BIOLOGY OF REPRODUCTION 57, 1275-1284 (1997)
On the Synthesis and Secretion of Rat Seminiferous Tubule Proteins In Vivo after
Ischemia and Germ Cell Loss'
T.T. Turner 2 ,3,4 and D.W. Miller3
Departments of Urology 3 and Cell Biology, 4 University of Virginia School of Medicine, Charlottesville, Virginia 22908
ABSTRACT
This study was undertaken to determine whether alterations
in Sertoli cell protein synthesis and secretion were important
precursors to germ cell loss after ischemic insult to the testis.
Ischemia was induced by a 1-h, 720 ° spermatic cord torsion,
and this was shown to cause a loss of germ cells over a 15-day
period. Seminiferous tubules were perifused in vivo with
[35S]methionine. Lumen fluid (LF) was collected by in vivo micropuncture, and seminiferous tubule extract (TE) was collected
after tubule homogenization and centrifugation. Electrophoresis
of proteins in these fluids followed by autoradiography of radiolabeled proteins allowed examination of synthesized, i.e., TE,
and secreted, i.e., LF proteins. No consistent changes were detected in synthesized or secreted proteins prior to the major loss
of germ cells; thus, major changes in the capacity of Sertoli cells
for protein assembly and transport are not a preliminary feature
of post-ischemia germ cell loss. Changes in specific protein synthesis and secretion were also modest in this in vivo environment after germ cell loss. Overall protein synthesis appeared
reduced as loss of germ cells progressed, but one protein whose
amino acid sequence confirmed identity with a testis-specific
stress protein (hst70) was up-regulated after ischemia and germ
cell loss.
INTRODUCTION
Sertoli cells provide vital anatomical and physiological
support for spermatogenesis [1]. The Sertoli cell or bloodtestis barrier divides the seminiferous epithelium into the
basal and adluminal compartments [2], and Sertoli cell secretions into those compartments provide the microenvironments necessary for spermatogenesis and for sperm viability in the tubule lumen [3, 4]. Sertoli cell secretions
include water, ions, and organic molecules [3]. It is well
established that proteins from Sertoli cells are important for
germ cell development [3], and recent reports have shown
that proteins secreted into the seminiferous tubule lumen
can also have a "lumicrine" influence on the function of
the downstream epididymal epithelium [5]. Thus, protein
synthesis and secretion by Sertoli cells can not only affect
spermatogenesis, but indirectly may affect sperm maturation as well. If that is the case, significant alterations in
Sertoli cell protein synthesis and secretion could compromise either spermatogenesis or sperm maturation.
The regulation of Sertoli cell protein synthesis and secretion is incompletely understood, though cyclic secretion
of many proteins on the template of the cycle of the seminiferous epithelium is well recognized [6]. Regulating factors may be from a combination of germ cells, peritubular
myoid cells, extracellular matrix, or Leydig cells on a background of stimulation by both steroid and peptide hormones
Accepted July 2, 1997.
Received May 14, 1997.
'Supported by NIH grant DK45179.
2
Correspondence: Terry T. Turner, Department of Urology, Medical
School Box 422, University of Virginia School of Medicine, Charlottesville, VA 22908. FAX: (804) 924-8311.
1275
[3, 4, 7]. Many studies of Sertoli cell regulation have been
performed in vitro, making it uncertain how important
some of these factors are in vivo. It is also unclear to what
degree protein synthesis and secretion is altered in clearly
dysfunctional seminiferous tubules in vivo. One might
speculate that alterations in Sertoli cell protein synthesis
and secretion are a primary part of adult seminiferous tubule failure at the cellular level; nevertheless, such speculations are without documentation. Testicular ischemia and
reperfusion are clinical concerns in patients with spermatic
cord torsion [8]. Testicular ischemia/reperfusion can result
in irreversible loss of spermatogenesis despite the survival
of Leydig cells and Sertoli cells [9, 10]. Previous reports
from this laboratory have demonstrated relatively robust
Leydig cell function in aspermatogenic testes at 1, 15 and
30 days after a period of ischemia sufficient to induce aspermatogenesis [10, 11]. Thus, Leydig cell insufficiency is
unlikely to be the cause for the post-ischemia loss of germ
cells. Subsequently, it was necessary to assess whether or
not the ability of Sertoli cells to complete complex metabolic tasks was compromised after ischemia and whether
such compromise precedes loss of spermatogenesis. We
have made such an assessment by examining both protein
synthesis and protein secretion in seminiferous tubules exposed to ischemia of sufficient duration to induce aspermatogenesis.
The initial focus of the study was on assessment of the
tubule's capacity for synthesizing and secreting a conventional panel of proteins in vivo after ischemic insult, rather
than on the synthesis and secretion of specific proteins. This
was because of our primary interest in making a broad assessment of Sertoli cell function after repair of testicular
torsion as had previously been done with Leydig cell function [11]. As a result of initial findings, our attention turned
to the only individual protein that was consistently up-regulated after torsion repair, and we have identified that protein by amino sequence analysis.
MATERIALS AND METHODS
In Vivo Microperifusion
Adult male Sprague-Dawley rats (450-550 g) were obtained from university vivarium sources and maintained on
a 12L:12D light cycle. These experiments were conducted
in accordance with the Guiding Principles for the Care and
Use of Research Animals promulgated by the Society for
the Study of Reproduction. Animals were anesthetized by
i.p. injection of urethane (100 mg/kg BW) and prepared for
in vivo micropuncture and microperifusion of seminiferous
tubules as described previously [12]. Seminiferous tubules
were perifused with [3 5 S]methionine (spec. act. 1200 Ci/
mmol; New England Nuclear, Boston, MA) in Minimum
Essential Medium. The final isotope concentration was 0.2
mCi/ml with a methionine specific activity of 1.5 Ci/mmol.
