Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, 45 Shattuck St., Boston, MA 02115, USA
CYCLIC AMP AND CYCLIC GMP ACCUMULATION IN HAMSTER PRE-OVULATORY FOLLICLES STIMULATED WITH LH AND FSH
By Anastasia Makris and Kenneth
J. Ryan
ABSTRACT
Cyclic AMP and cyclic GMP accumulation in hamster pre-ovulatory follicles was determined after in vitro stimulation by LH and FSH. Combined time course and dose response experiments determined that the acute response of the follicles (0\p=n-\30 min) to LH and FSH was similar with respect to cyclic AMP accumulation. The pattern of cyclic GMP accumulation was, however, distinctly different in LH and FSH stimulated follicles. LH increased follicular cyclic GMP only at the lowest dose (0.005 IU/ml), while higher doses of LH had no effect. In contrast, FSH at all doses stimulated cyclic GMP accumulation. The different cyclic AMP to cyclic relationships generated in the follicles by LH and FSH may be determinants in specificity of hormone action in pre\x=req-\ ovulatory follicles. LH and FSH control
some
aspects of follicular tissue metabolism via adenosine
3',5'-cyclic monophosphate (cyclic AMP) as their common messenger (Dorrington 8c Baggett 1969; Kolena 8c Channing 1972; Marsh 8c Savard 1966; Marsh et al. 1966; Miller 8c Keyes 1974; Tsafriri et al. 1972). Other intracellular mes¬ sengers generated by hormones may act separately from, in concert with, or antagonistic to cyclic AMP. Guanosine 3',5'-cyclic monophosphate (cyclic GMP), shown to be under hormonal control in several tissues (Ginge et al. 1970; Gold¬ berg et al. 1973; Kuehl et al. 1974; Rubin et al. 1977; Sharma et al. 1974), is also found in ovarian tissue. Differential effects by hormones on cyclic GMP in ovarian tissue from PMSG-primed immature rats has been demonstrated by
Ratner (1976). There was either no change (luteal ovarian homogenates) or a decrease (follicular ovarian homogenates) after both LH and FSH stimulation in cyclic GMP accumulation in vitro. Grinwich et al. (1976) reported an inverse relationship in cyclic AMP and cyclic GMP levels in luteal tissue of LH treated PMSG-primed immature rats. This inverse relationship of cyclic AMP to cyclic GMP is common to many tissues (Asakawa et al. 1975; Ginge et al. 1970; Goldberg et al. 1973; Kuehl el al. 1974; Rubin et al. 1977; Sharma et al.
1974). Hormonal control of cyclic GMP in pre-ovulatory follicles of normal cycling animals has not been reported. In order to obtain some insight as to the mechanism of specificity of LH and FSH effects on follicular tissue, we have studied the effects of these hormones on follicular cyclic GMP accumulation and its relationship to gonadotrophin-stimulated cyclic AMP.
MATERIALS AND METHODS Pro-oestrous hamsters (LAK:LVG[SYR]-Lakeview Hamster Colony) not yet exposed to the in vitro pre-ovulatory gonadotrophin surge, were used as a source of preovulatory follicles (Makris 8c Ryan 1977). The follicles were cleaned of adhering tissue and pooled prior to use. The ability of follicles to generate cyclic AMP and cyclic GMP was studied using combination dose response (0, 0.005, 0.05, 1.0 IU/ml LH or FSH) and time course experiments (0, 0.5, 2.0, 5.0, 15 and 30 min). The incubations were carried out at 37°C in a Dubonoff incubator under a 95 % air: 5 "la CO2 gas phase. The incubation medium (0.1 ml, containing 1 follicle) consisted of McCoy's medium (Grand Island Biological), 1 % Bovine Serum Albumin, Fraction V (Sigma), 1 % penicillin-streptomycin, IO-4 m l-methyl-3-isobutyl xanthine (MIX) ± LH (NIHLER 960, 4620 IU/mg) or FSH (NIH-LER-1575 C, 3600 IU/mg). At the end of incubation, the follicles were quickly separated from the culture medium, boiled for 3 min in 0.1 ml H»0, sonicated and frozen until assayed. Follicular content of cyclic AMP and cyclic GMP was assayed using the radioimmunoassay tech¬ nique of Steiner et al. (1973) as modified by Cailla et al. (1973). The sensitivity of the modified radioimmunoassay for cyclic AMP and cyclic GMP was as low as 1 fmol. The intra-assay coefficient of variation for cyclic AMP was 7 % and for cyclic GMP 8 °/o. Cyclic AMP and cyclic GMP reagents for the radioimmunoassay [I25I]antigens, standard and antisera) were purchased from Collaborative Research, Waltham, MA.
