Vol. 131. No. 6 I’rrntrd in I ‘S A
Leydig Cell Peroxisomes and Sterol Carrier Luteinizing Hormone-Deprived Rats* S. M. L. CHAMINDRANI MENDIS-HANDAGAMA, TERENCE J. SCALLEN
PAUL A. WATKINS,
SIGRID
Protein-2
in
J. GELBER,
AND
Department of Population Dynamics, The Johns Hopkins School of Hygiene and Public Health, Kennedy Institute (P.A. W.), and the Departments of Neurology and Pediatrics (S.J.G.), The Johns Hopkins School of Medicine, Baltimore, Maryland 21205; and the Department of Biochemistry, University of New Mexico School of Medicine (T.J.S.), Albuquerque, New Mexico 87131 ABSTRACT We investigated the effects of 8 days of LH withdrawal on rat Leydig cell peroxisomal volume, total and intraperoxisomal catalase and sterol carrier protein-2 (SCP,) contents, and LH-stimulated testosterone secretion in uitro. Three groups of adult male Sprague-Dawley rats, i.e. control, TE-implanted (testosterone-17P-estradiol-filled Silastic implants to suppress LH), and TELH-implanted (TE-implanted and LH replacement via Alzet mini osmotic pumps), were used. After 8 days, Leydig cell organelle volumes (stereology), intraperoxisomal catalase and SCP, contents (immunocytochemistry), LH-stimulated testosterone secretion by isolated Leydig cells in vitro (determined by RIA), and total catalase and SCP, contents in equal numbers of Leydig cells (immunoblot analyses) were determined. Results showed that the TELH-implanted rats were identical to controls in every parameter tested. Testis volume and Leydig cell number per testis in control and TE-implanted rats were not significantly different; however, reductions (P < 0.05) were observed in the average volume of a Leydig cell (one third of controls) and the volume of Leydig cells per testis. All Leydig
cell organelle volumes tested were significantly lower in TE-implanted rats than in the controls; however, the volumes of smooth endoplasmic reticulum (SER) and peroxisomes were the most reduced (lowered to one sixth of control values). LH-stimulated testosterone secretion per Leydig cell in vitro correlated well with these changes in the volumes of Leydig cell SER and peroxisomes. Intraperoxisomal catalase in Leydig cells was unchanged in TE-implanted rats, although immunoblotting demonstrated a loss of total catalase content (which reflected the reduction in the volume of peroxisomes). SCP, in Leydig cells of TE-implanted rats was undetectable with immunoblot analysis (explained by the reductions in Leydig cell peroxisome volume and intraperoxisomal SCP,). These results demonstrate that the organelles SER and peroxisomes and the protein SCP, in Leydig cells are more LH dependent than the other organelles (e.g. mitochondria, lysosomes) and protein catalase, respectively. Moreover, the findings of this study are consistent with the hypothesis that Leydig cell peroxisomes play a significant role in testosterone production. (Endocrinology 131: 2839-2845,1992)
T
more than what could be expected with the atrophy of individual Leydig cells) in Leydig cell peroxisomal volume and SCP2 content, which would correlate well with the degree of reduction in Leydig cell testosterone secretory capacity. Therefore, we designedthe present study to quantify the changes in volumes of Leydig cell organelles, total and intraperoxisomal catalaseand SCPZcontents in Leydig cells, and LH-stimulated testosterone secretory capacity in vitro in Leydig cells in responseto short term withdrawal of endogenousLH.
HE possibility that Leydig cell peroxisomesplay a role in cholesterol and androgen biosynthesisand/or catabolism was first speculatedupon by Reddy and Svoboda (1, 2). Our discovery of a positive and linear correlation between Leydig cell peroxisome volume and testosterone secretion by hamster, rat, and guinea pig testes perfused in vitro (3) was consistent with this speculation. We showed for the first time that sterol carrier protein-2 (SCP,) is concentrated in rat Leydig cell peroxisomes(4), as is also the situation in hepatocytes (5), and that a single injection of LH causesa rapid, but transient, increase in intraperoxisomal SCPz in Leydig cells (6). These findings have significance in understanding the possible role of peroxisomes in Leydig cells, because SCPz participates in microsomal conversion of lanosterol to cholesterol (7) in adrenal cortical cells as well as in the intracellular transport of cholesterol into mitochondria for steroid hormone biosynthesis by these cells (8, 9). Basedupon these previous observations we hypothesized that LH withdrawal would cause specific reductions (i.e. Received March 11, 1992. Address all correspondence and requests for reprints to: Dr. S. M. L. C. Mendis-Handagama, Department of Animal Science, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37901. * In memory of Larry L. Ewing. This work was supported by NIH Grant HD-07204 (to L.L.E.) and National Research Scientist Award F32DK-08312-02 (to S.J.G.).
Materials
and Methods
Animals Adult male Sprague-Dawley rats, chased from Dominion Laboratories tained under conditions of controlled (14 h of light, 10 h of darkness) and animals were fed with Agway Prolab water ad libitum and killed.
weighing 275-300 g, were pur(Dublin, VA). They were maintemperature (25 C) and lighting were housed four to a cage. The rat formula (Syracuse, NY) and
Hormones Testosterone and 17&estradiol were purchased from ton, NH). Ovine LH-25 was a gift from the National Pituitary Program, NIDDK.
Steraloids Hormone
2839
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(Wiland
LEYDIG
2840
CELL
PEROXISOMES
Treatments Rats were divided into three groups of five animals each. Control rats received no treatment. Subdermal implants of Silastic tubing (no. 302605, Dow-Corning, Midland, Ml) were filled with testosterone (2.5 cm) and 17/J-estradiol (0.3 cm) in the TE-implanted group (10). Implants were inserted under ether anesthesia. TELH-implanted rats received TE implants plus exogenous LH in 0.01 M borate buffer (pH 7.5) via Alzet miniosmotic pumps (model 2002, Clay Adams, Parsippany, NJ), which released LH continuously at a rate of 24 &day. These pumps were primed in normal saline at room temperature overnight before implantation SC. All rats were maintained for 8 days.
Tissue preparation stereology
for microscopy, histochemistry,
and
One testis from each rat was removed under ether anesthesia and weighed on a Mettler H54 balance to obtain fresh testis weight. The specific gravity of the fresh testis was determined by the flotation technique (11, 12) to determine fresh testis volume. The other testis of each rat was fixed by whole body perfusion; a cannula was passed through the ascending aorta and the left ventricle using a fixative containing 4% paraformaldehyde, 2.5% glutaraldehyde, and 0.1% picric acid buffered with 0.1 M cacodylate buffer (pH 7.4). The fixed testis was weighed, the specific gravity was measured, and the fixed testis volume was calculated. The fixed testis was cut into approximately l-mm cubes and processed for electron microscopic histochemistry and stereology (4) to localize catalase in peroxisomes. For immunocytochemical studies, one testis of each rat was fixed by cannulation of the ascending aorta through the left ventricle using a fixative containing 2.5% glutaraldehyde buffered with 0.1 M cacodylate buffer (pH 7.4) and processed as described previously (4).
