- Email: [email protected]
Stimulatory effects of gonadotropins on the formation of cyclic adenosine 3′,5′-monophosphate by porcine granulosa cells
BIOCHIMICAET BIOPHYSICAACTA
601
BBA Report BBA 21320
Stimulatory effects of gonadotropins on the formation of cyclic adenosine 3',5'-monophosphate by porcine granulosa cells JAROSLAV KOLENA~"and CORNELIA P. CHANNING Department o f Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213 (U.S.A.)
(Received November 2nd, 1971)
SUMMARY
The addition of luteinizing hormone (LH) or follicle stimulating hormone (FSH) to porcine granulosa cells stimulated cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation from intraceUularly labeled ATP. The LH stimulatory effect was larger than that of FSH. The effects of maximal doses of LH and FSH were not additive in the presence or absence of a phosphodiesterase inhibitor, aminophylline. In view of the previous finding that exogenous cyclic AMP can mimic the effect of gonadotropins upon luteinization of granulosa cells, these results indicate that cyclic AMP is a mediator of this action of gonadotropins.
Dibutyryl cyclic AMP as well as LH or FSH addition to rhesus monkey and porcine granulosa cell cultures causes morphological luteinization and a stimulation in progestin secretion 1,2. Since the effects of dibutyryl cyclic AMP and the two gonadotropins were similar it was postulated that LH and FSH exerted their luteinizing action via a stimulation in cyclic AMP production. In order to test this hypothesis the influence of LH and FSH upon the formation of cyclic AMP from intraceUularly labeled ATP in porcine granulosa cells was estimated. The porcine granulosa cell system is advantageous for these studies because it consists of a completely homogenous intact cell population 3 which is not disrupted by enzymatic digestion or other treatment. Ovaries were obtained from 4-6-month-old pigs from a local slaughterhouse and granulosa cells were harvested from 3-5-mm follicles as outlined previously except that the cells were aspirated with a syringe and 20 gauge needle instead of a platinum loop 3. Abbreviations: cyclic AMP, cyclic adenosine 3°,5S-monophosphate;dibutyryl cyclic AMP, N6-2-O-dibutyryl cyclic adenosine 3°,5°-monophosphate; LH, luteinizing hormone; FSH, follicle stimulating hormone. ~rpresent address: Institute of Experimental Endocrinology, Slovak Academy of Science, Limbova cesta, Bratislava, Czechoslovakia. Biochim. Biophys. Acra, 252 (1971) 601-606
602
BBA REPORT
Granulosa cells were preincubated at 37 ° for 1 h in a shaker with 1/~C/ml [14C8J adenine (spec. act., 52.6 mC/mmole; New England Nuclear Co., Boston, Mass.) in Eagle's medium with Earle's salts 4 (pH 7.4; Grand Island Biological Co., Grand Island, N.Y.). 5% CO2 in air was the gas phase. After preincubation, which brought on formation of intracellularly labeled ATP s, 6, the cells were separated by centrifugation at 700 rev./min in a Model UV International centrifuge and divided into incubation flasks. One flask contained about 107 cells per ml in 1 ml Eagle's medium. Cells were counted in a hemocytometer in 0.2% Lissamine green in Hanks' balanced salt solution which has been demonstrated previously to stain viable cells 7. Hanks' balanced salt solution 8 was obtained from Grand Island Biological Co. Hormones and aminophylline were added and the incubation was continued for an additional 20 rain. Conditions for the incubation were the same as for the preincubation except that no [14C8] adenine was added during the incubation and that hormones and other agents were present only during the incubation period. At the end of the incubation period the cells were centrifuged at 1000 rev./min and the supernatant incubation medium discarded. The cells were then sonicated in 0.5 ml 0.1 M HC1 containing 0.4 mM each of ATP, ADP, AMP, cyclic AMP, adenine and tracer amounts of cyclic [3H8] AMP (16 mC/mmole; Schwarz/Mann, Orangeburg, N.Y.) to monitor the recovery of cyclic AMP. The addition of unlabeled nucleotides enabled localization of these nucleotides as they came off the column by ultraviolet spectroscopy. Cyclic [ ~4C] AMP was separated from other interfering nucleotides by 4-fold ZnSO4-Ba(OH)2 precipitation and column chromatography on Dowex 50 - X4 (H + form) 9. The eluate containing labeled cyclic AMP was mixed with 15 ml of phosphor mixture and the radioactivity was measured in a Packard liquid scintillation spectrometer with a 55% detection efficiency for 14C and 28% for 3H. The results were corrected for cyclic [3H] AMP recovery. Nucleotide elution patterns obtained from the Dowex 50 (H + form; 200--400 mesh) columns were similar to those obtained by Krishna et al. 9. Adequate separation of cyclic AMP from ATP was monitored by chromatographing a mixture