RESULTS AND DISCUSSION
In the presence of
phosphodiesterase inhibitor (MIX), LH (Fig. 1 A) and FSH (Fig. A) generated approximately the same pattern of follicular cyclic AMP accumulation. The accumulation of follicular cyclic AMP was directly dose dependent for both hormones. Neither hormone at a dose of 0.005 IU/ml gene¬ rated a detectable rise in cyclic AMP. The immediate drop (evident at 0.5 min) in follicular cyclic AMP from zero time levels at 0.005 and 0.05 IU/ml was 2
a
LH DOSE RESPONSE
Effect of LH on cyclic AMP and cyclic GMP accumulation in follicles. Incubations were carried out for 0, 0.5, 2, 15 and 30 min, with LH at a concentration of 0 (•), 0.005 (A), 0.05 ( ) and 1.0 ( ) IU/ml. Each point represents the mean of 2 incubations + range. A. Cyclic AMP accumulation expressed as pmol/follicle. B. Cyclic GMP accumulation expressed as fmol/follicle.
similar to the
in control follicular cyclic AMP (Figs. 1 A and 2 A). This drop, present in control and gonadotrophin stimulated follicles, may have re¬ flected a transient increase in secretion to the medium due to temperature changes in the environment of the follicle. The rapid rise in follicular cyclic AMP (evident by 0.5 min) in both 1.0 IU/ml LH (Fig. 1 A) and FSH (Fig. 2 A) may have masked a similar loss of follicular cyclic AMP seen with the lower
drop
doses.
Cyclic AMP did not begin to rise in 0.05 IU/ml LH- or FSH-stimulated follicles until after a 0.5 min (LH) and 2 min (FSH). The increase continued until at least 15 min with no further increases seen at 30 min of incubation. In follicles stimulated with 1.0 IU/ml LH or FSH, cyclic AMP began to rise by a 0.5 min and peak levels were reached by 5 min. Follicular cyclic AMP content in both 1.0 IU/ml LH and FSH treated follicles had decreased at 15
SH DOSE RESPONSE
500r-
B.cGMP
Fig. 2. Effect of FSH on cyclic AMP and cyclic GMP accumulation in follicles. Incubations were carried out for 0, 0.5, 2, 15 and 30 min, with FSH at a concentration of 0 (•), 0.005 (A), 0.05 ( ) and 1.0 ( ) IU/ml. Each point represents the mean of 2 incubations ± range. A. Cyclic AMP accumulation expressed as pmol/follicle. B. Cyclic GMP accumulation expressed as fmol/follicle.
