| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Departments of * Urology,
Cell Biology,
Microbiology, and
Chemistry, University of Virginia,
Charlottesville, Virginia.
| Correspondence to: Dr Terry T. Turner, Department of Urology, University of Virginia School of Medicine, Charlottesville, VA 22908 (e-mail: ttt{at}virginia.edu). |
| Received for publication July 26, 2005; accepted for publication September 27, 2005. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Oxidative stress, testicular proteins
Interestingly, significant apoptosis has not been reported in Sertoli cells or Leydig cells after repair of testicular torsion, although ROS do disrupt Leydig cell steroidogenesis through perturbation of the mitochondrial membrane (Allen et al, 2004). This may be the reason for the temporary decline in steroidogenesis seen after torsion repair in the rat (Turner et al, 2005). Sertoli cells in the rat testis have also been reported to continue to secrete a relatively normal panel of proteins after torsion repair (Turner and Miller, 1997), although this is not to say that every protein's synthesis and secretion remain unaltered. It simply means that the Sertoli cell's synthetic and secretory apparatus remains intact.
Oxidative stress has been reported to be a factor in a growing number of circumstances associated with male infertility (Samanta and Chainy, 1998; Lucesoli and Fraga, 1999; Pasqualotto et al, 2000; Wellejus et al, 2004); therefore, the present study examined the alterations in the mouse testicular proteome after testicular torsion of a degree and duration sufficient to induce oxidative stress and germ cell apoptosis (Turner et al, 1997; Lysiak et al, 2001). In these studies we use a proteomic approach to assess global changes in testicular proteins 24 hours after IR and to identify by mass spectrometry selected, specific proteins that were consistently changed in relative concentration by that treatment. Further, phosphorylation status of testicular proteins was assessed globally with and without IR, as well as phosphorylation of specific, stress-related proteins. The overall hypothesis was that testicular torsion induces changes in specific, stress-related proteins in the testis that would help us identify key pathways involved in IR injury of that organ.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Protein Extraction and Electrophoresis
Extirpated testes were snap-frozen in liquid nitrogen and stored at
-80°C until use. At the time of protein extraction, whole testes were
ground with a mortar and pestle chilled in liquid nitrogen and the resultant
powder was resuspended in 1 mL of protein extraction/rehydration buffer (6 M
urea, 1 M thiourea, 0.5% 2-mercaptoethanol, 1% Triton X-100, 3%
3-[(3-cholmidopropyl)dimethylammonio]-1-propanesulfonate, 0.2% bio-lytes, 20
µg/mL aprotinin, 10 µM E-64, 100 µM leupeptin, and 1 mM
phenylmethylsulfonyl fluoride). The samples were placed on ice for 15 minutes
and then centrifuged at 14 000 x g for 15 minutes at 4°C.
The resultant supernatant was collected and an RC-DC protein assay (Bio-Rad,
Hercules, Calif) was performed according to the manufacturer instructions to
determine protein concentrations.
ReadyGel IPG strips (pH 4.0–7.0; Bio-Rad) were passively rehydrated with 350 µg of protein in 185 µL loading buffer. IPG strips were incubated for 45 minutes at room temperature, then covered with 2 mL of mineral oil to prevent evaporation and incubated overnight at room temperature. The IPG strips were drained of the mineral oil and transferred to the Protean isoelectric focusing (IEF) tray (Bio-Rad) according to manufacturer protocol. The IEF was performed with conditions of 25 000 volt hours for approximately 6 hours.
Following the IEF, the IPG strips were drained of mineral oil and placed in an equilibration buffer consisting of 6 M urea; 0.375 M Tris-HCl, pH 8.8; 20% glycerol; 2% sodium dodecyl sulfate (SDS); and 2% dithiothreitol for 15 minutes at room temperature on an orbital shaker. The first equilibration buffer was then discarded and the strips were incubated in a second equilibration buffer consisting of 6 M urea; 0.375 M Tris-HCl, pH 8.8; 20% glycerol; 2% SDS; and 2.5% iodoacetamide for 15 minutes at room temperature on an orbital shaker. The equilibrated IPG strips were rinsed in gel-running buffer (25 mM Tris; 192 mM glycine; 0.1% SDS, pH 8.3) and placed on precast, 10.5%–14% SDS polyacrylamide gels. Molecular weight standards were included and electrophoresis was carried out at 125 volts for 2 hours.
