.
Key words: Testicular oxidative stress, apoptosis, NF-B activation
Studies investigating the mechanisms of neutrophil recruitment to the
testis after IR have demonstrated that E-selectin is a critical endothelial
cell adhesion molecule responsible for neutrophil recruitment
(Lysiak et al, 2001).
E-selectin expressed on endothelial cells mediates the tethering and slow
rolling of neutrophils to endothelial cells
(Kunkel and Ley, 1996). Recent
studies examining the cell signaling pathway regulating E-selectin in the
testis after IR have found that there is an increase in the proinflammatory
cytokines, interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha
(TNF-), as early as 0.5 hour after IR
(Lysiak et al, 2003). This
increase in the proinflammatory cytokines is followed by the activation of the
c-jun N-terminal kinase (JNK) stress-related pathway, which in turn is
correlated with the expression of E-selectin mRNA and protein and the
retention of neutrophils in testicular venules. Direct injection of
proinflammatory cytokines into the testis demonstrated that IL-1ß but not
TNF- caused activation of JNK and the recruitment of neutrophils to the
testis. This indicates that IL-1ß is the mediating factor of neutrophil
recruitment to the testis after IR (Lysiak
et al, 2003).
It is well established that IL-1ß and TNF- can mediate their
effects through activation of nuclear factor kappa B (NF-B).
NF-B is a family of 5 proteins that are structurally and functionally
related and regulate the transcription of a variety of genes. Active
NF-B consists of 2 family members arranged to form either homodimers or
heterodimers that then bind to DNA with different specificities
(Baeuerle and Baltimore, 1996; Ghosh and Karin, 2002).
Under nonstimulating conditions NF-B is bound to a member of a
structurally related family of proteins termed inhibitors of NF-B
(IB). This family includes, IB, IBß,
IB
, IB
, IB
, and Bcl-3
(Mercurio and Manning, 1999;
Senftleben and Karin, 2002). IB's binding to NF-B physically masks the nuclear localization
sequences of NF-B and causes its retention in the cytoplasm. Activation
of NF-B occurs when IB is phosphorylated and degraded, thus
releasing NF-B for transport to the nucleus where it binds to DNA and
directs transcription of its target genes. This activation has been shown to
occur after stimulation by TNF- or IL-1ß
(Senftleben and Karin,
2002).
The existence of the multiple NF-B and IB family members is
thought to provide specificity of gene regulation to the many signals that can
activate NF-B. For example, different roles for the activation
IB and IBß have been described
(Baeuerle and Baltimore, 1996),
and both IB and IBß have been localized in the
murine testis (Budde et al,
2002) and may play different roles there.
The purpose of the present study was to determine whether IR of the testis
with its increase in proinflammatory cytokines alters the phosphorylation and
degradation of IB, thus indicating the activation of the
NF-B pathway. Further, we sought to determine whether testicular IR
would alter the expression of genes known to be NF-B target genes in
other tissues. These studies were conducted with the aim of detecting yet
unknown players in the pathway(s) to germ cell apoptosis.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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B (9242) were purchased from Cell Signaling Technology
(Beverly, Mass) and against phosphorylated IB (serine 32;
sc-8404) were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, Calif).
Anti--tubulin was purchased from Sigma.
Experimental Testicular Torsion
This work was conducted in accordance with the Society for the Study of
Reproduction's Guiding Principals of the Care and Use of Research
Animals. Adult male C57BL/6 mice were anesthetized with an
intraperitoneal injection of 0.01 mg/g body weight of sodium pentobarbital,
and the testis was rotated as described by Lysiak et al
(2001). Briefly, the testis
was exteriorized through a low midline laparotomy, the gubernaculum was
divided, and the testis was freed from the epididymo-testicular membrane. The
testis was rotated 720° for 2 hours, during which time it remained in the
abdomen with a closed incision. Following the 2 hours torsion the incision was
reopened, the testis was counterrotated to the natural position, the
gubernaculum was rejoined, and the testis was reinserted into the scrotum via
the inguinal canal. Testes were examined at the time of repair for the
apparent degree of ischemia and reperfusion. Sham operated animals were
treated identically except that upon completion of the torsion maneuver the
testis was immediately counterrotated.