and 30 min below that seen at 5 min, and approximated the content seen at 30 min in follicles stimulated with 0.05 IU/ml hormone. The rapid rise in follicular cyclic AMP seen with both 1.0 IU/ml LH and
FSH indicated that both hormones activated plasma membrane bound adenylate cyclase. In the intact follicle it is not possible to determine which cell type is activated by the gonadotrophins. LH binds to both theca and granulosa cells at pro-oestrous while FSH binding appears to be restricted to granulosa cells (Amsterdam et al. 1975; Channing 8c Kammerman 1974; Midgley 1973; Presl et al. 1972; Zeleznik et al. 1974). The binding data implies both common (FSH and LH on granulosa cells) and specific sites (LH on theca) of hormone action, although FSH-specific effects have been reported in theca (Hamberger et al. 1971; Makris & Ryan, subm. for publ.). The transient increase in cyclic AMP ac¬ cumulation in both LH and FSH stimulated follicles, seen in Figs. 1 A and 2 A,
either no further increase or a drop in follicular cyclic AMP content at 30 min. The drop in cyclic AMP content observed in our experi¬ ments was possibly due to secretion into the medium (Weiss el al. 1976), nonenzymatic breakdown and/or possibly enzymatic breakdown since 10^* M MIX does not inhibit phosphodiesterase completely. In addition, it has been shown that gonadotrophins rapidly and transiently increase follicular cyclic AMP with the follicle then becoming refractory to further hormone stimulation (Hunzicker-Dunn 8c Birnbaumer 1976; Lamprecht et al. 1973; Marsh et al. 1973). In contrast to similar effects by the gonadotrophins on cyclic AMP synthesis, LH and FSH were markedly different in the accumulation of cyclic GMP. LH effected a rapid and transient rise in follicular cyclic GMP (Fig. 1 B) only at the lowest dose (0.005 IU/ml). Follicular cyclic GMP peaked at 2 min and gradually decreased thereafter at 5, 15 and 30 min. Higher doses of LH had no effect on follicular cyclic GMP levels within the 30 min test period. The in¬ verse relationship of LH dose to cyclic accumulation was similar to that seen in ACTH stimulated rat adrenal cells in vitro reported by Sharma et al. (1974) and Rubin et al. (1977). FSH produced dose-related response in rate of cyclic GMP accumulation by the follicles (Fig. 2 B). Cyclic GMP levels began to rise between 5-15 min at 0.005 IU/ml, 2 and 5 min at 0.05 IU/ml, and by 0.5 min at 1.0 IU/ml. Cyclic GMP accumulation was the same and plateaued by 15 and 30 min with all of the doses of FSH. Very low amounts of FSH therefore, appeared to saturate receptors coupled to FSH-sensitive guanylate cyclase. Unlike the drop in FSH stimulated follicular cyclic AMP at 30 min (Fig. 2 A), cyclic GMP content plateaued at the peak levels obtained. The increase in FSH-stimulated follicular cyclic GMP is not in agreement with Ratner's (1976) observations that PMS-treated pre-pubertal rat follicular ovaries responded to FSH in vitro with decreased cyclic GMP synthesis. The difference in observations may be attributed to differences in hormone effects on primed pre-pubertal tissue and tissue from normal cycling pro-oestrous ani¬ mals. The data reported here have indicated that there are differences in the cyclic AMP and cyclic GMP relationships in the hamster pre-ovulatory fol¬ licle which are determined by the hormone and dose hormone used. Whether the differential effects of LH and FSH on follicular cyclic GMP are a deter¬ minant in hormone specificity of action, especially in granulosa cells where cyclic AMP is a common messenger to those hormones, remains to be elucidated. was
followed
by
ACKNOWLEDGMENTS This work was supported by grants from the Rockefeller Foundation (No. 65040) and the United States Public Health Service (HD 07923-04), and the Educational Foundation of America. We wish to thank Ms. Vicki McCord for typing the manuscript.
REFERENCES Amsterdam A., Koch Asakawa T., Ruiz J'.,
(1975)
]., Lieberman
Snyder R.,
M E. Se Lindner H. R.: J. Cell Biol. 67 (1975) 894. Scheinbaum T., Rüssel T. Se Ho R. J.: Fed. Proc. 34
2246.
Cailla H. L., Racine-Weisbuck M. S. Se
DeLaage
M. A.:
Analyt.
Biochem. 56
(1973)
394.
Channing C. P. Se Kammerman S.: Biol. Reprod. 70 (1974) 179. Dorrington J. F. Se Baggett B.: Endocrinology 84 (1969) 989. Ginge W. J., Paulsen J. B., O'Toole A. G. & Goldberg N. D.: Proc. nat. Acad. Sei. (USA) 66 (1970) 398. Goldberg N. D., O'Dea R. F. Se Haddox M. K In: Greengard P. and Robison G. A., Eds. Advances in Cyclic Nucleotide Research, Vol. 3. Raven Press (1973) 155. Grinwich D. L., Ham E. A., Hichens M. Se Behrman H. R.: Endocrinology 98 (1976) 146.
