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Inhibitory effect of gossypol on steroidogenic pathways in cultured bovine luteal cells
Vol. 169, No. 2, 1990 June 15, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 455-461
INHIBITORY EFFECTOF GOSSYPOL ON STEROIDOGENIC PATHWAYS IN CULTUREDBOVINE LUTEAL CELLS Y. Gu*, Y.C. Lin” and Y. Rikihisa”” Laboratory of Reproductive Endocrinology, Departments of *Veterinary Physiology and Pharmacology , and **Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210-1092 Received
April
20,
1990
Gossypol inhibits the reproductive s stem and steroidogenesis in both sexes. The present study investigated some possiti le sites subsequent to CAMP formation at which gossypol may inhibit progesterone biosynthesis. Bovine luteal cells were cultured with dibutyryl CAMP (dbcAMP), 25-OH cholesterol, or pregnenolone in the presence or absence of gossypol. Gossypol, at 17-34 gM, inhibited dbcAMP-induced progesterone secretion. Gossypol significantly inhibited the conversions of exogenous 25-OH cholesterol and pregnenolone to progesterone. However, the conversion of 25-OH cholesterol to pregnenolone was not significantly inhibited by gossypol at low doses (534 pM). These results suggest that gossypol inhibits progesterone synthesis in bovine luteal cells by suppressing steroidogenic enzyme activity. 0 1990Academic Press,fnc.
Gossypol, a natural polyphenolic compound, is a male antifertility agent and it has been reported that gossypol has an antifertility effect in females as well (1,2). We previously reported that gossypol interrupted normal estrous cycles (3) and inhibited embryo implantation(4,5) in rats. Recently, we also reported that gossypol inhibited human chorionic gonadotropin (hCG)-induced cyclic adenosine monophosphate (cAMP) formation
and progesterone secretion in cultured bovine
luteal cells (6). In the present study, we examined sites subsequent to hCG- induced CAMP formation at which gossypol might also inhibit progesterone synthesis in bovine luteal cells in vitro.
Materials and Methods Fetal bovine serum (FBS, lot:b79306) was purchased from Armour Pharmaceutical Co. (Kankakee, IL). 1,2,6,7,21-[3H(N)]-Progesterone (Lot:2370-059) was purchased from New England Nuclear (Boston, MA) and delta-5 I4,7-3Hl Pregnenolone (Batch 39) was purchased from Amersham (Arlington Heights, IL). Progesterone antiserum was obtained from Endocrine Sciences (Tarzana, CA) and pregnenolone antiserum was purchased from Scantibodies Laboratory Inc. (Lot: T463C, Santee, CA). Collagenase (type I, Lot:77380M) was urchased from Worthington Biochemical Co. (Freehold, NJ). Dulbecco’s Modifie d” Eagle’s Medium and Ham’s Nutrient Mixture F-12 and remaining reagents were purchased from Sigma Chemical Co. (St. Louis, MO). 0006-291XE'O 455
$1.50
Copyright 0 1990 by Academic Press, Inc. AN rights of reproduction in any form reserved.
Vol. 169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Preparation and culture of bovine luteal cells: Bovine ovaries containing corpora lutea were obtained from a local slaughterhouse and transported back to the laboratory in iced phosphate buffered saline (PBS) within an hour of slaughter. The corpora lutea with a size 14x14~12 mm3 were selected for luteal cell isolation. The corpora lutea were carefully dissected from ovaries on ice. The luteal cells were isolated as described by Poff et al. (7). Briefly, corpora lutea were sliced and minced to about 1 mm3. The luteal cells were dissociated at 35°C with collagenase (2000 U/g luteal tissue) and 0.5% bovine serum albumin (BSA) in Dulbecco’s Modified Eagle’s Medium and Ham’s nutrient mixture F-12 containing peniciflin G, 100 units/ml; streptom tin, 0.1 mg/ml; and amphotericin 8, 0.25 pg/ml (DME/F-12). After one hour milJ stirring, the cells were washed 4 times with fresh DME/F-12 containing no BSA by centrifu ation for 10 min in a series of descending speeds, from 150 to 100 x g at 4°C. The cef Is were counted with a hemocytometer. The viabilit of the cells was determined by the trypan blue exclusion method (8). B this metho dy, approximately 17.4 xl06 viable luteal cells per gram of tissue were yie Yded with a 20-30:80-70 ratio of large (>20 pm) to small (8-20 pm) cell population. The viability of yield cells were about 70%. All culture dishes were pretreated with 10% FBS in DME/F-12 to provide essential attachment factors. Approximately 106 viable bovine luteal cells were seeded in each dish with 5 ml of serum free, hormone supplemented DME/F-12 (DME/F-12/S), which contains insulin, 5 pg/ml; transferrin, 5p /ml; epidermal growth factor (EGF), 10 rig/ml; sodium selenite 10 ngiml. After 18 R our culture, all dishes were washed twice with DME/F-12 and an additional 3-hour culture was carried out in a total volume of 5 ml DME/F-12/S with different treatments. Treatment 1: the luteal cells were cultured with either 50 or 100 pM dbcAMP and gossypol (0, 17 and 34 pM). Treatment 2: the cells were cultured with various concentrations of 25-OH cholesterol and ossypol. Treatment 3: