0013-7227/90/1263-1504$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 3 Printed in U.S.A.
Atrial Natriuretic Peptide Inhibits Spontaneous Rat Oocyte Maturation* JAN TORNELL, BJORN CARLSSON, AND HAKAN BILLIG Department of Physiology, University of Gbteborg, Goteborg, Sweden
ABSTRACT. We report results of experiments demonstrating a dose-dependent inhibition of spontaneous maturation (resumption of meiosis) in rat oocyte-cumulus complexes by atrial natriuretic peptide (ANP). The inhibition was persistent over the time period studied. The ANP analog Tyr8-ANP, which mediates smooth muscle relaxation in other organs without elevating cGMP levels, did not inhibit the spontaneous maturation. ANP, but not Tyr8-ANP, dose-dependently stimulated cGMP accumulation in oocyte-cumulus complexes. Furthermore, sodium nitroprusside (SNP), that stimulates a soluble form of guanylate cyclase, inhibited spontaneous maturation in
A
TRIAL natriuretic peptide (ANP) is believed to act L via the cGMP system, and its primary targets include kidney, vascular smooth muscle, and adrenal cortex, where it induces natriuresis, causes vasodilatation, and inhibits aldosterone release, respectively (1). ANP is mainly produced in the heart atrial cardiocytes and released in response to increased tension in the atria (2). Also, glucocortocoids can increase plasma levels of ANP as well as ANP mRNA levels in the atria (3). Recently, a natriuretic peptide, brain natriuretic peptide (BNP), has been localized in the brain (4). BNP has a structure similar to that of ANP and interacts with the ANP receptor. Three forms of the ANP receptor are known, designated ANP-A, ANP-B, and ANP-C. The ANP-C receptor has no guanylate cyclase activity, in contrast to the ANP-A and ANP-B receptors. The ANPB receptor is more effectively stimulated by BNP than ANP. ANP and BNP are equally effective in stimulating cGMP accumulation when bound to the ANP-A receptor (5). The guanylate cyclase is an intracellular part of the receptor molecule (6, 7), which is activated when ANP is bound (8). In the ovary, local production of ANP or Received October 13, 1989. Address all correspondence and requests for reprints to: Jan Tornell, Department of Physiology, University of Goteborg, P.O. Box 33031, S400 33 Goteborg, Sweden. * This work was supported by grants from the Swedish Medical Research Council (no. B89-04x-27 and B-89-04x-05650), the Swedish Society for Medical Research, the Goteborg Medical Society, Swedish Medical Society, the Faculty of Medicine, University of Goteborg, and Handl Hjalmar Svenssons Research Funds.
oocyte-cumulus complexes and stimulated cGMP accumulation in oocyte-cumulus complexes. Neither ANP nor SNP stimulated cAMP accumulation. In oocytes where the surrounding cumulus cells had been removed neither ANP nor SNP inhibited the spontaneous maturation. These results demonstrate that cumulus cells, but not the oocyte itself, have ANP receptors and guanylate cyclases. Furthermore, ANP, via cGMP, can influence oocyte meiosis, suggesting a possible involvement of ANP and cGMP in the control of the meiotic process in rat oocytes. (Endocrinology 126: 1504-1508, 1990)
A N P - l i k e peptides h a s , to our knowledge, n o t yet been
demonstrated, but ANP stimulates LH-induced progesterone secretion from human cultured granulosa-lutein cells (9), indicating the presence of ANP receptors. A high concentration of binding sites has also been demonstrated in human preovulatory follicles (10). The oocyte undergoes meiotic divisions to reduce its genetic material before fusing with the sperm during fertilization. The first meiotic division in the oocyte starts during fetal life and becomes arrested at birth. The ovulatory LH surge leads to resumption of meiosis and completion of the first meiotic division (oocyte maturation) in the preovulatory follicle. The second meiotic division is not completed until the oocyte is fertilized. The fact that the oocyte matures spontaneously when liberated from the follicle (11) has led to the hypothesis that the oocyte is under constant inhibition when it is contained within the follicle. A number of theories have been presented to explain the mechanism of the intrafollicular meiotic arrest. cAMP has been shown, by several approaches, to be important for oocyte maturation. Experimentally, cAMP- or cAMP-elevating agents can prevent the spontaneous maturation in isolated oocytes surrounded by their cumulus cells (oocyte-cumulus complexes) and in oocytes where the cumulus cells were mechanically removed (denuded oocytes) (12, 13). In the mouse, cAMP levels decrease in the oocyte during the resumption of the meiosis (14, 15). Furthermore, injection of the catalytic subunit of cAMP-dependent protein
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ANP INHIBITS OOCYTE MATURATION kinase delays spontaneous mouse oocyte maturation (16). ANP does not increase cAMP accumulation. On the contrary, ANP can inhibit adenylate cyclase under some circumstances (1). However, cGMP can influence oocyte maturation. cGMP derivatives have been reported to inhibit the spontaneous maturation of oocyte-cumulus complexes in the hamster (17) and in denuded rat oocytes (18). Gonadotropins seem to influence the levels of cGMP, but in an inverse pattern compared to cAMP levels. In vitro, gonadotropins increase cAMP levels and decrease cGMP levels in isolated rat ovaries (19). In vivo, after the LH surge on proestrus, there is a decrease in cGMP levels and an increase in cAMP levels in whole rat ovaries (20) and isolated preovulatory hamster follicles (21). This temporal relationship between decreasing cGMP levels and resumption of meiosis after the LH surge could be of functional importance. Since cGMP derivatives inhibit spontaneous maturation, one may assume that if the oocyte-cumulus complex has ANP receptors, ANP may influence this process by increasing intracellular cGMP levels. Guanylate cyclase exists in several forms; besides the membrane-associated form there is also a soluble form which can be stimulated by sodium nitroprusside (SNP) (8). The aim of this study was to examine whether ANP could influence meiotic maturation and, in that case, by which mechanism.