The perifusion proceeded as described in detail elsewhere
[13]. Briefly, the transparent tunica albuginea was punc-
1276
TURNER AND MILLER
tured with a sharpened glass micropipette (50-pxm tip), and
the tip was left resting in the interstitial space between tubules. Tubules were perifused at a rate of 36 ix/min for 15
min, followed by a rate of 6 Il/min for 3 h. This procedure
maintains constant isotope concentrations around perifused
seminiferous tubules for the perifusion period.
At the end of the 3-h perifusion, another micropipette
was attached to a second micromanipulator, and seminiferous tubule luminal content was collected by micropuncture. A second collection pipette was then used to collect
the perifused interstitial fluid surrounding the tubule(s)
punctured. All samples were centrifuged for 20 min at 0°C,
10 000 x g, to obtain cell-free luminal fluid (LF) or interstitial fluid (IF). Perifused seminiferous tubules were excised, rinsed, blotted dry, and homogenized on ice for 1
min in a glass microhomogenizer. The tubule homogenate
g, to obtain
was centrifuged for 20 min at 0°C, 10 000
undiluted tubule extract (TE).
These techniques allow for the exposure of seminiferous
tubules to [ 3 5 S]methionine for 3 h in vivo and the subsequent collection of cell-free fluids containing all proteins
synthesized, i.e., TE, and cell-free fluids containing only
proteins secreted into the lumen, i.e., LF, during the 3 h
just prior to sample collection. IF was collected primarily
for determinations of free [3 5S]methionine concentrations
around the seminiferous tubules at the end of the experiment.
Some testes not subjected to tubule perifusion were used
for the collection of native LF and TE.
Electrophoresis and Autoradiography
Fluids were processed for determination of radiolabeled,
trichloroacetic acid (TCA)-precipitable protein concentrations and for one-dimensional SDS-PAGE and autoradiography as previously described [13] with two modifications:
all running gels were 10% polyacrylamide, and the exposure of x-ray film to the electrophoresed [3 5S]methioninelabeled proteins was for 4 days at room temperature.
Additionally, two-dimensional electrophoresis was performed by the method of O'Farrell [14] in a minigel system
(Mini-Protean II; Bio-Rad, Hercules, CA) with an effective
pH gradient of 4.9-7.0. Isoelectric focusing occurred at 750
volts over a 3.5-h time period. Focused gel-tubes were
equilibrated in SDS-containing buffer and then placed on a
10% polyacrylamide mini-slab gel (1.5 mm x 14 cm x 17
cm) with a 4% polyacrylamide stacking gel. The slab gels
were electrophoresed at 15 A per gel for 2 h. Gels were
stained with Coomassie blue, photographed, and dried on
a gel dryer (SG-100; Savant Instruments, Farmingdale, NY)
before exposure to x-ray film and development as described
previously [13] except that the exposure time was 20 days
at room temperature.
School of Medicine. The antibody has been shown to have
broad cross-species recognition sites for all three -tubulin
gene products (Bloodgood, personal communication). The
antibody was incubated with the electroblot overnight,
washed in 1% nonfat milk with Tris buffer, pH 7.5, incubated with horseradish peroxidase-conjugated secondary
antibody (goat anti-mouse IgG) for 2 h, and developed with
diaminobenzidine. Control blots were exposed to secondary
antibody only.
Torsion-Induced Testicular Ischemia and Reperfusion
Animals were divided into four groups; controls (n =
11) and those receiving -h, 720 ° testicular torsion followed
by repair and reperfusion either 1 day (n = 7), 7 days (n
= 6), or 15 days (n = 7) prior to study. Testicular torsion
was applied as previously described [10, 11]. Briefly, the
animals were anesthetized with 1% inspired halothane and
subjected to a midventral, aseptic laparotomy. Testes were
retracted through the inguinal canal, freed from the gubernacular attachment and from the epididymo-testicular
membrane, and rotated 720 ° on the testicular pedicle of
efferent ducts and testicular vasculature. The abdominal incision was closed for the 1-h time period, then reopened.
Each torsed testis was checked for maintenance of appropriate rotation. The torsion was repaired by counterrotation
and suturing of the gubernacular stumps (scrotal and testicular sides) to return and maintain the testis in the scrotum.
Testes were scored qualitatively 1-4 (1 = normal testis, 4
= dark purple testis) for ischemic appearance at the end of
the torsion period and upon relief of torsion were scored
1-4 (1 = no apparent reperfusion, 4 = rapid apparent reperfusion) for quality of reperfusion.
At the appropriate times, all animals were subjected to
the in vivo perifusion experiments and subsequent analysis
of protein synthesis and secretion as described above. An
additional four animals per group were committed for histology. Ipsilateral testes were imbedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Fifty random tubule profiles per complete testis cross
section in two, nonsequential cross sections per testis in
four ipsilateral testes of each group were qualitatively evaluated for disruption of spermatogenesis as noted by a range
of histological indicators. These indicators ranged in severity from a dissociation of germ cell/Sertoli cell contact
within the epithelium to a marked loss of germ cell types
within the epithelium to Sertoli cell-only tubule profiles.
Data Analysis
Within each experiment a protein synthesis index (PSI)
was determined by the equation:
PSI =
Western Blotting
To determine whether micropuncture of a seminiferous
tubule caused significant contamination of LF by intracellular proteins, [3-tubulin was probed for in control serum,
TE, and LF by the electroblot method of Towbin et al. [15].
Briefly, electrophoresed proteins were transferred onto nitrocellulose membranes at 40 mA constant current for 16
h. Nonspecific binding sites were blocked with 5% nonfat
milk and 0.5% Tween 20 in Tris buffer, pH 7.5. The primary antibody was an IgG1 mouse anti-guinea pig brain
[3-tubulin in ascites kindly provided by Dr. Robert Bloodgood, Department of Cell Biology, University of Virginia
TETCA
IFtotal - IFTCA
where TETCA = TCA-precipitable [3 5 S]methionine radioactivity in TE (cpm x 103/Kl).