Western Blot Analysis
Western blot analysis for total IB, phospho-IB,
and -tubulin were performed by isolating testicular proteins at 0.0,
0.5, 2.0, and 4.0 hours after reperfusion of the testis or after sham
operations. Briefly, the testes were removed, snap frozen in liquid nitrogen,
and stored at -80°C until use. Whole testes were ground with a mortar and
pestle chilled in liquid nitrogen. The resultant powder was resuspended in
RIPA buffer (0.1% sodium dodecylsulfide [SDS], 1 mM EDTA, 100 mM Tris, 0.15
NaCl, 1.0% deoxycholate, 1.0% triton X-100, pH 7.4) with the addition of
protease inhibitors (100 µM leupeptin, 1 mM PMSF, 10 µM E-64, 20 µg
aprotinin), and phosphatase inhibitor, (1 mM sodium orthovanadate). The
protein suspension was vortexed, incubated on ice for 15 minutes, and
centrifuged at 14 000 x g for 15 minutes at 4°C, and the
supernatant was collected. The protein concentration in the supernatant was
determined using the bicinchoninic assay kit (Pierce, Rockford, Ill), and 50
µg of protein per lane was subjected to SDS polyacrimide gel
electrophoresis (PAGE). The gel contents were electrotransfered to
nitro-cellulose membranes (Bio-Rad, Hercules, Calif); blocked with 5% nonfat
dried milk, 0.1% Tween 20 in phosphate-buffered saline (PBS); and incubated
overnight at 4°C with an antibody that recognized the form of the protein
specifically phosphorylated on serine 32 (1:100 dilution) or an antibody that
recognized the total protein (1:500 dilution). The next day membranes were
washed and incubated for 1 hour at room temperature with the appropriate
peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories,
West Grove, Pa). Immuncomplexes were detected with enhanced chemiluminescence
(SuperSignal West Pico; Pierce). Densitometry and ImageQuant analysis were
subsequently performed.
Immunohistochemistry
To obtain testes to be sectioned for immunohistochemistry for
IB, mice were sacrificed at 2 hours after reperfusion of the
testis. The testes were removed and placed in Bouin fixative for 6 hours and
subsequently paraffin embedded. Tissue sections were deparaffinized,
rehydrated, and washed in PBS. Slides were then placed in unmasking solution
(Vector Laboratories Inc; Burlinghame, Calif), microwaved for 20 minutes, and
allowed to cool for 1 hour at room temperature. Subsequently, endogenous
peroxidase activity was inhibited by incubating the sections in 3%
H2O2 methanol solution for 15 minutes. Slides were then
washed, blocked, and incubated with anti-IB antibody overnight
at 4°C. The next day slides were washed and incubated with the appropriate
biotinylated secondary antibody (Vector Laboratories Inc) for 1 hour at room
temperature. Immunocomplexes were visualized with avidin-biotin-peroxidase
complex (Vector Laboratories Inc) with diaminobenzidene (Sigma) as the
chromagen. All slides were lightly counterstained with hematoxylin.
Gene Array Analysis
NF-B gene array analysis was performed using the TranSignal
NF-B target gene array (Panomics, Redwood City, Calif). Briefly, RNA
was isolated from liver and from testes 2 hours after IR or sham operation
using Trizol reagent (Invitrogen, Carlsbad, Calif) according to the
manufacturer's instructions. Twenty micrograms of total testicular RNA was
incubated with 5 µl of the TranSignal NF-B primer mix for 2 minutes
at 70°C in a thermal cycler (Whatman Biometra, Gottingen, Germany). The
temperature was then reduced to 42°C. The cDNA synthesis mixture
consisting of labeling mix buffer, biotin-dUTP, reverse transcriptase, and
water was then added to the RNA and primer mix and incubated at 42°C for 2
hours. After the incubation, 3 µl of denaturing solution was added to each
sample and incubated at 68°C for 20 minutes. Samples were then incubated
at 72°C for 10 minutes in the presence of neutralization buffer. The
resultant biotin-label cDNA probes were then either used immediately or stored
at -20°C for future use.
TranSignal NF-B target gene arrays contain duplicate representations
of 107 different genes previously identified as NF-B target genes in
various tissues. Two array membranes representing sham and torsion testes,
respectively, were used in each experiment, and the experiment was performed 3
times. Membranes were placed in sterile 50-ml tubes and hydrated with water.
Prewarmed hybridization buffer was then added to each tube and incubated at
42°C for 2 hours in a hybridization oven (Hybaid, Franklin, Mass). The
labeled cDNA probes were then added to each bottle and hybridized overnight at
42°C. The next day the hybridization mixture was decanted from each bottle
and the membranes were washed twice with hybridization wash I buffer for 20
minutes at 42°C. This was followed by 2 washes with prewarmed
hybridization wash II buffer for 20 minutes at 42°C. Each membrane was
then removed from its hybridization bottle and transferred to a new container
containing 20 ml of blocking buffer for 15 minutes at room temperature. Twenty
microliters of streptavidin-horseradish peroxidase conjugate was then added
directly to the blocking solution, and the membranes were incubated for 15
minutes at room temperature. The membranes were subsequently washed 3 times
with wash buffer, each 8 minutes. Detection buffer was then added, and the
membranes were incubated for 5 minutes at room temperature. Hybridization
signals were detected with a solution of luminol enhancer and peroxide for 5
minutes at room temperature.