Materials and Methods Animals Twenty-six-day-old immature, female Sprague-Dawley rats (ALAB, Stockholm, Sweden) were injected with 10 IU PMSG (Sigma Chemical Co., St. Louis, MO), sc, to induce synchronized follicular development. The rats were housed under Standardized environmental conditions with lights on between 0500-1900 h. Water and food were supplied ad libitum. Tissue preparation and incubation procedures Twenty-eight-day-old rats were killed by cervical dislocation. The ovaries were dissected out, cleaned from adherent tissue, and placed in incubation medium. Large follicles were punctured with a hypodermic needle under a stereomicroscope. The oocyte-cumulus complexes were passed through three dishes to remove granulosa cells and follicular fluid before transfer to a final dish for incubation. Oocyte-cumulus complexes were incubated in 750 /xl Eagle's Minimum Essential Medium with Earle's salts (Gibco, Paisley, Scotland) buffered with 10 mM HEPES and supplemented with 0.1% BSA (Sigma) and Gentamicin (50 Mg/ml; Sigma). Oocytes from a pair of ovaries were incubated in humidified air for 1-6 h in each dish. Denuded oocytes were obtained by mechanically removing the cumulus cells by a series of mouth-controlled micropipettes of successively smaller diameter. Isolation and denudation of the oocytes were performed in medium M2 buffered with HEPES (22). Denuded oocytes were incubated in medium Mi6 (22) buffered
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with 25 mM HCO3. Both media contained 0.4% BSA (Sigma). Denuded oocytes were incubated under an humidified atmosphere of 5% CO2 in air for the indicated period of time. Chemicals and hormones Synthetic rat ANP (fragment 5-27), (Bu)2cAMP (dbcAMP), 8-bromo-cGMP (8BrcGMP) and SNP were purchased from Sigma. Tyr8-ANP-(5-27), an ANP analog that relaxes smooth muscle without affecting cGMP levels (23), was purchased from Peninsula Laboatories (Merseyside, United Kingdom). Oocyte examination The oocyte-cumulus complexes and denuded oocytes were collected from the culture dishes after incubation and examined by Nomarski optics to reveal stage of meiosis. Oocytes showing a germinal vesicle (GV) were considered still arrested in prophase I, and those with GV breakdown (GVB) to have resumed meiosis. Determination of cAMP and cGMP cAMP and cGMP analyses were performed with a RIA kit (Amersham, Radiochemical Centre, United Kingdom). The incubations were terminated by adding ice-cold ethanol to a final concentration of 60%. The samples were centrifuged, and pellets were rinsed with 750 ^1 60% ethanol and centrifugated. Total supernatant was evaporated, and dried extracts were acetylated according to the manufacturer's instructions. Fifteen to 20 oocyte-cumulus complexes were measured in each sample. The amounts of cAMP and cGMP in each sample were 4-10 and 5-40 fmol/tube, respectively. The sensitivity was 1.0 and 0.5 fmol/tube for the cAMP and cGMP assays, respectively. Cross-reactivity for the cAMP RIA to cGMP was less than 0.0004%, and that for the cGMP RIA to cAMP was less than 0.02% according to the manufacturer. Statistics The values are given as the mean ± SEM. Experiments were performed at least three times with similar results, except for the effect of ANP on cAMP accumulation that were performed twice with similar results. In the presented data on oocyte maturation n refers to number of oocytes, and the SE was obtained by a normal approximation of a binomial distribution. In data on levels of cyclic nucleotides n refers to number of samples. Statistical differences were calculated by analysis of variance followed by Student-Newman-Keuls test. P < 0.05 was considered statistically significant.
Results Effect of ANP on meiosis and cGMP accumulation in oocyte-cumulus complexes ANP inhibited spontaneous maturation of oocyte-cumulus complexes in a dose-dependent manner after 2 h of incubation in concentrations ranging from 1 nM (72% GVB) to 1 ixM (48% GVB) compared to the control group
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ANP INHIBITS OOCYTE MATURATION
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Endo • 1990 Vol 126 • No 3
lOO-i
80-
8 5
i
o 40
K
'
4-
32-
201-
0
10' 9
10"8
10"
10"6
0Jr
10"
10~
ANP (M)
TABLE 1. Effect of ANP, dbcAMP, SNP, or Tyr8-ANP on resumption of meiosis of cumulus-enclosed oocytes
Control ANP SNP Tyr8-ANP dbcAMP
10'8 10"7 10'6 10"
ANP (M)
FIG. 1. The effect of ANP on maturation of cumulus-enclosed oocytes after 2 h of incubation. The median number of oocytes in each group was 280. Data were pooled from three experiments. **, Significantly lower (P < 0.01) than control value.
Treatment
10'9
FIG. 3. The effect of ANP on cGMP accumulation after 1 h of incubation. The median number of oocytes in each group was 300. Data were pooled from four experiments. **, Significantly higher (P < 0.01) than control group. TABLE 2. Effect of SNP or Tyr8-ANP on cGMP accumulation from cumulus-enclosed oocytes after 1 h of incubation
Cone.
% Mature oocytes"
100 nM
83 ±2C 56±3
525 307
Treatment
Cone.
100 MM 10 nM 100 nM 1 mM
48 ± 3C
82 ± 3 83 ±2 6± 2C
284 159 326 217
Control SNP Tyr8-ANP
100 MM 100 nM
After 2 h of incubation the percentage of mature oocytes was evaluated. 0 Mean ± SEM. Data were pooled from three experiments. * Number of oocytes. c P < 0.01 vs. the control group.
cGMP accumulation (fmol/oocyte-cumulus complex)" 2.66 ± 0.38 5.50 ± 1.16C 1.39 ± 0.46
12 6 6
° Mean ± SEM. Data were pooled from three experiments. * Number of samples. c P < 0.01 us. the control group. TABLE 3. Effect of ANP (100 nM), 8BrcGMP (1 mM), or SNP (100 MM) on resumption of meiosis of denuded oocytes after 2 h of incubation
100-i
Treatment Control ANP SNP 8BrcGMP
80-
60>
% Mature oocytes" 76 ± 2 78 ± 2 74 ± 2 17 ± 2C
n" 400 265 331 360
° Mean ± SEM. Data were pooled from three experiments. Numbers of oocytes. c P < 0.01 us. the control group. b
20-
0-
TABLE 4. Effect of ANP on cAMP accumulation from cumulus-enclosed oocytes after 1 h of incubation 1 2
3
4
5
6
7
Hours
Treatment
FIG. 2. The effect ofANP (100 nM) on maturation of cumulus-enclosed oocytes after 1, 2, 4, and 6 h of incubation. The median number of oocytes in each group was 220. Data were pooled from three experiments. **, Significantly lower (P < 0.01) than corresponding control value.