IFtotaI = total [3 5 S]methionine in IF (cpm x 103/Rl) and
IFTCA = TCA-precipitable [3 5S]methionine in IF (cpm x
103/,ul).
This value relates how much [3 5 S]methionine was incorporated into precipitable proteins by each tubule sample
relative to the amount of free [ 35 S]methionine available in
the fluids surrounding the tubules.
A protein secretion index (PCI) was determined by the
equation:
PROTEIN SYNTHESIS AND SECRETION IN VIVO
PCI
=
LFTCA
TETCA
where LFTCA = TCA-precipitable [ 3 5 S]methionine radioactivity in LF (cpm x 103/,ul) and TETCA = TCA-precipitable [3 5S]methionine in TE (cpm x 103/,ul).
This value relates how much labeled protein was secreted into the tubule lumen relative to the total amount of
synthesized protein present in the TE. The PSI and PCI
values give indications of relative protein synthesis and relative protein secretory activity, respectively, in the seminiferous tubules that had or had not experienced testicular
ischemia and subsequent loss of germ cells.
Within groups and fluid types, the mean + SE TCAprecipitable radioactivity in cell-free fluids and the mean +
SE PSI and PCI values were determined. The TCA-precipitable radioactivity data, PSI, and PCI were analyzed for
between-group differences by the Kruskal-Wallis test for
nonparametric data (oa = 0.05). Protein electrophoretic patterns in LF and TE in one-dimensional electrophoretograms
of control animals and those examined either 1 day, 7 days,
or 15 days after repair of a l-h, 720 ° testicular torsion were
examined for number and apparent molecular mass of consistently appearing protein bands (bands appearing in a
minimum of 5 of 6 gels in each group). The total number
of protein bands evident and the number of prominent or
"major" bands evident were determined in each gel lane.
Banding patterns of proteins in Coomassie blue-stained gels
were compared to banding patterns in autoradiograms of
the same gel for estimation of the contribution of recent
synthesis and secretion to the total proteins present. Banding patterns of LF (total secreted) proteins were compared
to those of TE (total synthesized) proteins of the same gels.
Differences between treatment groups within gel lanes were
noted.
Two-dimensional electrophoretograms and autoradiograms of TE and LF were examined for alterations in protein synthesis and secretion patterns not evident on onedimensional electrophoretograms in control animals and
those examined 15 days after torsion repair. Total protein
spots and major spots were counted. Gels and electrophoretograms were examined for appearance or disappearance
of spots indicating treatment-induced alterations in protein
synthesis and secretion patterns.
Identification of Up-Regulated Protein 71/6.26
The amino acid sequence of a protein of molecular mass
71 kDa and isoelectric point (pI) 6.26 was determined using
a modification of previously described techniques [16]. The
protein spot of interest was cut from the dried gel with
minimal margins to minimize remaining polyacrylamide.
The gel spot was cut into smaller pieces and destained in
500 Rl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 50 1 10 mM dithiothreitol in 0.1 M ammonium bicarbonate, and reduced
at 55°C for 1 h. The dithiothreitol solution was removed,
and the sample was alkylated in 50 Il 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature
for 1 h in the dark. The reagent was removed and the gel
pieces were washed with 100 pl 0.1 M ammonium bicarbonate and dehydrated in 100 pIl acetonitrile for 5 min. The
acetonitrile was removed, and the gel pieces were rehydrated in 100 pl 0.1 M ammonium bicarbonate. The pieces
were again dehydrated in 100 Il acetonitrile, the acetonitrile was removed, and the pieces were completely dried by
vacuum centrifugation. The gel pieces were rehydrated in
1277
12.5 ng/lxl trypsin in 50 mM ammonium bicarbonate and
incubated on ice for 45 min. Excess trypsin was removed,
and 20 IlI 50 mM ammonium bicarbonate was added. The
sample was digested overnight at 37C, and the peptides
released were extracted from the polyacrylamide twice with
200 Rl 50% acetonitrile, 50% formic acid. These extracts
were combined and evaporated to < 20 Il1 for liquid chromatography-mass spectrometry (LC-MS) analysis.
The LC-MS system consisted of a TSQ7000 (Finnigan
MAT, San Jose, CA) with an electrospray ion source interfaced to a 10-cm x 75-txm i.d. POROS:10 RC (Perceptive
Biosystems, Cambridge, MA) reversed-phase capillary column. One-microliter volumes of the extract were injected
and the peptides were eluted from the column by an acetonitrile-0.1 M acetic acid gradient at a flow rate of 0.6 Ul/
min. The electrospray ion source was operated at 4.5 kV
with a 1.2 Ril/min coaxial sheath liquid flow of 70% methanol, 30% water, and 0.125% acetic acid. Coaxial nitrogen
flow was adjusted as needed for optimum sensitivity. Capillary LC-electrospray MS determined the molecular
weights of the peptides present in the sample digest, and
peptide sequences were determined by collisionally activated dissociation (CAD) using argon as the collision gas.
Total Protein Concentration in LF
In 11 control testes not subjected to perifusion, LF was
collected by micropuncture and analyzed for total protein
concentration using a Bradford assay ([17]; Bio-Rad, Richmond, CA) adapted for use with microsamples.
RESULTS
Western Blot Detection of /3-Tubulin
Western blots of LF and TE for -tubulin were negative
and positive, respectively (Fig. 1A). The faint band evident
in the serum lane of Figure 1A was also faintly evident in
the negative control blot (Fig. 1B), indicating a nonspecific
reaction of the secondary antibody.