Gels (n = 5/group) were incubated with a fluorescent stain (Sypro-Ruby; Molecular Probes, Eugene, Oreg) and viewed under ultraviolet light. Digital images were captured and analyzed using PDQuest software (Bio-Rad).
Image Analysis
Gel images were analyzed with PDQuest 7 (Bio-Rad). Image analysis of
multiple gels is most effective when the gels are excellent replicates with
regard to overall mobility dimensions, intensity of overall staining, clarity
of individual protein spots, and absence of artifacts. Thus, image analysis
was done on the 3 gel pairs (the paired sham and torsion testes from 3
different animals) that provided the highest replication of those general
characteristics.
One of the gels in the sham group was assigned as the reference gel, and an identification number (IDN) was assigned to each protein spot. The spots in all other gels were compared to spots on the reference gel and, where matched by molecular weight and pI location, were assigned the same IDN. The PDQuest software determined the number of protein spots in each gel and the number and IDN of spots common to other gels within the sham group and then the torsion group. This spot identification system was augmented by 1) manual addition of occasional faint spots, which were ignored by the software in some gels but were visually detected in all gels of a group, 2) removal of occasional spots in some gels that were confounded with streaking or detergent micelles, and 3) manual matching when in occasional, complex situations the software could not make a match because of protein smearing or streaking. Individual spot intensities in all 6 gels representing proteins from 3 sham testes and 3 testes 24 hours after torsion repair were determined by the PDQuest software.
Spot quantification data were exported into Excel (Microsoft, Richmond, Wash) and analyzed to verify the average intensity of each protein spot in the gels of the sham and torsion groups. Proteins with average spot intensities that increased or decreased by a twofold measure or more after repair of torsion were noted as being increased or decreased, respectively. Any protein selected for further study appeared in all gels of the sham-operated group.
All gels originally stained with Sypro-Ruby were restained with commassie blue to allow direct visualization. Six proteins (3 that had increased after IR and 3 that had decreased after IR) were selected for identification by mass spectrometry based on the spot's intensity in the commassie-stained gels, the expectation that the spot could be cored from the gel without contamination from adjacent spots, and the further requirement that a protein selected for analysis should be present in every commassie-stained gel examined.
Protein Identification by Mass Spectrometry
Proteins to be identified were cored from gels with the use of a microblade
while under magnification. The cored samples were stored in distilled water at
4°C until analysis. The selected gel spots were subjected to in-gel
proteolysis as described previously
(Shevchenko et al, 1966). All
digest samples were analyzed by nanoflow high-performance liquid
chromatography/micro-electrospray ionization on a Thermo LCQ ion trap (San
Jose, Calif) operating in the positive ion mode. Peptides were loaded onto
home-built, 8-cm–long microcapillary columns (360-µm outer diameter
[o.d.], 75-µm inside diameter fused silica) packed with YMC brand 5-µm
C18 particles (Waters, Milford, Mass). Peptides were eluted into the mass
spectrometer using an acetonitrile/100-mM acetic acid linear gradient
(1%–70% acetonitrile over 17 minutes) through an integrated electrospray
emitter tip at a flow rate of approximately 60 nL/min.
(Martin et al, 2000). The mass
spectrometer was operated in the data-dependent mode, which allowed for the
acquisition of 1 full-scan mass spectrum followed by 5 sequential MS/MS scans
(3-Da window; precursor m/z ± 1.5 Da, collision
energy set to 35%, dynamic exclusion time of 1 minute) every 10–15
seconds of the most abundant ions present in the earlier full-scan mass
spectrum. All acquired MS/MS spectra were searched against a mouse/rat protein
database using the Sequest computer algorithm
(Eng et al, 1994), and peptide
sequence assignments were accepted if 
Cn score was at least 0.08;
Xcorr for fully tryptic peptides was equal to or greater than 1.8,
2.4, or 3.5 for charge states +1, +2, or +3, respectively; and
Xcorr for partially tryptic peptides was equal to or greater than
3.0 (+2 state) or 4.0 (+3 state).