(83% GVB; Fig. 1). For comparison, after 2 h of incubation in the presence of 1 mM dbcAMP 6% of the oocytes showed GVB (Table 1). The inhibitory effect on oocyte
Control ANP
Cone.
0.1 nM 1 nM 10 nM 100 nM
1000 nM 0 6
cAMP accumulation (fmol/oocyte-cumulus complex)" 3.16 ± 2.49 ± 2.55 ± 2.49 ± 3.01 ± 2.96 ±
0.39 0.15 0.24 0.44 0.58 0.11
Mean ± SEM. Data were pooled from two experiments. Number of samples.
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9 4 4
9 8 8
ANP INHIBITS OOCYTE MATURATION
maturation of ANP (100 nM) was evident after 1 h of incubation and persisted at least 6 h (Fig. 2). Furthermore, when the ANP analog Tyr8-ANP was used, no effect was seen after 2 h with 10 or 100 nM of the compound (82% and 83% GVB, respectively) compared to the control (83%; Table 1). ANP dose-dependently stimulated cGMP accumulation in oocyte-cumulus complexes after 1 h of incubation. ANP significantly stimulated cGMP accumulation at 100 nM and higher concentrations (Fig. 3), but Tyr8-ANP did not increase the levels of cGMP (Table 2). Effect of SNP on meiosis and cGMP levels in oocytecumulus complexes To test the possibility that the inhibition of oocyte maturation by ANP was mediated via cGMP, the oocytecumulus complexes were incubated in the presence of SNP, which is believed to activate the soluble guanylate cyclase. After 2 h of incubation with 100 /*M SNP, 48% of the oocytes matured compared to 83% in the control group (Table 1). SNP (100 /*M) also stimulated cGMP accumulation in oocyte-cumulus complexes after 1 h of incubation (Table 2). Effect of ANP and SNP on meiosis in denuded oocytes The role of the cumulus cells was studied by incubating denuded oocytes for 2 h in the presence of the different compounds. Compared to control (76% GVB) neither SNP (100 MM; 74% GVB) nor ANP (100 nM; 78% GVB) inhibited the resumption of meiosis. However, 8Br cGMP (1 mM; 17% GVB) significantly inhibited the maturation rate in the denuded oocytes compared to the control value (Table 3). Effect of ANP and SNP on cAMP levels in oocytecumulus complexes The level of cAMP in oocyte-cumulus complex was not increased by ANP (0.1-1000 nM) after 1 h incubation (Table 4). Neither SNP (100 /xM; 1.42 ± 0.25 fmol cAMP/ oocyte-cumulus complex) nor Tyr8-ANP (100 nM; 4.4 ± 1.1 fmol cAMP/oocyte-cumulus complex) stimulated cAMP accumulation after 1 h of incubation compared to the control value (3.2 ± 0.83 fmol cAMP/oocyte-cumulus complex. Discussion Our results demonstrate that ANP dose-dependently inhibited spontaneous rat oocyte maturation. The inhibition was persistent over the time studied. We also demonstrate that ANP increased cGMP accumulation in oocyte-cumulus complexes without elevating cAMP
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levels. Preliminary data from our laboratory indicate an inhibition of spontaneous maturation by ANP in mouse oocyte-cumulus complexes. The ANP analog Tyr8-ANP could differentiate between two different ANP receptors that mediated relaxation of smooth muscle, only one of them with a concomitant increase in cGMP (23). In our experiments this ANP analog did not affect spontaneous oocyte maturation or cGMP accumulation in oocytecumulus complexes. To further support the possibility that the ANP-induced cGMP levels could influence oocyte maturation we stimlated the soluble form of the guanylate cyclase with SNP. Also in this instance spontaneous maturation was inhibited, and cGMP accumulation was increased without a rise in cAMP levels. This indicated that both the membrane-associated and the soluble form of the guanylate cyclase are present in the oocyte-cumulus complex. Taken together, these data suggest that oocyte-cumulus complexes have ANP receptors and that increased levels of cGMP inhibit spontaneous oocyte maturation in the rat. The inhibition was related to guanylate cyclase activation, but we have no evidence suggesting whether the ANP-A or the ANP-B receptor (5) mediated the effect. The concentrations of ANP (1 nM to 1 JLIM) used in this study were in the same range as those used in other studies to achieve progesterone secretion from granulosa cells (9) or cGMP accumulation in cultured fibroblasts (8). However, if ANP-B receptor was activated, an effect might be accomplished with lower concentrations of BNP or a putative substance of ovarian origin similar to ANP. ANP and SNP had effects on oocyte-cumulus complexes. In denuded oocytes, where the cumulus cells had been mechanically removed, neither ANP nor SNP inhibited spontaneous maturation. This suggests that the oocyte lacks both ANP receptors mediating the inhibition and a soluble guanylate cyclase. However, 8BrcGMP inhibited the spontaneous maturation of denuded oocytes, in accordance to earlier findings (18), suggesting that cGMP does have an effect in the oocyte, but is produced in cumulus cells. Recently, we have shown that levels of cGMP decrease in the spontaneously maturing oocyte in the rat, parallel to and of the same magnitude as cAMP (Tornell, J., H. Billig, and O. T. Hillens, submitted for publication). We have also shown that the resumption of meiosis is delayed in rat oocytes microinjected with cGMP in a similar way as oocytes injected with equimolar amounts of cAMP (Tornell, J., H. Billig, and O. T. Hillens, submitted), suggesting that, at least in the rat, cGMP level has a temporal relation to resumption of meiosis and may play a functional role in this process. Several experiments support the hypothesis that decreased levels of cAMP in the oocyte are crucial for the resumption of meiosis (12-16). Extracts from mouse
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ANP INHIBITS OOCYTE MATURATION
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oocytes contain a cyclic nucleotide phosphodiesterase (PDE) that can be inhibited by cGMP (24) in concentrations found in the rat oocyte (Tornell, J., H. Billig, and 0. T. Hillens, submitted). Consequently, it may be suggested that when cGMP levels decrease in the oocyte, the activity of the cGMP-inhibited PDE may be increased, and the level of cAMP decreased. Another possibility is that cGMP may stimulate the cGMP-dependent protein kinase which phosphorylates putative proteins important to hold the oocyte arrested in meiotic prophase. Partly contradictionary data suggest that ANP in vitro could increase the maturation rate in hamster oocytes at certain incubation times and concentrations. The authors speculated that this increase in maturation rate could be an effect of cGMP stimulating PDE activity in the oocyte (25). The presented data demonstrate that the cumulus cells surrounding the oocyte have ANP receptors, a membrane-associated and a soluble guanylate cyclase, while the oocyte lacks ANP receptors that produce cGMP and a soluble guanylate cyclase. Furthermore, that stimulation of both guanylate cyclase in cumulus cells inhibits spontaneous oocyte maturation, suggesting a possible involvement of cGMP in the meiotic arrest. A physiological role for ANP in the ovary has not yet been established. However, a possible local production of ANP-like factors similar to BNP production in the brain is plausible, but an influence of circulating ANP cannot at present be ruled out.