of trace amounts of cyclic [3 Hs] AMP and [14C8] ATP (16 mC/mmole; Schwarz/Mann). Evidence for radiochemical purity of cyclic AMP isolated according to the method of Krishna et al. has been presented by others s, 6,9. Evidence that the column chromatography plus the ZnSO4-Ba(OH)2 precipitation achieved radiochemical purity of the cyclic [14C] AMP produced by granulosa cells from intracellularly labeled ATP was confirmed in the following experiments. Dowex column cyclic AMP fractions from several incubations were pooled and subjected to a 4-fold ZnSO4-Ba(OH)2 precipitation, A portion of the supernatant was mixed with 50 mg carrier cyclic AMP, dissolved by heating in boiling water and crystallized by cooling. The crystals were removed by centrifugation and washed with ethanol and counted and the process repeated 3 times. The 14C to 3H ratio of the successive crystals was similar to the Ba(OH)2-ZnSO4 supernatant starting material (Table I). Parallel experiments have been carried out using similar incubation conditions and a similar method of isolation and column purification of cyclic AMP except that no [~4C] adenine substrate was used and the amount of intracellular cyclic AMP was measured according to the method of Kaneko and Field 1°. Unlabeled cyclic AMP produced by control or LH-treated cells was eluted from the Dowex columns in the same region as was cyclic [14C] AMP in the present experiments 11, 12. In both types of experiments cyclic [3H]AMP was used as a marker. Biochim. Biophys. Acta, 252 (1971) 601-606
BBA REPORT
603
TABLE I CRYSTALLIZATION OF CYCLIC [14Cl AMP PRODUCED BY PORCINE GRANULOSA CELLS FROM INTRACELLULARLY LABELED [ 14C]ATP The Ba(OH)z-ZnSO4 supernatant fluid containing tracer cyclic [3HI AMP and cyclic [14C] AMP produced by two samples of porcine granulosa cells was crystallized in water. Initially Sample 1 contained 2570 counts/min 3H and 1620 counts/min 14C and Sample 2 contained 3650 counts/min 3H and 1020 counts/rain 14C. Counts~rain 14C/counts/min aH
Before crystallization First crystallization Second cry stallization Third crystallization Fourth crystallization
Sample 1
Sample 2
0.63 0.66 0.59 0.56 0.59
0.28 0.36 0.33 0.33 0.33
Evidence that the cells do produce intracellular [14C]ATP from adenine during the preincubation period was obtained and it is likely that this is used as substrate for cyclic AMP production during the subsequent incubation period. Aliquots of granulosa cells were subjected to premcubation alone and the 0.1 M HCI extract of the cells chromatographed on the Dowex columns. The ATP region was collected, counted, pooled, mixed with carrier ATP and crystallized 3 times as above. After the first crystallization which removed an impurity which accounted for less than 20% of the ~4C, the specific activity of successive crystallizations did not change. A similar experiment was done after subsequent incubation with nothing or 2 pg LH for 20 rain. [14C] ATP levels after the 20-min incubation with nothing or LH were about half the levels at the end of the preincubation. Porcine FSH (0.76 X NIH-FSH-S1) which contained 0.0075 NIH-LH-S1 units/rag and ovine LH, NIH-LH-S 13, (0.93 × NIH-LH-S 1) which contained less than 0.014 NIH-FSH-S1 units/mg, were used in each incubation. Hormones were kept frozen at a concentration of 1 mg/ml and diluted immediately prior to use. As shown in Figs. 1-3, granulosa cells converted intracellularly labeled ATP to cyclic AMP and the levels of this nucleotide were markedly increased by addition of 2.0 ~tg/ml ovine LH or porcine FSH. In additional experiments the minimally effective doses of LH and FSH in stimulation of cyclic AMP production were less than 0.02 and 0.2 ~g/ml, respectively. The maximal doses of LH and FSH were 0.2 and 2 vg/ml, respectively. LH at a dose of 0.2, 2 or 5 #g/ml produced a similar stimulation. Such doses were similar to those which stimulated cyclic AMP levels in porcine granulosa cells in the absence of exogenous precursors 11,12 and stimulated morphological luteinization and progestin secretion by porcine granulosa cell cultures 3. Dorrington and Baggett 13, who observed that 10 tag/ml LH stimulated adenyl cyclase in homogenates of rabbit ovarian tissue, were unable to observe stimulatory effects of 10/ag/ml FSH. Fontaine et al. ~4 using prepubertal rat ovarian homogenates, however, were able to observe a stimulatory effect of both LH and FSH upon adenyl cyclase which is in agreement with the present results. Although the FSH preparationwas contaminated with LH (0.0075 NIH-LH-S 1 units/rag) the effectiveness of the FSH in stimulation of cyclic AMP formation was not only due to its LH contaminant. Biochim. Biophys. Acta, 252 (1971) 601-606
604
BBA REPORT
Ld 44 Z
20001
~J
__0
E
I000
0 u o_
U
_.1 >-
CONTROl..