AND
SCP,
Endo. 1992 Vol131.No6
(3 X 10’) of Leydig cells were incubated with 100 rig/ml LH at 34 C for 3 h (18). Testosterone concentrations in the incubation medium were measured by RIA (ICN Diagnostic kits, Irvine, CA) and expressed as secretion per Leydig cell.
Immunocytochemistry Catalase and SCP2 in Leydig cells were immunolocalized via the protein-A-gold technique. With each antibody incubation, a control incubation was performed using preimmune serum as a control. The affinity-purified anticatalase was a gift from Dr. T Hashimoto (Shinsu University, Japan) and the anti-SCP, was prepared in Dr. T. Scallen’s laboratory (9). The gold particle density (defined as the number of gold particles per unit area of the organelle) in Leydig cell peroxisomes for the above two proteins was determined (4).
Immunoblot analysis for catalase and SCP, in Leydig cells of control, TE-implanted, and TELH-implanted rats Highly purified Leydig cells were isolated (18). Aliquots of 3 x 10’ cells from control, TE-implanted, and TELH-implanted rats were resuspended in 0.25 M sucrose containing 1 rnM Tris, pH 7.5, and 1 rnM EDTA and homogenized, and the homogenates were electrophoresed on a 12.5% polyacrylamide gel (6). Proteins were transferred to a nitrocellulose membrane (from Schleicher and Schuell, Keene, NH), as previously described (6). Immunoblot analyses using antibodies to catalase and SCP, were performed as previously described (6), except that bound antibody was detected using the Enhanced Chemihiminescence Immunoblotting System protocol (ECL, Amersham, Arlington Heights, IL) instead of the Protoblot Immunoblotting System protocol used in our previous study (6).
Light microscopic stereology
Statistical
Serial sections, each 1 Grn thick, were cut from the testicular tissue blocks prepared for stereology using a LKB III ultramicrotome and glass knives. These sections were stained with 1% toluidine blue in 1% borax (13). The volume density of Leydig cells (volume of Leydig cells per unit volume of testicular tissue) was obtained via point counting (3), and the numerical density of Leydig cells (number per unit volume) was obtained via the disector method (14), as described previously (12). The average volume of a Leydig cell was obtained by dividing the volume density by the numerical density (3). The number of Leydig cells per testis was calculated by multiplying the numerical density by the fresh testis volume (3, 12). Tissue samples were examined and photographed with a Zeiss Standard LAB 16 light microscope.
The results are expressed as the mean and SEM (in parentheses). Significant differences (P < 0.05) between means were determined by Duncan’s multiple range test after analysis of variance.
Electron
microscopic
stereology
Thin sections exhibiting pale gold to silver interference colors (90-60 nm) were cut using a Sapphatome (Sakura, Japan) and picked up on Formvarand carbon-coated 200.mesh copper grids. These sections were stained with aqueous uranyl acetate (15) and lead citrate (16), and examined and photographed by a Hitachi HU-11F electron microscope (Tokyo, Japan) operating at 75 kV. The volume density of each Leydig cell organelle type was determined by point counting (3), using a transparent test overlay containing 418 test points. A total of 250 micrographs/group (50/testis) were counted to determine the volume density of smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), mitochondria, peroxisomes, negative bodies, lysosomes, and nuclei in Leydig cells from all treatment groups. Summation average graphs (17) were drawn to determine sampling adequacy. The absolute volumes of organelles per Leydig cell were determined by multiplying the volume density of each organelle type by the average volume of a Leydig cell (3).
Testosterone
secretory capacity of purified
Highly purified (95%) Leydig cells were control, TE-implanted, and TELH-implanted
Leydig cells
isolated (18) from testes of rats (n = 6). Equal aliquots
analysis
Results Effect of LH on testis volume, Leydig cell number per testis, absolute volume of Leydig cells per testis, and averagevolume of a Leydig cell
The average testis volume, Leydig cell number per testis, absolute volume of Leydig cells per testis, and average volume of a Leydig cell in control, TE-implanted, and TELHimplanted rats are shown in Fig. 1. The average testicular volume and number of Leydig cells per testis in control, TEimplanted, and TELH-implanted rats were not significantly different (Fig. 1, A and B). The absolute volume of Leydig cells per testis and the average volume of a Leydig cell were significantly reduced to one third of control values after 8 days of LH withdrawal (i.e. TE implantation); however, these reductions were prevented with exogenousLH (Fig. 1, C and D). Effect of LH on organelle
volumes in Leydig cells
Table 1 shows the absolute volumes of Leydig cell cytoplasmic organelles in control, TE-implanted, and TELH-implanted rats and the degree of volume reduction with LH deprival for 8 days. The absolute volumes of all organelles measured (SER, peroxisomes, negative bodies, RER, mitochondria, lysosomes,and nuclei) were significantly reduced
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LEYDIG
A
a
CELL
PEROXISOMES
a
AND
2841
SCP,
one secretion per Leydig cell was 7-fold lower (P < 0.05) in Leydig cells from TE-implanted rats than in control rats. Immunocytochemistry
B
a
a
CONTROL
TE
FIG. 1. Effects of TE implants and TELH Leydig cell number per testis, volume of average volume of a Levdie cell in adult control group did not receive any implants. .I
”
I
TELH
implants on testis volume, Leydig cells per testis, and Snrague-Dawlev rats. The n = 5 rats/group. Error bar _
I
= SEM.
in volume per cell and per testis in the TE-implanted group, but were unchanged in TELH-implanted rats compared to controls. The absolute volumes of Leydig cell SER, peroxisomes,and negative bodies were decreasedto one sixth of the control values after 8 days of LH deprivation. This degree of volume reduction is in excessof the degree of atrophy of Leydig cells after TE treatment for 8 days (seeFig. 1). However, the absolute volumes of RER, mitochondria, and lysosomeswere decreasedto one fourth, one third, and one third of control values, respectively, and were similar to the reduction in the average volume of a Leydig cell after 8 days of TE deprivation. The least affected Leydig cell organelle was the nucleus, which was two thirds of the control value after 8 days of TE treatment. Testosterone
Immunocytochemical localization of catalase and SCPZ Leydig cell peroxisomes revealed the changes in the intraperoxisomal contents of both of these proteins after LH deprivation. Catalase labeling was exclusively observed in Leydig cell peroxisomesin all three treatment groups. Figure 3 shows representative Leydig cell peroxisomes immunolabeled for catalase via the protein-A-gold technique. Qualitatively, no difference in gold labeling was observed between Leydig cell peroxisomesof control, TE-implanted, and TELHimplanted rats. Quantification of gold particles representing catalasein these Leydig cell peroxisomesis shown in Fig. 4. No significant differences (P > 0.05) were observed in gold particle density (number per unit area) in Leydig cell peroxisomesfor catalasein the three groups. Immunolabeling for SCP2 was observed in Leydig cell peroxisomes and mitochondria in both control and TELHimplanted rats (results not shown). However, SCP, was observed only in peroxisomesin Leydig cells of TE-implanted rats. Figure 5 shows representative Leydig cell peroxisomes in control, TE-implanted, and TELH-implanted rats immunolabeled for SCP2. A marked reduction in gold particles labeling SCPZ was observed in the TE-implanted group. However, in both control and TELH-implanted groups, no obvious difference was observed in gold labeling for SCP, in Leydig cell peroxisomes.Quantification of gold particles representing SCP, in these Leydig cell peroxisomesis shown in Fig. 6. No significant difference was observed between control ( 146/pm2)and TELH-implanted (130/pm2) groups. However, the TE-implanted (69/pm2) group showed a significant reduction (P < 0.05) in gold particle density labeling of SCP2. Immunoblot
analysis of total catalase and SCP, in Leydig cells
Immunoblot analysisrevealed the changesin total catalase and SD2 contents in equal numbers of Leydig cells from control, TE-implanted, and TELH-implanted rats after 8 days. Figure 7 shows immunoblots for catalase in Leydig cells from the three groups. The levels of total catalasewere similar in equal numbers of Leydig cells from control and TELH-implanted rats. However, the samenumber of Leydig cellsin TE-implanted rats showed very little catalasein them. Figure 8 shows immunoblots for SCP2in Leydig cells from the three groups. Equal numbers of Leydig cells in control and TELH-implanted rats showed no differences in their total SCP2 contents. However, we were unable to detect SCP, in Leydig cells from TE-implanted rats with immunoblot analysis.