Acknowledgment We would like to thank Dr. Torbjorn Hillensjo for valuable suggestions and discussions.
References 1. Cantin M, Genest J 1985 The heart and atrial natriuretic factor. Endocr Rev 6:107 2. Ledsome J, Wilson N, Courneya C, Rankin A 1985 Release of atrial natriuretic peptide by atrial distension. Can J Physiol 63:739 3. Gardner D, Hane S, Trachewsky D, Schenk D, Baxter J 1986 Atrial natriuretic peptide mRNA is regulated by glucocorticoids in vivo. Biochem Biophys Res Commun 139:1047 4. Sudoh T, Kangawa K, Minamino N, Matsuo H 1988 A new natriuretic peptide in porcine brain. Nature 332:78 5. Chang M, Lowe DG, Lewis M, Hellmiss R, Chen E, Goeddel DV 1989 Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases. Nature 341:68 6. Lowe D, Chang M, Hellmiss R, Chen E, Singh S, Garbers D, Goeddel D 1989 Human atrial natriuretic peptide defines a new paradigm for second messenger signal transduction. EMBO J
Endo«1990 Voll26«No3
8:1377 7. Chinkers M, Garbers D, Chang M, Lowe D, Chin H, Goeddel D, Schultz S 1989 A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature 338:78 8. Leitman D, Agnost V, Tuan J, Andresen J, Murad F 1987 Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase. Biochem J 244:69 9. Pandey K, Osteen K, Inagami T 1987 Specific receptor-mediated of progesterone secretion and cCMP accumulation by rat atrial natriuretic factor in cultured human granulosa-lutein (G-L) cells. Endocrinology 121:1195 10. Usuki S, Hosokawa A, Ichikawa Y, Kubota S, Usuki Y, Shinjo M, Kim S, Miyazaki H, Murakami K, Binding sites for atrial natriuretic peptide in high concentrations in human ovarian follicles. Kyoto Symposium on ANP, 1988, poster 39, 44 (Abstract) 11. Pincus G, Enzmann E 1935 The comparative behavior of mammalian eggs in vivo and in vitro. The activation of ovarian eggs. J Exp Med 62:665 12. Cho W, Stern S, Biggers J 1974 Inhibitory effect of dibuturyl cAMP on mouse oocyte maturation in vitro. J Exp Zool 187:383 13. Magnusson C, Hillensjo T 1977 Inhibition of maturation and metabolism in rat oocytes by cyclic AMP. J Exp Zool 201:139 14. Schultz R, Montgomery R, Belanoff J 1983 Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis. Dev Biol 97:264 15. Vivarelli E, Conti M, DeFelici M, Siracusa G 1983 Meiotic resumption and intracellular cAMP levels in mouse oocytes treated with compounds which act on cAMP metabolism. Cell Differentiation 12:271 16. Bornslaeger E, Mattei P, Schultz R 1986 Involvement of cAMPdependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. Dev Biol 114:453 17. Hubbard C, Terranova P 1982 Inhibitory action of cyclic guanosine 5'-phosphoric acid (GMP) on oocyte maturation: dependence on an intact cumulus. Biol Reprod 26:628 18. Tornell J, Brannstrom M, Hillensjo T 1984 Different effects of cyclic nucleotide derivatives upon the fat oocyte cumulus complex in vitro. Acta Physiol Scand 122:507 19. Ratner A 1976 Effects of follicle stimulating hormone and luteinizing hormone upon cyclic AMP and cyclic GMP levels in rat ovaries in vitro. Endocrinology 99:1496 20. Ratner A, Sanborn C 1980 Effect of endogenous LH secretion on ovarian cyclic AMP and cyclic GMP levels in the rat. Life Sci 26:439 21. Hubbard C, Greenwald G 1982 Cyclic nucleotides, DNA, and steroid levels in ovarian follicles and corpora lutea of the cyclic hamster. Biol Reprod 26:230 22. Hogan B, Costantini F, Lacy E 1986 In vitro culture of eggs, embryos and teratocarcinoma cells. In: Hogan B, Costantini F, Lacy E (eds) Manipulating the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, p 245 23. Budzik G, Firestone S, Bush E, Connolly P, Rockway T, Sarin V, Holleman W 1987 Divergence of ANF analogs in smooth muscle cell cGMP response and aorta vasorelaxation: evidence for receptor subtypes. Biochem Biophys Res Commun 144:422 24. Bornslaeger E, Wilde M, Schultz R 1984 Regulation of mouse oocyte maturation: involvement of cyclic AMP phosphodiesterase and calmodulin. Dev Biol 105:488 25. Hubbard C, Price J 1988 The effects of follicle-stimulating hormone and cyclic guanosine 3',5'-monophosphate on cyclic adenosine 3',5'-monophosphatephosphodiesterase and resumption of meiosis in hamster cumulus-oocyte complexes. Biol Reprod 39:829
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Vol. 126, No. 3 Printed in U.S.A.
