0013-7227/91/l291-0256$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 1 Printed in U.S.A.
Effects of Interleukin-6, Interleukin-2, and Tumor Necrosis Factor a on Transferrin Release from Sertoli Cells in Culture* F. R. BOOCKFOR AND L. K. SCHWARZ Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston, South Carolina 29425-2204
segments, in addition to those segments which are normally responsive without pretreatment (III-V and VII segments). Further experiments revealed that IL-6 also had a chronic influence on the proportions of TF secretors present in certain staged cultures. Treatment for 24 h with IL-6 markedly reduced the percentage of TF secretors in cultures from stage XIII segments and resulted in a slight increase in TF cells for stage VII cultures. However, no chronic influences in TF secretors were detected with either IL-2 or tumor necrosis factor a treatment. Our results demonstrate very clearly that certain cytokines acting in a stagespecific manner have acute and/or chronic influences on the release of TF from Sertoli cells. These findings, when viewed in light of reports of the presence of these factors in the testis, suggest strongly that cytokines or cytokine-like substances, by modulating the release of Sertoli cell substances, may play an important role in testis function. {Endocrinology 129: 256-262, 1991)
ABSTRACT. Recent studies demonstrate that several cytokines are potent modulators of steroid release from the testis. In an attempt to determine whether these agents may influence other types of secreted substances, we used plaque assays to measure the effect of interleukin-6 (IL-6), interleukin-2 (IL-2), and tumor necrosis factor a on transferrin (TF) release from Sertoli cells in culture. Because Sertoli cells from different parts of the tubule respond differently to modulatory factors, we used cultures obtained by microdissection from stages III-V, VII, IXXI, and XIII of the cycle of the seminiferous epithelium. Our results revealed that each agent increased the rate of TF plaque formation from cultures of IX-XI, and XIII staged segments but not from those staged III-V and VII. Moreover, IL-6, but not the other cytokines, modified the response of Sertoli cells to another regulator, FSH. This was evidenced by our findings that pretreatment with IL-6 for 1 h resulted in FSH-induced increases in the rate of plaque formation for cells from IX-XI
T
HERE is a growing body of evidence to suggest that cytokines or immune cell factors influence hormone secretion from gonadal tissue. Progesterone, estradiol, and androgen released by ovarian cells have been shown to be modified by several different interleukins (1-5). In testis tissue, various cytokines including interleukin-1 (IL-1), interleukin-2 (IL-2), and tumor necrosis factor a (TNF) for rats (6-9) and interferon-7 (10, 11) and IL-1 for pigs were reported to inhibit Leydig cell steroidogenesis. A brief study suggests that interferon may also inhibit estradiol secretion from Sertoli cells (12). In contrast, Warren et al. (13) reported that IL-1 and interferon have a stimulatory influence on testosterone production. Although it is unclear whether these agents act in a stimulatory or inhibitory manner, these findings, when coupled with reports of the presence of several
different immune cell types in the ovary and testis (1421) suggest that cytokines may be involved in gonadal function. Many of the investigations of cytokine action in gonadal tissue, especially the testis, have been concerned with steroid release. Little information is available on the effect of these factors on other secreted substances. Sertoli cells, the source of many factors that are vital for proper germ cell development in the testis (22-27), would be a potentially important target for cytokines. Even though Sertoli cells do not come in direct contact with immune cells found in the interstitial spaces, they are exposed to interstitial fluid. Because most, if not all, cytokines identified have been shown to be released from immune cells, it would be reasonable to expect that these products would be secreted into the interstitial fluid. In light of the potent manner in which these agents act and the probable access they have to Sertoli cells, it is quite possible that cytokines may modulate the release of certain substances from this cell population, which may have a direct effect on spermatogenesis. In order to investigate this possibility, we assessed the
Received February 4,1991. Address all correspondence and requests for reprints to: Dr. F. R. Boockfor, Department of Anatomy and Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2204. * This work was supported in part by NIH NICHHD Grant HD25090 (to F.R.B.).
256 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 08:19 For personal use only. No other uses without permission. . All rights reserved.