secretion per Leydig cell
Figure 2 shows testosterone secretion per Leydig cell in vitro after 3 h of incubation with 100 rig/ml LH in control, TE-implanted, and TELH-implanted rats after 8 days of treatment. Testosterone secretion was similar in Leydig cells
from control and TELH-implanted rats. However, testoster-
Discussion
In the present study, treatment of adult rats with TE implants for 8 days did not significantly alter testis volume or Leydig cell number, but did significantly reduce (P c 0.05) the average volume of a Leydig cell. These results are con-
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2842 TABLE
LEYDIG
CELL
PEROXISOMES
AND
Endo. 1992 Voll31 -No 6
SCP,
1. Leydig cell organelles: volume per cell (pm3) and per testis ( lo6 pm3) Organelle SER Per cell Per testis Peroxisomes Per cell Per testis Negative bodies Per cell Per testis RER Per cell Per testis Mitochondria Per cell Per testis Lysosomes Per cell Per testis Nucleus Per cell Per testis
Control
TE
735 (60) 16,558 (226)
133 (8)” 2,604 (78)”
11.91 (0.98)
TELH
TE/control
767 (58) 16,720 (475)
13.99 (1.06)
2.02 (0.03) a
268 (3.5)
41.92 (1.3)”
15.39 (1.26) 347 (4.68)
50 (3.1)”
18.56 (0.87) 397 (6.1)
89 (3.3)”
460 (7.8)
114 114
67 (4)” 1,262 (37)”
234 (18) 5,121 (146)
l/3 113
237 (19) 5,338 (72)
2.41 (0.03)”
16.95 (1.29) 370 (10.5)
4.19 (0.02)”
13.74 (1.1) 309 (4.2)
21.49 (1.1)
18.86 (1.43) 412 (12)
4.54 (0.3)” 94 (2.9)” 148 (9)” 3,080 (52)”
218 (17) 4,904 (67)
305 (8.8)
234 (17) 5,116 (145)
116 116
113 l/3 213 213
Values are the mean, with the SE in parentheses (n = 5 rats/group). ’ Significantly different at P < 0.01, by Duncan’s multiple range test after analysis of variance.
CONTROL
TE
TELH
FIG. 2. LH-stimulated testosterone secretion per Leydig cell in vitro in control, TE-implanted, and TELH-implanted rats. Error bar = SEM. n = 3 incubations/group. P c 0.01.
sistent with the earlier studiesof Ewing et al. (19) and Keeney et al. (20), in which no significant differences were observed in Leydig cell number after LH deprival for 5 and 7 days, respectively.
In the present study we found that TE implants in vim for 8 days reduced the LH-stimulated Leydig cell testosterone secretory capacity in vitro to one seventh the control value. However, in vitro Leydig cell testosterone secretory capacity in response to LH stimulation was not different between control and TELH-implanted rats. These results are in agreement with those of previous studieson Leydig cell testosterone secretion in perfused testes of TE rats and TELH rats (19, 21). The impaired testosteronesecretory capacity in Leydig cells of TE-implanted rats after 8 days may be explained partly by the atrophy of Leydig cells with LH deprivation. The results of the present study showed that the average volume of a Leydig cell of a TE-implanted rat became one third of the control value after 8 days of TE treatment. By contrast, the reduction in the testosteronesecretory capacity of Leydig cells in vitro was lowered to one seventh of the control value after 8 days of TE implantation. This observation raised the question of whether there are specific reductions in Leydig cell organelles involved in testosterone biosynthesis after 8 days of TE treatment. The existence of a relationship between Leydig cell SER
peroxisomes FIG. 3. Representative from control, TE-implanted, and TELH-implanted rats immunolabeled for catalase. Magnification, ~20,000. Bar = 0.5 urn.
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LEYDIG >
CELL
PEROXISOMES
1 CATALASE
C TE TELH FIG. 4. Quantification of intraperoxisomal catalase in Leydig cell peroxisomes of control, TE-implanted, and TELH-implanted rats. P > 0.05. n = 5 rats/group.