Hornberger L., Hamberger
A. Se Herlitz H.: Acta endocr. (Kbh.) Suppl. 753 (1971) 41. Hunzicker-Dunn M. Se Birnbaumer L.: Endocrinology 99 (1976) 185. Kolena J. Se Channing C. P.: Endocrinology 90 (1972) 1543. Kuehl F. A., Jr., Ham E. A., Zanetli M. E., Sanford C. H., Nicol S. E. 8c Goldberg N. D.: Proc. nat. Acad. Sei. (USA) 77 #5 (1974) 1866. Lamprecht S. A., Zor U'., Tsafriri A. Se Lindner H. R.: J. Endocr. 57 (1973) 217. Makris A. Se Ryan K. J.: Steroids 29 (1977) 65. Marsh J. M., Butcher R. W'., Savard K. Se Sutherland E. W.: J. biol. Chem. 241 (1966) 1596. Marsh J. M., Mills T. M. 8c Le Maire W. J.: Biochim. biophys. Acta (Amst.) 304 (1973) 197.
J. M. Se Savard K.: Steroids cS (1966) 133. Midgley A. R., Jr.: Advanc. exp. Med. Biol. 36 (1973) 365. Miller J. B. Se Keyes P. L.: Endocrinology 95 (1974) 253. Presl J., Popsil V., Figorova V. Se Frabic Z.: Endocr. exp. 8 (1972) 2. Ratner A.: Endocrinology 99 (1976) 1496. Rubin R. P., Laycock S. G. 8c End D. W.: Biochim. biophys. Acta (Amst.) Marsh
496
(1977)
329. Sharma K. R., Ahmed W. K., Sutliff L. S. Se Brush J. S.: FEBS Letters 45 (1974) 107. Steiner A. L., Parker C. W. Se Kipnis D. M.: J. biol. Chem. 247 (1973) 1106. Tsafriri A., Lindner H. R., Zor U. Se Lamprecht S. A.: J. Reprod. Fértil. 37 (1972) 39. Weiss T. J., Seamark R. F., Mclntosh J. E. A. 8c Moor R. M.: J. Reprod. Fértil. 46 (1976) 347. Zeleznick A. J., Midgley A. R., Jr. 8c Reichert L. E., Jr.: Endocrinology 95 (1974) 818.
Received
on
July
8th. 1977.
CYCLIC AMP AND CYCLIC GMP ACCUMULATION IN HAMSTER PRE-OVULATORY FOLLICLES STIMULATED WITH LH AND FSH
By Anastasia Makris and Kenneth
J. Ryan
ABSTRACT
Cyclic AMP and cyclic GMP accumulation in hamster pre-ovulatory follicles was determined after in vitro stimulation by LH and FSH. Combined time course and dose response experiments determined that the acute response of the follicles (0\p=n-\30 min) to LH and FSH was similar with respect to cyclic AMP accumulation. The pattern of cyclic GMP accumulation was, however, distinctly different in LH and FSH stimulated follicles. LH increased follicular cyclic GMP only at the lowest dose (0.005 IU/ml), while higher doses of LH had no effect. In contrast, FSH at all doses stimulated cyclic GMP accumulation. The different cyclic AMP to cyclic relationships generated in the follicles by LH and FSH may be determinants in specificity of hormone action in pre\x=req-\ ovulatory follicles. LH and FSH control
some
aspects of follicular tissue metabolism via adenosine
3',5'-cyclic monophosphate (cyclic AMP) as their common messenger (Dorrington 8c Baggett 1969; Kolena 8c Channing 1972; Marsh 8c Savard 1966; Marsh et al. 1966; Miller 8c Keyes 1974; Tsafriri et al. 1972). Other intracellular mes¬ sengers generated by hormones may act separately from, in concert with, or antagonistic to cyclic AMP. Guanosine 3',5'-cyclic monophosphate (cyclic GMP), shown to be under hormonal control in several tissues (Ginge et al. 1970; Gold¬ berg et al. 1973; Kuehl et al. 1974; Rubin et al. 1977; Sharma et al. 1974), is also found in ovarian tissue. Differential effects by hormones on cyclic GMP in ovarian tissue from PMSG-primed immature rats has been demonstrated by
Ratner (1976). There was either no change (luteal ovarian homogenates) or a decrease (follicular ovarian homogenates) after both LH and FSH stimulation in cyclic GMP accumulation in vitro. Grinwich et al. (1976) reported an inverse relationship in cyclic AMP and cyclic GMP levels in luteal tissue of LH treated PMSG-primed immature rats. This inverse relationship of cyclic AMP to cyclic GMP is common to many tissues (Asakawa et al. 1975; Ginge et al. 1970; Goldberg et al. 1973; Kuehl el al. 1974; Rubin et al. 1977; Sharma et al.
