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From the * Center for Reproductive Biology,
Washington State University, Pullman, Washington; and the
Division of Reproductive Biology, Department
of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg
School of Public Health, Baltimore, Maryland.
| Correspondence to: Dr Matthew D. Anway, Center for Reproductive Biology, Washington State University, Pullman, WA 99164-4231 (e-mail: manway{at}mail.wsu.edu). |
| Received for publication December 29, 2004; accepted for publication April 9, 2005. |
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Abstract |
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Key words: Estradiol, androgen, testis, protease inhibitor
PCI has been shown to be expressed in several human and rodent tissues, including the seminal vesicles, prostate, epididymis gland, and testis (Laurell et al, 1992; Wakita et al, 1998). Mice lacking a functional PCI protein are infertile (Uhrin et al, 2000). Disrupted Sertoli cell-tight junctions and apoptotic germ cells at all stages of the cycle of the seminiferous epithelium characterize the testes of these mice, suggesting that PCI expression has a vital function in spermatogenesis (Uhrin et al, 2000). PCI (-/-) mice have been reported to have a twofold increase in urokinase-like proteolytic activity in testis extracts when compared to control mice, an effect that was reversed with purified recombinant human PCI (Uhrin et al, 2000). On the basis of these observations, Uhrin et al (2000) ascribed the infertility observed in the PCI (-/-) mice to disorganization of germ cell development within the seminiferous epithelium resulting from increased proteolytic activity.
It is not yet known whether the expression of the PCI gene is sensitive to androgen levels. On the one hand, Wakita et al (1998) reported that PCI gene expression in the rat seminal vesicle depends on the presence of testosterone in blood serum. This conclusion was based on the observation that within 48 hours following castration, PCI mRNA levels, relatively high in untreated rats, became undectectable, and this effect was reversed upon the replacement of testosterone. Consistent with this, the administration of estradiol to rats, which decreases serum testosterone levels, resulted in a time-dependent decrease in PCI mRNA levels in the seminal vesicles (Wakita et al, 1998). However, this treatment had no effect on PCI mRNA levels in the testis (Wakita et al, 1998), suggesting that testicular PCI gene expression is not sensitive to androgen levels. Intratesticular testosterone levels were not measured, however, and, as pointed out by Wakita et al (1998), the administration of estradiol for only 7 days may not have been adequate to reduce intratesticular testosterone levels. Therefore, it is not known whether PCI within the testis is responsive to intratesticular testosterone concentration or, alternatively, whether PCI in the testis responds differently to reduce testosterone than does PCI within other components of the reproductive tract.
We recently published a method for isolating adult Sertoli cells rapidly and with high purity (Anway et al, 2003). In the current study, we used this method to conduct an ex vivo study designed to assess the effect of reduced and repleted intratesticular testosterone on the expression of PCI mRNA by freshly isolated rat Sertoli cells. Our intent was to test the hypothesis that the expressions of PCI mRNA and protein in the testis are responsive to intratesticular androgen concentration.
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Materials and Methods |
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Intratesticular Testosterone
To experimentally reduce intratesticular testosterone, rats were
administered subdermal 2.5 cm of testosterone (T)/0.1 cm of estradiol
(E)-filled polydimethylsiloxane (Silastic, Dow Corning, Midland, Md) capsules,
or empty capsules as controls, according to methods previously described
(Stratton et al, 1973; Ewing et al, 1977). To study
the effects of reduced intratesticular testosterone on PCI expression, rats
were implanted with TE capsules for 7, 14, 28, and 56 days. To study the
effects of testosterone repletion, rats that had received TE treatment for 56
days were implanted with 3-x 8-cm (24 cm total) capsules for 5-7 (T7) or
14-15 (T14) days. Some rats received 3-x 8-cm testosterone-filled
capsules for 56 days (TM). This dose was shown to maintain quantitative
spermatogenesis, although at lower than normal intratesticular testosterone
concentration (Zirkin et al,
1989). All protocols were approved by the Johns Hopkins University
School of Public Health Animal Care and Use Committee.