CYTOKINE EFFECT ON SERTOLI CELL FUNCTION influence of three different cytokines (IL-6, IL-2, and TNF) on transferrin (TF) release, which is not only important for proper germ cell development but is also an excellent marker of Sertoli cell function. Because not all Sertoli cells along the tubule respond to modulatory factors in a similar manner (28-30) we used cells from several different stage-associated segments of the seminiferous epithelium to determine the responsiveness to these agents. By using plaque assays for TF, we found that each of these cytokines influence the acute release of TF in a stage-specific manner. Moreover, IL-6 not only modified the acute response of certain cultures to FSH, but alone had a chronic stage-associated effect on the proportions of TF secretors present. Materials and Methods Animal and cell culture Sertoli cells from stage-specific seminiferous tubule segments of adult rats were obtained as described previously (30). In short, testes from rats (>70 days; Holtzman Laboratory Animals, Madison, WI) were decapsulated and placed into an enzyme solution. This solution consisted of collagenase/dispase (0.03%, Boehringer-Mannheim Biochemicals, Indianapolis, IN) and hyaluronidase (0.05%, Type 1-S, Sigma Chemical Co., St. Louis, MO) in Minimum Essential Medium (MEM+) which contained BSA (0.01%, Sigma) and antibiotics [penicillin-G (100 U/ml), streptomycin (100 Mg/ml), and gentamicin (50 ^g/ ml)]. After gently agitating for 90 min, the tubules from each testis were transferred to a large Petri dish and subjected to a microdissection procedure employing transilluminated light as described by Parvinen and Ruokonen (31). Using distinct shading patterns described by these authors, we obtained segments that correlated with stages III-V, VII, IX-XI, and XIII of the seminiferous epithelial cycle. These segments were then subjected to an additional enzyme solution (0.02% collagenase III, Worthington Biochemical Corp., Freehold, NJ; and 0.07% hyaluronidase in MEM+) for 30 min at 34.5 C. The tubule fragments obtained were then reduced in size by repeated passage through the tip of a flame-polished pipette. The cells were resuspended in Dulbecco's Modified Eagle's Medium and Ham's F-12 (1:1 mixture) with 4% fetal bovine serum and antibiotics. Tissue culture dishes (35 mm) were each seeded with approximately 4.5 x 106 cells and incubated for 48 h at 34.5 C in a water-saturated atmosphere of 5% CO2/95% O2. During this period, the cells attached to the floor of the culture chambers. Germ cells and debris, which did not attach, were removed by washing the cultures vigorously three times with MEM+ at the end of the 48-h incubation period. For acute experiments, control medium was placed into the dishes and the cultures allowed to incubate for an additional 24 h before being subjected to TF plaque assays. For chronic studies, control medium, alone or containing human recombinant cytokines, [IL-6 (20 ng/ml; Amgen, Thousand Oaks, CA), IL-2 (5 ng/ml; Bachem Inc., Torrance, CA), or TNF (1 ng/ml; Amgen)] each with bioactivity at 1 X 107 U/mg or greater was added to the dishes. These concentrations were found in preliminary
257
experiments to be maximal. The cultures were then incubated for 24 h before being assayed. Reverse hemolytic plaque assays for TF Reverse hemolytic plaque assays were performed as reported initially (32, 33), except that modifications were made to enable detection of TF (34). On the day of an experiment, cells obtained from each tubule segment were exposed to a dilute solution of trypsin (0.025% in MEM+) for 5 min. The cells were detached by directing the enzymes onto the monolayer and monodispersed by passing them back and forth through the tip of a flame-polished Pasteur pipette. They were then washed and diluted in assay medium (Dulbecco's Modified Eagle's Medium with 0.1% BSA and antibiotics) to obtain a concentration of 800,000 cells/ml. An aliquot of this preparation was mixed with an equal volume of protein A-coated ovine red blood cells (9% suspension) and infused into poly-L-lysinetreated slide chambers. Following a 45-min period during which time the cells attached to the chamber floors, TF antibody (antirat TF; 4.0 mg antibody protein/ml; U.S. Biochemical Corp., Cleveland, OH), diluted further in assay medium (1:80), was added. As described by the manufacturer, immunoelectrophoresis performed simultaneously using separate samples of whole serum and purified rat TF revealed that this antiserum identified only TF and did not cross-react with other blood components. The slide chambers were then incubated for various lengths of time in a water-saturated atmosphere at 34.5 C. In some experiments, ovine FSH (NIDDK