1974). Hormonal control of cyclic GMP in pre-ovulatory follicles of normal cycling animals has not been reported. In order to obtain some insight as to the mechanism of specificity of LH and FSH effects on follicular tissue, we have studied the effects of these hormones on follicular cyclic GMP accumulation and its relationship to gonadotrophin-stimulated cyclic AMP.
MATERIALS AND METHODS Pro-oestrous hamsters (LAK:LVG[SYR]-Lakeview Hamster Colony) not yet exposed to the in vitro pre-ovulatory gonadotrophin surge, were used as a source of preovulatory follicles (Makris 8c Ryan 1977). The follicles were cleaned of adhering tissue and pooled prior to use. The ability of follicles to generate cyclic AMP and cyclic GMP was studied using combination dose response (0, 0.005, 0.05, 1.0 IU/ml LH or FSH) and time course experiments (0, 0.5, 2.0, 5.0, 15 and 30 min). The incubations were carried out at 37°C in a Dubonoff incubator under a 95 % air: 5 "la CO2 gas phase. The incubation medium (0.1 ml, containing 1 follicle) consisted of McCoy's medium (Grand Island Biological), 1 % Bovine Serum Albumin, Fraction V (Sigma), 1 % penicillin-streptomycin, IO-4 m l-methyl-3-isobutyl xanthine (MIX) ± LH (NIHLER 960, 4620 IU/mg) or FSH (NIH-LER-1575 C, 3600 IU/mg). At the end of incubation, the follicles were quickly separated from the culture medium, boiled for 3 min in 0.1 ml H»0, sonicated and frozen until assayed. Follicular content of cyclic AMP and cyclic GMP was assayed using the radioimmunoassay tech¬ nique of Steiner et al. (1973) as modified by Cailla et al. (1973). The sensitivity of the modified radioimmunoassay for cyclic AMP and cyclic GMP was as low as 1 fmol. The intra-assay coefficient of variation for cyclic AMP was 7 % and for cyclic GMP 8 °/o. Cyclic AMP and cyclic GMP reagents for the radioimmunoassay [I25I]antigens, standard and antisera) were purchased from Collaborative Research, Waltham, MA.
RESULTS AND DISCUSSION
In the presence of
phosphodiesterase inhibitor (MIX), LH (Fig. 1 A) and FSH (Fig. A) generated approximately the same pattern of follicular cyclic AMP accumulation. The accumulation of follicular cyclic AMP was directly dose dependent for both hormones. Neither hormone at a dose of 0.005 IU/ml gene¬ rated a detectable rise in cyclic AMP. The immediate drop (evident at 0.5 min) in follicular cyclic AMP from zero time levels at 0.005 and 0.05 IU/ml was 2
a
LH DOSE RESPONSE
Effect of LH on cyclic AMP and cyclic GMP accumulation in follicles. Incubations were carried out for 0, 0.5, 2, 15 and 30 min, with LH at a concentration of 0 (•), 0.005 (A), 0.05 ( ) and 1.0 ( ) IU/ml. Each point represents the mean of 2 incubations + range. A. Cyclic AMP accumulation expressed as pmol/follicle. B. Cyclic GMP accumulation expressed as fmol/follicle.
similar to the
in control follicular cyclic AMP (Figs. 1 A and 2 A). This drop, present in control and gonadotrophin stimulated follicles, may have re¬ flected a transient increase in secretion to the medium due to temperature changes in the environment of the follicle. The rapid rise in follicular cyclic AMP (evident by 0.5 min) in both 1.0 IU/ml LH (Fig. 1 A) and FSH (Fig. 2 A) may have masked a similar loss of follicular cyclic AMP seen with the lower
drop
doses.