and testosteronebiosynthesis has been described in qualitative (22-24) and quantitative (3, 19, 25) ultrastructural studies. As treatment of rats with TE implants causedthe atrophy of Leydig cells to one third of control values, it was not surprising to observe reductions in the volumes of all organelles in Leydig cells in TE-implanted rats. Nevertheless, the stereological data presented herein demonstrate for the first time that all Leydig cell organellesare not reduced in volume to the samedegree of magnitude in responseto withdrawal of LH, i.e. someorganellesappear to be more LH dependent than others. The absolute volumes of organelles such as mitochondria and lysosomesin Leydig cells of TE-implanted rats were reduced to one third of control values. However, our findings revealed that the absolute volumes of SER and peroxisomes in Leydig cells of TE-implanted rats were reduced to one sixth of control values. Thus, the decreasein the volume of peroxisomes and SER was twice the overall reduction in average Leydig cell volume. To our knowledge, the present study is the first to demonstrate such variations in trophic effects of LH on different types of Leydig cell organelles. Moreover, the magnitude of the reduction in the absolute volume of SER and peroxisomes approximates the reduction in the LH-stimulated testosterone secretory capacity of these Leydig cells in vitro. The greater reduction in the absolute volume of SER associatedwith reduced testosterone secretory capacity in Leydig cells of TE-implanted rats is in agreement with the fact that SER contains LH-regulated steroidogenic enzymes that are required to convert pregnenolone to testosterone(26, 27). This striking lossof Leydig cell peroxisome volume and its correlation with the reduction in testosterone secretion in LH-deprived rats is consistent with
AND
SCP,
2843
our previous finding of a specific increase in Leydig cell peroxisome volume after acute LH treatment (6) and our hypothesis that peroxisomes are involved in the steroidogenesis in Leydig cells (3), respectively. In the present study we correlated Leydig cell SER and peroxisome volume determined in viva with the LH-stimulated testosterone production in vitro using isolated Leydig cells. It should be mentioned that the yield (counted on a hemocytometer) of Leydig cells obtained from TE-implanted rats were the sameas in the controls, and the purity (determined by S/3-hydroxysteroid dehydrogenase staining) (28) was 93-95% in control and treated groups. As Leydig cells from control and treated groups were handled in exactly the samemanner, we are confident of the fact that the comparisonsmade among these treatment groups are valid. The negative bodies in Leydig cells, the single membranebound organelles negative for catalase but otherwise morphologically identical to peroxisomes (4), still remain an enigma. In an earlier study (4) we ruled out the possibility that negative bodies are artifacts. The absolute volume of negative bodies was reduced 6-fold after 8 days of LH withdrawal, revealing that the effects of LH on negative bodies are similar to the effects on SER and peroxisomes. Previously, we have shown that the absolute volume of negative bodies was increased 3-fold 0.5 h after a single LH injection (6). This strengthens our hypothesis that negative bodies may be peroxisomes that are devoid of catalase. Experiments are underway to test this hypothesis. Previously, we have demonstrated that Leydig cell peroxisomescontain SCPp(4) and that acute LH treatment causes specific, but transient, increases in intraperoxisomal SCP, content in Leydig cells (6). By contrast, intraperoxisomal catalase content in Leydig cells was unchanged with this treatment (6). The results of the present study revealed that LH withdrawal causesa distinct decline in the intraperoxisomal SCPp content without any change in the content of intraperoxisomal catalase.However, as the total peroxisomal volume in Leydig cells of the TE-implanted rat was reduced, the total amount of catalasein Leydig cells of this treatment group showed a decreasein the immunoblot analysis. Despite the fact that the technique of enhanced chemiluminescence that we used is highly sensitive, we were unable to demonstrate the presence of SCP, in Leydig cells of TEimplanted rats. This is not surprising for two reasons.First, we observed a reduction in the absolute volume of Leydig cell peroxisomes(the organelle that has the highest concen-
FIG. 5. Representative peroxisomes from control, TE-implanted, and TELH-implanted rats immunolabeled for SCPp. Magnification, ~20,000. Bar = 0.5 pm.
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2844
LEYDIG
CELL PEROXISOMES
AND
SCP,
Endo. Voll31.
1992 No 6
-66 -45 -6 -9 9 -24 -2Od 0
C
TE
TELH
-l4m2
FIG. 6. Quantification of intraperoxisomal SCP, in Leydig cell peroxisomes of control, TE-implanted, and TELH-implanted rats. P < 0.05. n = 5 rats/group.
123 FIG. 8. Immunoblots for SCPz (14 kilodaltons) in Leydig cells (3 x 105/group) of control, TE-implanted, and TELH-implanted rats (lanes 1,2, and 3 respectively). We previously demonstrated that the antibody to SCPZ detected a rat liver protein whose electrophoretic mobility was identical to that of the Leydig cell protein (6). When protein from E. co2i, which does not contain SCP,, was immunoblotted with the SCPz antibody, no 14-kilodalton protein was detected (data not shown).
-45 -36 -29 -24
References
-20-I -14-2 FIG. 7. Immunoblots for catalase (60 kilodaltons) in Leydig cells (3 x NY/group) of control, TE-implanted, and TELH-implanted rats (lanes 1,2, and 3, respectively). We previously demonstrated that the antibody to catalase detected a rat liver protein whose electrophoretic mobility was identical to that of the Leydig cell protein (6).
tration of SCP, in control Leydig cells) in TE-implanted rats. Second, the intraperoxisomal SCP, content (determined by immunolabeling) was also decreased in this treatment group (by contrast, the intraperoxisomal catalase was not decreased). Therefore, the reduction in the absolute volume of Leydig cell peroxisomes together with the reduction in the intraperoxisomal SCP2 content should cause a greater reduction in the total SCP, content in Leydig cells of TE-implanted rats compared to the total Leydig cell catalase content. This explains why we were unable to detect SCPp in Leydig cells of TE-implanted rats with immunoblot analysis. Acknowledgments We thank Dr. Takashi Hashimoto, Shinsu University (Japan), providing us the affinity-purified anticatalase used in this study, the late Dr. Larry Ewing for his guidance and encouragement.
for and
1. Reddy JK, Svoboda D 1972 Microbodies (peroxisomes): identification in interstitial cells of the testis. J Histochem Cytochem 20:140142 2. Reddy JK, Svoboda D 1972 Microbodies (peroxisomes) in the interstitial cells of rodent testes. Lab Invest 26:657-665 3. Mendis-Handagama SMLC, Zirkin BR, Ewing LL 1988 Comparison of components of the testis interstitium with testosterone secretion in hamster, rat and guinea pig testes perfused in vitro. Am J Anat 181:12-22 4. Mendis-Handagama SMLC, Zirkin BR, Scallen TJ, Ewing LL 1990 Studies on peroxisomes of the adult rat Leydig cell. J Androll1:270278 5. Keller GA, Scallen TJ, Clarke D, Maher PA, Krisans SK, Singer SJ 1989 Subcellular localization of sterol carrier protein-2 in rat hepatocytes. J Cell Biol 108:1353-1361 6. Mendis-Handagama SML C, Watkins PA, Gelber SJ, Scallen TJ, Zirkin BR, Ewing LL 1990 Luteinizing hormone causes rapid and transient changes in rat Leydig cell peroxisome volume and intraperoxisomal sterol carrier protein-2 content. Endocrinology 127~2947-2954 7. Noland BJ, Arebalo RE, Hansbury E, Scallen TJ 1980 Purification and properties of sterol carrier protein-2. J Biol Chem 255:42824289 8. Chanderbhan R, Tanaka T, Strauss JF, Irwin D, Noland BJ, Scallen TJ 1983 Evidence for sterol carrier protein-2-like activity in hepatic, adrenal, and ovarian cytosol. Biochem Biophys Res Commun 117:702-709 9. Vahounv GV. Dennis P. Chanderbhan R. Fiskum G. Noland BI. Scallen ?J 1984 Sterol cdrrier protein-2 (S&&mediated transfer c? cholesterol to mitochondrial inner membranes. Biochem Biophys Res Commun 122:509-515 1 0. Ewing LL, Desjardins C, Irby DC, Robaire B 1977 Synergistic interaction of testosterone and estradiol inhibits spermatogenesis in rats. Nature 269:409-411 1. Mori H, Christensen AK 1980 Morphometric analysis of Leydig cells in the normal rat testis. J Cell Biol 84:340-354 2. Mendis-Handagama SMLC, Ewing LL 1990 Sources of error in
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LEYDIG the estimation of Leydig mamalian testes. J Microsc
cell numbers 159:73-82
in control
CELL and
PEROXISOMES
atrophied
KC, Larett L, Finke EH 1960 Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 35:313-323 14. Sterio DC 1984 The unbiased estimation of number and sizes of arbitrary particles using the disector. J Microsc 134:127-16 15. Watson ML 1958 Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475-478 16. Reynolds ES 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208-212 17. Aherne WA, Dunnill MS 1982 Preparation of tissues: sampling. In: Morphometry. Edward Arnold, London, p 19 18. Klinefelter GR, Hall PF, Ewing LL 1987 Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by multistep procedure. Biol Reprod 36:760-783 13.