the cells were cultured with various concentrations o9 pregnenolone and ossypol. Gossypol and steroids were first dissolved in 100% ethanol and then di7 uted so that the maximal concentration of ethanol in the culture medium was 0.25Oh. This concentration of ethanol did not affect progesterone secretion (data not shown). All treatments were performed in triplicate. At the end of each treatment, the medium was collected and stored at 20°C until steroid content determination. Progesterone and pregnenolone determination: The collected culture medium was extracted with 1:7 (V/V) ratio of medium to ethyl ether. Ether phase was separated from aqueous phase by flash freezing. The ether phase was evaporated under N2 and reconstituted with PBS containin 0.1% elatin. The progesterone and pre nenolone contents were determine 8 by ra%ioimmunoassay (RIA) as describe % by Stouffer et al. (9). The intra- and interassay coefficients of variation were 4.3% and 14.2% for progesterone RIA, and were 4.9O/6 and 16Oh for pre nenolone RIA, respectively. The sensitivity of assay was about 64 fmoles/tube for %0th RIAs. The cross-reactivity of pregnenolone antiserum with progesterone was 1%. The cross-reactivity of progesterone antiserum with pre nenolone was 15Oh,using 5Ooh inhibition of binding method, as described by Abra R am (10). Since the steroids in our measurements were not chromatographically separated, the values of steroids in all measurements may be higher than actual values expressed. All standards and unknowns were performed in duplicate. The secretions of progesterone and pregnenolone were expressed as pmoles or nmoles/hour/lO6 cells. Statistical analysis: Differences between treatment means were measured by analysis of variance and Duncan or Bonferroni multiple comparison procedures. In all comparisons, differences were considered significant at P
Results dbcAMP-induced progesterone secretion: Progesterone secretion by either 50 or 100 PM dbcAMP-stimulated bovine luteal cells was significantly (P
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No dbcAMP lOO/kM dbcAMP 50 fl dbcAMP
3 f ” 2.0 “0 > f 1.5 4 E 5 1.0
m
NO gossypol
I
170 /AM gossypol
i 0 0.5 D r a’ 0 Control
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Gossypol
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12.5
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25-OH
25 cholesterol
50
100
(IrM) in medium
Fi ure 1. Effect of gossypol on dbcAMP-induced progesterone secretion in cultured 8---r ovme uteal cells. dbcAMP (50 and 100 PM) significantly stimulated progesterone secretion. Gossypol(17 and 34 PM) inhibited the stimulative effect of dbcAMP. Fiaure 2. Effect of gossypol on 25-OH cholesterol-enhanced progesterone secretion in cultured bovine luteal cells. Gossypol (170 pM) completely abolished the conversion of 25-OH cholesterol to progesterone (P
inhibited Although
pM dbcAMP-stimulated progesterone secretion in luteal cells. gossypol diminished the stimulative effect of 50 pM dbcAMP (Fig. l), the 100
inhibition was not statistically significant. Our previous data showed that higher concentrations of gossypol (>34 PM) did not result in further inhibition of progesterone secretion (data not shown). Conversion of 25OH cholesterol to progesterone: Addition of exogenous 25OH cholesterol (12.5 to 100 PM) markedly enhanced progesterone secretion by the luteal
cells in a dose-dependent
manner
(Fig. 2).
When
170 pM
of gossypol
was
added, the conversion of 25-OH cholesterol to progesterone by the luteal cells was
3g ” “0
Mean f SD. 50
m I
No 25-OH cholestero 100 MM 25-OH cholesterol
3 t” “0
KL 40 f i 30 s t ; g z a’
03
Mean f S.D.
10 0 c.antrcA
inhibited
0
0
12.5
50 &A) in medium
Effect of gossypol on 25-OH cholesterol-enhanced progesterone bovine luteal cells. Progesterone secretion was significantly by gossypol at concentration of 17 pM and higher dose.
secretion (P
17
34
25-OH
25 cholesterol
Gossypol 0 4.25 @A) in medium a.5
4
170 M gossyp0l NO goasypol
75
5 f 4 50 E & z + 25 : 6 h 0
20
I m
Fi ure 4. Effect of gossypol on the conversion of 25-OH cholesterol to pre nenolone +-In cu tured bovine luteal cells. Gossypol (170 PM) completely abo Prshed the conversion. Pregnenolone in culture medium was measured by RIA. 451
100
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Maon
c H
f
BIOCHEMICAL
= I
SD.
N-3
No 25-OH cholesterol 100 jd.4 25-OH cholesterol
30
ContraI
05
0
4.25
Gossypol
a.5
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17
I m
34
0
12.5
06
in medium
Pregnenolone
25
170 phi gossypol NO goS.ypo,
50
100
(JAM) in medium
Fi ure 5. Effect of ossypol on the conversion of 25-OH cholesterol to pregnenolone --In-r cu tured bovine 9 uteal cells. The conversion was slightly reduced by ossypol, but the differences were not statistically significant compared with t il at In 0 pM gossypol-treated cells (P>O.O5). Fi ure 6. Effect of gossypol on the conversion of pre nenolone to progesterone -7-x. cu ture bovme luteal cells. Gossypol (170 PM) sign1 .Pscantly (P
in the
completely (P
of gossypol.