oFSH-17, National Hormone and Pituitary Program, Baltimore, MD) or IL-6, IL2, and TNF (each at doses cited above and found to be maximal) were included in the assay mixture. After incubation, a solution of complement (1:40 guinea pig serum in assay medium) was infused into the assay chambers to initiate plaque formation. The reaction was terminated by addition of fixative (1.8% glutaraldehyde, in isotonic saline). The cells were stained with toluidine blue, and the ratios of plaque formers to nonplaque formers were determined. This was accomplished by microscopic evaluation of at least three different chamber areas that were chosen randomly. Specificity controls were performed as reported previously (34). We found that preabsorption of 200 ix\ of a working dilution of antiserum (1:80) with purified rat TF (1 Mg/ml; Sigma) for 2 h at 34.5 C completely abolished plaque formation. Additionally, plaques did not form if any assay component was deleted from the assay procedure. Plaque assays were to be used in two ways in the following studies. First, changes in the rate of plaque formation were assessed by performing plaque assays for 1, 2, and 8 h in the absence or presence of a putative modulator of secretion. An increase or decrease in the rate of plaque formation indicated a stimulatory or inhibitory acute influence, respectively. Second, the overall proportions of TF releasing cells in Sertoli cell cultures were determined by using an 8-h incubation period which, under the conditions of our assay, enables the maximal detection of TF plaque formers. In this manner, an influence on the proportions of TF releasing cells in culture after chronic exposure to an agent can be detected.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 08:19 For personal use only. No other uses without permission. . All rights reserved.
CYTOKINE EFFECT ON SERTOLI CELL FUNCTION
258 Statistics
Analysis of variance followed by Tukey's multiple range test was employed in experiments requiring multiple comparisons such as those in which the effects of various agents on the rate of plaque formation were assessed. Differences between treatment groups were determined by paired Student's t tests for experiments in which single comparisons were needed (35).
Results The effects of IL-6, IL-2, and TNF on the rate of TF release from cultures of cells from various tubule segments are presented in Fig. 1. As shown for controls of each segment, the proportion of TF plaque formers increased gradually with time reaching a maximum by at least 8 h of incubation. Performance of plaque assays in
Endo'1991 Voll29«Nol
the presence of IL-6, IL-2, or TNF accelerated the rate at which plaque formation occurred from cells from section IX-XI and XIII but had no effect on cells from sections III-V or VII. Our use of 10-fold higher concentrations of these agents did not further alter the rate of plaque formation from cells of any segment (data not shown). The influence of IL-6 pretreatment of cells from various stages on FSH responsiveness is presented in Fig. 2. Without preexposure, (Fig. 2A), the rate of TF plaque formation is enhanced markedly with FSH for cells from segments III-V and VII. However, untreated cells from stage IX-XI or XIII sections are not responsive to this gonadotropin. In contrast, pretreatment of cells with IL6 for 1 h resulted in FSH-induced increases in the rate of plaque formation for cells from IX-XI segments, in SECTION VII
SECTIONS III-V 60 T
60-i
3
•*-*• • -o-
IL-6 IL-2 TNF CONTROL
UJ
SO-
•*• IL-6 -*- |L-2
2
40H
•«H TNF •O- CONTROL
u.
30-
u o
2 DC O
2010-
2
2
4 6 INCUBATION TIME (h)
4 6. INCUBATION TIME (h)
SECTION XIII
SECTIONS IX-XI 60-
3 Ui
O O oc o
u.
50*
40-
Y
30-
Ui
3
o 20GL
100^
t
0
2
**[
••• IL-6 -A- VLrZ
-*• TNF -o- CONTROL
ft
"
i
4
*
6
INCUBATION TIME (h)
v
II
8
1
0
2
4
6
INCUBATION TIME (h)
FIG. 1. The influence of IL-2, IL-6, and TNF on the rate of TF plaque formation from different staged segments of the seminiferous epithelium. Seminiferous tubule segments corresponding to stage III-V, VII, IX-XI, or XIII of the cycle of the seminiferous epithelium were obtained by microdissection, dispersed with enzymes, and the cells placed into culture. After 3 days, plaque assays for TF were performed in the absence or the presence of IL-6 (20 ng/ml), IL-2 (5 ng/ml), or TNF (1 ng/ml) for various periods of time. Shown here are the results (mean ± SE) of five separate experiments. Within each experiment, at least 250 cells were counted at each time point for every treatment. *, P < 0.01; -sir, P < 0.05 for each agent tested compared to controls.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 08:19 For personal use only. No other uses without permission. . All rights reserved.