Sertoli Cell Isolation
Sertoli cells were isolated following methods previously described
(Anway et al, 2003) but
omitting the 10-minute trypsin digestion. Briefly, 2 decapsulated testes were
incubated in 0.5 mg/mL of collagenase in Hanks balanced salt solution (HBSS,
pH 7.4) at 34°C, with shaking for 15 minutes to eliminate the interstitial
cells. After 3 washes, the seminiferous tubules were incubated in a mixture of
enzymes (0.1% collagenase, 0.2% hyaluronidase, 0.04% DNase I, and 0.03%
trypsin inhibitor in HBSS, pH 7.4) at 34°C, with shaking for 40 minutes.
The Sertoli cells were pelleted by centrifugation, and the resulting
supernatant was collected for peritubular myoid cell cultures (see below). The
Sertoli cell pellet was washed in HBSS and repelleted by centrifugation. This
step was repeated a total of 3 times. The Sertoli cells then were resuspended
in HBSS, subjected to hypotonic shock in a dilute HBSS solution (1:3.5
HBSS/water), collected by centrifugation, resuspended in HBSS, and filtered
through 53-µM nylon mesh. The cells were washed and resuspended in Ham
media and Dulbecco modified Eagle medium mixed 1:1 (F12/DMEM).
Cell viability, assessed by trypan blue exclusion, consistently was greater than 98%. Sertoli cells were identified by their distinct morphology (Griswold et al, 1988). The total number of isolated Sertoli cells per testis was approximately 4-7 million, as estimated by hemacytometer counts. Isolated Sertoli cells were stained for vimentin protein, and purity was assessed by determining the percentage of stained cells, as previously described (Anway et al, 2003). Purity was approximately 75%-80% for all Sertoli cell preparations. The contaminants were peritubular myoid cells (approximately 10%) and germ cells (10%-15%).
Peritubular Myoid Cell Isolation
Peritubular myoid cells, contained in the supernatants after pelleting
Sertoli cells, were collected by centrifugation (1000 x g for 2
minutes). The myoid cells were plated on 100-mm plates in DMEM plus 5% fetal
calf serum and allowed to proliferate for 3 days. The purity of the
peritubular myoid cells was estimated to be greater than 95% by staining for
smooth muscle bactin, as previously described
(Anway et al, 2003).
Germ Cell Isolation
Pachytene spermatocytes, round spermatids, and elongating spermatids were
isolated from the testes of 12-week-old rats by unit gravity sedimentation
(Staput) according to methods previously described
(Aguilar-Mahecha et al, 2001).
Purity was determined by cell morphology under phase-contrast microscopy. The
purities of pachytene spermatocytes, round spermatids, and elongating
spermatids were estimated to be 90%, 90%, and 85%, respectively.
Leydig Cell Isolation
Leydig cells were isolated as previously described
(Klinefelter et al, 1987). Briefly, the testicular artery was cannulated and perfused with collagenase (1
mg/mL) in dissociation buffer (199 medium with 2.2 mg/mL HEPES, 1.0 mg/mL
bovine serum albumin, 25 mg/L trypsin inhibitor, and 0.7 mg/mL sodium
bicarbonate, pH 7.4) to clear blood from the testes. Testes were decapsulated
and digested in collagenase (0.25 mg/mL) at 34°C, with shaking for 15
minutes. The dissociated cells were subjected to centrifugal elutriation and
Percoll gradient centrifugation purification, as previously described
(Klinefelter et al, 1987). The
final purity of the Leydig cells obtained this way, determined by staining the
cells for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), consistently was
95%.