FSH
LH
L. H F~'H
Fig. 1. Effect of LH (2.0 pg/ml) and FSH (2.0 tzg/ml) alone compared to a combination of LH and FSH upon formation of cyclic AMP in porcine granulosa ceils. N=4, In this figure and in those following the asterick (~) indicates that the values shown are significantly greater than control values (P < 0.01) using a Fishers t test. Here and in subseque,nt experiments incubations with hormones were carried out for 20 min. P
300~
F
.9.0
1
2:00(
.E
8
IO00
Q.
_o
,...i '0 L,,)
T o C~I'tY~L
AMINOPH.
AM.~.INOPH. FSH
~OPI'I.
LH
AMINOPH.
L+H
Fig. 2. The stimulation of cyclic AMP formation in porcine granulosa cells by gonadotropins (2.0 #/ml) in the presence of 1.5 mM aminophyllineo N=4.
Biochim. Biophys. Acta, 252 (1971) 601-606
BBA REPORT '~, i,d
605
200C
"t4 Z ,< bJ
._c E
Z
ioo(
g u
<
r-d. v U
a
CONTROL
L H
AMINOPH.
L.,H AMINOPH. r
Fig. 3. Effect of 1.5 mM aminophylline and 2.0 ~g/ml LH upon formation of cyclic AMP in porcine granulosa ceUs.N=4, In the presence of more than a maximal dose of LH (2.0/sg/ml) addition of 2.0/ag/ml FSH produced no further stimulation in cyclic AMP formation by ,porcine granulosa cells in the absence (Fig. 1) as well as in the presence (Fig. 2) of 1.5 mM aminophyUine. This rules out apparent synergism between LH and FSH and may indicate that, at maximal doses, both gonadotropins stimulate adenyl cyclase in a similar or closely related manner. The addition of aminophylline alone or with LH did not increase the formation of cyclic AMP from intracellularly labeled ATP (Fig. 3). However, when the total content of cyclic AMP was determinated, without labeled precursor, it was found that aminophylline potentiated the effect of gonadotropins 11,12. This is to be compared to studies on cultures of granulosa cells in which it was observed that addition of 0.5 mM aminophylline with or without cyclic AMP stimulated progestin secretion 1,1 s These results, along with the previous demonstration that exogenous cyclic AMP can mimic the action of gonadotropins on progestin synthesis in cultures of granulosa cells ]' 2, 16 and that LH and FSH stimulate cyclic AMP levels in porcine granulosa cells 11,12 indicate that cyclic AMP is an intermediate in the action of gonadotropins upon granulosa cells. One of the early actions of LH and FSH on porcine granulosa cells is a stimulation of cyclic AMP formation somewhere between adenine and cyclic AMP specifically between ATP and cyclic AMP. The cyclic nucleotide subsequently can act as an intracellular mediator of gonadotropic induced luteinization. This investigation was supported by Contract NIH-70-2256 and