CYTOKINE EFFECT ON SERTOLI CELL FUNCTION
259
SECTION III-V
SECTION VII
• • - FSH •o- CONTROL
FSH CONTROL
2
2 4 6 INCUBATION TIME (h)
4 6. INCUBATION TIME (h)
SECTIONS IX-XI
2
2
4 6 INCUBATION TIME (h)
4
SECTION III-V
SECTION VII
3 S 50o 1 40-
•m- FSH -o- CONTROL
OC
2
30-
UJ
3
j
20-
P
10-
6
INCUBATION TIME (h)
IE FORMINQ CELLS
FlG. 2. The influence of IL-6 on the responsiveness to FSH of cultured Sertoli cells obtained from different staged segments. Cultures were prepared as described in the legend to Fig. 1. On the day of an assay, cells were preincubated for 1 h in the absence (A) or the presence (B) of IL-6 and then subjected to plaque assays conducted with or without FSH (500 ng/ml). The results (mean ± range) of this experiment are representative of 2 others. Within every experiment, at least 250 cells were counted on each of two slides for every incubation period, both with FSH and without FSH. -fr, P < 0.05 for FSH compared to control.
SECTION XIII
o
60so -
-o-
40-
FSH CONTROL
3020-
^/Z^-
a.
10*
0< 1)
0 2
4 6 INCUBATION TIME (h)
)
0
2
4 6 INCUBATION TIME (h)
SECTIONS IX-XI
0
SECTION XIII
6050-
W 50-
40-
t
30-
4030-
20
-O- CONTROL
• • • FSH •O- CONTROL
20-
10
0
Vol. 129, No. 1 Printed in U.S.A.
Effects of Interleukin-6, Interleukin-2, and Tumor Necrosis Factor a on Transferrin Release from Sertoli Cells in Culture* F. R. BOOCKFOR AND L. K. SCHWARZ Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston, South Carolina 29425-2204
segments, in addition to those segments which are normally responsive without pretreatment (III-V and VII segments). Further experiments revealed that IL-6 also had a chronic influence on the proportions of TF secretors present in certain staged cultures. Treatment for 24 h with IL-6 markedly reduced the percentage of TF secretors in cultures from stage XIII segments and resulted in a slight increase in TF cells for stage VII cultures. However, no chronic influences in TF secretors were detected with either IL-2 or tumor necrosis factor a treatment. Our results demonstrate very clearly that certain cytokines acting in a stagespecific manner have acute and/or chronic influences on the release of TF from Sertoli cells. These findings, when viewed in light of reports of the presence of these factors in the testis, suggest strongly that cytokines or cytokine-like substances, by modulating the release of Sertoli cell substances, may play an important role in testis function. {Endocrinology 129: 256-262, 1991)
ABSTRACT. Recent studies demonstrate that several cytokines are potent modulators of steroid release from the testis. In an attempt to determine whether these agents may influence other types of secreted substances, we used plaque assays to measure the effect of interleukin-6 (IL-6), interleukin-2 (IL-2), and tumor necrosis factor a on transferrin (TF) release from Sertoli cells in culture. Because Sertoli cells from different parts of the tubule respond differently to modulatory factors, we used cultures obtained by microdissection from stages III-V, VII, IXXI, and XIII of the cycle of the seminiferous epithelium. Our results revealed that each agent increased the rate of TF plaque formation from cultures of IX-XI, and XIII staged segments but not from those staged III-V and VII. Moreover, IL-6, but not the other cytokines, modified the response of Sertoli cells to another regulator, FSH. This was evidenced by our findings that pretreatment with IL-6 for 1 h resulted in FSH-induced increases in the rate of plaque formation for cells from IX-XI
T
HERE is a growing body of evidence to suggest that cytokines or immune cell factors influence hormone secretion from gonadal tissue. Progesterone, estradiol, and androgen released by ovarian cells have been shown to be modified by several different interleukins (1-5). In testis tissue, various cytokines including interleukin-1 (IL-1), interleukin-2 (IL-2), and tumor necrosis factor a (TNF) for rats (6-9) and interferon-7 (10, 11) and IL-1 for pigs were reported to inhibit Leydig cell steroidogenesis. A brief study suggests that interferon may also inhibit estradiol secretion from Sertoli cells (12). In contrast, Warren et al. (13) reported that IL-1 and interferon have a stimulatory influence on testosterone production. Although it is unclear whether these agents act in a stimulatory or inhibitory manner, these findings, when coupled with reports of the presence of several