Cyclic AMP did not begin to rise in 0.05 IU/ml LH- or FSH-stimulated follicles until after a 0.5 min (LH) and 2 min (FSH). The increase continued until at least 15 min with no further increases seen at 30 min of incubation. In follicles stimulated with 1.0 IU/ml LH or FSH, cyclic AMP began to rise by a 0.5 min and peak levels were reached by 5 min. Follicular cyclic AMP content in both 1.0 IU/ml LH and FSH treated follicles had decreased at 15
SH DOSE RESPONSE
500r-
B.cGMP
Fig. 2. Effect of FSH on cyclic AMP and cyclic GMP accumulation in follicles. Incubations were carried out for 0, 0.5, 2, 15 and 30 min, with FSH at a concentration of 0 (•), 0.005 (A), 0.05 ( ) and 1.0 ( ) IU/ml. Each point represents the mean of 2 incubations ± range. A. Cyclic AMP accumulation expressed as pmol/follicle. B. Cyclic GMP accumulation expressed as fmol/follicle.
and 30 min below that seen at 5 min, and approximated the content seen at 30 min in follicles stimulated with 0.05 IU/ml hormone. The rapid rise in follicular cyclic AMP seen with both 1.0 IU/ml LH and
FSH indicated that both hormones activated plasma membrane bound adenylate cyclase. In the intact follicle it is not possible to determine which cell type is activated by the gonadotrophins. LH binds to both theca and granulosa cells at pro-oestrous while FSH binding appears to be restricted to granulosa cells (Amsterdam et al. 1975; Channing 8c Kammerman 1974; Midgley 1973; Presl et al. 1972; Zeleznik et al. 1974). The binding data implies both common (FSH and LH on granulosa cells) and specific sites (LH on theca) of hormone action, although FSH-specific effects have been reported in theca (Hamberger et al. 1971; Makris & Ryan, subm. for publ.). The transient increase in cyclic AMP ac¬ cumulation in both LH and FSH stimulated follicles, seen in Figs. 1 A and 2 A,
either no further increase or a drop in follicular cyclic AMP content at 30 min. The drop in cyclic AMP content observed in our experi¬ ments was possibly due to secretion into the medium (Weiss el al. 1976), nonenzymatic breakdown and/or possibly enzymatic breakdown since 10^* M MIX does not inhibit phosphodiesterase completely. In addition, it has been shown that gonadotrophins rapidly and transiently increase follicular cyclic AMP with the follicle then becoming refractory to further hormone stimulation (Hunzicker-Dunn 8c Birnbaumer 1976; Lamprecht et al. 1973; Marsh et al. 1973). In contrast to similar effects by the gonadotrophins on cyclic AMP synthesis, LH and FSH were markedly different in the accumulation of cyclic GMP. LH effected a rapid and transient rise in follicular cyclic GMP (Fig. 1 B) only at the lowest dose (0.005 IU/ml). Follicular cyclic GMP peaked at 2 min and gradually decreased thereafter at 5, 15 and 30 min. Higher doses of LH had no effect on follicular cyclic GMP levels within the 30 min test period. The in¬ verse relationship of LH dose to cyclic accumulation was similar to that seen in ACTH stimulated rat adrenal cells in vitro reported by Sharma et al. (1974) and Rubin et al. (1977). FSH produced dose-related response in rate of cyclic GMP accumulation by the follicles (Fig. 2 B). Cyclic GMP levels began to rise between 5-15 min at 0.005 IU/ml, 2 and 5 min at 0.05 IU/ml, and by 0.5 min at 1.0 IU/ml. Cyclic GMP accumulation was the same and plateaued by 15 and 30 min with all of the doses of FSH. Very low amounts of FSH therefore, appeared to saturate receptors coupled to FSH-sensitive guanylate cyclase. Unlike the drop in FSH stimulated follicular cyclic AMP at 30 min (Fig. 2 A), cyclic GMP content plateaued at the peak levels obtained. The increase in FSH-stimulated follicular cyclic GMP is not in agreement with Ratner's (1976) observations that PMS-treated pre-pubertal rat follicular ovaries responded to FSH in vitro with decreased cyclic GMP synthesis. The difference in observations may be attributed to differences in hormone effects on primed pre-pubertal tissue and tissue from normal cycling pro-oestrous ani¬ mals. The data reported here have indicated that there are differences in the cyclic AMP and cyclic GMP relationships in the hamster pre-ovulatory fol¬ licle which are determined by the hormone and dose hormone used. Whether the differential effects of LH and FSH on follicular cyclic GMP are a deter¬ minant in hormone specificity of action, especially in granulosa cells where cyclic AMP is a common messenger to those hormones, remains to be elucidated. was
followed
by
ACKNOWLEDGMENTS This work was supported by grants from the Rockefeller Foundation (No. 65040) and the United States Public Health Service (HD 07923-04), and the Educational Foundation of America. We wish to thank Ms. Vicki McCord for typing the manuscript.