Research Grant HD 03315 from the National Institutes of Health, U.S. Public Health Service.J.K. is the recipient of a Population Council Post-Doctoral Fellowship. We thank the National Institutes of Health for a generous supply of porcine FSH and ovine LH. The technical assistance of Mrs. Viki Tsai, Miss Haina Tu and Mr. Thilbert North is appreciated. Biochim. Biophy~ Acta, 252 (1971) 601-606
606
BBA REPOR]
REI:ERENCES 1 C.P. Channing and J.F. Seymour, Endocrinology, 87 (1970) 165. 2 C.P. Channing, Recent Prog. Horm. Res., 26 (1970) 589. 3 C.P. Channing, Endocrinology, 87 (1970) 156 4 H. Eagle, Science, 130 (1959) 432. 5 J.L. ltumes, M. Rounbehler and I:.A. Kuehl, Jr.,Anal. Biochem., 32 (1969) 210. 6 J.l;. Kuo and I.K. Dill, Biochem. Biophys. Res. Commun., 32 (1968) 333. 7 C.P. Channing, J. Endocrinol., 43 (1969) 381. 8 J.H. Hanks and R.E. Wallace, Proc. Soc. Exp. Biol., 71 (1949) 196. 9 G. Krishna, 13. Weiss and B.B. Brodie, J. Pharmacol. Exp. Ther., 163 (1968) 379. 10 T. Kaneko and J.B. Field, J. Lab. 67in. Med., 74 (1969) 682. 11 J. Kolena and C.P. Channing, 53rd Meeting of the Endocrine Society, Salt Francisco, Calif. 1971, Abstr. 151. 12 J. Kolena and C.P. Channing, l£ndocrinology, ( 1971 ) in the press. 13 J. Dorrington and B. Baggett, Endocrinology, 84 (1969) 989. 14 Y.-A. Fontaine, E. Fontaine-Bertrand, M.C. Sahnon and N. Delerue-Lebelle, 6\ R. Acad. Sci. Ser. D, 272 (1971) 1137. 15 C.P. Channing, Proc. ConJl on Regulation of Mammalian Reproduction. National Institutes of ttealth. Bethesda, Md., 1971, in the press. 16 V.J. Cirillo, O.t'. Anderson, E.A. Ham and R.B.L. Gwatkin, Exp. CelIRes., 57 (1969) 39.
Biochim. Biophys. Acta, 252 (1971) 6 0 1 - 6 0 6
601
BBA Report BBA 21320
Stimulatory effects of gonadotropins on the formation of cyclic adenosine 3',5'-monophosphate by porcine granulosa cells JAROSLAV KOLENA~"and CORNELIA P. CHANNING Department o f Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213 (U.S.A.)
(Received November 2nd, 1971)
SUMMARY
The addition of luteinizing hormone (LH) or follicle stimulating hormone (FSH) to porcine granulosa cells stimulated cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation from intraceUularly labeled ATP. The LH stimulatory effect was larger than that of FSH. The effects of maximal doses of LH and FSH were not additive in the presence or absence of a phosphodiesterase inhibitor, aminophylline. In view of the previous finding that exogenous cyclic AMP can mimic the effect of gonadotropins upon luteinization of granulosa cells, these results indicate that cyclic AMP is a mediator of this action of gonadotropins.