RNA Isolation and Northern Blot Analyses
Total RNA was purified from rat testis, cultured peritubular myoid cells,
freshly isolated Leydig cells, Sertoli cells, pachytene spermatocytes, round
spermatids, and elongating spermatids by the Trizol method (Invitrogen
Corporation, Carlsbad, Calif). The cDNA clone for PCI was generated for
Northern blot analyses by reverse transcriptase-polymerase chain reaction
(RT-PCR). First, total RNA was isolated from the testis of a 4-month-old rat.
Testis total RNA (2 µg) was reverse transcribed in a 20-µL reaction at
46°C for 60 minutes, using 0.2 U of Superscript II (Invitrogen) and 50 ng
of oligo-dT primer in single-strength first-strand synthesis buffer, according
to manufacturer's specifications. PCR was performed in a reaction volume of
50:1 containing 0.5:1 of cDNA, single-strength buffer, 20 µM deoxy
nucleotide triphosphates (dNTPs), 1.5 mM MgCl2, 300 nM antisense
primer, 300 nM sense primer, and 0.5 U of AmpliTaqR DNA Polymerase (Perkin
Elmer, Boston, Mass). The PCR conditions were 30 cycles at 94°C for 30
seconds, 60°C for 30 seconds, and 72°C for 1 minute and a final
extension at 72°C for 2 minutes. PCR products were cloned into p-GemT Easy
Vector (Promega, Madison, Wis) according to manufacturer's specifications and
sequenced to verify the product. PCI cDNA was generated using sense primer
5'-GGACTCCTTCTGAAACTCA-3' and antisense primer
5'-AACTGCAGTCAGCCAGGTT-3' corresponding to base pairs 1272-1942 of
the cDNA GenBank accession number NM_022957.
Total RNA (10 µg) from isolated germ and Sertoli cell preparations was
fractionated in a 1% agarose/formaldehyde gel, transferred overnight to a
nylon membrane (HybondTM-N; Amersham Pharmacia, Piscataway, NJ), and
ultraviolet (UV) cross-linked (UV Stratagene 1800, Stratagene Inc, La Jolla,
Calif). PCI, clusterin (Collard and
Griswold, 1987), protamine-2
(Anway et al, 2002), full-length transferrin (which detects both Sertoli cell transferrin) and germ
cell hemiferrin mRNA (Huggenvik et al,
1987), and ribosomal protein S2 (ChoB; used as a control for RNA
loading) (Mukherjee et al,
1996) cDNAs were radiolabeled with (
-32P) deoxy
adenine triphosphate (dATP), using the Rad Prime DNA Labeling Kit
(Invitrogen). Northern blots were hybridized overnight at 65°C with
labeled cDNA probes in ExpressHyb hybridization solution (Clontech, Palo Alto,
Calif). Following hybridization, blots were washed in 2x saline sodium
citrate (SSC) with 1.0% sodium dodecyl sulfate (SDS) for 30 minutes at
65°C; 1x SSC with 0.5% SDS for 30 minutes at 65°C; and 0.1% SSC
with 0.1% SDS for 30 minutes at 65°C. After blots were washed, they were
placed in a phosphor screen cassette for 8-12 hours. The signals were detected
using a Typhoon 9200 and quantified using ImageQuant software (Amersham
Pharmacia). Northern blots were normalized to the mRNA levels of clusterin,
with the control ratios given a numerical value of 1.0 and treatment groups
represented as a percentage of the control. Normalization was to clusterin
mRNA, because previous studies have shown that clusterin mRNA levels do not
change following androgen withdrawal
(Roberts et al, 1992;
Turner et al, 2001).
Radioimmunossays
Interstitial fluid (IF) was collected according to a previously described
method (Turner et al, 1984).
In brief, an 18-gauge needle was used to puncture 3 holes in the tunica at one
end of a testis. The testis was then placed in a tube and centrifuged at 400
x g to separate the IF from the tissue
(Turner et al, 1984). All
samples were stored at -80°C until assayed for testosterone. IF
testosterone concentrations were determined in duplicate for each sample as
previously described (Turner et al,
1984). Testosterone was assayed by radioimmunoassay with a
testosterone antibody from ICN (Costa Mesa, Calif) and 3H-T from
NEN (Boston, Mass). The sensitivity of the assay was 10 pg per tube.