REFERENCES Amsterdam A., Koch Asakawa T., Ruiz J'.,
(1975)
]., Lieberman
Snyder R.,
M E. Se Lindner H. R.: J. Cell Biol. 67 (1975) 894. Scheinbaum T., Rüssel T. Se Ho R. J.: Fed. Proc. 34
2246.
Cailla H. L., Racine-Weisbuck M. S. Se
DeLaage
M. A.:
Analyt.
Biochem. 56
(1973)
394.
Channing C. P. Se Kammerman S.: Biol. Reprod. 70 (1974) 179. Dorrington J. F. Se Baggett B.: Endocrinology 84 (1969) 989. Ginge W. J., Paulsen J. B., O'Toole A. G. & Goldberg N. D.: Proc. nat. Acad. Sei. (USA) 66 (1970) 398. Goldberg N. D., O'Dea R. F. Se Haddox M. K In: Greengard P. and Robison G. A., Eds. Advances in Cyclic Nucleotide Research, Vol. 3. Raven Press (1973) 155. Grinwich D. L., Ham E. A., Hichens M. Se Behrman H. R.: Endocrinology 98 (1976) 146.
Hornberger L., Hamberger
A. Se Herlitz H.: Acta endocr. (Kbh.) Suppl. 753 (1971) 41. Hunzicker-Dunn M. Se Birnbaumer L.: Endocrinology 99 (1976) 185. Kolena J. Se Channing C. P.: Endocrinology 90 (1972) 1543. Kuehl F. A., Jr., Ham E. A., Zanetli M. E., Sanford C. H., Nicol S. E. 8c Goldberg N. D.: Proc. nat. Acad. Sei. (USA) 77 #5 (1974) 1866. Lamprecht S. A., Zor U'., Tsafriri A. Se Lindner H. R.: J. Endocr. 57 (1973) 217. Makris A. Se Ryan K. J.: Steroids 29 (1977) 65. Marsh J. M., Butcher R. W'., Savard K. Se Sutherland E. W.: J. biol. Chem. 241 (1966) 1596. Marsh J. M., Mills T. M. 8c Le Maire W. J.: Biochim. biophys. Acta (Amst.) 304 (1973) 197.
J. M. Se Savard K.: Steroids cS (1966) 133. Midgley A. R., Jr.: Advanc. exp. Med. Biol. 36 (1973) 365. Miller J. B. Se Keyes P. L.: Endocrinology 95 (1974) 253. Presl J., Popsil V., Figorova V. Se Frabic Z.: Endocr. exp. 8 (1972) 2. Ratner A.: Endocrinology 99 (1976) 1496. Rubin R. P., Laycock S. G. 8c End D. W.: Biochim. biophys. Acta (Amst.) Marsh
496
(1977)
329. Sharma K. R., Ahmed W. K., Sutliff L. S. Se Brush J. S.: FEBS Letters 45 (1974) 107. Steiner A. L., Parker C. W. Se Kipnis D. M.: J. biol. Chem. 247 (1973) 1106. Tsafriri A., Lindner H. R., Zor U. Se Lamprecht S. A.: J. Reprod. Fértil. 37 (1972) 39. Weiss T. J., Seamark R. F., Mclntosh J. E. A. 8c Moor R. M.: J. Reprod. Fértil. 46 (1976) 347. Zeleznick A. J., Midgley A. R., Jr. 8c Reichert L. E., Jr.: Endocrinology 95 (1974) 818.
Received
on
July
8th. 1977.