Effect of Torsion-Induced Ischemia on Tubule Morphology
One-hour, 720 ° torsion induced severe ischemia (average
ischemia score = 3.6) followed by good reperfusion (average reperfusion score = 3.6) that was not significantly
different in any of the groups studied (data not shown). The
1-h torsion resulted in a progressive loss of spermatogenesis over the 15-day experimental period (Fig. 2). Tissues
from control testes (Fig. 2A) and those 1 day after torsion
(Fig. 2B) appeared similar except for a few focal tubules
(< 10% of tubule profiles) in which disruption had been
initiated. This beginning disruption consisted primarily of
a loosening of cell-cell contacts in tubules perhaps due to
Sertoli or germ cell shrinkage. The proportion of tubule
with disrupted spermatogenesis progressed to 30-40% of
tubule profiles by 7 days after torsion repair (Fig. 2C) and
to 70-80% of tubule profiles by 15 days after torsion repair
(Fig. 2D). By 15 days after surgery, virtually all tubules
evidencing disruption of spermatogenesis were those that
showed marked loss of germ cells or that contained Sertoli
cells only (Fig. 2D).
Synthesis and Secretion of 3 5sS-Labeled Proteins by
Seminiferous Tubules
Free [3 5S]methionine in IF surrounding the tubule (approximately 12.5 x 103 cpm/ul) at the end of the experi-
1278
TURNER AND MILLER
TABLE 1. TCA-precipitable [3S]methionine-labeled proteins (cpm x
1013/l; mean ± SE) in fluid from seminiferous and epididymal tubules
after perifusion for 3 h in vivo.
Group*
n
Control
1/1
1/7
1/15
11
7
6
7
IF
1.2
0.9
1.2
0.9
+ 0.2a
0.1'
+ 0.1 a
+ 0.1'
t
LF
TE
173.0 +
187.6
+
108.7
109.5 ±
19.8'
223.9'
b
21.3
b
10.2
34.2 + 3.9,
30.4 _ 6.0"
31.7 + 8.3,i
22.2 + 3.0'
* The first number for each group is the number of hours the testis was
kept in 720 torsion; the second number is the number of days after torsion repair the testes were studied.
,'hWithin columns, means sharing the same superscript are not significantly different (p < 0.05).
FIG. 1. Western blot analysis of 13-tubulin in rat seminiferous tubule LF,
cytoplasmic extracts of entire tubules (TE), and peripheral serum (SE). A)
Electroblot probed with primary and secondary antibodies. Note marked
staining in TE for -tubulin at the appropriate molecular mass. No 3-tubulin was evident in LF. B) Electroblot probed with a secondary antibody
only.
mental period did not vary significantly from group to
group (data not shown).
TCA-precipitable proteins. TCA precipitation removed
all detectable proteins from the collected fluids. LF and TE
samples were electrophoresed after TCA treatment, and no
remaining proteins were detected either with Coomassie
blue staining of gels or by autoradiography (not shown).
FIG. 2. Histology of control rat testes (A)
°
and those subjected to 1-h, 720 torsion to
revascular
by
induce ischemia followed
perfusion. All sections were stained with
hematoxylin and eosin. Testes were examined 1 day (B), 7 days (C), and 15 days
(D) after torsion. In B, an arrowhead indicates an epithelium showing initial disruption of the seminiferous epithelium evident
on original stained sections. The proportion of disrupted tubules increased from 1
day after ischemia/reperfusion to 15 days
after, and the loss of germ cells was dramatic. x75 (reproduced at 88%).
[35S]Methionine incorporation into seminiferous tubule
proteins of control testes was robust. Mean TCA-precipitable [3 5S]methionine in TE (173 x 103 cpm/lLl; Table 1)
35
was approximately 115% of the mean free [ S]methionine
3
concentrations in IF (150 x 10 cpm/,l), and TCA-precipitable [3 5S]methionine concentrations in control LF were
approximately 20% of those in TE (Table 1). There was a
significant decline in TE [3 5S]methionine incorporation 7
and 15 days after torsion repair. LF values were not significantly altered by treatment, though there was a trend
toward declining LF values at longer times after the ischemic period (Table 1).
PSI values 7 and 15 days after torsion repair tended to
be lower than control PSI values, but this trend was not
statistically significant (Table 2). PCI values demonstrated
that approximately 20% of the radiolabeled proteins synthesized within the 3-h experimental period were secreted
by the end of the period (Table 2), but secretion as a proportion of total synthesis was not significantly affected at
any time after torsion repair (Table 2).
One-dimensional electrophoresis and autoradiography.
Coomassie blue-stained one-dimensional electrophoretograms demonstrated which protein bands were present in
TE and LF at the time of micropuncture collection, while
PROTEIN SYNTHESIS AND SECRETION IN VIVO
1279
TABLE 2. PSI and PCI of seminiferous tubules in vivo after a 3-h perifusion with [35Slmethionine (mean
SE).
Group*
n
Control
1/1
1/7
1/15
11
7
6
7
PSI
1.21
1.51
0.81
1.02
± 0.16,,b
_ 0.14,
+ 0.10b
± 0.08b
PCI
0.20 ±
0.16 _
0.29 0.23 +
0.024
0.02 a
0.07a
0.03'
* Numbers explained in Table 1.
a,b As in Table 1.
banding patterns in autoradiograms illustrated which of
these proteins were synthesized (TE) or secreted (LF) within the 3-h perifusion period. In any given gel or autoradiogram, particular protein bands might vary in staining intensity from another sample, or a band might appear in one
sample but not another; but in order for a change in synthesis or secretion to be claimed in this study the alteration
had to be consistent-that is, the change had to be seen in
at least 5 of the gels or autoradiograms of the group.
Also, all lanes were loaded with equal volumes (1 ll)
of TE or LF so that a direct comparison of the proteins in
the two different fluids could be made and the amount of
secretion relative to synthesis could be observed. Thus,
4-day exposure of autoradiograms (necessary for development of control LF lanes) caused overdevelopment of many
TE samples (Fig. 3). These samples, though dark, could
still be viewed over a light box and the number and distribution of protein bands determined. Also, reexposure of TE
for only 2 days, when necessary (e.g., Fig. 3, TE/2), allowed for a clearer comparison of synthesized bands versus
secreted bands and illustrates that the majority of synthesized proteins detected in TE were secreted into the tubule
lumen.