Atrial Natriuretic Peptide Inhibits Spontaneous Rat Oocyte Maturation* JAN TORNELL, BJORN CARLSSON, AND HAKAN BILLIG Department of Physiology, University of Gbteborg, Goteborg, Sweden
ABSTRACT. We report results of experiments demonstrating a dose-dependent inhibition of spontaneous maturation (resumption of meiosis) in rat oocyte-cumulus complexes by atrial natriuretic peptide (ANP). The inhibition was persistent over the time period studied. The ANP analog Tyr8-ANP, which mediates smooth muscle relaxation in other organs without elevating cGMP levels, did not inhibit the spontaneous maturation. ANP, but not Tyr8-ANP, dose-dependently stimulated cGMP accumulation in oocyte-cumulus complexes. Furthermore, sodium nitroprusside (SNP), that stimulates a soluble form of guanylate cyclase, inhibited spontaneous maturation in
A
TRIAL natriuretic peptide (ANP) is believed to act L via the cGMP system, and its primary targets include kidney, vascular smooth muscle, and adrenal cortex, where it induces natriuresis, causes vasodilatation, and inhibits aldosterone release, respectively (1). ANP is mainly produced in the heart atrial cardiocytes and released in response to increased tension in the atria (2). Also, glucocortocoids can increase plasma levels of ANP as well as ANP mRNA levels in the atria (3). Recently, a natriuretic peptide, brain natriuretic peptide (BNP), has been localized in the brain (4). BNP has a structure similar to that of ANP and interacts with the ANP receptor. Three forms of the ANP receptor are known, designated ANP-A, ANP-B, and ANP-C. The ANP-C receptor has no guanylate cyclase activity, in contrast to the ANP-A and ANP-B receptors. The ANPB receptor is more effectively stimulated by BNP than ANP. ANP and BNP are equally effective in stimulating cGMP accumulation when bound to the ANP-A receptor (5). The guanylate cyclase is an intracellular part of the receptor molecule (6, 7), which is activated when ANP is bound (8). In the ovary, local production of ANP or Received October 13, 1989. Address all correspondence and requests for reprints to: Jan Tornell, Department of Physiology, University of Goteborg, P.O. Box 33031, S400 33 Goteborg, Sweden. * This work was supported by grants from the Swedish Medical Research Council (no. B89-04x-27 and B-89-04x-05650), the Swedish Society for Medical Research, the Goteborg Medical Society, Swedish Medical Society, the Faculty of Medicine, University of Goteborg, and Handl Hjalmar Svenssons Research Funds.
oocyte-cumulus complexes and stimulated cGMP accumulation in oocyte-cumulus complexes. Neither ANP nor SNP stimulated cAMP accumulation. In oocytes where the surrounding cumulus cells had been removed neither ANP nor SNP inhibited the spontaneous maturation. These results demonstrate that cumulus cells, but not the oocyte itself, have ANP receptors and guanylate cyclases. Furthermore, ANP, via cGMP, can influence oocyte meiosis, suggesting a possible involvement of ANP and cGMP in the control of the meiotic process in rat oocytes. (Endocrinology 126: 1504-1508, 1990)
A N P - l i k e peptides h a s , to our knowledge, n o t yet been
demonstrated, but ANP stimulates LH-induced progesterone secretion from human cultured granulosa-lutein cells (9), indicating the presence of ANP receptors. A high concentration of binding sites has also been demonstrated in human preovulatory follicles (10). The oocyte undergoes meiotic divisions to reduce its genetic material before fusing with the sperm during fertilization. The first meiotic division in the oocyte starts during fetal life and becomes arrested at birth. The ovulatory LH surge leads to resumption of meiosis and completion of the first meiotic division (oocyte maturation) in the preovulatory follicle. The second meiotic division is not completed until the oocyte is fertilized. The fact that the oocyte matures spontaneously when liberated from the follicle (11) has led to the hypothesis that the oocyte is under constant inhibition when it is contained within the follicle. A number of theories have been presented to explain the mechanism of the intrafollicular meiotic arrest. cAMP has been shown, by several approaches, to be important for oocyte maturation. Experimentally, cAMP- or cAMP-elevating agents can prevent the spontaneous maturation in isolated oocytes surrounded by their cumulus cells (oocyte-cumulus complexes) and in oocytes where the cumulus cells were mechanically removed (denuded oocytes) (12, 13). In the mouse, cAMP levels decrease in the oocyte during the resumption of the meiosis (14, 15). Furthermore, injection of the catalytic subunit of cAMP-dependent protein
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ANP INHIBITS OOCYTE MATURATION kinase delays spontaneous mouse oocyte maturation (16). ANP does not increase cAMP accumulation. On the contrary, ANP can inhibit adenylate cyclase under some circumstances (1). However, cGMP can influence oocyte maturation. cGMP derivatives have been reported to inhibit the spontaneous maturation of oocyte-cumulus complexes in the hamster (17) and in denuded rat oocytes (18). Gonadotropins seem to influence the levels of cGMP, but in an inverse pattern compared to cAMP levels. In vitro, gonadotropins increase cAMP levels and decrease cGMP levels in isolated rat ovaries (19). In vivo, after the LH surge on proestrus, there is a decrease in cGMP levels and an increase in cAMP levels in whole rat ovaries (20) and isolated preovulatory hamster follicles (21). This temporal relationship between decreasing cGMP levels and resumption of meiosis after the LH surge could be of functional importance. Since cGMP derivatives inhibit spontaneous maturation, one may assume that if the oocyte-cumulus complex has ANP receptors, ANP may influence this process by increasing intracellular cGMP levels. Guanylate cyclase exists in several forms; besides the membrane-associated form there is also a soluble form which can be stimulated by sodium nitroprusside (SNP) (8). The aim of this study was to examine whether ANP could influence meiotic maturation and, in that case, by which mechanism.