19.
Richardson
Ewing LL, Wing T-Y, Cochran RC, Kromann N, Zirkin BR 1983 Effects of luteinizing hormone terone secretion. Endocrinology
20.
21.
Keeney DS, Mendis-Handagama
on Leydig cell structure 112: 1763-l 769
SML C, Zirkin
and testos-
22.
23.
24.
25.
26. 27.
BR, Ewing LL
1988 Effect of long term deprivation of luteinizing hormone on Leydig cell volume, Leydig cell number, and steroidogenic capacity of the rat testis. Endocrinology 123:2906-2915
28.
AND
SCP,
Wing T-Y, Ewing LL, Zirkin BR 1984 Effects
of luteinizing hormone withdrawal on Leydig cell smooth endoplasmic reticulum and steroidogenic reactions which convert pregnenolone to testosterone. Endocrinology 115:2290 de Kretser DM 1967 Changes in the fine structure of the human testicular interstitial cells after treatment with human gonadotrophins. Z Zellforsch 83:344-358 Aoki A, Massa EM 1972 Early responses of testicular interstitial cells to stimulation by interstitial-cell-stimulating hormone. Am J Anat 134:239-262 Neaves WB 1973 Changes in testicular Leydig cells and in plasma testosterone levels among seasonally breeding rock hyrax. Biol Reprod 8:451-466 Wing T-Y, Ewing LL, Zegeye B, Zirkin BR 1985 Restoration effects of exogenous luteinizing hormone on the testicular steroidogenesis and Leydig cell ultrastructure. Endocrinology 117:1779 Tamaoki TB 1973 Steroidogenesis and cell structure. Biochemical pursuit of sites of steroid biosynthesis. J Steroid Biochem 4:89-l 18 Samuels LT, Bussmann L, Matsumoto K, Huseby RA 1975 Organization of androgen biosynthesis in the testis. J Steroid Biochem 6:291-296 Levy H, Deane HW, Rubin BL 1959 Visualization of steroid 3&01dehydrogenase activity in tissues of intact and hypophysectomized rats. Endocrinology 42:73-90
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Leydig Cell Peroxisomes and Sterol Carrier Luteinizing Hormone-Deprived Rats* S. M. L. CHAMINDRANI MENDIS-HANDAGAMA, TERENCE J. SCALLEN
PAUL A. WATKINS,
SIGRID
Protein-2
in
J. GELBER,
AND
Department of Population Dynamics, The Johns Hopkins School of Hygiene and Public Health, Kennedy Institute (P.A. W.), and the Departments of Neurology and Pediatrics (S.J.G.), The Johns Hopkins School of Medicine, Baltimore, Maryland 21205; and the Department of Biochemistry, University of New Mexico School of Medicine (T.J.S.), Albuquerque, New Mexico 87131 ABSTRACT We investigated the effects of 8 days of LH withdrawal on rat Leydig cell peroxisomal volume, total and intraperoxisomal catalase and sterol carrier protein-2 (SCP,) contents, and LH-stimulated testosterone secretion in uitro. Three groups of adult male Sprague-Dawley rats, i.e. control, TE-implanted (testosterone-17P-estradiol-filled Silastic implants to suppress LH), and TELH-implanted (TE-implanted and LH replacement via Alzet mini osmotic pumps), were used. After 8 days, Leydig cell organelle volumes (stereology), intraperoxisomal catalase and SCP, contents (immunocytochemistry), LH-stimulated testosterone secretion by isolated Leydig cells in vitro (determined by RIA), and total catalase and SCP, contents in equal numbers of Leydig cells (immunoblot analyses) were determined. Results showed that the TELH-implanted rats were identical to controls in every parameter tested. Testis volume and Leydig cell number per testis in control and TE-implanted rats were not significantly different; however, reductions (P < 0.05) were observed in the average volume of a Leydig cell (one third of controls) and the volume of Leydig cells per testis. All Leydig
cell organelle volumes tested were significantly lower in TE-implanted rats than in the controls; however, the volumes of smooth endoplasmic reticulum (SER) and peroxisomes were the most reduced (lowered to one sixth of control values). LH-stimulated testosterone secretion per Leydig cell in vitro correlated well with these changes in the volumes of Leydig cell SER and peroxisomes. Intraperoxisomal catalase in Leydig cells was unchanged in TE-implanted rats, although immunoblotting demonstrated a loss of total catalase content (which reflected the reduction in the volume of peroxisomes). SCP, in Leydig cells of TE-implanted rats was undetectable with immunoblot analysis (explained by the reductions in Leydig cell peroxisome volume and intraperoxisomal SCP,). These results demonstrate that the organelles SER and peroxisomes and the protein SCP, in Leydig cells are more LH dependent than the other organelles (e.g. mitochondria, lysosomes) and protein catalase, respectively. Moreover, the findings of this study are consistent with the hypothesis that Leydig cell peroxisomes play a significant role in testosterone production. (Endocrinology 131: 2839-2845,1992)
T
more than what could be expected with the atrophy of individual Leydig cells) in Leydig cell peroxisomal volume and SCP2 content, which would correlate well with the degree of reduction in Leydig cell testosterone secretory capacity. Therefore, we designedthe present study to quantify the changes in volumes of Leydig cell organelles, total and intraperoxisomal catalaseand SCPZcontents in Leydig cells, and LH-stimulated testosterone secretory capacity in vitro in Leydig cells in responseto short term withdrawal of endogenousLH.
HE possibility that Leydig cell peroxisomesplay a role in cholesterol and androgen biosynthesisand/or catabolism was first speculatedupon by Reddy and Svoboda (1, 2). Our discovery of a positive and linear correlation between Leydig cell peroxisome volume and testosterone secretion by hamster, rat, and guinea pig testes perfused in vitro (3) was consistent with this speculation. We showed for the first time that sterol carrier protein-2 (SCP,) is concentrated in rat Leydig cell peroxisomes(4), as is also the situation in hepatocytes (5), and that a single injection of LH causesa rapid, but transient, increase in intraperoxisomal SCPz in Leydig cells (6). These findings have significance in understanding the possible role of peroxisomes in Leydig cells, because SCPz participates in microsomal conversion of lanosterol to cholesterol (7) in adrenal cortical cells as well as in the intracellular transport of cholesterol into mitochondria for steroid hormone biosynthesis by these cells (8, 9). Basedupon these previous observations we hypothesized that LH withdrawal would cause specific reductions (i.e. Received March 11, 1992. Address all correspondence and requests for reprints to: Dr. S. M. L. C. Mendis-Handagama, Department of Animal Science, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37901. * In memory of Larry L. Ewing. This work was supported by NIH Grant HD-07204 (to L.L.E.) and National Research Scientist Award F32DK-08312-02 (to S.J.G.).