The number of protein bands in Coomassie blue-stained
TE lanes of control testes and the number of protein bands
in their autoradiograms were 50.6 + 1.4 and 48.1
1.7,
FIG. 3. Autoradiogram of rat seminiferous TE and LF after 4-day exposure of x-ray film to the electrophoresed proteins. A repeat exposure of
the TE lane for only 2 days (TE/2) allowed better visualization of the TE
protein bands and a more direct comparison of the TE proteins to those
secreted into the tubule lumen (LF).
FIG. 4. One-dimension separation of proteins of the rat seminiferous
tubule after in vivo perifusion with [3S]lmethionine for 3 h. The four panels
illustrate representative Coomassie blue-stained gels (COOM) and their
autoradiograms (ATRD) from control testes (A) and those that had experienced ischemia during a 1-h, 720 ° testicular torsion 1 day (B), 7 days
(C), or 15 days (D) previously. Stained gels indicate all detected proteins
present in seminiferous TE and those present in LF. Autoradiograms indicate total detected proteins synthesized during the last 3 h (TE) and those
synthesized proteins secreted (LF) during the same time period. The position of molecular weight standards (x 10 -3) is shown on the left. None
of the apparent changes in specific protein band synthesis or secretion
(ATRD) were consistent within each group. There was an overall apparent
reduction in protein synthesis and secretion in the later time periods after
torsion.
respectively. These numbers for control LF lanes were 36.3
+ 2.7 and 41.0 ± 2.3, respectively. Neither the number of
protein bands nor the banding pattern in control TE and LF
(Fig. 4A) was consistently altered 1 day after repair of torsion (Fig. 4B) when disturbance of spermatogenesis had
already begun (Fig. 2). There was consistent evidence of
less overall synthesis and less total secretion in both the
7-day and 15-day groups (Fig. 4, C and D) because of fainter development in the TE and LF autoradiograms. Nevertheless, the number of protein bands detectable remained
unchanged from control values except for LF 15 days after
torsion repair. In those samples, only eight [35S]methioninelabeled protein bands were consistently detected (Fig. 4D).
No individual protein bands in either the TE or LF at any
time after torsion repair were consistently and markedly
affected in a manner not shared by all proteins detected in
those groups. No new protein bands were consistently detected with one-dimensional electrophoresis at any time after torsion repair.
Two-dimensional electrophoresis and autoradiography.
TE and LF proteins in 1-tl samples from control testes and
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TURNER AND MILLER
TABLE 3. Molecular mass (kDa) and isoelectric point (pl) estimates of
the eight most prominent synthesized proteins in control seminiferous
tubules of adult rats.
Protein no.
kDa
pl
1
2
3
4
5
6
7
8
97
73
70
64
65
58
59
43
5.9-6.2
6.3
6.4
6.4
6.4
5.9
6.0
6.2
were still being synthesized and secreted by tubules 15 days
after torsion repair despite severe disruption of spermatogenesis (Fig. 2) and remained the prominent proteins in all
gels (Fig. 5B). One minor protein in control tissues (71
kDa, pi 6.26, Fig. 5A, arrow) was consistently up-regulated
to become a prominent protein in tubules 15 days after
torsion repair (Fig. 5B, arrow).
Identification of Up-Regulated Protein 71/6.26
Eight peptides were detected from the gel extraction process. Molecular masses and amino acid sequences (Table
4) were obtained for all eight. Database searches using
CAD spectral information (SEQUEST; National Center for
Biotechnology Information [NCBI], Washington, DC) and
partial peptide sequences (MS-Edman and BLAST; NCBI)
identified all these peptides to be in the sequence of a rat
heat shock protein in the NCBI database (NCBInr.1.30.97,
accession number 123658, molecular mass 69.5 kDa). All
peptides detected were exact matches for the database protein sequence and covered approximately 20% of the entire
sequence.
FIG. 5. Autoradiograms of two-dimensional electrophoresis of proteins
synthesized by rat seminiferous tubules. TE was collected after 3 h in vivo
perifusion of seminiferous tubules with [3Slmethionine. A) Control. Eight
synthesized proteins were consistently prominent (1-8). A minor protein
(arrow) was up-regulated after torsion (see below). The relatively small
protein easily apparent in this autoradiogram (27 kDa, pl 6.5) but missing
in B did not appear consistently in either control or post-ischemia samples. B) Fifteen days after ischemia produced by 1-h, 720 ° testicular torsion. The panel of proteins detected did not appear to be different from
controls. One protein (arrow) was consistently up-regulated in these testes
after torsion. This panel illustrates the most intensely reactive autoradiogram of the 15-day post-torsion group in order to demonstrate the abundance of proteins 1-8 relative to each other after ischemia and the relative
up-regulation of the 71-kDa/pl 6.26 protein (arrow).
samples collected 15 days after repair of a -h, 720 ° torsion
were also visualized by two-dimensional electrophoresis
and subsequent autoradiography. TE autoradiograms from
control tubules exhibited approximately 170 proteins (Fig.
5A). Fifteen days after torsion repair, seminiferous tubules
synthesized approximately 120 proteins visible under the
same conditions. The decline in the number of synthesized
proteins detected by [3SS]methionine incorporation was corroborated by a decrease in the Coomassie blue staining intensity in the gels themselves (not shown).