Detection of Phosphorylated Proteins
Alterations in protein phosphorylation patterns were first investigated
with Western blot analysis of proteins separated in 2 dimensions as above and
probed with anti-phosphotyrosine (Transduction Labs, Lexington, Ky) and
anti-phosphoserine/threonine antibodies (Cell Signaling Technology, Beverly,
Mass). Briefly, electrophoresed proteins were electrotransferred to
nitrocellulose membranes (Bio-Rad) using standard protocols and blocked with
5% nonfat dried milk and 0.1% Tween 20 in phosphate-buffered saline. The
membranes were then incubated overnight at 4°C with either the
phosphoserine/threonine or phosphotyrosine antibody (n = 4–7, each, for
sham-operated and torsion testes). The samples were subsequently washed and
incubated for 1 hour with appropriate secondary antibody, and immunocomplexes
were recognized with enhanced chemiluminescence (Super-Signal West Pico,
Pierce Chemical, Rockford, Ill).
Also, phosphorylation of specific signal transduction proteins was analyzed using the Bio-PlexTM bead suspension system (Bio-Rad). This is a multiplexing system allowing the assay of multiple proteins in a single sample. The system utilizes a liquid suspension array of multiple sets of beads internally dyed with different ratios of 2 spectrally distinct fluorophores. This assigns each bead a unique spectral address, and each bead is conjugated to a capture molecule (an antibody directed against a specific protein). The conjugated beads are mixed and incubated with protein samples in a 96-well microplate. Biotin-labeled reporter antibodies directed toward a different epitope on each of the captured proteins are added and samples are drawn into the Bio-Plex array reader. Precise fluid dynamics align the beads in single file through a flow cell in which 2 lasers excite the beads and allow the beads to be identified by their spectral address. The biotin-labeled reporter antibodies bind to the proteins bound to the beads and are detected by high-speed digital signal processors. The fluorescent signals from the rapidly passing beads are translated into quantifiable data by onboard software (Bio-Rad).
In the present experiments, testes were removed 24 hours after the repair
of torsion or sham operation and were immediately processed for the Bio-Plex
analysis. Briefly, testes were homogenized in 500 µL of lysis solution
(Bio-Rad), vortexed, and set on ice. Forty microliters of 500-mM
phenylmethanesulfonylfluoride was added, and the tissue homogenate was
transferred to a clean microcentrifuge tube and frozen at -70°C. The
samples were then thawed, centrifuged at 10 000 x g for 4
minutes and the supernatant collected. Protein concentration was determined
using the RC/DC method (Bio-Rad) according to manufacturer instructions. The
lysates were adjusted to 500 µg/mL for use in an assay for 6 different
phosphorylated proteins. The phospho-6-plex kit (Bio-Rad) contained antibodies
directed against phosphorylated c-Jun NH2-terminal kinase (JNK),
phosphorylated activating transcription factor 2 (ATF2), phosphorylated p38
mitogen-activated kinase (p38 MAPK), phosphorylated signal transducer and
activator of transcription 3 (STAT3), phosphorylated extracellular
signal–regulated kinases 1 and 2 (ERK1&2), and phosphorylated
inhibitor of nuclear factor 
B
(IB).