Conversion of 2IOH cholesterol to pregnenolone: Addition of exogenous 25OH cholesterol (12.5 to 100 pM) also significantly increased pregnenolone level in the culture medium in a dose- dependent manner (Fig. 4). Gossypol, 170 pM, again completely abolished (PXO.01) the conversion (Fig. 4). However, gossypol at low doses (4.25 to 34 pM) did not significantly inhibit the conversion of 25-OH cholesterol to pregnenolone (Fig. 5).
3 s mi 800.
Yean *so. N-3
= I
3 c acetl B
No pngnanolone 25 +I pregnenolons
* .
4 400. B 1
*oo-
8
OJ
.
control
0 4.25 Gcdypol @I)
8.5 in medium
?7
34
Fi we 7. Effect of gossypolon the conversion of pregnenolone to progesterone in luteal cells. Gossypol inhibited the conversion in a dose-dependent
cGi73izabovine
manner. Gossypolsignificantly (P
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Conversion pregnenolone secretion conversion inhibited inhibited
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AND
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to progesterone:
(12.5 to 100 pM) significantly
(PgO.01)
RESEARCH
COMMUNICATIONS
Addition
of exogenous
increased
progesterone
by luteal cells (Fig. 6), but when 170 pM gossypol was added, the of exogenous pregnenolone to progesterone was significantly (P
(P
Discussion Corpora lutea are the primary site of progesterone biosynthesis during the secretory phase or luteal phase in normal cycling females and during early stages of pregnancy. Progesterone is necessary to regulate a normal cycle, control embryo implantation and maintain normal pregnancy in females. Gossypol, initially considered as a male antifertility agent, has been shown to possess inhibitory effects on testicular steroidogenesis in both mature (11,12) and immature rats (13,14). Recently, the successful treatments of menorrhagia and endometriosis in China (15) indicated that gossypol might alter the endocrine function of the reproductive system in females as well. Direct evidence was later reported by Wang et al. (16) that gossypol inhibited hCG-stimulated CAMP formation, and hCG- and CAMP-stimulated progesterone production in cultured rat luteal cells. We have reported that gossypol inhibited hCG- and forskolin-induced intracellular cAMP formation and progesterone secretion in bovine luteal cells in vitro (6). The present study illustrates that gossypol also inhibits steroidogenesis at a site subsequent to CAMP formation
in
cultured luteal cells. Luteal tissue utilizes cholesterol asa major source of substrate for progesterone synthesis (17). Cholesterol is first converted to pregnenolone by side-chain cleavage enzyme complex (18). This is considered to be a rate-limiting step in luteal steroidogenesis (19,20). Pregnenolone is later converted to progesterone in luteal cells by 3&hydroxysteroid dehydrogenase-isomerase complex (3I3-HSD). Although 3B-HSD is not considered a rate-limiting step for steroidogenesis (21), it plays a key role in the synthesis of progesterone in luteal cells. During progesterone synthesis in luteal cells, the availability of intracellular cholesterol and the activities of steroidogenic enzymes, particularly side-chain cleavage enzyme complex, are believed to be mediated by hormones and cAMP (22). Addition of exogenous dbcAMP significantly stimulated progesterone secretion in present study. However, gossypol arrested the stimulative effect of dbcAMP completely (Fig. 1). This result demonstrates that gossypol indeed affects steps along the steroidogenic pathway subsequent to cAMP formation in luteal ceiij, as proposed by other investigators (16,23).
Pearce et al. (23) reported that gossypol, at concentrations similar to that employed in our study, inhibited dbcAMP-stimulated testosterone secretion in 459
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2, 1990
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AND
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purified mouse Leydig cell suspension, but it had little pregnenoloneand 25-OH cholesterol-enhanced testosterone
COMMUNICATIONS
effect on exogenous secretion. Therefore,
they concluded that gossypol exerted its inhibitory effect at a site subsequent CAMP formation, but prior to the conversion of cholesterol to pregnenolone. contrast, our study clearly demonstrated that gossypol stimulated progesterone secretion, but also significantly OH cholesterol The differences
and pregnenolone in results between
to In
not only inhibited dbcAMPinhibited conversions of 25-
to progesterone the two studies
in cultured bovine luteal cells. may be due to the variation of
animal species, cell types and assay conditions. steroidogenesis and the sensitivity of responsiveness
The control mechanism of to chemicals, such as gossypol,
in bovine luteal cells may also differ from that in mouse Leydig cells. The presence
of
BSA (0.1 Oh) in assay suspension may contribute an inactivation of gossypol in Pearce’s study since serum protein prevents the biological activity of gossypol (24-26). Addition
of exogenous
25-OH cholesterol
enhanced
progesterone
secretion
in
a dose-dependent manner (Fig. 2). Gossypol, at 17 pM or higher concentration, significantly inhibited the substrate-enhanced conversion (Fig. 2 & 3). These results demonstrates of cholesterol
that gossypol inhibits to progesterone.