To obtain semiquantitative numerical data, the resultant films were scanned on a densitometer and imported into ImageQuant. Numerical values corresponding to the optical density were determined for each target gene, and values were corrected for background using the application's local average method. The local average method determines the average density of pixels surrounding the target gene hybridization spot and subsequently corrects the volume report for background. The background corrected volume report for each gene on the arrays was then normalized to ubiquitin expression as a load control. Genes were assigned to 1 of 3 groups, those genes that were highly expressed in sham testes (upper third of pixel intensities), those genes that were moderately expressed (middle third of intensities), and those that were less abundantly expressed (lower third of intensities). Within these groups data were examined to identify only those genes that were consistently up- or down-regulated in triplicate experiments.
Statistical Analysis
All statistical evaluations were either by analysis of variance followed by
the Tukey range test or the Student's t test (P < .05)
after evaluation of each data set by the Chauvenet criterion for
homogeneity.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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B After IR of the TestisB pathway in the testis after IR was examined
indirectly by Western blot analysis for phosphorylation and degradation of the
NF-B inhibitory protein, IB. Phosphorylated
IB was detected as a single band of approximately 47 kd in
Western blots of electrophoresed proteins
(Figure 1A). A significant
increase in phosphorylated IB occurred by 2 hours after IR, but
this phosphorylated product was not detectable 4 hours after IR
(Figure 1A and B).
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Western blot analysis for total IB revealed a major band at
33 kd that decreased at both 0.5 and 2 hours after IR of the testis
(Figure 2A and B). The decrease
in this 33-kd band corresponds in time with the appearance of a 47-kd band
(Figure 2A), which agrees with
the 47-kd phosphorylated IB band in
Figure 1A. This shift in
molecular weight most likely represents the phosphorylation and ubiquitination
of the 33-kd form.
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Localization of IB in the Testis
Immunohistochemical analysis of total IB in the testis
revealed a pattern of specific immunoreactivity resembling that of Sertoli
cells in the seminiferous tubules (Figure
3). Other cell types in the testis were not immunoreactive.
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Activation of NF-B Target Genes
NF-B target gene arrays were used to determine whether IR caused the
alteration of NF-B target gene expression in concert with the
phosphorylation and degradation of IB. To ensure specificity of
the NF-B target gene array, liver RNA was compared with normal testis
RNA. Numerous target gene expressions were different between testis and liver.
For example, ferritin heavy chain (Fth) gene was expressed more in
the liver than in the testis and the prostaglandin-endoperoxide synthase-2
(Ptgs2) gene was less in the liver than the testis
(Figure 4). Other genes like
ubiquitin (ubic) were expressed equivalently in both tissues
(Figure 4). Major consistent
gene expressions on the arrays using sham testis RNA included genes like
gapdh, pdgfb, vegfc, and wt-1
(Figure 5), the genes for
glyceraldehydes-3-phosphate dehydrogenase (GAPDH), platelet-derived growth
factor B (PDGF-B), vascular endothelial growth factor (VEGF) isoform c, and
Wilms Tumor (WT)-1 protein, respectively. Of the 3 genes on the array to be
used as load controls, ß-actin (ß-actin), gapdh,
and ubic (Figure 5),
only ubic was relatively unchanged between arrays representing sham
and IR testes; therefore, all data were normalized to the average value for
ubic for each array.
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Of the 107 genes on the NF-B targeted gene array, many exhibited
changes, either positive or negative, in individual runs of the experiment,
but the standard was maintained that in order to be a gene of interest the
gene had to change in a consistent direction in 3 out of 3 experiments. With
that standard, the potential NF-B target genes in the testis were
reduced to 9 (Figure 6). Of the
genes that were considered highly expressed, 4 changed in expression after IR.
The mRNA for high-mobility group nucleosomal binding domain 1 (hmgn1)
consistently increased by approximately 40%, and the mRNAs for gapdh,
pdgf-1 (platlet-derived growth factor B), and wt-1 (the mouse
Wilms tumor homolog) were consistently decreased in expression by an average
45%, 25%, and 26%, respectively.