Materials
and Methods
Animals Adult male Sprague-Dawley rats, chased from Dominion Laboratories tained under conditions of controlled (14 h of light, 10 h of darkness) and animals were fed with Agway Prolab water ad libitum and killed.
weighing 275-300 g, were pur(Dublin, VA). They were maintemperature (25 C) and lighting were housed four to a cage. The rat formula (Syracuse, NY) and
Hormones Testosterone and 17&estradiol were purchased from ton, NH). Ovine LH-25 was a gift from the National Pituitary Program, NIDDK.
Steraloids Hormone
2839
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(Wiland
LEYDIG
2840
CELL
PEROXISOMES
Treatments Rats were divided into three groups of five animals each. Control rats received no treatment. Subdermal implants of Silastic tubing (no. 302605, Dow-Corning, Midland, Ml) were filled with testosterone (2.5 cm) and 17/J-estradiol (0.3 cm) in the TE-implanted group (10). Implants were inserted under ether anesthesia. TELH-implanted rats received TE implants plus exogenous LH in 0.01 M borate buffer (pH 7.5) via Alzet miniosmotic pumps (model 2002, Clay Adams, Parsippany, NJ), which released LH continuously at a rate of 24 &day. These pumps were primed in normal saline at room temperature overnight before implantation SC. All rats were maintained for 8 days.
Tissue preparation stereology
for microscopy, histochemistry,
and
One testis from each rat was removed under ether anesthesia and weighed on a Mettler H54 balance to obtain fresh testis weight. The specific gravity of the fresh testis was determined by the flotation technique (11, 12) to determine fresh testis volume. The other testis of each rat was fixed by whole body perfusion; a cannula was passed through the ascending aorta and the left ventricle using a fixative containing 4% paraformaldehyde, 2.5% glutaraldehyde, and 0.1% picric acid buffered with 0.1 M cacodylate buffer (pH 7.4). The fixed testis was weighed, the specific gravity was measured, and the fixed testis volume was calculated. The fixed testis was cut into approximately l-mm cubes and processed for electron microscopic histochemistry and stereology (4) to localize catalase in peroxisomes. For immunocytochemical studies, one testis of each rat was fixed by cannulation of the ascending aorta through the left ventricle using a fixative containing 2.5% glutaraldehyde buffered with 0.1 M cacodylate buffer (pH 7.4) and processed as described previously (4).
AND
SCP,
Endo. 1992 Vol131.No6
(3 X 10’) of Leydig cells were incubated with 100 rig/ml LH at 34 C for 3 h (18). Testosterone concentrations in the incubation medium were measured by RIA (ICN Diagnostic kits, Irvine, CA) and expressed as secretion per Leydig cell.
Immunocytochemistry Catalase and SCP2 in Leydig cells were immunolocalized via the protein-A-gold technique. With each antibody incubation, a control incubation was performed using preimmune serum as a control. The affinity-purified anticatalase was a gift from Dr. T Hashimoto (Shinsu University, Japan) and the anti-SCP, was prepared in Dr. T. Scallen’s laboratory (9). The gold particle density (defined as the number of gold particles per unit area of the organelle) in Leydig cell peroxisomes for the above two proteins was determined (4).
Immunoblot analysis for catalase and SCP, in Leydig cells of control, TE-implanted, and TELH-implanted rats Highly purified Leydig cells were isolated (18). Aliquots of 3 x 10’ cells from control, TE-implanted, and TELH-implanted rats were resuspended in 0.25 M sucrose containing 1 rnM Tris, pH 7.5, and 1 rnM EDTA and homogenized, and the homogenates were electrophoresed on a 12.5% polyacrylamide gel (6). Proteins were transferred to a nitrocellulose membrane (from Schleicher and Schuell, Keene, NH), as previously described (6). Immunoblot analyses using antibodies to catalase and SCP, were performed as previously described (6), except that bound antibody was detected using the Enhanced Chemihiminescence Immunoblotting System protocol (ECL, Amersham, Arlington Heights, IL) instead of the Protoblot Immunoblotting System protocol used in our previous study (6).
Light microscopic stereology
Statistical
Serial sections, each 1 Grn thick, were cut from the testicular tissue blocks prepared for stereology using a LKB III ultramicrotome and glass knives. These sections were stained with 1% toluidine blue in 1% borax (13). The volume density of Leydig cells (volume of Leydig cells per unit volume of testicular tissue) was obtained via point counting (3), and the numerical density of Leydig cells (number per unit volume) was obtained via the disector method (14), as described previously (12). The average volume of a Leydig cell was obtained by dividing the volume density by the numerical density (3). The number of Leydig cells per testis was calculated by multiplying the numerical density by the fresh testis volume (3, 12). Tissue samples were examined and photographed with a Zeiss Standard LAB 16 light microscope.
The results are expressed as the mean and SEM (in parentheses). Significant differences (P < 0.05) between means were determined by Duncan’s multiple range test after analysis of variance.
Electron
microscopic
stereology
Thin sections exhibiting pale gold to silver interference colors (90-60 nm) were cut using a Sapphatome (Sakura, Japan) and picked up on Formvarand carbon-coated 200.mesh copper grids. These sections were stained with aqueous uranyl acetate (15) and lead citrate (16), and examined and photographed by a Hitachi HU-11F electron microscope (Tokyo, Japan) operating at 75 kV. The volume density of each Leydig cell organelle type was determined by point counting (3), using a transparent test overlay containing 418 test points. A total of 250 micrographs/group (50/testis) were counted to determine the volume density of smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), mitochondria, peroxisomes, negative bodies, lysosomes, and nuclei in Leydig cells from all treatment groups. Summation average graphs (17) were drawn to determine sampling adequacy. The absolute volumes of organelles per Leydig cell were determined by multiplying the volume density of each organelle type by the average volume of a Leydig cell (3).