The conversion
of cholesterol
steroidogenesis
to pregnenolone
at step(s)
within
the conversion
is catalyzed
by side-chain
cleavage enzyme complex in luteal cells. Addition of exogenous 25-OH cholesterol resulted in an increase of pregnenolone level in the culture medium in a substrate concentration-dependent manner (Fig. 4). Gossypol, at 170 PM, significantly inhibited the conversion (Fig. 4). However, at low doses (4.25 to 34 PM), gossypol did not significantly inhibit this substrate-enhanced conversion (Fig. 5). The inhibited conversion of exogenous 25-OH cholesterol to pregnenolone with a high dosage of gossypol may lie on the cytotoxic effect. It has been reported that gossypol at high concentrations cells (26,27).
and prolonged culture times in vitro was cytotoxic for many types of However, as indicated by the trypan blue exclusion method in the
present study, the viability of luteal cells treated with gossypol, even at 170 PM, did not show an obvious difference from that of the controls. Therefore, we assume that antisteroidogenic effect of gossypol in cultured bovine luteal cells may not be caused by the cytotoxic effect of gossypol. Pregnenolone is finally converted to progesterone by 3&HSD in luteal cells. Addition of exogenous pregnenolone, as a substrate, greatly increased progesterone secretion in the luteal cells (Fig. 6). In general, 3l3-HSD is not a ratelimit enzyme in steroidogenesis and it is present in great excess in luteal cells. Nevertheless, gossypol significantly inhibited this substrate-enhanced (Fig 6), even at the dose as low as 8.5 PM (Fig. 7). These results strongly gossypol inhibits the activity of 38-HSD. 460
conversion suggest that
Vol. 169, No. 2, 1990
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AND BIOPHYSICAL
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Our results clearly illustrate that gossypol inhibits dbcAMP-stimulated progesterone synthesis in bovine luteal cells. The inhibitory effect of gossypol is exerted subsequent to CAMP formation.
The inhibited conversions of exogenous 25-
OH cholesterol and pregnenolone to progesterone strongly suggest that gossypol inhibits the activity of 3&HSD, but probably not side-chain cleavage enzyme complex. Based on the data from previous and present studies, it is reasonable to conclude that gossypol exerts its inhibitory bovine luteal cells at multiple sites.
effect on progesterone biosynthesis in
Acknowledgments This study was supported by grants from March of Dimes Birth Defects Foundation and the Food and Drug Administration (FD-U-000-354). The authors wish to thank Falter Herman Packing Company, Columbus, Ohio for supplying the bovine ovaries used for our study. References Yang Y.Q. and Wu X.Y. (1987) J. Reprod. Fert. 80,425-429. Yuan Q.X., Gao D.W., AND Li C.Z. (1983) Reprod. Contraception 3,25-30. 3: Gu Y. and Anderson N.O. (1985) Contraception 32,491-496. 4. Lin Y.C., Fukaya T., and Rikihisa Y. (1984) Biol. Reprod. 30 (Suppl 1),103. Lin Y.C., Fukaya T., Rikihisa Y., and Walton A. (1985) Life Sciences 37,39-47. Z: Gu Y., Chang C.J.G., Rikihisa Y., and Lin Y.C. (1989) FASEB J. 3(3), 3717A. 7. Poff J.P., Fairchild D.L., and Condon W.A. (1988) J. Reprod. Fert. 82, 135-143. Tennant J.R. (1964) Transplantation 2,685-694. ii: Stouffer R.L., Nixon W.E., Gulyas B.J., Johnson D.K., and Hodgen G.D. (1976) Steroids 27(4), 1106-I 113. 10. Abraham G.E. (1969) J. Clin. Endocrinol. Metab. 29,866-870. 11. Hadley M.A., Lin Y.C., and Dym M. (1981) J. Androl. 2,190-199. 12. Lin T., Murono E.P., Osterman J., Nankin H.R., and Coulson P.B. (1981) Fert. Steril. 35, 563-566. 13. Kalla N.R., Weinbauer G.F., Rovan E., and Frick J. (1983) J. Androl. 4,331-335. 14. Lin Y.C., Fukaya T., Rikihisa Y., and DeSanto T. (1985) Adv. Contracept. Deliv. System. Monograph 2,200-206. 15. Cheng K.F., Wu W.Y., Tang M.Y., and Chu P.T. (1980) Am. J. Obstet. Gynecol. 138,1227-1229. 16. Wang N.G., Guan M.Z., and Lei H.P. (1987) J. Ethnopharmacol. 20,45-51. 17. Azhar 5. and Menon K.M.J. (1981) J. Biol. Chem. 256,6548-6555. 18. Savard K. (1973) Biol. Reprod. 8,1 83-202. 19. Stone D. and Hechter 0. (1954) Arch. Biochem. Biophys. 51,457-469. Hall P.F. and Koritz S.B. (1964) Biochemistry 30, 129-134. 57: Caffrey J.L., NettT.M., Abel Jr. J.H., and Niswender G.D. (1979) Biol. Reprod. 20, 279-287. Hoyer P.B. and Niswender G.D. (1985) Can. J. Physiol. Pharmacol. 63,240-248. S f : Pearce S., Sufi S.B., O’Shaughnessy P.J., Donaldson A., and Jeffcoate S.L. (1986) J. Steroid. Biochem. 25,683-687. 24. Whaley K.J., Sampath D.S., and Balaram P. (1984) Biochimica et Biophysics Acta :-
801,127-130. 25. f S:
Haspel H.C., Ren Y.F., Watanabe K.A., Sonenberg M., and Corin R.E., (1983) J. Pharm. Exp. Therap. 229,218-225. Joseph A.E.A., Matlin S.A., and Knox P. (1986) Br. J. Cancer 54, 51 l-513. Ye W.S., Liang J.C., and Hsu T.C. (1983) In Vitro 19,53-57.