Materials and Methods Animals Twenty-six-day-old immature, female Sprague-Dawley rats (ALAB, Stockholm, Sweden) were injected with 10 IU PMSG (Sigma Chemical Co., St. Louis, MO), sc, to induce synchronized follicular development. The rats were housed under Standardized environmental conditions with lights on between 0500-1900 h. Water and food were supplied ad libitum. Tissue preparation and incubation procedures Twenty-eight-day-old rats were killed by cervical dislocation. The ovaries were dissected out, cleaned from adherent tissue, and placed in incubation medium. Large follicles were punctured with a hypodermic needle under a stereomicroscope. The oocyte-cumulus complexes were passed through three dishes to remove granulosa cells and follicular fluid before transfer to a final dish for incubation. Oocyte-cumulus complexes were incubated in 750 /xl Eagle's Minimum Essential Medium with Earle's salts (Gibco, Paisley, Scotland) buffered with 10 mM HEPES and supplemented with 0.1% BSA (Sigma) and Gentamicin (50 Mg/ml; Sigma). Oocytes from a pair of ovaries were incubated in humidified air for 1-6 h in each dish. Denuded oocytes were obtained by mechanically removing the cumulus cells by a series of mouth-controlled micropipettes of successively smaller diameter. Isolation and denudation of the oocytes were performed in medium M2 buffered with HEPES (22). Denuded oocytes were incubated in medium Mi6 (22) buffered
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with 25 mM HCO3. Both media contained 0.4% BSA (Sigma). Denuded oocytes were incubated under an humidified atmosphere of 5% CO2 in air for the indicated period of time. Chemicals and hormones Synthetic rat ANP (fragment 5-27), (Bu)2cAMP (dbcAMP), 8-bromo-cGMP (8BrcGMP) and SNP were purchased from Sigma. Tyr8-ANP-(5-27), an ANP analog that relaxes smooth muscle without affecting cGMP levels (23), was purchased from Peninsula Laboatories (Merseyside, United Kingdom). Oocyte examination The oocyte-cumulus complexes and denuded oocytes were collected from the culture dishes after incubation and examined by Nomarski optics to reveal stage of meiosis. Oocytes showing a germinal vesicle (GV) were considered still arrested in prophase I, and those with GV breakdown (GVB) to have resumed meiosis. Determination of cAMP and cGMP cAMP and cGMP analyses were performed with a RIA kit (Amersham, Radiochemical Centre, United Kingdom). The incubations were terminated by adding ice-cold ethanol to a final concentration of 60%. The samples were centrifuged, and pellets were rinsed with 750 ^1 60% ethanol and centrifugated. Total supernatant was evaporated, and dried extracts were acetylated according to the manufacturer's instructions. Fifteen to 20 oocyte-cumulus complexes were measured in each sample. The amounts of cAMP and cGMP in each sample were 4-10 and 5-40 fmol/tube, respectively. The sensitivity was 1.0 and 0.5 fmol/tube for the cAMP and cGMP assays, respectively. Cross-reactivity for the cAMP RIA to cGMP was less than 0.0004%, and that for the cGMP RIA to cAMP was less than 0.02% according to the manufacturer. Statistics The values are given as the mean ± SEM. Experiments were performed at least three times with similar results, except for the effect of ANP on cAMP accumulation that were performed twice with similar results. In the presented data on oocyte maturation n refers to number of oocytes, and the SE was obtained by a normal approximation of a binomial distribution. In data on levels of cyclic nucleotides n refers to number of samples. Statistical differences were calculated by analysis of variance followed by Student-Newman-Keuls test. P < 0.05 was considered statistically significant.
Results Effect of ANP on meiosis and cGMP accumulation in oocyte-cumulus complexes ANP inhibited spontaneous maturation of oocyte-cumulus complexes in a dose-dependent manner after 2 h of incubation in concentrations ranging from 1 nM (72% GVB) to 1 ixM (48% GVB) compared to the control group
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ANP INHIBITS OOCYTE MATURATION
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Endo • 1990 Vol 126 • No 3
lOO-i
80-
8 5
i
o 40
K
'
4-
32-
201-
0
10' 9
10"8
10"
10"6
0Jr
10"
10~
ANP (M)
TABLE 1. Effect of ANP, dbcAMP, SNP, or Tyr8-ANP on resumption of meiosis of cumulus-enclosed oocytes
Control ANP SNP Tyr8-ANP dbcAMP
10'8 10"7 10'6 10"
ANP (M)
FIG. 1. The effect of ANP on maturation of cumulus-enclosed oocytes after 2 h of incubation. The median number of oocytes in each group was 280. Data were pooled from three experiments. **, Significantly lower (P < 0.01) than control value.
Treatment
10'9
FIG. 3. The effect of ANP on cGMP accumulation after 1 h of incubation. The median number of oocytes in each group was 300. Data were pooled from four experiments. **, Significantly higher (P < 0.01) than control group. TABLE 2. Effect of SNP or Tyr8-ANP on cGMP accumulation from cumulus-enclosed oocytes after 1 h of incubation
Cone.
% Mature oocytes"
100 nM
83 ±2C 56±3
525 307
Treatment
Cone.
100 MM 10 nM 100 nM 1 mM
48 ± 3C
82 ± 3 83 ±2 6± 2C
284 159 326 217
Control SNP Tyr8-ANP
100 MM 100 nM
After 2 h of incubation the percentage of mature oocytes was evaluated. 0 Mean ± SEM. Data were pooled from three experiments. * Number of oocytes. c P < 0.01 vs. the control group.
cGMP accumulation (fmol/oocyte-cumulus complex)" 2.66 ± 0.38 5.50 ± 1.16C 1.39 ± 0.46
12 6 6
° Mean ± SEM. Data were pooled from three experiments. * Number of samples. c P < 0.01 us. the control group. TABLE 3. Effect of ANP (100 nM), 8BrcGMP (1 mM), or SNP (100 MM) on resumption of meiosis of denuded oocytes after 2 h of incubation
100-i
Treatment Control ANP SNP 8BrcGMP
80-
60>
% Mature oocytes" 76 ± 2 78 ± 2 74 ± 2 17 ± 2C
n" 400 265 331 360
° Mean ± SEM. Data were pooled from three experiments. Numbers of oocytes. c P < 0.01 us. the control group. b
20-
0-
TABLE 4. Effect of ANP on cAMP accumulation from cumulus-enclosed oocytes after 1 h of incubation 1 2
3
4
5
6
7
Hours
Treatment
FIG. 2. The effect ofANP (100 nM) on maturation of cumulus-enclosed oocytes after 1, 2, 4, and 6 h of incubation. The median number of oocytes in each group was 220. Data were pooled from three experiments. **, Significantly lower (P < 0.01) than corresponding control value.