Testosterone
secretory capacity of purified
Highly purified (95%) Leydig cells were control, TE-implanted, and TELH-implanted
Leydig cells
isolated (18) from testes of rats (n = 6). Equal aliquots
analysis
Results Effect of LH on testis volume, Leydig cell number per testis, absolute volume of Leydig cells per testis, and averagevolume of a Leydig cell
The average testis volume, Leydig cell number per testis, absolute volume of Leydig cells per testis, and average volume of a Leydig cell in control, TE-implanted, and TELHimplanted rats are shown in Fig. 1. The average testicular volume and number of Leydig cells per testis in control, TEimplanted, and TELH-implanted rats were not significantly different (Fig. 1, A and B). The absolute volume of Leydig cells per testis and the average volume of a Leydig cell were significantly reduced to one third of control values after 8 days of LH withdrawal (i.e. TE implantation); however, these reductions were prevented with exogenousLH (Fig. 1, C and D). Effect of LH on organelle
volumes in Leydig cells
Table 1 shows the absolute volumes of Leydig cell cytoplasmic organelles in control, TE-implanted, and TELH-implanted rats and the degree of volume reduction with LH deprival for 8 days. The absolute volumes of all organelles measured (SER, peroxisomes, negative bodies, RER, mitochondria, lysosomes,and nuclei) were significantly reduced
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LEYDIG
A
a
CELL
PEROXISOMES
a
AND
2841
SCP,
one secretion per Leydig cell was 7-fold lower (P < 0.05) in Leydig cells from TE-implanted rats than in control rats. Immunocytochemistry
B
a
a
CONTROL
TE
FIG. 1. Effects of TE implants and TELH Leydig cell number per testis, volume of average volume of a Levdie cell in adult control group did not receive any implants. .I
”
I
TELH
implants on testis volume, Leydig cells per testis, and Snrague-Dawlev rats. The n = 5 rats/group. Error bar _
I
= SEM.
in volume per cell and per testis in the TE-implanted group, but were unchanged in TELH-implanted rats compared to controls. The absolute volumes of Leydig cell SER, peroxisomes,and negative bodies were decreasedto one sixth of the control values after 8 days of LH deprivation. This degree of volume reduction is in excessof the degree of atrophy of Leydig cells after TE treatment for 8 days (seeFig. 1). However, the absolute volumes of RER, mitochondria, and lysosomeswere decreasedto one fourth, one third, and one third of control values, respectively, and were similar to the reduction in the average volume of a Leydig cell after 8 days of TE deprivation. The least affected Leydig cell organelle was the nucleus, which was two thirds of the control value after 8 days of TE treatment. Testosterone
Immunocytochemical localization of catalase and SCPZ Leydig cell peroxisomes revealed the changes in the intraperoxisomal contents of both of these proteins after LH deprivation. Catalase labeling was exclusively observed in Leydig cell peroxisomesin all three treatment groups. Figure 3 shows representative Leydig cell peroxisomes immunolabeled for catalase via the protein-A-gold technique. Qualitatively, no difference in gold labeling was observed between Leydig cell peroxisomesof control, TE-implanted, and TELHimplanted rats. Quantification of gold particles representing catalasein these Leydig cell peroxisomesis shown in Fig. 4. No significant differences (P > 0.05) were observed in gold particle density (number per unit area) in Leydig cell peroxisomesfor catalasein the three groups. Immunolabeling for SCP2 was observed in Leydig cell peroxisomes and mitochondria in both control and TELHimplanted rats (results not shown). However, SCP, was observed only in peroxisomesin Leydig cells of TE-implanted rats. Figure 5 shows representative Leydig cell peroxisomes in control, TE-implanted, and TELH-implanted rats immunolabeled for SCP2. A marked reduction in gold particles labeling SCPZ was observed in the TE-implanted group. However, in both control and TELH-implanted groups, no obvious difference was observed in gold labeling for SCP, in Leydig cell peroxisomes.Quantification of gold particles representing SCP, in these Leydig cell peroxisomesis shown in Fig. 6. No significant difference was observed between control ( 146/pm2)and TELH-implanted (130/pm2) groups. However, the TE-implanted (69/pm2) group showed a significant reduction (P < 0.05) in gold particle density labeling of SCP2. Immunoblot
analysis of total catalase and SCP, in Leydig cells
Immunoblot analysisrevealed the changesin total catalase and SD2 contents in equal numbers of Leydig cells from control, TE-implanted, and TELH-implanted rats after 8 days. Figure 7 shows immunoblots for catalase in Leydig cells from the three groups. The levels of total catalasewere similar in equal numbers of Leydig cells from control and TELH-implanted rats. However, the samenumber of Leydig cellsin TE-implanted rats showed very little catalasein them. Figure 8 shows immunoblots for SCP2in Leydig cells from the three groups. Equal numbers of Leydig cells in control and TELH-implanted rats showed no differences in their total SCP2 contents. However, we were unable to detect SCP, in Leydig cells from TE-implanted rats with immunoblot analysis.
secretion per Leydig cell
Figure 2 shows testosterone secretion per Leydig cell in vitro after 3 h of incubation with 100 rig/ml LH in control, TE-implanted, and TELH-implanted rats after 8 days of treatment. Testosterone secretion was similar in Leydig cells
from control and TELH-implanted rats. However, testoster-
Discussion
In the present study, treatment of adult rats with TE implants for 8 days did not significantly alter testis volume or Leydig cell number, but did significantly reduce (P c 0.05) the average volume of a Leydig cell. These results are con-
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2842 TABLE
LEYDIG
CELL
PEROXISOMES
AND
Endo. 1992 Voll31 -No 6
SCP,
1. Leydig cell organelles: volume per cell (pm3) and per testis ( lo6 pm3) Organelle SER Per cell Per testis Peroxisomes Per cell Per testis Negative bodies Per cell Per testis RER Per cell Per testis Mitochondria Per cell Per testis Lysosomes Per cell Per testis Nucleus Per cell Per testis
Control
TE
735 (60) 16,558 (226)
133 (8)” 2,604 (78)”
11.91 (0.98)
TELH
TE/control
767 (58) 16,720 (475)
13.99 (1.06)
2.02 (0.03) a
268 (3.5)
41.92 (1.3)”
15.39 (1.26) 347 (4.68)
50 (3.1)”
18.56 (0.87) 397 (6.1)
89 (3.3)”
460 (7.8)
114 114
67 (4)” 1,262 (37)”
234 (18) 5,121 (146)
l/3 113
237 (19) 5,338 (72)
2.41 (0.03)”
16.95 (1.29) 370 (10.5)
4.19 (0.02)”
13.74 (1.1) 309 (4.2)
21.49 (1.1)
18.86 (1.43) 412 (12)
4.54 (0.3)” 94 (2.9)” 148 (9)” 3,080 (52)”
218 (17) 4,904 (67)
305 (8.8)
234 (17) 5,116 (145)
116 116
113 l/3 213 213
Values are the mean, with the SE in parentheses (n = 5 rats/group). ’ Significantly different at P < 0.01, by Duncan’s multiple range test after analysis of variance.
CONTROL
TE
TELH
FIG. 2. LH-stimulated testosterone secretion per Leydig cell in vitro in control, TE-implanted, and TELH-implanted rats. Error bar = SEM. n = 3 incubations/group. P c 0.01.
sistent with the earlier studiesof Ewing et al. (19) and Keeney et al. (20), in which no significant differences were observed in Leydig cell number after LH deprival for 5 and 7 days, respectively.