Eight major proteins between the molecular masses of
97 kDa and 40 kDa and pIs of 5.9 and 6.4 were consistently
synthesized by control tubules (Fig. 5A, Table 3) and secreted into LF (not shown). All of these major proteins
Variability in Protein Synthesis and Secretion by
Individual Seminiferous Tubules
Micropuncture collection of LF was markedly easier in
approximately 20% of control tubules than in the remainder. Ease of collection was associated with the presence of
apparent flow activity within the tubule visible to the micropuncturist at the time of operation. It was also associated
with a lower rate of [ 35 S]methionine incorporation into TE
and LF proteins. For example, in control animals the group
PSI was 1.21
0.16 (Table 2), but the mean PSI for the
"low-flow" and the "high-flow" samples within the group
were 1.42 + 0.2 and 1.05 ± 0.1, respectively. This pattern
was evident with PCI data as well (not shown) and with
the autoradiograms of TE and LF proteins (Fig. 6). The
high-flow TE autoradiograms (Fig. 6A) were less intense
than the typical, or low-flow, autoradiogram (Fig. 6B), and
the LF of high-flow tubules evidenced little secretion of
labeled proteins (Fig. 6A).
LF samples from control, unperifused testes were collected from low-flow and high-flow tubules and assayed for
total protein concentration. The LF protein concentration
(mean ± SE) for the entire group (n = 11) was 22.2 +_5.0
mg/ml; however, the protein concentration in the high-flow
LF was only 7.8 + 1.4 mg/ml, and the protein concentration in the low-flow LF was 33.7 + 3.8 mg/ml.
DISCUSSION
Western blot analysis was used to probe LF and TE for
3-tubulin because it was important to determine whether
1281
PROTEIN SYNTHESIS AND SECRETION IN VIVO
TABLE 4.
Peptide
number
Peptide sequences from upregulated protein 71/6.26.
Measured
molecular massa
Computed
Peptide sequence by CADb
1
2
1083.2
1098.5
XXQDFFNGK
PAYFNDSQR
3
1230.8
--GTXTGXNVXR
4
5
6
7
8
1255.0
1488.6
1680.8
1790.0
2279.0
FEEXNADXFR
--PSYVAFTDTER
--VAM(o)NPTNTXFDDAK
--NEPTAAA...
--AXGX...
Peptide sequence from databaseb
LLQDFFNGK
PAYFNDSQR
DAGTITGLNVLR
FEELNADLFR
TTPSYVAFTDTER
NQVAM(o)NTPNTXFDAK
IINEPTAAAIAYGLDKK
GPAIGIDLGTTYSC*VGVFQHGK
molecular mass
1082.3 a
1098.2
1230.4
1254.4
1488.6
1680.9
1789.1
2278.6
Daltons.
X designates I or L that cannot be distinguished by low-energy CAD; M (o) designates oxidized M; C* designates carbamidomethyl c; - designates a
single unknown amino acid; ... designates an unknown number of unknown amino acids.
b
LF proteins, said to be secreted, were appreciably contaminated by cytosol possibly arising from Sertoli cell rupture
during micropuncture. -Tubulin, present in seminiferous
tubule cytosol (Fig. 1A, TE), does not appear in LF (Fig.
1A) at this level of detection. This and previous evidence
using radiolabeled inert molecules in the intravascular and
interstitial compartments indicates that LF collected by micropuncture is minimally contaminated by constituents
from other compartments [18, 19].
Previous studies have demonstrated that -h, 720 ° torsion leads to a Sertoli cell-only condition by 30 and 60
days after torsion repair [10], but earlier time points after
repair of torsion have not been examined histologically.
The present results demonstrate that the tubular injury after
reperfusion is progressive over a 2-wk period (Fig. 2). If
seminiferous tubule dysfunction after torsion repair were
due to massive reperfusion injury [20], the lesion would
have been expected to include the entire ischemic organ
soon after torsion repair. The present findings indicate that
some more complex mechanism must be important in the
cellular response to testicular torsion. Nevertheless, the experimental design provided a schedule of testes with progressive seminiferous tubular injury and allowed examination of Sertoli cell protein synthesis and secretion in control testes and in testes after ischemic insult and increasing
germ cell loss.
TCA-precipitable [35S]methionine-labeled proteins in TE
were significantly reduced 7 and 15 days after ischemic
injury (Table 1), but the PSI values, which adjust for variations in free [3 5 S]methionine, showed that a tendency toward loss of overall protein synthesis was not statistically
significant (Table 2). Surprisingly, the reduction in radiolabeled proteins in TE or PSI seemed very modest over the
post-torsion period relative to the degree of tubular injury
(Fig. 2).
It is the case that the PSI valve could potentially misreport overall protein synthesis if the free, unlabeled methionine pool in the testis changed drastically due to treatment. That such a possibility affects the present results is
unlikely because 1) the free methionine pools are relatively
similar (50-100 jIm) in all testis compartments [21], and
2) free, unlabeled methionine in the perifusion fluid (100
iM) was in molar excess of the labeled methionine (125
nM)-thus only massive changes in the unlabeled methionine pool would more than modestly change the specific
activity of the total available methionine.
TCA-precipitable [ 35S]methionine-labeled proteins in LF
did not decline significantly during the experimental period,
though there was a tendency toward reduction by 15 days
after torsion repair (Table 1). PCI values were not significantly altered during the experimental period (Table 2), in-
dicating that for the amount of new proteins synthesized,
the amount secreted did not change over the experimental
period. Thus the secretory apparatus of Sertoli cells, separate from the synthesis apparatus, was not changed by either the ischemic period or the absence of germ cells.
The data mentioned above speak to overall protein synthesis and secretion but do not allow examination of individual proteins, which could potentially change dramatically without significant changes in the total protein synthesis and secretion data. Examination of one-dimensional
autoradiograms of TE and LF from animals examined 1
day, 7 days, and 15 days after torsion repair showed no
consistent changes in individual protein bands that were not
the result of an overall reduction in band density (Fig. 4).
Between-group comparison of [35S]methionine-labeled proteins in any given lane (e.g., LF, Figure 4, A and B) will
show minor differences in staining intensity of a particular
band or bands that are not apparently due simply to an
overall decrease in band densities within a given lane, and
such variations were observed even within groups. This is
to be expected in complex, physiological systems; thus con-
FIG. 6. Autoradiograms of proteins
flow seminiferous tubule (A) and TE
tubule (B). Protein synthesis appeared
and there is little evidence of protein
in TE and LF collected from highand LF collected from a low-flow
decreased in the high-flow tubules
secretion into the tubule lumen.