The bead/antibody complexes were vortexed and added to the wells of a 96-well filter plate. Fifty microliters of the tissue lysates as well as positive control samples provided in the analysis kit were added to the wells in duplicate and the plate was incubated for 15 hours with constant agitation. The plate was then vacuum filtered and washed 3 times. Twenty-five microliters of detection antibodies were then added to the wells and the plate was incubated for 30 minutes at room temperature. The plate was again vacuum filtered and washed. Streptavidin-PE was added to each well for 10 minutes, after which time the plate was vacuum filtered, rinsed 3 times, and 125 µL resuspension buffer was added. The 96-well plate was then placed in the BioPlex reader and the data were collected and analyzed as described previously.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
The image analysis software also produced schematic maps of the composite images to better identify overlapping, but separately detected, proteins and to allow clear identification of consistently increased (Figure 2A) and decreased (Figure 2B) proteins. Seven hundred seventeen different proteins consistently appeared within or between groups and were subject to this analysis. Among these proteins, 23 were detected as having been consistently increased 24 hours after torsion repair (Figure 2A).
|
Based on the intensity of Sypro-Ruby staining, 48 proteins decreased by more than or equal to 50% 24 hours after torsion repair (Figure 2B), with 10 of them being completely extinguished. The proteins that disappeared in the IR group had typically been very lightly stained, though consistently appearing, even in the sham group. Based on spot intensity in the commassie blue–stained gels from the sham-operated testis and the additional requirement that the cored spot appear in all 5 gels of the sham-control group, 3 of the proteins (Figure 2B; spots 3701, 8604, and 9505) were selected for identification by mass spectrometry.
The commassie blue–stained proteins consistently increased 24 hours after torsion repair were ß-tublin (IDN#2601), and two different isoforms of a serum albumin precursor (5808 and 6807; Table 1). The 3 decreased proteins identified from the commassie blue–stained gels were vimentin (3701), chaperonin containing TCP1, subunit 2ß (8604), and phosphoglycerate kinase 2 (PGK2; 9505; Table 1).
|
Protein Phosphorylation
Phosphorylation of testicular proteins was altered 24 hours after IR, as
shown by both Western blot and Bio-Plex analyses. Western blot analysis of
proteins from the sham-operated testis typically detected 5 to 6 proteins that
were phosphorylated on serine/threonine residues
(Figure 3A). A similar number
of phosphorylated proteins were detected after IR
(Figure 3B), but the
phosphorylation associated with some spots had increased, while others had
decreased. There was considerable sample-to-sample variation, but the
consistent changes induced by IR are illustrated in
Figure 3. Proteins 1 to 5
showed little or no phosphorylation signal in sham-treated testes
(Figure 3A), but the
phosphorylation signal in those spots increased in all 4 samples examined
after IR (Figure 3B). Protein 6
appeared in 3 out of 4 blots of sham-treated and IR testes and was not
affected by the treatment. Spots 8, 9, and 10 consistently showed reduced
phosphorylation signal after IR. Four proteins (7, 11, 12, and 13) are typical
of those appearing in only 1 or 2 blots after testicular IR but were never
present in sham-operated testes.
|
B are each phosphorylated on serine or threonine, but JNK and
ATF-2 were significantly increased (P < .05) after IR by an
average 53% and 50%, respectively, while IB phosphorylation
remained virtually unchanged (Table
2).
|
Tyrosine phosphorylation patterns (not shown) were much more complex than the serine/threonine phosphorylation patterns (eg, 30–40 phosphorylated proteins were detected on each protein blot from sham-operated testes, and there was also more variability between blots). As with the serine/threonine phosphorylations, IR increased detectable tyrosine phosphorylation of some proteins, while it decreased tyrosine phosphorylation in others. A difference was that a high proportion of proteins with tyrosine phosphorylation occurred in only 1 gel pair (sham vs IR within the same animal); thus, these individual phosphorylations did not consistently occur within the groups of testes. Overall, tyrosine phosphorylations typically appeared decreased rather than increased after torsion repair (not shown).