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Of the genes that were moderately expressed, the mRNAs for cd80 (CD80 antigen) and bcl2-A1a (Bcl-2 related protein A1a) were consistently down-regulated in testes after IR by an average 38% and 27%, respectively. In the category of genes that showed low expression, the mRNAs for apoc3 (apolipoprotein C-III) and lyzs (lysozyme) were consistently up-regulated in the testis after IR (an average 13-fold and twofold increase over sham values, respectively), and galr1 (Galinin receptor-1) was consistently down-regulated and reduced by an average 70% between IR and sham testes.
There were numerous genes that changed in the same direction in only 2 of the 3 experiments. An interesting example is gstp1 (glutathione-S-transferase-1), a prominently expressed gene (Figure 6). This gene increased by an average 62% in 2 experiments, but declined in the third.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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(Lysiak et al 2003). A role
for IL-1ß has been demonstrated in the activation of the stress-related
kinase, JNK, ultimately leading to E-selectin expression in testicular
endothelial cells and neutrophil recruitment
(Lysiak et al, 2003). The
recruitment of neutrophils to the testis after IR is essential for the
observed germ cell-specific apoptosis
(Lysiak et al, 2001). Since
these proinflammatory cytokines can also activate the NF-B pathway, we
sought to determine whether NF-B is activated after testicular IR and,
if so, what genes are affected by its activation. Results of the present study
demonstrate that IB is phosphorylated and shifts in molecular
weight, suggesting that it is ubiquinated 2 hours after IR of the murine
testis (Figures 1 and
2). Further, total
IB was immunolocalized in Sertoli cells, suggesting NF-B
activation in that cell type after IR
(Figure 3). IB
phosphorylation is an indicator of NF-B activation, and that activation
was confirmed by employing an NF-B gene target array (Figures
4,
5,
6).
In most cells NF-B remains sequestered in the cytoplasm until
activation by certain stimuli such as proinflammatory cytokines, bacterial and
viral products, physical stress, oxidative stress, phorbol esters, protein
kinase C, or cell mitogens (Baeuerle and
Baltimore, 1996). Upon binding to specific cell surface receptors,
TNF- and IL-1ß can induce NF-B activation by activating
members of the mitogen activated protein kinase/ERK kinase kinase
(MEKK)-related family including NF-B-inducing kinase (NIK) and MEKK1.
Activation of NIK and MEKK1 phosphorylate and activate 2 major components of
the IB kinase complex, IB kinase and ß (IKK
and IKKß), but biochemical as well as targeted gene knockout studies have
determined that IKKß and not IKK is the target for cytokine
induced NF-B activation (Senftleben
and Karin, 2002). IB is recruited to the complex and
subsequently phosphorylated on serine residues at positions 32 and 36
(DiDonato et al, 1997). This
phosphorylation keys a rapid ubiquitination and degradation through the
proteasome pathway.
Following IR of the murine testis IB is phosphorylated on
serine residue 32 (Figure 1)
and most likely ubiquitinated (Figures
1 and
2). We have previously
demonstrated that TNF- and IL-1ß mRNA are up-regulated 0.5 hour
after IR of the testis (Lysiak et al,
2003); thus, as hypothesized, phosphorylation of IB
follows the production of the proinflammatory cytokines.
This is not the first report of IB modulation in the testis, but it
is the first to report localization in the Sertoli cells. Budde et al
(2002) described the presence
of both IB and IBß in the murine testis and reported
that IB is less expressed than IBß. These
investigators went on to show that IBß is localized to haploid
spermatids, but they did not localize IB. We have examined for
IB specifically, since it is the form induced by the
proinflammatory cytokines of interest in IR injury of the testis.
IB-specific staining was detected only in Sertoli cells
(Figure 3); thus, NF-B
activation in the testis may be regulated in different cell types (spermatids
and Sertoli cells) by the specific expression of different IB family
members.
Previous results from our laboratory have demonstrated that IR of the testis results in germ cell-specific apoptosis (Turner et al, 1997; Lysiak et al, 2000b). A significant increase in the number of apoptotic germ cells occurs by 24 hours after IR of the murine testis, but a trend toward increased apoptosis begins by 4 hours (Lysiak et al, 2001).
Since phosphorylation of IB is an indirect assessment of
NF-B activation, we performed NF-B targeted gene array analysis
on RNA from sham testes and testes 2 hours after IR. Identifying changes in
NF-B target genes at 2 hours after IR of the testis would not only
provide further evidence that NF-B is activated, but screening those
downstream genes would suggest targets for further study in their potential
role in germ cell-specific apoptosis or Sertoli cell survival. Clearly, gene
arrays hybridized with whole-testis RNA will reflect contributions from the
entire testis, but IKB was localized to Sertoli cells and the timing of
the RNA extraction relative to IR was chosen to match the time of maximum
IKB activation (Figure
1); thus, activation of NF-B target genes is very likely in
Sertoli cells.