1282
TURNER AND MILLER
sistent treatment-induced, protein-specific changes were not
observed in the one-dimensional autoradiograms.
A decline in overall protein synthesis over time after
torsion repair that was only hinted at by the PSI calculations (Table 2) was consistently observed in the autoradiograms by the lighter development of radiolabeled proteins
from testes 7 and 15 days after torsion repair (Fig. 4).
The absence of major, consistent changes in synthesized
or secreted proteins seemed remarkable in light of the many
previous studies demonstrating that germ cells play an important role in regulating Sertoli cell protein synthesis and
secretion [22-25]. The present one-dimensional electrophoresis studies showed not only no major specific changes in
detectable Sertoli cell synthesis and secretion due to ischemia prior to germ cell loss, but no major specific changes
after germ cell loss. Of course, cells synthesize thousands
of proteins, and autoradiograms of proteins separated in
only one dimension would be expected to detect only major
changes in major proteins. This is what we were examining
for; nevertheless, anticipating that important changes in
Sertoli cell synthesis and secretion were being missed by
one-dimensional electrophoresis, we expanded the study to
include two-dimensional electrophoresis of proteins collected from control testes and those collected 15 days after
1-h testicular torsion, i.e., collected from testes after the
loss of the vast majority of developing germ cells (Fig. 2).
Of approximately 170 proteins visible on the minigel
autoradiograms of control TE, eight prominent proteins (Table 3) not only appeared consistently, but appeared consistently relative to each other and the other proteins detected
(Fig. 5A). Apparent clusterin and testibumin isoforms
[1,4,26] are present but sufficiently faint on the autoradiograms to not reproduce well (Fig. 5A) and are not among
these eight proteins.
There are fewer total protein spots detectable in the TE
samples collected 15 days after torsion repair, but again,
the absent proteins appeared to be due to the overall reduction in protein synthesis detectable on the autoradiograms (Fig. 5B), not to down-regulation of specific proteins relative to others in the same gel. Proteins 1-8 still
appeared prominently and appeared unchanged relative to
each other in both TE (Fig. 5) and LF (not shown).
One protein was consistently up-regulated in the twodimensional electrophoretograms of TE and LF 15 days
after torsion repair (Fig. 5). This protein, 71 kDa, pI 6.3 on
our determination, was successfully sequenced, and eight
fragment peptides matched perfectly with database sequences of a member of the hsp70 (heat shock protein,
approximately 70 kDa molecular mass) family of proteins.
This hsp70 protein has previously been identified as a testis-specific heat shock protein termed hst70 to indicate its
testis specificity [27]. The rat hst70 protein is a non-heatinducible stress protein associated with spermatogenesis,
and its mRNA has been reported to vary across a broad
spectrum of the stages of the spermatogenic cycle (low expression in stages late VII-XI, higher expression in stages
XII-XIV and I-early VII) [28]. In situ hybridization results
have been consistent with hst70's being a product of spermatocytes or spermatids [28]. The nucleotide sequence of
the hst70 gene is similar to that of the mouse hsp70.2 gene
[29]. The function of the hst70 protein is unknown, though
speculations have ranged from the broad (involved in the
transition of spermatocytes to spermatids [28]) to the specific (involved in the formation of the synaptonemal complex during meiosis [27, 29]). Our findings that this stress
protein is up-regulated in TE after ischemia and severe
germ cell loss (Figs. 2 and 5) appear inconsistent with the
unique presence of hst70 in germ cells [28]. While it is
possible that the few remaining germ cells (certainly
stressed, mostly degenerating) in the testis at 15 days after
torsion repair produced the hst70, stressed Sertoli cells are
also present and their protein synthesis pathways are demonstrably active.
The present results demonstrate that massive disarray in
the Sertoli cell's protein synthetic and secretory pathways
are not preludes to the degeneration of spermatogenesis.
Rather, changes in protein synthesis and secretion in vivo
appear to follow the loss of germ cells rather than the ischemic insult. Surprisingly, the most striking effect of ischemia and germ cell loss on seminiferous tubule protein synthesis and secretion was an overall decline in protein synthesis. Changes in the synthesis or secretion of specific proteins relative to other proteins in the same gels were not
detected except for the up-regulation of the hst70 protein.
This result seemed remarkable given the many previous
studies demonstrating that germ cells play an important role
in regulating Sertoli cell protein synthesis and secretion
[22-25]. Since cells synthesize thousands of proteins, since
these contain varying amounts of methionine, and since less
than 200 proteins were detectable on our minigel autoradiograms, it might be thought that specific changes in protein synthesis and secretion were simply not detected in our
system. Alternatively, if the changes in specific protein synthesis and secretion by Sertoli cells after germ cell loss
were as profound as is generally taken from the literature,
one would hardly expect all the changes to take place with
proteins invisible to this system and all the non-changes
with proteins visible. The difference between the present
results and previous results might, in large part, be explained by the differences in cell function in vivo and in
vitro, a difference that has been previously addressed [13]
and is substantial.
Studies demonstrating germ cell effects on Sertoli cell
protein synthesis and secretion have been typically performed using Sertoli cells in vitro, though some investigations have used seminiferous tubules from testes rendered
germ cell-free either in utero or by drug treatment in the
adult. Unfortunately, Sertoli cells resulting from in utero
treatments undergo development in abnormal testes, and
comparing their protein synthesis and secretion to that of
normal Sertoli cells may speak to developmental factors in
addition to the absence of germ cells. There are some studies in which treatments are administered in vivo but the
seminiferous tubular synthesis and secretion are examined
in vitro. In these studies, the removal of tubules from their
normal vascular and neural inputs as well as from their
normal cell neighbors is a problem. The effects of isolating
cells from their native environment for hours and having
them instead adapt to their new in vitro environments are
not often considered but could contribute to cells' performing differently in vitro than they do in vivo. Also, the difference between mRNA expression and actual protein secretion can also be substantial, and the difference is sometimes ignored.