461
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 455-461
INHIBITORY EFFECTOF GOSSYPOL ON STEROIDOGENIC PATHWAYS IN CULTUREDBOVINE LUTEAL CELLS Y. Gu*, Y.C. Lin” and Y. Rikihisa”” Laboratory of Reproductive Endocrinology, Departments of *Veterinary Physiology and Pharmacology , and **Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210-1092 Received
April
20,
1990
Gossypol inhibits the reproductive s stem and steroidogenesis in both sexes. The present study investigated some possiti le sites subsequent to CAMP formation at which gossypol may inhibit progesterone biosynthesis. Bovine luteal cells were cultured with dibutyryl CAMP (dbcAMP), 25-OH cholesterol, or pregnenolone in the presence or absence of gossypol. Gossypol, at 17-34 gM, inhibited dbcAMP-induced progesterone secretion. Gossypol significantly inhibited the conversions of exogenous 25-OH cholesterol and pregnenolone to progesterone. However, the conversion of 25-OH cholesterol to pregnenolone was not significantly inhibited by gossypol at low doses (534 pM). These results suggest that gossypol inhibits progesterone synthesis in bovine luteal cells by suppressing steroidogenic enzyme activity. 0 1990Academic Press,fnc.
Gossypol, a natural polyphenolic compound, is a male antifertility agent and it has been reported that gossypol has an antifertility effect in females as well (1,2). We previously reported that gossypol interrupted normal estrous cycles (3) and inhibited embryo implantation(4,5) in rats. Recently, we also reported that gossypol inhibited human chorionic gonadotropin (hCG)-induced cyclic adenosine monophosphate (cAMP) formation
and progesterone secretion in cultured bovine
luteal cells (6). In the present study, we examined sites subsequent to hCG- induced CAMP formation at which gossypol might also inhibit progesterone synthesis in bovine luteal cells in vitro.
Materials and Methods Fetal bovine serum (FBS, lot:b79306) was purchased from Armour Pharmaceutical Co. (Kankakee, IL). 1,2,6,7,21-[3H(N)]-Progesterone (Lot:2370-059) was purchased from New England Nuclear (Boston, MA) and delta-5 I4,7-3Hl Pregnenolone (Batch 39) was purchased from Amersham (Arlington Heights, IL). Progesterone antiserum was obtained from Endocrine Sciences (Tarzana, CA) and pregnenolone antiserum was purchased from Scantibodies Laboratory Inc. (Lot: T463C, Santee, CA). Collagenase (type I, Lot:77380M) was urchased from Worthington Biochemical Co. (Freehold, NJ). Dulbecco’s Modifie d” Eagle’s Medium and Ham’s Nutrient Mixture F-12 and remaining reagents were purchased from Sigma Chemical Co. (St. Louis, MO). 0006-291XE'O 455
$1.50
Copyright 0 1990 by Academic Press, Inc. AN rights of reproduction in any form reserved.
Vol. 169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Preparation and culture of bovine luteal cells: Bovine ovaries containing corpora lutea were obtained from a local slaughterhouse and transported back to the laboratory in iced phosphate buffered saline (PBS) within an hour of slaughter. The corpora lutea with a size 14x14~12 mm3 were selected for luteal cell isolation. The corpora lutea were carefully dissected from ovaries on ice. The luteal cells were isolated as described by Poff et al. (7). Briefly, corpora lutea were sliced and minced to about 1 mm3. The luteal cells were dissociated at 35°C with collagenase (2000 U/g luteal tissue) and 0.5% bovine serum albumin (BSA) in Dulbecco’s Modified Eagle’s Medium and Ham’s nutrient mixture F-12 containing peniciflin G, 100 units/ml; streptom tin, 0.1 mg/ml; and amphotericin 8, 0.25 pg/ml (DME/F-12). After one hour milJ stirring, the cells were washed 4 times with fresh DME/F-12 containing no BSA by centrifu ation for 10 min in a series of descending speeds, from 150 to 100 x g at 4°C. The cef Is were counted with a hemocytometer. The viabilit of the cells was determined by the trypan blue exclusion method (8). B this metho dy, approximately 17.4 xl06 viable luteal cells per gram of tissue were yie Yded with a 20-30:80-70 ratio of large (>20 pm) to small (8-20 pm) cell population. The viability of yield cells were about 70%. All culture dishes were pretreated with 10% FBS in DME/F-12 to provide essential attachment factors. Approximately 106 viable bovine luteal cells were seeded in each dish with 5 ml of serum free, hormone supplemented DME/F-12 (DME/F-12/S), which contains insulin, 5 pg/ml; transferrin, 5p /ml; epidermal growth factor (EGF), 10 rig/ml; sodium selenite 10 ngiml. After 18 R our culture, all dishes were washed twice with DME/F-12 and an additional 3-hour culture was carried out in a total volume of 5 ml DME/F-12/S with different treatments. Treatment 1: the luteal cells were cultured with either 50 or 100 pM dbcAMP and gossypol (0, 17 and 34 pM). Treatment 2: the cells were cultured with various concentrations of 25-OH cholesterol and ossypol. Treatment 3: the cells were cultured with various concentrations o9 