The Bio-Plex analysis showed that STAT3, p38 MAPK, and ERK1&2, which are all phosphorylated on tyrosine residues, were variably affected by torsion (Table 2). Average STAT3 and ERK1&2 phosphorylation values were increased by 23% and 20%, respectively, 24 hours after torsion repair, but only the increase in STAT3 phosphorylation was statistically significant (P < .05). p38 MAPK remained essentially unchanged (Table 2).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Previous reports from this lab have partially detailed an across-compartment pathway leading to germ cell apoptosis after IR. Early in this pathway, cytokine induction of stress-related kinases leads to E-selectin expression by the testicular vascular endothelium (Lysiak et al, 2003). E-selectin expression is associated with the neutrophil recruitment into the testicular interstitial compartment that occurs after torsion repair (Turner et al, 1997; Lysiak et al, 2001), which is contemporaneous with intratesticular ROS production (Turner et al, 1997), increases in Bax and caspase expression (Lysiak et al, 2000), and germ cell apoptosis inside the seminiferous tubule compartment (Turner et al, 1997; Lysiak et al, 2001).
We have previously used the rat testis to examine for testicular proteins, the synthesis of which is consistently and significantly increased or decreased after IR (Turner and Miller, 1997). We found a limited number of proteins in which synthesis was consistently altered after torsion repair. This was interpreted to mean that Sertoli cell function remains relatively robust after IR. On a related note, Sertoli cells persist in seminiferous tubules after periods of torsion that eliminate germ cells through apoptosis, and Sertoli cells have not themselves been seen to undergo apoptosis after torsion.
Despite the fact that the synthesis of detected proteins was little altered by degrees and durations of torsion sufficient to induce aspermatogenesis in the rat (Turner and Miller, 1997), it remained possible that activation or inhibition of existing proteins (binding proteins, enzymes, etc) is an important aspect of the testicular injury that occurs after IR (Lysiak et al, 2003, 2005), even though those changes may not be specific to the IR injury. We have continued our studies in the mouse because of the greater molecular biology resources available in that species.
An average of over 1100 proteins were detected in individual gels separating proteins of the sham-operated mouse testis (Figure 1), many of them faintly stained and not consistently appearing in replicate gels. A total of 717 spots were consistently present and used for the comparison between the sham and torsion groups to determine the increased and decreased proteins in this mouse model of testicular torsion (Figure 2). As was the case with the results of Turner and Miller (1997), relatively few proteins were consistently and significantly altered after torsion repair. Of the 23 proteins with increased spot densities 24 hours after IR, 3 were chosen for identification based on the ability to confidently identify and isolate them from commassie blue–stained gels. One of the proteins with increased spot density after IR was identified as a ß2-tubulin, a major microtubule constituent that increased by a 2.4-fold measure (Table 1). A major source of testicular tubulin is likely to be the condensing spermatids and spermatozoa in the seminiferous tubules, but the increase in the protein occurs despite the fact that germ cells are being lost through apoptosis 24 hours after torsion repair (Lysiak et al, 2001). The reason for this discrepancy is obscure, but a developmental "switch" in some ß-tubulin genes has been reported (Slaughter et al, 1987), and it is possible that the testicular dystrophy induced by IR has recapitulated such a switch "on," leading to the increased abundance of the ß2-tubulin protein, although the cell source of such a switch is unknown.
Two of the proteins that increased after IR were isoforms of serum albumin precursor, which increased by 2.4-fold and 2.3-fold measures, respectively (Table 1). Albumin is not known to be synthesized in the testis, although in the rat testis a related protein, testibumin, is (Shaha et al, 1988). Interestingly, Melsert et al (1991) reported that albumin stimulates Leydig cell steroid production in a dose-dependent manner in vitro when added to testicular fluid. While serum albumin is known to be distributed in the interstitial space in the testis (Christensen et al, 1985), it has been presumed to be there as a transudate from serum, and we are unaware of any suggestion of local synthesis. Serum albumin is known to be synthesized in places other than the liver, and in other cell types it has been postulated to be part of the cellular redox sensing apparatus (Holderman et al, 2002). Thus, the presence of serum albumin precursor in testis extracts indicates that testicular cells (type unknown) synthesize albumin, either as a part of their protection mechanism against oxidative stress or as a facilitator of Leydig cell steroidogenesis under stress conditions.