Of the 107 genes in the NF-B targeted gene array, 9 demonstrated
consistent changes 2 hours after IR of the testis. Hmgn1 was
consistently up-regulated. Its protein product is a member of a family of
proteins that regulate DNA-related activities like transcription, replication,
and repair (Bustin, 1999).
Since Hmgn1 is involved in these DNA-related activities it is not
surprising that it would be increased when a transcription factor like
NF-B is activated. Both PDGF-B, a growth factor with roles in cell
proliferation and differentiation (Hwang
et al, 2003), and WT1, a transcription factor known primarily for
controlling gene expression during embryonic kidney development
(Armstrong et al, 1993), were
down-regulated after IR of the testis. Interestingly, both PDGF-B
(Loveland et al, 1995) and WT1
(Del Rio-Tsonis et al, 1996)
have been reported to be produced by Sertoli cells.
Loveland et al (1995) not
only found PDGF-B in Sertoli cells, but found PDGF receptor subunits as well,
and neither ligand nor receptor was detected in germ cells. This suggests that
PDGF-B may not play a direct role in germ cell survival but may have a
paracrine mechanism of action. Indeed, a role for PDGF-B in gonocyte
proliferation has been previously described
(Li et al, 1997). WT1 gene
expression in Sertoli cells has been found associated with specific states of
germ cell maturation and has been shown to regulate sex-determining gene (SRY;
Hossain and Saunders, 2001), suggesting that the WT1 gene product may have a role in spermatogenesis.
Further, even though NF-B is usually thought to increase gene
transcription, there is evidence that activation of NF-B can result in
inhibition of specific gene transcription
(Todorov et al, 2004).
Of the genes that were moderately expressed in the testis cd80, whose product is typically expressed on immune cells, and bcl2a1a, whose product has previously been described to have an antiapoptotic role in hematopoietic cells (Zong et al, 1999), were both decreased after IR. In the less abundantly expressed category ApocIII, which encodes a protein associated with chylomicrons and low- and high-density lipoproteins (Aalto-Setala et al, 1996), and Lyzs, which encodes a small protein that cleaves carbohydrate chains and is important in antibacterial responses (Mir, 1977), were both up-regulated after IR. Galr1 in the less abundantly expressed group encodes a rhodospin-like, G protein-coupled receptor that binds the neuropeptide galinin (Iismaa and Shine, 1999). This gene was down-regulated after IR. None of these latter genes have been previously reported in the testis, and their roles in germ cell survival or death remains unknown.
Activation of the NF-B pathway in Sertoli cells is suggested by the
immunolocalization of IB in those cells. Sertoli cells remain
present and active in the hours and even days after IR injury to the rodent
testis (Turner and Miller,
1997); thus, their role is presumably an indirect one in
channeling a variety of cell signals that will potentially lead to germ cell
apoptosis. Another possibility is that the pathways involved may also play
important roles in Sertoli cell survival after IR. The present results direct
our interest to gene expressions heretofore unknown in the testis and to their
potential activities subsequent to IR injury, whether the ultimate effect is
on Sertoli cells or germ cells.
IR of the testis results in germ cell-specific apoptosis. Testicular torsion of the murine testis followed by repair is an excellent model system to study the many facets of this pathology. Dissecting the intracellular signaling pathways activated after testicular IR, including discovering target genes of selected transcription factors, will not only aid in understanding the molecular pathway(s) to germ cell apoptosis after IR but may well provide insight into IR injury in general. Our goal is to exploit these pathways for the development of therapies to protect the at-risk organ after an IR episode.
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Footnotes |
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 R. J. Rasoulpour and K. Boekelheide NF-kappaB Activation Elicited by Ionizing Radiation Is Proapoptotic in Testis Biol Reprod, February 1, 2007; 76(2): 279 - 285. [Abstract] [Full Text] [PDF]
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T. T. Turner, J. J. Lysiak, J. D. Shannon, Q. A. T. Nguyen, and C. R. Bazemore-Walker Testicular Torsion Alters the Presence of Specific Proteins in the Mouse Testis as Well as the Phosphorylation Status of Specific Proteins J Androl, March 1, 2006; 27(2): 285 - 293. [Abstract] [Full Text] [PDF]
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