Germ cell regulation of Sertoli cell function is convenient to the idea that Sertoli cell function operates on the
template of the cycle of the seminiferous epithelium. It
should be kept in mind, however, that the alteration of protein synthesis within the cycle is often quite modest [6],
and whether these alterations appear as a significant part of
the sperm's environment in the LF is unknown. An argument can be made that important Sertoli cell proteins are
PROTEIN SYNTHESIS AND SECRETION IN VIVO
transported directly to germ cells in the adluminal compartment and that these proteins might not appear in the
tubule cytosols (TE) or in the LF, i.e., might have been
missed in this study. This seems an unlikely reason for the
present findings, since previous investigators have freely
used supernatants from tissue culture to study and claim
germ cell-dependent protein synthesis in vitro.
It has been reported that several LF proteins arise from
germ cells [7]. Many tubule cross sections in the present
study still contained a few germ cells even in the 15-day
post-ischemia testes (Fig. 2); thus, we did not totally eliminate the possibility that Sertoli cells continued to secrete
proteins relatively normally because traces of product from
a few germ cells were present in the tubule lumen. We think
it unlikely, but even if germ cell-derived luminal factors
maintained Sertoli cell protein synthesis and secretion, this
would still contradict the idea that conventional germ cell
associations regulate Sertoli cell protein synthesis. In the
tubules with severely disrupted spermatogenesis, there are
no recognizable stages of the cycle of the seminiferous epithelium (Fig. 2D), yet synthesis of proteins detected in this
system remained relatively normal (Fig. 5).
The recent report of Clouthier et al. [30] demonstrates
that rat spermatogenesis can be supported in mouse seminiferous tubules despite the fact that the cycles of the seminiferous epithelia of these two species differ in the number
of stages, in the duration of various spermatogenic processes within the cycle (e.g., meiotic prophase, spermatogenesis), and in the overall duration of spermatogenesis
[31, 32]. This could be interpreted to mean that germ cells
are totipotent in controlling Sertoli cell function, even to
the point of being able to take command of Sertoli cells of
another species. Alternatively, it could mean that the germ
cell-Sertoli relationship is less exquisitely tuned than generally accepted and that Sertoli cell-germ cell signaling
pathways are redundant enough to allow great leeway in
what is sufficient to support spermatogenesis. The results
of Clouthier et al. show that Sertoli cells are at least somewhat promiscuous; that is, they are not particularly discerning about the germ cells they are intimate with. Interestingly, the present results show that changing the rat germ
cell population in rat seminiferous tubules has remarkably
little effect on in vivo protein synthesis and secretion other
than a general reduction in synthesis. This also demonstrates that Sertoli cells are not really so sensitive to the
germ cells present with them. This does not mean that Sertoli cell-germ cell communication is unimportant or that
stages of the epithelial cycle are completely irrelevant. It
does mean that germ cell-Sertoli cell relationships are more
broadly controlled than generally appreciated.
Additionally, an observation made during these studies
provides an explanation for an old conflict in seminiferous
tubule physiology. Reports of protein concentrations in
seminiferous tubule fluid are few and have varied between
two poles: approximately 6 g/txl [33] and approximately
38 jig/pl [34]. It was noted in the present study that some
tubules were exhibiting a period of rapid intraluminal fluid
flux when the micropuncture collection was in process. It
is well established that intraluminal fluid can move rapidly
through individual seminiferous tubules [33]. Subsequent
electrophoresis and autoradiography of the collected proteins demonstrated that the protein content of the LF from
the high-flow tubules was very low (Fig. 6). Also, LF samples of low-flow and high-flow tubules were analyzed for
total protein content, and the two types of LF contained
approximately 34 jig/txl and 8 g/Ll, respectively. The
1283
samples used by Setchell et al. [33] were collected preferentially from high-flow tubules (B.T. Hinton, personal communication), whereas those used by Turner et al. [34] were
not; thus, it seems that the previously reported results
[19,28] differed only because of the characteristics of the
tubules sampled.
The source of the fluid flux in a given seminiferous tubule remains unknown, but the lower protein concentration
in the high-flow fluid suggests possible reflux from the rete
testis, where protein concentrations are approximately 1 g/
tlI [35, 36]. The idea of such a reflux was first proposed by
Setchell and colleagues, but it was later abandoned (see
[19]). If such reflux does occur, it could be an assistance to
spermiation and a way of flushing released sperm from an
individual tubule.
In conclusion, it has been previously determined that
permanent loss of spermatogenesis after acute experimental
torsion is not due to major incapacity of Leydig cells to
produce testosterone [11]. Likewise, the present study has
shown that post-torsion degeneration of spermatogenesis is
not due to a preceding disruption of the Sertoli cell's capacity to carry out the complex work of protein synthesis
and secretion. While alterations in synthesis or secretion of
specific proteins may occur and may have been undetected
in this study, such individual changes would not be the
same thing as major changes indicative of cell pathology.
This leaves open the question of the cellular mechanism by
which acute experimental torsion permanently eliminates
spermatogenesis. Present investigations in our laboratory
are focusing on the post-torsion induction of germ cellspecific apoptosis.
ACKNOWLEDGMENTS
We gratefully acknowledge the services of the Cell Science Core of
the Center for Research in Reproduction (NIH P30-HD28934) and the
W.M. Keck Biomedical Mass Spectroscopy Laboratory funded by the
W.M. Keck Foundation, both of the University of Virginia. We thank Dr.
Robert Bloodgood for the donation of the -tubulin antibody and Dr. Michael Kinter for his expertise in performing the amino acid sequence analysis.
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