(83% GVB; Fig. 1). For comparison, after 2 h of incubation in the presence of 1 mM dbcAMP 6% of the oocytes showed GVB (Table 1). The inhibitory effect on oocyte
Control ANP
Cone.
0.1 nM 1 nM 10 nM 100 nM
1000 nM 0 6
cAMP accumulation (fmol/oocyte-cumulus complex)" 3.16 ± 2.49 ± 2.55 ± 2.49 ± 3.01 ± 2.96 ±
0.39 0.15 0.24 0.44 0.58 0.11
Mean ± SEM. Data were pooled from two experiments. Number of samples.
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9 4 4
9 8 8
ANP INHIBITS OOCYTE MATURATION
maturation of ANP (100 nM) was evident after 1 h of incubation and persisted at least 6 h (Fig. 2). Furthermore, when the ANP analog Tyr8-ANP was used, no effect was seen after 2 h with 10 or 100 nM of the compound (82% and 83% GVB, respectively) compared to the control (83%; Table 1). ANP dose-dependently stimulated cGMP accumulation in oocyte-cumulus complexes after 1 h of incubation. ANP significantly stimulated cGMP accumulation at 100 nM and higher concentrations (Fig. 3), but Tyr8-ANP did not increase the levels of cGMP (Table 2). Effect of SNP on meiosis and cGMP levels in oocytecumulus complexes To test the possibility that the inhibition of oocyte maturation by ANP was mediated via cGMP, the oocytecumulus complexes were incubated in the presence of SNP, which is believed to activate the soluble guanylate cyclase. After 2 h of incubation with 100 /*M SNP, 48% of the oocytes matured compared to 83% in the control group (Table 1). SNP (100 /*M) also stimulated cGMP accumulation in oocyte-cumulus complexes after 1 h of incubation (Table 2). Effect of ANP and SNP on meiosis in denuded oocytes The role of the cumulus cells was studied by incubating denuded oocytes for 2 h in the presence of the different compounds. Compared to control (76% GVB) neither SNP (100 MM; 74% GVB) nor ANP (100 nM; 78% GVB) inhibited the resumption of meiosis. However, 8Br cGMP (1 mM; 17% GVB) significantly inhibited the maturation rate in the denuded oocytes compared to the control value (Table 3). Effect of ANP and SNP on cAMP levels in oocytecumulus complexes The level of cAMP in oocyte-cumulus complex was not increased by ANP (0.1-1000 nM) after 1 h incubation (Table 4). Neither SNP (100 /xM; 1.42 ± 0.25 fmol cAMP/ oocyte-cumulus complex) nor Tyr8-ANP (100 nM; 4.4 ± 1.1 fmol cAMP/oocyte-cumulus complex) stimulated cAMP accumulation after 1 h of incubation compared to the control value (3.2 ± 0.83 fmol cAMP/oocyte-cumulus complex. Discussion Our results demonstrate that ANP dose-dependently inhibited spontaneous rat oocyte maturation. The inhibition was persistent over the time studied. We also demonstrate that ANP increased cGMP accumulation in oocyte-cumulus complexes without elevating cAMP
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levels. Preliminary data from our laboratory indicate an inhibition of spontaneous maturation by ANP in mouse oocyte-cumulus complexes. The ANP analog Tyr8-ANP could differentiate between two different ANP receptors that mediated relaxation of smooth muscle, only one of them with a concomitant increase in cGMP (23). In our experiments this ANP analog did not affect spontaneous oocyte maturation or cGMP accumulation in oocytecumulus complexes. To further support the possibility that the ANP-induced cGMP levels could influence oocyte maturation we stimlated the soluble form of the guanylate cyclase with SNP. Also in this instance spontaneous maturation was inhibited, and cGMP accumulation was increased without a rise in cAMP levels. This indicated that both the membrane-associated and the soluble form of the guanylate cyclase are present in the oocyte-cumulus complex. Taken together, these data suggest that oocyte-cumulus complexes have ANP receptors and that increased levels of cGMP inhibit spontaneous oocyte maturation in the rat. The inhibition was related to guanylate cyclase activation, but we have no evidence suggesting whether the ANP-A or the ANP-B receptor (5) mediated the effect. The concentrations of ANP (1 nM to 1 JLIM) used in this study were in the same range as those used in other studies to achieve progesterone secretion from granulosa cells (9) or cGMP accumulation in cultured fibroblasts (8). However, if ANP-B receptor was activated, an effect might be accomplished with lower concentrations of BNP or a putative substance of ovarian origin similar to ANP. ANP and SNP had effects on oocyte-cumulus complexes. In denuded oocytes, where the cumulus cells had been mechanically removed, neither ANP nor SNP inhibited spontaneous maturation. This suggests that the oocyte lacks both ANP receptors mediating the inhibition and a soluble guanylate cyclase. However, 8BrcGMP inhibited the spontaneous maturation of denuded oocytes, in accordance to earlier findings (18), suggesting that cGMP does have an effect in the oocyte, but is produced in cumulus cells. Recently, we have shown that levels of cGMP decrease in the spontaneously maturing oocyte in the rat, parallel to and of the same magnitude as cAMP (Tornell, J., H. Billig, and O. T. Hillens, submitted for publication). We have also shown that the resumption of meiosis is delayed in rat oocytes microinjected with cGMP in a similar way as oocytes injected with equimolar amounts of cAMP (Tornell, J., H. Billig, and O. T. Hillens, submitted), suggesting that, at least in the rat, cGMP level has a temporal relation to resumption of meiosis and may play a functional role in this process. Several experiments support the hypothesis that decreased levels of cAMP in the oocyte are crucial for the resumption of meiosis (12-16). Extracts from mouse
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ANP INHIBITS OOCYTE MATURATION
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oocytes contain a cyclic nucleotide phosphodiesterase (PDE) that can be inhibited by cGMP (24) in concentrations found in the rat oocyte (Tornell, J., H. Billig, and 0. T. Hillens, submitted). Consequently, it may be suggested that when cGMP levels decrease in the oocyte, the activity of the cGMP-inhibited PDE may be increased, and the level of cAMP decreased. Another possibility is that cGMP may stimulate the cGMP-dependent protein kinase which phosphorylates putative proteins important to hold the oocyte arrested in meiotic prophase. Partly contradictionary data suggest that ANP in vitro could increase the maturation rate in hamster oocytes at certain incubation times and concentrations. The authors speculated that this increase in maturation rate could be an effect of cGMP stimulating PDE activity in the oocyte (25). The presented data demonstrate that the cumulus cells surrounding the oocyte have ANP receptors, a membrane-associated and a soluble guanylate cyclase, while the oocyte lacks ANP receptors that produce cGMP and a soluble guanylate cyclase. Furthermore, that stimulation of both guanylate cyclase in cumulus cells inhibits spontaneous oocyte maturation, suggesting a possible involvement of cGMP in the meiotic arrest. A physiological role for ANP in the ovary has not yet been established. However, a possible local production of ANP-like factors similar to BNP production in the brain is plausible, but an influence of circulating ANP cannot at present be ruled out.