pregnenolone and ossypol. Gossypol and steroids were first dissolved in 100% ethanol and then di7 uted so that the maximal concentration of ethanol in the culture medium was 0.25Oh. This concentration of ethanol did not affect progesterone secretion (data not shown). All treatments were performed in triplicate. At the end of each treatment, the medium was collected and stored at 20°C until steroid content determination. Progesterone and pregnenolone determination: The collected culture medium was extracted with 1:7 (V/V) ratio of medium to ethyl ether. Ether phase was separated from aqueous phase by flash freezing. The ether phase was evaporated under N2 and reconstituted with PBS containin 0.1% elatin. The progesterone and pre nenolone contents were determine 8 by ra%ioimmunoassay (RIA) as describe % by Stouffer et al. (9). The intra- and interassay coefficients of variation were 4.3% and 14.2% for progesterone RIA, and were 4.9O/6 and 16Oh for pre nenolone RIA, respectively. The sensitivity of assay was about 64 fmoles/tube for %0th RIAs. The cross-reactivity of pregnenolone antiserum with progesterone was 1%. The cross-reactivity of progesterone antiserum with pre nenolone was 15Oh,using 5Ooh inhibition of binding method, as described by Abra R am (10). Since the steroids in our measurements were not chromatographically separated, the values of steroids in all measurements may be higher than actual values expressed. All standards and unknowns were performed in duplicate. The secretions of progesterone and pregnenolone were expressed as pmoles or nmoles/hour/lO6 cells. Statistical analysis: Differences between treatment means were measured by analysis of variance and Duncan or Bonferroni multiple comparison procedures. In all comparisons, differences were considered significant at P
Results dbcAMP-induced progesterone secretion: Progesterone secretion by either 50 or 100 PM dbcAMP-stimulated bovine luteal cells was significantly (P
Vol.
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
No dbcAMP lOO/kM dbcAMP 50 fl dbcAMP
3 f ” 2.0 “0 > f 1.5 4 E 5 1.0
m
NO gossypol
I
170 /AM gossypol
i 0 0.5 D r a’ 0 Control
0
0
1
Gossypol
17 (!A)
34
0
0
12.5
2
in medium
25-OH
25 cholesterol
50
100
(IrM) in medium
Fi ure 1. Effect of gossypol on dbcAMP-induced progesterone secretion in cultured 8---r ovme uteal cells. dbcAMP (50 and 100 PM) significantly stimulated progesterone secretion. Gossypol(17 and 34 PM) inhibited the stimulative effect of dbcAMP. Fiaure 2. Effect of gossypol on 25-OH cholesterol-enhanced progesterone secretion in cultured bovine luteal cells. Gossypol (170 pM) completely abolished the conversion of 25-OH cholesterol to progesterone (P
inhibited Although
pM dbcAMP-stimulated progesterone secretion in luteal cells. gossypol diminished the stimulative effect of 50 pM dbcAMP (Fig. l), the 100
inhibition was not statistically significant. Our previous data showed that higher concentrations of gossypol (>34 PM) did not result in further inhibition of progesterone secretion (data not shown). Conversion of 25OH cholesterol to progesterone: Addition of exogenous 25OH cholesterol (12.5 to 100 PM) markedly enhanced progesterone secretion by the luteal
cells in a dose-dependent
manner
(Fig. 2).
When
170 pM
of gossypol
was
added, the conversion of 25-OH cholesterol to progesterone by the luteal cells was
3g ” “0
Mean f SD. 50
m I
No 25-OH cholestero 100 MM 25-OH cholesterol
3 t” “0
KL 40 f i 30 s t ; g z a’
03
Mean f S.D.
10 0 c.antrcA
inhibited
0
0
12.5
50 &A) in medium
Effect of gossypol on 25-OH cholesterol-enhanced progesterone bovine luteal cells. Progesterone secretion was significantly by gossypol at concentration of 17 pM and higher dose.
secretion (P
17
34
25-OH
25 cholesterol
Gossypol 0 4.25 @A) in medium a.5
4
170 M gossyp0l NO goasypol
75
5 f 4 50 E & z + 25 : 6 h 0
20
I m
Fi ure 4. Effect of gossypol on the conversion of 25-OH cholesterol to pre nenolone +-In cu tured bovine luteal cells. Gossypol (170 PM) completely abo Prshed the conversion. Pregnenolone in culture medium was measured by RIA. 451
100
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169, No. 2, 1990
Maon
c H
f
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= I
SD.
N-3
No 25-OH cholesterol 100 jd.4 25-OH cholesterol
30
ContraI
05
0
4.25
Gossypol
a.5
@i)
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
17
I m
34
0
12.5
06
in medium
Pregnenolone
25
170 phi gossypol NO goS.ypo,
50
100
(JAM) in medium
Fi ure 5. Effect of ossypol on the conversion of 25-OH cholesterol to pregnenolone --In-r cu tured bovine 9 uteal cells. The conversion was slightly reduced by ossypol, but the differences were not statistically significant compared with t il at In 0 pM gossypol-treated cells (P>O.O5). Fi ure 6. Effect of gossypol on the conversion of pre nenolone to progesterone -7-x. cu ture bovme luteal cells. Gossypol (170 PM) sign1 .Pscantly (P
in the
completely (P
of gossypol.