Dibutyryl cyclic AMP as well as LH or FSH addition to rhesus monkey and porcine granulosa cell cultures causes morphological luteinization and a stimulation in progestin secretion 1,2. Since the effects of dibutyryl cyclic AMP and the two gonadotropins were similar it was postulated that LH and FSH exerted their luteinizing action via a stimulation in cyclic AMP production. In order to test this hypothesis the influence of LH and FSH upon the formation of cyclic AMP from intraceUularly labeled ATP in porcine granulosa cells was estimated. The porcine granulosa cell system is advantageous for these studies because it consists of a completely homogenous intact cell population 3 which is not disrupted by enzymatic digestion or other treatment. Ovaries were obtained from 4-6-month-old pigs from a local slaughterhouse and granulosa cells were harvested from 3-5-mm follicles as outlined previously except that the cells were aspirated with a syringe and 20 gauge needle instead of a platinum loop 3. Abbreviations: cyclic AMP, cyclic adenosine 3°,5S-monophosphate;dibutyryl cyclic AMP, N6-2-O-dibutyryl cyclic adenosine 3°,5°-monophosphate; LH, luteinizing hormone; FSH, follicle stimulating hormone. ~rpresent address: Institute of Experimental Endocrinology, Slovak Academy of Science, Limbova cesta, Bratislava, Czechoslovakia. Biochim. Biophys. Acra, 252 (1971) 601-606
602
BBA REPORT
Granulosa cells were preincubated at 37 ° for 1 h in a shaker with 1/~C/ml [14C8J adenine (spec. act., 52.6 mC/mmole; New England Nuclear Co., Boston, Mass.) in Eagle's medium with Earle's salts 4 (pH 7.4; Grand Island Biological Co., Grand Island, N.Y.). 5% CO2 in air was the gas phase. After preincubation, which brought on formation of intracellularly labeled ATP s, 6, the cells were separated by centrifugation at 700 rev./min in a Model UV International centrifuge and divided into incubation flasks. One flask contained about 107 cells per ml in 1 ml Eagle's medium. Cells were counted in a hemocytometer in 0.2% Lissamine green in Hanks' balanced salt solution which has been demonstrated previously to stain viable cells 7. Hanks' balanced salt solution 8 was obtained from Grand Island Biological Co. Hormones and aminophylline were added and the incubation was continued for an additional 20 rain. Conditions for the incubation were the same as for the preincubation except that no [14C8] adenine was added during the incubation and that hormones and other agents were present only during the incubation period. At the end of the incubation period the cells were centrifuged at 1000 rev./min and the supernatant incubation medium discarded. The cells were then sonicated in 0.5 ml 0.1 M HC1 containing 0.4 mM each of ATP, ADP, AMP, cyclic AMP, adenine and tracer amounts of cyclic [3H8] AMP (16 mC/mmole; Schwarz/Mann, Orangeburg, N.Y.) to monitor the recovery of cyclic AMP. The addition of unlabeled nucleotides enabled localization of these nucleotides as they came off the column by ultraviolet spectroscopy. Cyclic [ ~4C] AMP was separated from other interfering nucleotides by 4-fold ZnSO4-Ba(OH)2 precipitation and column chromatography on Dowex 50 - X4 (H + form) 9. The eluate containing labeled cyclic AMP was mixed with 15 ml of phosphor mixture and the radioactivity was measured in a Packard liquid scintillation spectrometer with a 55% detection efficiency for 14C and 28% for 3H. The results were corrected for cyclic [3H] AMP recovery. Nucleotide elution patterns obtained from the Dowex 50 (H + form; 200--400 mesh) columns were similar to those obtained by Krishna et al. 9. Adequate separation of cyclic AMP from ATP was monitored by chromatographing a mixture of trace amounts of cyclic [3 Hs] AMP and [14C8] ATP (16 mC/mmole; Schwarz/Mann). Evidence for radiochemical purity of cyclic AMP isolated according to the method of Krishna et al. has been presented by others s, 6,9. Evidence that the column chromatography plus the ZnSO4-Ba(OH)2 precipitation achieved radiochemical purity of the cyclic [14C] AMP produced by granulosa cells from intracellularly labeled ATP was confirmed in the following experiments. Dowex column cyclic AMP fractions from several incubations were pooled and subjected to a 4-fold ZnSO4-Ba(OH)2 precipitation, A portion of the supernatant was mixed with 50 mg carrier cyclic AMP, dissolved by heating in boiling water and crystallized by cooling. The crystals were removed by centrifugation and washed with ethanol and counted and the process repeated 3 times. The 14C to 3H ratio of the successive crystals was similar to the Ba(OH)2-ZnSO4 supernatant starting material (Table I). Parallel experiments have been carried out using similar incubation conditions and a similar method of isolation and column purification of cyclic AMP except that no [~4C] adenine substrate was used and the amount of intracellular cyclic AMP was measured according to the method of Kaneko and Field 1°. Unlabeled cyclic AMP produced by control or LH-treated cells was eluted from the Dowex columns in the same region as was cyclic [14C] AMP in the present experiments 11, 12. In both types of experiments cyclic [3H]AMP was used as a marker. Biochim. Biophys. Acta, 252 (1971) 601-606