The number of proteins with decreased spot density after IR (48) was more than double the number with increased density (23). The 3 protein spots chosen for identification were vimentin, t-complex protein 1ß, and testis-specific PGK2. Vimentin is an intermediate filament protein that has been reported to have a role in Leydig cell testosterone secretion, and that role has been speculated to be through its function in the transport of cholesterol to the outer mitochondrial membrane (Bilinska et al, 1997). Vimentin was reduced by approximately 54% in the mouse testis 24 hours after IR. That reduction in intermediate filament may be associated with an acute decrease in testicular venous testosterone concentrations in the mouse, such as that previously reported for the rat testis 24 hours after torsion repair (Turner et al, 2005). Further, the afore-mentioned increase in serum albumin precursor may be a part of the general mechanism attempting to compensate for or repair this loss of Leydig cell function.
The t-complex protein 1ß is an actin-binding chaperonin that was reduced by 52% after IR of the mouse testis. This protein is one of many t-complex proteins in germ cells, and its reduction may be a reflection of the disturbance to spermatogenesis and the loss of germ cells by apoptosis that occurs by 24 hours after torsion repair. Finally, PGK2 was reduced by 55% after IR. PGK2 is an enzyme localized to spermatocytes and spermatids and is involved in the metabolism of glucose and fructose (Naz and Vanek, 1998). The reason for a testis-specific isoform and the specific role of that isoform in spermatogenesis is unknown, but the decline in the protein in testis extracts after IR may well be due to the loss of healthy germ cells, as indicated above.
While the identified proteins from the 2-dimensional (2-D) electrophoresis were other than well-known stress-related pathway molecules, they could be common, end-point proteins bearing the result of such pathway activations. Enzyme activation is commonly by phosphorylation of the inactive molecule, and these phosphorylations can be on serine, threonine, or serine residues of the native protein. Thus, in addition to the identification of proteins from among those that had increased or decreased after torsion repair, changes in protein phosphorylation were also studied using Western blots with anti–phosphoserine/threonine and anti-phosphotyrosine antibodies as probes. Very few proteins were consistently phosphorylated on serine/threonine residues (Figure 3). While over 8 times more proteins were phosphorylated on tyrosine than on serine/threonine (not shown), those results were also highly variable, making interpretation difficult. Phosphorylations detected on specific residues may vary depending on the antibody used, but with the present antibodies, in no case did the phosphorylated proteins identifiably co-migrate with spots on the Sypro-Ruby gels discussed previously. Alternatively, Bio-Plex technology was used to determine quantitative changes in the phosphorylation of 6 specific stress-related kinases, 3 of them phosphorylated on serine/threonine, 3 of them on tyrosine (Table 2).
The most prominent quantitative changes in protein phosphorylation occurred with JNK and ATF2, both of them threonine phosphorylations. Detection of JNK and ATF2 phosphorylation increased by an average of 53% and 50%, respectively. Both increases were statistically significant (P < .05). ATF2 is a member of the cAMP-response element-binding protein family and is a leucine zipper protein involved in cellular stress responses; further, ATF2 is a known target of JNK in the mediation of those cell-stress responses (Hayakawa et al, 2003). The more modest changes in phosphorylated STAT3 and ERK1&2 may indicate a stimulation of cell-repair events via pathways involving those molecules.
In summary, IR injury induced by testicular torsion causes significant changes in the presence of selected testicular proteins, but among an average of over 1100 proteins detectable on 2-D electrophoresis, relatively few were consistently altered in spot density 24 hours after torsion repair. The increases or decreases (depending) of 6 identified proteins from those gels, vimentin, ß2-tubulin, PGK2, t-complex protein 1ß, and 2 isoforms of serum albumin precursors, could be rationalized, but they gave little information regarding the predominant pathway(s) within 24 hour of IR injury. Protein phosphorylation studies demonstrated that phosphorylation patterns among the testicular proteins were highly variable, although typically more tyrosine phosphorylation was detected than serine/threonine phosphorylation. None of the phosphorylated proteins on 2-D gels matched spot locations with consistently appearing proteins on Sypro-Ruby–stained gels. On the other hand, when specific protein phosphorylations were studied by Bio-Plex analysis, 2 proteins phosphorylated on threonine residues; JNK and ATF2 were quantitatively the most predominant. JNK and ATF2 have previously been shown to undergo activation within minutes of testicular reperfusion (Lysiak et al, 2003), but the present results demonstrate a prolonged activation still detectable 24 hours later. Whether this is a persistence of the acute process detected earlier by Western blot analysis (Lysiak et al, 2003), but now seen at a longer time point through use of the more sensitive Bio-Plex analysis, or whether it is a rebound activation particularly influencing delayed events is yet to be determined. By whatever means, it is likely an extension of the pathophysiology of the testis after IR injury, and identification of proteins altered either by increases or decreases in expression or by phosphorylation status will provide insight into specific pathways affected in the testis after torsion. These proteins and pathways may offer targets for potential therapies for the rescue of the testis after torsion or other oxidative stress injuries to the testis.