Acknowledgment We would like to thank Dr. Torbjorn Hillensjo for valuable suggestions and discussions.
References 1. Cantin M, Genest J 1985 The heart and atrial natriuretic factor. Endocr Rev 6:107 2. Ledsome J, Wilson N, Courneya C, Rankin A 1985 Release of atrial natriuretic peptide by atrial distension. Can J Physiol 63:739 3. Gardner D, Hane S, Trachewsky D, Schenk D, Baxter J 1986 Atrial natriuretic peptide mRNA is regulated by glucocorticoids in vivo. Biochem Biophys Res Commun 139:1047 4. Sudoh T, Kangawa K, Minamino N, Matsuo H 1988 A new natriuretic peptide in porcine brain. Nature 332:78 5. Chang M, Lowe DG, Lewis M, Hellmiss R, Chen E, Goeddel DV 1989 Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases. Nature 341:68 6. Lowe D, Chang M, Hellmiss R, Chen E, Singh S, Garbers D, Goeddel D 1989 Human atrial natriuretic peptide defines a new paradigm for second messenger signal transduction. EMBO J
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8:1377 7. Chinkers M, Garbers D, Chang M, Lowe D, Chin H, Goeddel D, Schultz S 1989 A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature 338:78 8. Leitman D, Agnost V, Tuan J, Andresen J, Murad F 1987 Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase. Biochem J 244:69 9. Pandey K, Osteen K, Inagami T 1987 Specific receptor-mediated of progesterone secretion and cCMP accumulation by rat atrial natriuretic factor in cultured human granulosa-lutein (G-L) cells. Endocrinology 121:1195 10. Usuki S, Hosokawa A, Ichikawa Y, Kubota S, Usuki Y, Shinjo M, Kim S, Miyazaki H, Murakami K, Binding sites for atrial natriuretic peptide in high concentrations in human ovarian follicles. Kyoto Symposium on ANP, 1988, poster 39, 44 (Abstract) 11. Pincus G, Enzmann E 1935 The comparative behavior of mammalian eggs in vivo and in vitro. The activation of ovarian eggs. J Exp Med 62:665 12. Cho W, Stern S, Biggers J 1974 Inhibitory effect of dibuturyl cAMP on mouse oocyte maturation in vitro. J Exp Zool 187:383 13. Magnusson C, Hillensjo T 1977 Inhibition of maturation and metabolism in rat oocytes by cyclic AMP. J Exp Zool 201:139 14. Schultz R, Montgomery R, Belanoff J 1983 Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis. Dev Biol 97:264 15. Vivarelli E, Conti M, DeFelici M, Siracusa G 1983 Meiotic resumption and intracellular cAMP levels in mouse oocytes treated with compounds which act on cAMP metabolism. Cell Differentiation 12:271 16. Bornslaeger E, Mattei P, Schultz R 1986 Involvement of cAMPdependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. Dev Biol 114:453 17. Hubbard C, Terranova P 1982 Inhibitory action of cyclic guanosine 5'-phosphoric acid (GMP) on oocyte maturation: dependence on an intact cumulus. Biol Reprod 26:628 18. Tornell J, Brannstrom M, Hillensjo T 1984 Different effects of cyclic nucleotide derivatives upon the fat oocyte cumulus complex in vitro. Acta Physiol Scand 122:507 19. Ratner A 1976 Effects of follicle stimulating hormone and luteinizing hormone upon cyclic AMP and cyclic GMP levels in rat ovaries in vitro. Endocrinology 99:1496 20. Ratner A, Sanborn C 1980 Effect of endogenous LH secretion on ovarian cyclic AMP and cyclic GMP levels in the rat. Life Sci 26:439 21. Hubbard C, Greenwald G 1982 Cyclic nucleotides, DNA, and steroid levels in ovarian follicles and corpora lutea of the cyclic hamster. Biol Reprod 26:230 22. Hogan B, Costantini F, Lacy E 1986 In vitro culture of eggs, embryos and teratocarcinoma cells. In: Hogan B, Costantini F, Lacy E (eds) Manipulating the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, p 245 23. Budzik G, Firestone S, Bush E, Connolly P, Rockway T, Sarin V, Holleman W 1987 Divergence of ANF analogs in smooth muscle cell cGMP response and aorta vasorelaxation: evidence for receptor subtypes. Biochem Biophys Res Commun 144:422 24. Bornslaeger E, Wilde M, Schultz R 1984 Regulation of mouse oocyte maturation: involvement of cyclic AMP phosphodiesterase and calmodulin. Dev Biol 105:488 25. Hubbard C, Price J 1988 The effects of follicle-stimulating hormone and cyclic guanosine 3',5'-monophosphate on cyclic adenosine 3',5'-monophosphatephosphodiesterase and resumption of meiosis in hamster cumulus-oocyte complexes. Biol Reprod 39:829
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