BBA REPORT
603
TABLE I CRYSTALLIZATION OF CYCLIC [14Cl AMP PRODUCED BY PORCINE GRANULOSA CELLS FROM INTRACELLULARLY LABELED [ 14C]ATP The Ba(OH)z-ZnSO4 supernatant fluid containing tracer cyclic [3HI AMP and cyclic [14C] AMP produced by two samples of porcine granulosa cells was crystallized in water. Initially Sample 1 contained 2570 counts/min 3H and 1620 counts/min 14C and Sample 2 contained 3650 counts/min 3H and 1020 counts/rain 14C. Counts~rain 14C/counts/min aH
Before crystallization First crystallization Second cry stallization Third crystallization Fourth crystallization
Sample 1
Sample 2
0.63 0.66 0.59 0.56 0.59
0.28 0.36 0.33 0.33 0.33
Evidence that the cells do produce intracellular [14C]ATP from adenine during the preincubation period was obtained and it is likely that this is used as substrate for cyclic AMP production during the subsequent incubation period. Aliquots of granulosa cells were subjected to premcubation alone and the 0.1 M HCI extract of the cells chromatographed on the Dowex columns. The ATP region was collected, counted, pooled, mixed with carrier ATP and crystallized 3 times as above. After the first crystallization which removed an impurity which accounted for less than 20% of the ~4C, the specific activity of successive crystallizations did not change. A similar experiment was done after subsequent incubation with nothing or 2 pg LH for 20 rain. [14C] ATP levels after the 20-min incubation with nothing or LH were about half the levels at the end of the preincubation. Porcine FSH (0.76 X NIH-FSH-S1) which contained 0.0075 NIH-LH-S1 units/rag and ovine LH, NIH-LH-S 13, (0.93 × NIH-LH-S 1) which contained less than 0.014 NIH-FSH-S1 units/mg, were used in each incubation. Hormones were kept frozen at a concentration of 1 mg/ml and diluted immediately prior to use. As shown in Figs. 1-3, granulosa cells converted intracellularly labeled ATP to cyclic AMP and the levels of this nucleotide were markedly increased by addition of 2.0 ~tg/ml ovine LH or porcine FSH. In additional experiments the minimally effective doses of LH and FSH in stimulation of cyclic AMP production were less than 0.02 and 0.2 ~g/ml, respectively. The maximal doses of LH and FSH were 0.2 and 2 vg/ml, respectively. LH at a dose of 0.2, 2 or 5 #g/ml produced a similar stimulation. Such doses were similar to those which stimulated cyclic AMP levels in porcine granulosa cells in the absence of exogenous precursors 11,12 and stimulated morphological luteinization and progestin secretion by porcine granulosa cell cultures 3. Dorrington and Baggett 13, who observed that 10 tag/ml LH stimulated adenyl cyclase in homogenates of rabbit ovarian tissue, were unable to observe stimulatory effects of 10/ag/ml FSH. Fontaine et al. ~4 using prepubertal rat ovarian homogenates, however, were able to observe a stimulatory effect of both LH and FSH upon adenyl cyclase which is in agreement with the present results. Although the FSH preparationwas contaminated with LH (0.0075 NIH-LH-S 1 units/rag) the effectiveness of the FSH in stimulation of cyclic AMP formation was not only due to its LH contaminant. Biochim. Biophys. Acta, 252 (1971) 601-606
604
BBA REPORT
Ld 44 Z
20001
~J
__0
E
I000
0 u o_
U
_.1 >-
CONTROl..
FSH
LH
L. H F~'H
Fig. 1. Effect of LH (2.0 pg/ml) and FSH (2.0 tzg/ml) alone compared to a combination of LH and FSH upon formation of cyclic AMP in porcine granulosa ceils. N=4, In this figure and in those following the asterick (~) indicates that the values shown are significantly greater than control values (P < 0.01) using a Fishers t test. Here and in subseque,nt experiments incubations with hormones were carried out for 20 min. P
300~
F
.9.0
1
2:00(
.E
8
IO00
Q.
_o
,...i '0 L,,)
T o C~I'tY~L
AMINOPH.
AM.~.INOPH. FSH
~OPI'I.
LH
AMINOPH.
L+H
Fig. 2. The stimulation of cyclic AMP formation in porcine granulosa cells by gonadotropins (2.0 #/ml) in the presence of 1.5 mM aminophyllineo N=4.
Biochim. Biophys. Acta, 252 (1971) 601-606
BBA REPORT '~, i,d
605
200C
"t4 Z ,< bJ
._c E
Z
ioo(
g u
<
r-d. v U
a
CONTROL
L H
AMINOPH.