Western Blot Analyses
For some studies, testes or isolated cells were homogenized in protein
suspension buffer (100 mM NaCl, 10 mM Tris, pH 7.6, 1 mM EDTA, 0.1% Triton
X-100, and 0.5% protease inhibitor cocktail [P8340; Sigma]). For others,
seminiferous tubule fluid (STF) was obtained from rat testes by a method
previously described (Turner et al,
1984). Briefly, an incision was made in the tunica albuginea of
the distal pole of the testis, and the testis was centrifuged at 54 x
g at 0°C for 15 minutes to collect IF, as above. The testis was
then decapsulated, and the seminiferous tubules were rinsed to remove residual
IF. The seminiferous tubules were extruded through the hub of a syringe and
then centrifuged at 6000 x g at 0°C for 15 minutes to
collect the supernatant above the collapsed seminiferous tubules, which was
composed of STF. The STF was placed in a new 1.5-mL tube, and protein
concentration was measured. For all samples, the protein concentrations,
determined by the BCA method (Pierce, Rockford, Ill), were similar, ranging
from 0.47 to 0.62 mg/µL.
Protein samples were sonicated for 45 seconds and then centrifuged at 13 000 x g for 5 minutes at room temperature to eliminate insoluble material. The supernatant was added to an equal volume of 2x loading buffer (100 mM Tris, pH 6.8, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) and stored at -20°C until analyzed. Prior to analyses, samples were reduced with 0.1% beta-mercaptoethanol, boiled for 2 minutes, and loaded on a 10% SDS-polyacrylamide gel as previously described (Laemmli, 1970). Protein was transferred to Protran Nitrocellulose (Schleicher & Schuell, Keene, NH) with a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, Calif), according to manufacturer's specifications.
PCI protein was detected on Western blots using anti-PCI antibody (Uhrin et al, 2000). Membranes were blocked for 1 hour with blocking solution (5% nonfat dry milk in 1x TBSS (25 mM Tris, 137 mM NaCl, and 3 mM KCl) and 0.1% Tween 20 at room temperature, followed by anti-PCI antibody (1:1000) overnight in blocking solution at room temperature. After 3 washes in 1x TBSS plus 0.1% Tween 20, membranes were incubated with secondary anti-rabbit horseradish peroxidase (HRP)-linked immunoglobulin G (IgG; 1:3000, NA 931; Amersham Pharmacia) for 1 hour in 1x TBSS at room temperature. The membranes were washed 3 times in 1x TBSS plus 0.1% Tween 20 and then 3 times in 1x TBSS (standard wash following all secondary antibody incubations). All signals were detected using a SuperSignal WestPico Chemiluminescent kit (Pierce) according to manufacturer's specifications. Membranes were stripped using Restore Western Blot Stripping Solution (Pierce) according to manufacturer's specifications. Membranes with total testis protein were blocked and probed with anti-beta actin antibody (1:1000, A5441; Sigma) for 3 hours in blocking solution, followed by anti-mouse HRP-linked IgG (1:3000, NA 931; Amersham Pharmacia) for 1 hour at room temperature. A duplicate set of membranes with STF protein was generated and probed with anti-clusterin antibody (1:1000) overnight, followed by anti-rabbit HRP-linked IgG for 1 hour. All films were digitized and intensities quantified by MacBAS software version 2.2 (Fuji Photo Film Co, Edison, NJ). Intensities were normalized to actin expression; control ratios were given a numerical value of 1.0, and treatment groups were represented as a percentage of the control.
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Statistical Analysis
Data are expressed as the mean ± SEM of 3-5 experiments. Sample
differences were analyzed by 1-way analysis of variance (ANOVA). If ANOVA
showed P < .05, group mean differences were determined by the
Scheffe multiple range test. Means were considered significantly different at
P < .05.