different immune cell types in the ovary and testis (1421) suggest that cytokines may be involved in gonadal function. Many of the investigations of cytokine action in gonadal tissue, especially the testis, have been concerned with steroid release. Little information is available on the effect of these factors on other secreted substances. Sertoli cells, the source of many factors that are vital for proper germ cell development in the testis (22-27), would be a potentially important target for cytokines. Even though Sertoli cells do not come in direct contact with immune cells found in the interstitial spaces, they are exposed to interstitial fluid. Because most, if not all, cytokines identified have been shown to be released from immune cells, it would be reasonable to expect that these products would be secreted into the interstitial fluid. In light of the potent manner in which these agents act and the probable access they have to Sertoli cells, it is quite possible that cytokines may modulate the release of certain substances from this cell population, which may have a direct effect on spermatogenesis. In order to investigate this possibility, we assessed the
Received February 4,1991. Address all correspondence and requests for reprints to: Dr. F. R. Boockfor, Department of Anatomy and Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2204. * This work was supported in part by NIH NICHHD Grant HD25090 (to F.R.B.).
256 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 08:19 For personal use only. No other uses without permission. . All rights reserved.
CYTOKINE EFFECT ON SERTOLI CELL FUNCTION influence of three different cytokines (IL-6, IL-2, and TNF) on transferrin (TF) release, which is not only important for proper germ cell development but is also an excellent marker of Sertoli cell function. Because not all Sertoli cells along the tubule respond to modulatory factors in a similar manner (28-30) we used cells from several different stage-associated segments of the seminiferous epithelium to determine the responsiveness to these agents. By using plaque assays for TF, we found that each of these cytokines influence the acute release of TF in a stage-specific manner. Moreover, IL-6 not only modified the acute response of certain cultures to FSH, but alone had a chronic stage-associated effect on the proportions of TF secretors present. Materials and Methods Animal and cell culture Sertoli cells from stage-specific seminiferous tubule segments of adult rats were obtained as described previously (30). In short, testes from rats (>70 days; Holtzman Laboratory Animals, Madison, WI) were decapsulated and placed into an enzyme solution. This solution consisted of collagenase/dispase (0.03%, Boehringer-Mannheim Biochemicals, Indianapolis, IN) and hyaluronidase (0.05%, Type 1-S, Sigma Chemical Co., St. Louis, MO) in Minimum Essential Medium (MEM+) which contained BSA (0.01%, Sigma) and antibiotics [penicillin-G (100 U/ml), streptomycin (100 Mg/ml), and gentamicin (50 ^g/ ml)]. After gently agitating for 90 min, the tubules from each testis were transferred to a large Petri dish and subjected to a microdissection procedure employing transilluminated light as described by Parvinen and Ruokonen (31). Using distinct shading patterns described by these authors, we obtained segments that correlated with stages III-V, VII, IX-XI, and XIII of the seminiferous epithelial cycle. These segments were then subjected to an additional enzyme solution (0.02% collagenase III, Worthington Biochemical Corp., Freehold, NJ; and 0.07% hyaluronidase in MEM+) for 30 min at 34.5 C. The tubule fragments obtained were then reduced in size by repeated passage through the tip of a flame-polished pipette. The cells were resuspended in Dulbecco's Modified Eagle's Medium and Ham's F-12 (1:1 mixture) with 4% fetal bovine serum and antibiotics. Tissue culture dishes (35 mm) were each seeded with approximately 4.5 x 106 cells and incubated for 48 h at 34.5 C in a water-saturated atmosphere of 5% CO2/95% O2. During this period, the cells attached to the floor of the culture chambers. Germ cells and debris, which did not attach, were removed by washing the cultures vigorously three times with MEM+ at the end of the 48-h incubation period. For acute experiments, control medium was placed into the dishes and the cultures allowed to incubate for an additional 24 h before being subjected to TF plaque assays. For chronic studies, control medium, alone or containing human recombinant cytokines, [IL-6 (20 ng/ml; Amgen, Thousand Oaks, CA), IL-2 (5 ng/ml; Bachem Inc., Torrance, CA), or TNF (1 ng/ml; Amgen)] each with bioactivity at 1 X 107 U/mg or greater was added to the dishes. These concentrations were found in preliminary