Conversion of 2IOH cholesterol to pregnenolone: Addition of exogenous 25OH cholesterol (12.5 to 100 pM) also significantly increased pregnenolone level in the culture medium in a dose- dependent manner (Fig. 4). Gossypol, 170 pM, again completely abolished (PXO.01) the conversion (Fig. 4). However, gossypol at low doses (4.25 to 34 pM) did not significantly inhibit the conversion of 25-OH cholesterol to pregnenolone (Fig. 5).
3 s mi 800.
Yean *so. N-3
= I
3 c acetl B
No pngnanolone 25 +I pregnenolons
* .
4 400. B 1
*oo-
8
OJ
.
control
0 4.25 Gcdypol @I)
8.5 in medium
?7
34
Fi we 7. Effect of gossypolon the conversion of pregnenolone to progesterone in luteal cells. Gossypol inhibited the conversion in a dose-dependent
cGi73izabovine
manner. Gossypolsignificantly (P
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Conversion pregnenolone secretion conversion inhibited inhibited
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of pregnenolone
AND
BIOPHYSICAL
to progesterone:
(12.5 to 100 pM) significantly
(PgO.01)
RESEARCH
COMMUNICATIONS
Addition
of exogenous
increased
progesterone
by luteal cells (Fig. 6), but when 170 pM gossypol was added, the of exogenous pregnenolone to progesterone was significantly (P
(P
Discussion Corpora lutea are the primary site of progesterone biosynthesis during the secretory phase or luteal phase in normal cycling females and during early stages of pregnancy. Progesterone is necessary to regulate a normal cycle, control embryo implantation and maintain normal pregnancy in females. Gossypol, initially considered as a male antifertility agent, has been shown to possess inhibitory effects on testicular steroidogenesis in both mature (11,12) and immature rats (13,14). Recently, the successful treatments of menorrhagia and endometriosis in China (15) indicated that gossypol might alter the endocrine function of the reproductive system in females as well. Direct evidence was later reported by Wang et al. (16) that gossypol inhibited hCG-stimulated CAMP formation, and hCG- and CAMP-stimulated progesterone production in cultured rat luteal cells. We have reported that gossypol inhibited hCG- and forskolin-induced intracellular cAMP formation and progesterone secretion in bovine luteal cells in vitro (6). The present study illustrates that gossypol also inhibits steroidogenesis at a site subsequent to CAMP formation
in
cultured luteal cells. Luteal tissue utilizes cholesterol asa major source of substrate for progesterone synthesis (17). Cholesterol is first converted to pregnenolone by side-chain cleavage enzyme complex (18). This is considered to be a rate-limiting step in luteal steroidogenesis (19,20). Pregnenolone is later converted to progesterone in luteal cells by 3&hydroxysteroid dehydrogenase-isomerase complex (3I3-HSD). Although 3B-HSD is not considered a rate-limiting step for steroidogenesis (21), it plays a key role in the synthesis of progesterone in luteal cells. During progesterone synthesis in luteal cells, the availability of intracellular cholesterol and the activities of steroidogenic enzymes, particularly side-chain cleavage enzyme complex, are believed to be mediated by hormones and cAMP (22). Addition of exogenous dbcAMP significantly stimulated progesterone secretion in present study. However, gossypol arrested the stimulative effect of dbcAMP completely (Fig. 1). This result demonstrates that gossypol indeed affects steps along the steroidogenic pathway subsequent to cAMP formation in luteal ceiij, as proposed by other investigators (16,23).
Pearce et al. (23) reported that gossypol, at concentrations similar to that employed in our study, inhibited dbcAMP-stimulated testosterone secretion in 459
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2, 1990
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AND
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RESEARCH
purified mouse Leydig cell suspension, but it had little pregnenoloneand 25-OH cholesterol-enhanced testosterone
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effect on exogenous secretion. Therefore,
they concluded that gossypol exerted its inhibitory effect at a site subsequent CAMP formation, but prior to the conversion of cholesterol to pregnenolone. contrast, our study clearly demonstrated that gossypol stimulated progesterone secretion, but also significantly OH cholesterol The differences
and pregnenolone in results between
to In
not only inhibited dbcAMPinhibited conversions of 25-
to progesterone the two studies
in cultured bovine luteal cells. may be due to the variation of
animal species, cell types and assay conditions. steroidogenesis and the sensitivity of responsiveness
The control mechanism of to chemicals, such as gossypol,
in bovine luteal cells may also differ from that in mouse Leydig cells. The presence
of
BSA (0.1 Oh) in assay suspension may contribute an inactivation of gossypol in Pearce’s study since serum protein prevents the biological activity of gossypol (24-26). Addition
of exogenous
25-OH cholesterol
enhanced
progesterone
secretion
in
a dose-dependent manner (Fig. 2). Gossypol, at 17 pM or higher concentration, significantly inhibited the substrate-enhanced conversion (Fig. 2 & 3). These results demonstrates of cholesterol
that gossypol inhibits to progesterone.