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Allen JA, Diemer T, Janus P, Hales KH, Hales DB. Bacterial endotoxin lipopolysaccharide and reactive oxygen species inhibit Leydig cell steroidogenesis via perturbation of mitochondria. Endocrine. 2004; 25: 265 -275.[CrossRef][Medline]
Anderson JB, Williamson RCN. The fate of the human testis following unilateral torsion of the spermatic cord. Br J Urol. 1986; 58: 698 -704.[Medline]
Bilinska B, Genissel C, Carreau S. Paracrine effect of seminiferous tubule factors on rat Leydig cell testosterone production: role of cytoskeleton. Biol Cell. 1997; 89: 435 -442.[Medline]
Christensen AK, Komorowski TE, Wilson BJ, Ma SF, Stevens RW. The distribution of serum albumin in the rat testis studied by electron microscope immunocytochemistry on ultrathin frozen sections. Endocrinology. 1985; 116: 1983 -1996.[Abstract]
Eng J, McCormack AL, Yates JR. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectr. 1994; 5: 976 -985.[CrossRef]
Hayakawa J, Depatie C, Ohmichi M, Mercola D. The activation of
c-Jun NH2-terminal kinase (JNK) by DNA-damaging agents serves to promote drug
resistance via activating transcription factor 2 (ATF2)–dependent
enhanced DNA repair. J Biol Chem. 2003; 278: 20582
-20592.
Holderman MT, Miller KP, Dangott LJ, Ramos KS. Identification of
albumin precursor protein, phi AP3, and -smooth muscle actin as novel
components of redox sensing machinery in vascular smooth muscle cells.
Mol Pharmacol. 2002; 61: 1183
.
Lucesoli F, Fraga CG. Oxidative stress in testes of rats subjected
to chronic ion intoxication and 
-tocopherol supplementation.
Toxicology. 1999; 132: 179
-186.[CrossRef][Medline]
Lysiak JJ, Bang HJ, Nguyen QAT, Turner TT. Activation of the
nuclear factor kappa B pathway following ischemia-reperfusion of the murine
testis. J Androl. 2005; 26: 129
-135.
Lysiak JJ, Nguyen QAT, Kirby JL, Turner TT. Ischemia-reperfusion of
the murine testis stimulates the expression of proinflammatory cytokines and
activation of c-jun N-terminal kinase in a pathway to E-selectin expression.
Biol Reprod. 2003; 69: 202
-210.
Lysiak JJ, Turner SD, Nguyen QAT, Singbartl K, Ley K, Turner TT.
Essential role of neutrophils in germ cell–specific apoptosis following
ischemia/reperfusion injury of the mouse testis. Biol
Reprod. 2001; 65: 718
-725.
Lysiak JJ, Turner SD, Turner TT. Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis. Biol Reprod. 2000;63: 1464 -1472.
Martin SE, Shavanowitz J, Hunt DF, Marto JA. Subfemtamole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI Fourier Transform ion cyclotron resonance mass spectrometry. Anal Chem. 2000; 72: 4266 -4274.[Medline]
Melsert R, Bos OJ, van der Linden RF, Fischer MJ, Wilting J, Janssen LH, Hoogerbrugge JW, Rommerts FF. The stimulatory effect of albumin on luteinizing hormone-stimulated Leydig cell steroid production depends on its fatty acid content and correlates with conformational changes. Mol Cell Endocrinol. 1991;82: 23 -32.[Medline]
Naz RK, Vanek CM. Testis specific proteins and their role in contraceptive vaccine development. Frontiers Biosci. 1998; 3: 19 -48.