L.,H AMINOPH. r
Fig. 3. Effect of 1.5 mM aminophylline and 2.0 ~g/ml LH upon formation of cyclic AMP in porcine granulosa ceUs.N=4, In the presence of more than a maximal dose of LH (2.0/sg/ml) addition of 2.0/ag/ml FSH produced no further stimulation in cyclic AMP formation by ,porcine granulosa cells in the absence (Fig. 1) as well as in the presence (Fig. 2) of 1.5 mM aminophyUine. This rules out apparent synergism between LH and FSH and may indicate that, at maximal doses, both gonadotropins stimulate adenyl cyclase in a similar or closely related manner. The addition of aminophylline alone or with LH did not increase the formation of cyclic AMP from intracellularly labeled ATP (Fig. 3). However, when the total content of cyclic AMP was determinated, without labeled precursor, it was found that aminophylline potentiated the effect of gonadotropins 11,12. This is to be compared to studies on cultures of granulosa cells in which it was observed that addition of 0.5 mM aminophylline with or without cyclic AMP stimulated progestin secretion 1,1 s These results, along with the previous demonstration that exogenous cyclic AMP can mimic the action of gonadotropins on progestin synthesis in cultures of granulosa cells ]' 2, 16 and that LH and FSH stimulate cyclic AMP levels in porcine granulosa cells 11,12 indicate that cyclic AMP is an intermediate in the action of gonadotropins upon granulosa cells. One of the early actions of LH and FSH on porcine granulosa cells is a stimulation of cyclic AMP formation somewhere between adenine and cyclic AMP specifically between ATP and cyclic AMP. The cyclic nucleotide subsequently can act as an intracellular mediator of gonadotropic induced luteinization. This investigation was supported by Contract NIH-70-2256 and Research Grant HD 03315 from the National Institutes of Health, U.S. Public Health Service.J.K. is the recipient of a Population Council Post-Doctoral Fellowship. We thank the National Institutes of Health for a generous supply of porcine FSH and ovine LH. The technical assistance of Mrs. Viki Tsai, Miss Haina Tu and Mr. Thilbert North is appreciated. Biochim. Biophy~ Acta, 252 (1971) 601-606
606
BBA REPOR]
REI:ERENCES 1 C.P. Channing and J.F. Seymour, Endocrinology, 87 (1970) 165. 2 C.P. Channing, Recent Prog. Horm. Res., 26 (1970) 589. 3 C.P. Channing, Endocrinology, 87 (1970) 156 4 H. Eagle, Science, 130 (1959) 432. 5 J.L. ltumes, M. Rounbehler and I:.A. Kuehl, Jr.,Anal. Biochem., 32 (1969) 210. 6 J.l;. Kuo and I.K. Dill, Biochem. Biophys. Res. Commun., 32 (1968) 333. 7 C.P. Channing, J. Endocrinol., 43 (1969) 381. 8 J.H. Hanks and R.E. Wallace, Proc. Soc. Exp. Biol., 71 (1949) 196. 9 G. Krishna, 13. Weiss and B.B. Brodie, J. Pharmacol. Exp. Ther., 163 (1968) 379. 10 T. Kaneko and J.B. Field, J. Lab. 67in. Med., 74 (1969) 682. 11 J. Kolena and C.P. Channing, 53rd Meeting of the Endocrine Society, Salt Francisco, Calif. 1971, Abstr. 151. 12 J. Kolena and C.P. Channing, l£ndocrinology, ( 1971 ) in the press. 13 J. Dorrington and B. Baggett, Endocrinology, 84 (1969) 989. 14 Y.-A. Fontaine, E. Fontaine-Bertrand, M.C. Sahnon and N. Delerue-Lebelle, 6\ R. Acad. Sci. Ser. D, 272 (1971) 1137. 15 C.P. Channing, Proc. ConJl on Regulation of Mammalian Reproduction. National Institutes of ttealth. Bethesda, Md., 1971, in the press. 16 V.J. Cirillo, O.t'. Anderson, E.A. Ham and R.B.L. Gwatkin, Exp. CelIRes., 57 (1969) 39.
Biochim. Biophys. Acta, 252 (1971) 6 0 1 - 6 0 6