257
experiments to be maximal. The cultures were then incubated for 24 h before being assayed. Reverse hemolytic plaque assays for TF Reverse hemolytic plaque assays were performed as reported initially (32, 33), except that modifications were made to enable detection of TF (34). On the day of an experiment, cells obtained from each tubule segment were exposed to a dilute solution of trypsin (0.025% in MEM+) for 5 min. The cells were detached by directing the enzymes onto the monolayer and monodispersed by passing them back and forth through the tip of a flame-polished Pasteur pipette. They were then washed and diluted in assay medium (Dulbecco's Modified Eagle's Medium with 0.1% BSA and antibiotics) to obtain a concentration of 800,000 cells/ml. An aliquot of this preparation was mixed with an equal volume of protein A-coated ovine red blood cells (9% suspension) and infused into poly-L-lysinetreated slide chambers. Following a 45-min period during which time the cells attached to the chamber floors, TF antibody (antirat TF; 4.0 mg antibody protein/ml; U.S. Biochemical Corp., Cleveland, OH), diluted further in assay medium (1:80), was added. As described by the manufacturer, immunoelectrophoresis performed simultaneously using separate samples of whole serum and purified rat TF revealed that this antiserum identified only TF and did not cross-react with other blood components. The slide chambers were then incubated for various lengths of time in a water-saturated atmosphere at 34.5 C. In some experiments, ovine FSH (NIDDK oFSH-17, National Hormone and Pituitary Program, Baltimore, MD) or IL-6, IL2, and TNF (each at doses cited above and found to be maximal) were included in the assay mixture. After incubation, a solution of complement (1:40 guinea pig serum in assay medium) was infused into the assay chambers to initiate plaque formation. The reaction was terminated by addition of fixative (1.8% glutaraldehyde, in isotonic saline). The cells were stained with toluidine blue, and the ratios of plaque formers to nonplaque formers were determined. This was accomplished by microscopic evaluation of at least three different chamber areas that were chosen randomly. Specificity controls were performed as reported previously (34). We found that preabsorption of 200 ix\ of a working dilution of antiserum (1:80) with purified rat TF (1 Mg/ml; Sigma) for 2 h at 34.5 C completely abolished plaque formation. Additionally, plaques did not form if any assay component was deleted from the assay procedure. Plaque assays were to be used in two ways in the following studies. First, changes in the rate of plaque formation were assessed by performing plaque assays for 1, 2, and 8 h in the absence or presence of a putative modulator of secretion. An increase or decrease in the rate of plaque formation indicated a stimulatory or inhibitory acute influence, respectively. Second, the overall proportions of TF releasing cells in Sertoli cell cultures were determined by using an 8-h incubation period which, under the conditions of our assay, enables the maximal detection of TF plaque formers. In this manner, an influence on the proportions of TF releasing cells in culture after chronic exposure to an agent can be detected.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 08:19 For personal use only. No other uses without permission. . All rights reserved.
CYTOKINE EFFECT ON SERTOLI CELL FUNCTION
258 Statistics
Analysis of variance followed by Tukey's multiple range test was employed in experiments requiring multiple comparisons such as those in which the effects of various agents on the rate of plaque formation were assessed. Differences between treatment groups were determined by paired Student's t tests for experiments in which single comparisons were needed (35).