In the present study we found that TE implants in vim for 8 days reduced the LH-stimulated Leydig cell testosterone secretory capacity in vitro to one seventh the control value. However, in vitro Leydig cell testosterone secretory capacity in response to LH stimulation was not different between control and TELH-implanted rats. These results are in agreement with those of previous studieson Leydig cell testosterone secretion in perfused testes of TE rats and TELH rats (19, 21). The impaired testosteronesecretory capacity in Leydig cells of TE-implanted rats after 8 days may be explained partly by the atrophy of Leydig cells with LH deprivation. The results of the present study showed that the average volume of a Leydig cell of a TE-implanted rat became one third of the control value after 8 days of TE treatment. By contrast, the reduction in the testosteronesecretory capacity of Leydig cells in vitro was lowered to one seventh of the control value after 8 days of TE implantation. This observation raised the question of whether there are specific reductions in Leydig cell organelles involved in testosterone biosynthesis after 8 days of TE treatment. The existence of a relationship between Leydig cell SER
peroxisomes FIG. 3. Representative from control, TE-implanted, and TELH-implanted rats immunolabeled for catalase. Magnification, ~20,000. Bar = 0.5 urn.
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LEYDIG >
CELL
PEROXISOMES
1 CATALASE
C TE TELH FIG. 4. Quantification of intraperoxisomal catalase in Leydig cell peroxisomes of control, TE-implanted, and TELH-implanted rats. P > 0.05. n = 5 rats/group.
and testosteronebiosynthesis has been described in qualitative (22-24) and quantitative (3, 19, 25) ultrastructural studies. As treatment of rats with TE implants causedthe atrophy of Leydig cells to one third of control values, it was not surprising to observe reductions in the volumes of all organelles in Leydig cells in TE-implanted rats. Nevertheless, the stereological data presented herein demonstrate for the first time that all Leydig cell organellesare not reduced in volume to the samedegree of magnitude in responseto withdrawal of LH, i.e. someorganellesappear to be more LH dependent than others. The absolute volumes of organelles such as mitochondria and lysosomesin Leydig cells of TE-implanted rats were reduced to one third of control values. However, our findings revealed that the absolute volumes of SER and peroxisomes in Leydig cells of TE-implanted rats were reduced to one sixth of control values. Thus, the decreasein the volume of peroxisomes and SER was twice the overall reduction in average Leydig cell volume. To our knowledge, the present study is the first to demonstrate such variations in trophic effects of LH on different types of Leydig cell organelles. Moreover, the magnitude of the reduction in the absolute volume of SER and peroxisomes approximates the reduction in the LH-stimulated testosterone secretory capacity of these Leydig cells in vitro. The greater reduction in the absolute volume of SER associatedwith reduced testosterone secretory capacity in Leydig cells of TE-implanted rats is in agreement with the fact that SER contains LH-regulated steroidogenic enzymes that are required to convert pregnenolone to testosterone(26, 27). This striking lossof Leydig cell peroxisome volume and its correlation with the reduction in testosterone secretion in LH-deprived rats is consistent with
AND
SCP,
2843
our previous finding of a specific increase in Leydig cell peroxisome volume after acute LH treatment (6) and our hypothesis that peroxisomes are involved in the steroidogenesis in Leydig cells (3), respectively. In the present study we correlated Leydig cell SER and peroxisome volume determined in viva with the LH-stimulated testosterone production in vitro using isolated Leydig cells. It should be mentioned that the yield (counted on a hemocytometer) of Leydig cells obtained from TE-implanted rats were the sameas in the controls, and the purity (determined by S/3-hydroxysteroid dehydrogenase staining) (28) was 93-95% in control and treated groups. As Leydig cells from control and treated groups were handled in exactly the samemanner, we are confident of the fact that the comparisonsmade among these treatment groups are valid. The negative bodies in Leydig cells, the single membranebound organelles negative for catalase but otherwise morphologically identical to peroxisomes (4), still remain an enigma. In an earlier study (4) we ruled out the possibility that negative bodies are artifacts. The absolute volume of negative bodies was reduced 6-fold after 8 days of LH withdrawal, revealing that the effects of LH on negative bodies are similar to the effects on SER and peroxisomes. Previously, we have shown that the absolute volume of negative bodies was increased 3-fold 0.5 h after a single LH injection (6). This strengthens our hypothesis that negative bodies may be peroxisomes that are devoid of catalase. Experiments are underway to test this hypothesis. Previously, we have demonstrated that Leydig cell peroxisomescontain SCPp(4) and that acute LH treatment causes specific, but transient, increases in intraperoxisomal SCP, content in Leydig cells (6). By contrast, intraperoxisomal catalase content in Leydig cells was unchanged with this treatment (6). The results of the present study revealed that LH withdrawal causesa distinct decline in the intraperoxisomal SCPp content without any change in the content of intraperoxisomal catalase.However, as the total peroxisomal volume in Leydig cells of the TE-implanted rat was reduced, the total amount of catalasein Leydig cells of this treatment group showed a decreasein the immunoblot analysis. Despite the fact that the technique of enhanced chemiluminescence that we used is highly sensitive, we were unable to demonstrate the presence of SCP, in Leydig cells of TEimplanted rats. This is not surprising for two reasons.First, we observed a reduction in the absolute volume of Leydig cell peroxisomes(the organelle that has the highest concen-
FIG. 5. Representative peroxisomes from control, TE-implanted, and TELH-implanted rats immunolabeled for SCPp. Magnification, ~20,000. Bar = 0.5 pm.
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2844
LEYDIG
CELL PEROXISOMES
AND
SCP,
Endo. Voll31.
1992 No 6
-66 -45 -6 -9 9 -24 -2Od 0
C
TE
TELH
-l4m2
FIG. 6. Quantification of intraperoxisomal SCP, in Leydig cell peroxisomes of control, TE-implanted, and TELH-implanted rats. P < 0.05. n = 5 rats/group.
123 FIG. 8. Immunoblots for SCPz (14 kilodaltons) in Leydig cells (3 x 105/group) of control, TE-implanted, and TELH-implanted rats (lanes 1,2, and 3 respectively). We previously demonstrated that the antibody to SCPZ detected a rat liver protein whose electrophoretic mobility was identical to that of the Leydig cell protein (6). When protein from E. co2i, which does not contain SCP,, was immunoblotted with the SCPz antibody, no 14-kilodalton protein was detected (data not shown).
-45 -36 -29 -24
References
-20-I -14-2 FIG. 7. Immunoblots for catalase (60 kilodaltons) in Leydig cells (3 x NY/group) of control, TE-implanted, and TELH-implanted rats (lanes 1,2, and 3, respectively). We previously demonstrated that the antibody to catalase detected a rat liver protein whose electrophoretic mobility was identical to that of the Leydig cell protein (6).
tration of SCP, in control Leydig cells) in TE-implanted rats. Second, the intraperoxisomal SCP, content (determined by immunolabeling) was also decreased in this treatment group (by contrast, the intraperoxisomal catalase was not decreased). Therefore, the reduction in the absolute volume of Leydig cell peroxisomes together with the reduction in the intraperoxisomal SCP2 content should cause a greater reduction in the total SCP, content in Leydig cells of TE-implanted rats compared to the total Leydig cell catalase content. This explains why we were unable to detect SCPp in Leydig cells of TE-implanted rats with immunoblot analysis. Acknowledgments We thank Dr. Takashi Hashimoto, Shinsu University (Japan), providing us the affinity-purified anticatalase used in this study, the late Dr. Larry Ewing for his guidance and encouragement.
for and
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