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Results |
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Discussion |
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Our results show a 50% reduction in PCI mRNA levels associated with a twofold increase in protease activity within the seminiferous tubules, suggesting that protease activity is inversely related to PCI expression levels in the testis. Although the function(s) of PCI in the male reproductive tract is (are) unknown, its functions in other systems suggest that it is involved in regulating protease activity within the testis (Uhrin et al, 2000). PCI is likely to interact with proteases secreted from Sertoli cells, such as urokinase and/or tissue plasminogen activators (Espana et al, 1993); germ cells, such as acrosin (Hermans et al, 1994; Zheng et al, 1996); and/or from Leydig cells, such as protein C (He et al, 1995; Odet et al, 2004). For example, urokinase and kallikrein-like protease activities were shown to be increased by 3.1- and 1.8-fold, respectively, in the testes of the mice lacking PCI protein, an effect that was inhibited by the addition of recombinant PCI (Uhrin et al, 2000). Uhrin et al (2000) demonstrated that male mice lacking a functional PCI protein were infertile and that this infertility was associated with a 2.2-fold increase in proteolytic activity when compared to control mice. PCI also has been suggested to play a role in sperm motility and fertilization; lack of PCI, or non-functional PCI protein, was shown to result in decreased sperm motility in ejaculated semen as well as inhibition of sperm-egg binding in vitro (Moore et al, 1993; Zheng et al, 1996; Elisen et al, 1998).
The present study supports the contention that PCI is regulated, at least in part, by testosterone. Thus, PCI mRNA was reduced significantly from control levels in the Sertoli cells in response to reduced intratesticular testosterone, suggesting that PCI mRNA is sensitive to intratesticular testosterone levels, responds to the loss of germ cells resulting from reduced intratesticular testosterone, or both. Previous studies have shown that changes in the germ cell population with which Sertoli cells are associated can alter Sertoli cell gene expression (Chung et al, 1998; Zabludoff et al, 2001). However, the observation that PCI mRNA levels in TE-treated rats increased in response to 7 days of exogenously administered testosterone supports the contention that PCI mRNA responds to intratesticular testosterone levels, because germ cells would not have been restored in 7 days. This observation does not rule out the possibility that PCI mRNA also responds to changes in germ cell content or number, however.
The method used to reduce intratesticular testosterone in the present study, a combination of testosterone and estradiol, requires discussion, because estrogen receptors are expressed in the testis, making it conceivable that estrogen might, itself, directly affect PCI expression. Although we cannot completely rule this out, it seems unlikely for the following reasons. Neither the human nor the rodent PCI promoters have estrogen response elements, but both contain multiple androgen receptor response elements (unpublished data). Additionally, the administration of estradiol to rats for 8 days had no effect on the testis expression of PCI, and although the seminal vesicle PCI mRNA levels did decrease, this occurred only after serum testosterone levels decreased (Wakita et al, 1998). The conclusion that PCI mRNA responds to testosterone is consistent with reports of decreased PCI gene expression in the seminal vesicles following castration and of increased expression following the administration of testosterone to castrated rats (Wakita et al, 1998; Suzuki and Hayashi, 2000). Additionally, microarray studies have shown that testosterone supplementation administered to hypogonadal mice resulted in an almost twofold increase in the expression of PCI by 24 hours (Sadate-Ngatchou et al, 2004).
In summary, we have shown that Sertoli cell expression of PCI decreases following reduced levels of intratesticular testosterone and that it can be restored by the administration of testosterone alone to rats in which intratesticular testosterone was reduced experimentally. Although the function of PCI within the testis remains uncertain, the association of reduced PCI with increased proteolytic activity suggests that PCI is involved in the protease/protease inhibitor balance within the seminiferous epithelium.
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Acknowledgments |
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Footnotes |
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