The conversion
of cholesterol
steroidogenesis
to pregnenolone
at step(s)
within
the conversion
is catalyzed
by side-chain
cleavage enzyme complex in luteal cells. Addition of exogenous 25-OH cholesterol resulted in an increase of pregnenolone level in the culture medium in a substrate concentration-dependent manner (Fig. 4). Gossypol, at 170 PM, significantly inhibited the conversion (Fig. 4). However, at low doses (4.25 to 34 PM), gossypol did not significantly inhibit this substrate-enhanced conversion (Fig. 5). The inhibited conversion of exogenous 25-OH cholesterol to pregnenolone with a high dosage of gossypol may lie on the cytotoxic effect. It has been reported that gossypol at high concentrations cells (26,27).
and prolonged culture times in vitro was cytotoxic for many types of However, as indicated by the trypan blue exclusion method in the
present study, the viability of luteal cells treated with gossypol, even at 170 PM, did not show an obvious difference from that of the controls. Therefore, we assume that antisteroidogenic effect of gossypol in cultured bovine luteal cells may not be caused by the cytotoxic effect of gossypol. Pregnenolone is finally converted to progesterone by 3&HSD in luteal cells. Addition of exogenous pregnenolone, as a substrate, greatly increased progesterone secretion in the luteal cells (Fig. 6). In general, 3l3-HSD is not a ratelimit enzyme in steroidogenesis and it is present in great excess in luteal cells. Nevertheless, gossypol significantly inhibited this substrate-enhanced (Fig 6), even at the dose as low as 8.5 PM (Fig. 7). These results strongly gossypol inhibits the activity of 38-HSD. 460
conversion suggest that
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BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Our results clearly illustrate that gossypol inhibits dbcAMP-stimulated progesterone synthesis in bovine luteal cells. The inhibitory effect of gossypol is exerted subsequent to CAMP formation.
The inhibited conversions of exogenous 25-
OH cholesterol and pregnenolone to progesterone strongly suggest that gossypol inhibits the activity of 3&HSD, but probably not side-chain cleavage enzyme complex. Based on the data from previous and present studies, it is reasonable to conclude that gossypol exerts its inhibitory bovine luteal cells at multiple sites.
effect on progesterone biosynthesis in
Acknowledgments This study was supported by grants from March of Dimes Birth Defects Foundation and the Food and Drug Administration (FD-U-000-354). The authors wish to thank Falter Herman Packing Company, Columbus, Ohio for supplying the bovine ovaries used for our study. References Yang Y.Q. and Wu X.Y. (1987) J. Reprod. Fert. 80,425-429. Yuan Q.X., Gao D.W., AND Li C.Z. (1983) Reprod. Contraception 3,25-30. 3: Gu Y. and Anderson N.O. (1985) Contraception 32,491-496. 4. Lin Y.C., Fukaya T., and Rikihisa Y. (1984) Biol. Reprod. 30 (Suppl 1),103. Lin Y.C., Fukaya T., Rikihisa Y., and Walton A. (1985) Life Sciences 37,39-47. Z: Gu Y., Chang C.J.G., Rikihisa Y., and Lin Y.C. (1989) FASEB J. 3(3), 3717A. 7. Poff J.P., Fairchild D.L., and Condon W.A. (1988) J. Reprod. Fert. 82, 135-143. Tennant J.R. (1964) Transplantation 2,685-694. ii: Stouffer R.L., Nixon W.E., Gulyas B.J., Johnson D.K., and Hodgen G.D. (1976) Steroids 27(4), 1106-I 113. 10. Abraham G.E. (1969) J. Clin. Endocrinol. Metab. 29,866-870. 11. Hadley M.A., Lin Y.C., and Dym M. (1981) J. Androl. 2,190-199. 12. Lin T., Murono E.P., Osterman J., Nankin H.R., and Coulson P.B. (1981) Fert. Steril. 35, 563-566. 13. Kalla N.R., Weinbauer G.F., Rovan E., and Frick J. (1983) J. Androl. 4,331-335. 14. Lin Y.C., Fukaya T., Rikihisa Y., and DeSanto T. (1985) Adv. Contracept. Deliv. System. Monograph 2,200-206. 15. Cheng K.F., Wu W.Y., Tang M.Y., and Chu P.T. (1980) Am. J. Obstet. Gynecol. 138,1227-1229. 16. Wang N.G., Guan M.Z., and Lei H.P. (1987) J. Ethnopharmacol. 20,45-51. 17. Azhar 5. and Menon K.M.J. (1981) J. Biol. Chem. 256,6548-6555. 18. Savard K. (1973) Biol. Reprod. 8,1 83-202. 19. Stone D. and Hechter 0. (1954) Arch. Biochem. Biophys. 51,457-469. Hall P.F. and Koritz S.B. (1964) Biochemistry 30, 129-134. 57: Caffrey J.L., NettT.M., Abel Jr. J.H., and Niswender G.D. (1979) Biol. Reprod. 20, 279-287. Hoyer P.B. and Niswender G.D. (1985) Can. J. Physiol. Pharmacol. 63,240-248. S f : Pearce S., Sufi S.B., O’Shaughnessy P.J., Donaldson A., and Jeffcoate S.L. (1986) J. Steroid. Biochem. 25,683-687. 24. Whaley K.J., Sampath D.S., and Balaram P. (1984) Biochimica et Biophysics Acta :-
801,127-130. 25. f S:
Haspel H.C., Ren Y.F., Watanabe K.A., Sonenberg M., and Corin R.E., (1983) J. Pharm. Exp. Therap. 229,218-225. Joseph A.E.A., Matlin S.A., and Knox P. (1986) Br. J. Cancer 54, 51 l-513. Ye W.S., Liang J.C., and Hsu T.C. (1983) In Vitro 19,53-57.
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