Ozkan DU, Boran C, Kilinc M, Garipardic M, Kurutas EB. The effect of zinc aspartate pretreatment on ischemia-reperfusion injury and early changes of blood and tissue antioxidant enzyme activities after unilateral testicular torsion-detorsion. J Pediatr Surg. 2004; 391: 91 -95.
Pasqualotto FF, Sharma RK, Potts JM, Nelson DR, Thomas AJ, Agarwal A. Seminal oxidative stress in patients with chronic prostatitis. Urology. 2000;55: 881 -885.[CrossRef][Medline]
Powell JD, Elshtein R, Forest DJ, Palladino MA. Stimulation of
hypoxia-inducible factor-1 (HIF-1) protein in the adult rat
testis following ischemia injury occurs without an increase in HIF-
messenger RNA expression. Biol Reprod. 2002; 67: 995
-1002.
Samanta L, Chainy GB. Comparison of hexachlorocyclohexane-induced oxidative stress in the testis of immature and adults rats. Comp Biochem Physiol C. 1998;118: 319 -327.
Shaha C, Cheng CY, Phillips DM, Talwar GP, Bardin CW. Localization of immunoreactive testibumin in the testis and epididymis of adult rats. Int J Androl. 1988; 11: 547 -559.[Medline]
Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1966; 68: 850 -858.[CrossRef]
Shiraishi K, Naito K, Yoshida K. Nitric oxide promotes germ cell
necrosis in the delayed phase after experimental testicular torsion of rat.
Biol Reprod. 2001; 65: 514
-521.
Slaughter GR, Needleman DS, Means AR. Developmental regulation of calmodulin, actin, and tubulin RNAs during rat testis differentiation. Biol Reprod. 1987; 37: 1259 -1270.[Abstract]
Smith-Harrison LI, Koontz WW. Torsion of the testis: changing concepts. AUA Update. 1990; 9: 250 -254.
Thomas WEG, Cooper MJ, Crane GA, Lee G, Williamson RCN. Testicular exocrine malfunction after torsion. Lancet. 1984; 2: 1357 -1360.[Medline]
Turner TT. Acute testicular torsion: no effect on the contralateral testis. J Androl. 1984; 6: 65 -72.
Turner TT, Bang HJ, Lysiak JL. The molecular pathology of experimental testicular torsion suggests an adjunct therapy to surgical repair. J Urol. 2004; 172: 2574 -2578.[CrossRef][Medline]
Turner TT, Bang HJ, Lysiak JJ. Experimental testicular torsion: reperfusion blood flow and subsequent testicular venous plasma testosterone concentrations. Urology. 2005; 65: 390 -394.[CrossRef][Medline]
Turner TT, Miller DW. On the synthesis and secretion of rat seminiferous tubule proteins in vivo after ischemia and germ cell loss. Biol Reprod. 1997; 57: 1275 -1284.[Abstract]
Turner TT, Tung KSK, Tomomasa H, Wilson LH. Acute testicular ischemia results in germ cell–specific apoptosis in the rat. Biol Reprod. 57; 1997 : 1267-1274.[Abstract]
Uz E, Sogut S, Sahin S, Var A, Ozyurt H, Gulec M, Akyol O. The protective role of caffeic acid phenethyl ester (CAPE) on testicular tissue after testicular torsion and detorsion. World J Urol. 2002; 20: 264 -260.[Medline]
Wellejus A, Bornholdt J, Vogel UB, Risom L, Wiger R, Loft S. Cell-specific oxidative DNA damage induced by estrogen in rat testicular cells in vitro. Toxicol Lett. 2004; 150: 317 -323.[Medline]
Williamson RCN. The continuing conundrum of testicular torsion. Br J Surg. 1985; 72: 509 -510.[Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