Results The effects of IL-6, IL-2, and TNF on the rate of TF release from cultures of cells from various tubule segments are presented in Fig. 1. As shown for controls of each segment, the proportion of TF plaque formers increased gradually with time reaching a maximum by at least 8 h of incubation. Performance of plaque assays in
Endo'1991 Voll29«Nol
the presence of IL-6, IL-2, or TNF accelerated the rate at which plaque formation occurred from cells from section IX-XI and XIII but had no effect on cells from sections III-V or VII. Our use of 10-fold higher concentrations of these agents did not further alter the rate of plaque formation from cells of any segment (data not shown). The influence of IL-6 pretreatment of cells from various stages on FSH responsiveness is presented in Fig. 2. Without preexposure, (Fig. 2A), the rate of TF plaque formation is enhanced markedly with FSH for cells from segments III-V and VII. However, untreated cells from stage IX-XI or XIII sections are not responsive to this gonadotropin. In contrast, pretreatment of cells with IL6 for 1 h resulted in FSH-induced increases in the rate of plaque formation for cells from IX-XI segments, in SECTION VII
SECTIONS III-V 60 T
60-i
3
•*-*• • -o-
IL-6 IL-2 TNF CONTROL
UJ
SO-
•*• IL-6 -*- |L-2
2
40H
•«H TNF •O- CONTROL
u.
30-
u o
2 DC O
2010-
2
2
4 6 INCUBATION TIME (h)
4 6. INCUBATION TIME (h)
SECTION XIII
SECTIONS IX-XI 60-
3 Ui
O O oc o
u.
50*
40-
Y
30-
Ui
3
o 20GL
100^
t
0
2
**[
••• IL-6 -A- VLrZ
-*• TNF -o- CONTROL
ft
"
i
4
*
6
INCUBATION TIME (h)
v
II
8
1
0
2
4
6
INCUBATION TIME (h)
FIG. 1. The influence of IL-2, IL-6, and TNF on the rate of TF plaque formation from different staged segments of the seminiferous epithelium. Seminiferous tubule segments corresponding to stage III-V, VII, IX-XI, or XIII of the cycle of the seminiferous epithelium were obtained by microdissection, dispersed with enzymes, and the cells placed into culture. After 3 days, plaque assays for TF were performed in the absence or the presence of IL-6 (20 ng/ml), IL-2 (5 ng/ml), or TNF (1 ng/ml) for various periods of time. Shown here are the results (mean ± SE) of five separate experiments. Within each experiment, at least 250 cells were counted at each time point for every treatment. *, P < 0.01; -sir, P < 0.05 for each agent tested compared to controls.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 08:19 For personal use only. No other uses without permission. . All rights reserved.
CYTOKINE EFFECT ON SERTOLI CELL FUNCTION
259
SECTION III-V
SECTION VII
• • - FSH •o- CONTROL
FSH CONTROL
2
2 4 6 INCUBATION TIME (h)
4 6. INCUBATION TIME (h)
SECTIONS IX-XI
2
2
4 6 INCUBATION TIME (h)
4
SECTION III-V
SECTION VII
3 S 50o 1 40-
•m- FSH -o- CONTROL
OC
2
30-
UJ
3
j
20-
P
10-
6
INCUBATION TIME (h)
IE FORMINQ CELLS
FlG. 2. The influence of IL-6 on the responsiveness to FSH of cultured Sertoli cells obtained from different staged segments. Cultures were prepared as described in the legend to Fig. 1. On the day of an assay, cells were preincubated for 1 h in the absence (A) or the presence (B) of IL-6 and then subjected to plaque assays conducted with or without FSH (500 ng/ml). The results (mean ± range) of this experiment are representative of 2 others. Within every experiment, at least 250 cells were counted on each of two slides for every incubation period, both with FSH and without FSH. -fr, P < 0.05 for FSH compared to control.
SECTION XIII
o
60so -
-o-
40-
FSH CONTROL
3020-
^/Z^-
a.
10*
0< 1)
0 2
4 6 INCUBATION TIME (h)
)
0
2
4 6 INCUBATION TIME (h)
SECTIONS IX-XI
0
SECTION XIII
6050-
W 50-
40-
t
30-
4030-
20
-O- CONTROL
• • • FSH •O- CONTROL
20-
10
0
