Acta Physiol Scund 1990, 139, 511-517
Resumption of rat oocyte meiosis is paralleled by a decrease in guanosine 3’,5’-cyclic monophosphate (cGMP) and is inhibited by microinjection of cGMP J. T O R N E L L , H. B I L L I G and T. H I L L E N S J O * Departments of Physiology and *Obstetrics and Gynecology, University of Goteborg, Sweden TORNELL, J., BILLIG,H. & HILLENSJO, T. 1990. Resumption of rat oocyte meiosis is paralleled by a decrease in guanosine 3’,5‘-cyclic monophosphate (cGMP) and is inhibited by microinjection of cGMP. Acta Physiol Scund 139, 511-517. Received 24 January 1990, accepted 6 March 1990. ISSN 0001-6772. Departments of Physiology and Obstetrics and Gynecology, University of Goteborg, Sweden.
The aim of the present study was to measure the level of cyclic GMP (cGMP) compared with the level of cyclic AMP (CAMP) in rat oocytes during resumption of meiosis I (oocyte maturation) and to microinject these cyclic nucleotides into the oocyte to study their effects on oocyte maturation. Immature oocytes were obtajned from prepubertal rats primed with pregnant mare’s serum gonadotropin. Oocytes were isolated and shortterm cultured under conditions enabling spontaneous maturation. The levels of cGMP and cAMP decreased in the oocyte during spontaneous maturation (from 0.41 to 0.25 and from 0.64 to 0.42 fmol per oocyte respectively). The decrease was observed during the first hour of culture, and no further decline was seen after 2 h. Microinjection of cGMP or cAMP into isolated immature oocytes delayed the spontaneous maturation, cGMP being slightly more effective than CAMP, whereas 2-deoxy-CAMP (which does not stimulate protein kinase A) did not. These results demonstrate for the first time that the level of cGMP decreases in the oocyte parallel to spontaneous meiosis, as already shown for CAMP. This suggests that cGMP, as well as CAMP, may be of importance for regulating this process. This assumption is further supported by data demonstrating a delay in the maturation of oocytes injected with cGMP. Key words: CAMP, cGMP, meiosis, oocyte, rat.
T h e mammalian oocyte matures, i.e. resumes meiosis I, spontaneously when liberated from the follicle and cultured in vitro (Pincus & Enzmann 1935). I t has therefore been suggested that the oocyte is under constant inhibition within the follicle, preventing it from undergoing meiosis prematurely. A number of substances have been proposed to be intrafollicular inhibitors of oocyte maturation. Spontaneous maturation of isolated porcine, rat and mouse oocytes is prevented by addition of follicular Correspondence : Jan Tornell, Department of Physiology, University of Goteborg, PO Box 33031, S-400 33 Goteborg, Sweden.
fluid (FFl) or by co-culture with granulosa cells (Tsafriri & Channing 1975, Tsafriri et al. 1977, Chari et al. 1983, Downs & Eppig 1984). A partially purified inhibitor of oocyte maturation (OMI) has been isolated from porcine FF1 (Tsafriri et al. 1976), but its chemical nature is still unknown. Hypoxanthine in concentrations found in mouse FF1 is inhibitory to spontaneous mouse oocyte maturation (Downs et al. 1985). Moreover, cyclic AMP (CAMP) can inhibit spontaneous oocyte maturation in several species (Cho et al. 1974, Magnusson & Hillensjo 1977). Experimental studies on the effects of cAMP on meiosis are complicated by the fact that there is
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a compartmentalization of cAMP levels within the follicle. In the mouse the levels of cAMP decrease in the oocyte and increase in granulosa cells prior to the resumption of meiosis in response to the luteinizing hormone (LH) surge (Schultz et al. 1983). Under certain conditions cAMP derivatives actually stimulate meiosis in follicle-enclosed rat oocytes (Hillensjo et al. 1978). However, microinjection of cAMP or the catalytic subunit of protein kinase A (PKA) into mouse oocytes inhibits their spontaneous maturation. Oocytes injected with a PKA inhibitor undergo spontaneous maturation in spite of the presence of dibuturyl-CAMP or the phosphodiesterase inhibitor isobutyl-metb.ylxanthine (IBMX; Bornslaeger et al. 1986). Therefore, the direct role of cAMP in the oocyte is generally believed to be inhibitory to meiosis. Ovarian cyclic G M P (cGMP) levels are also influenced by gonadotropins. Whole rat ovaries exposed to gonadotropins in vitro respond with decreased levels of cGMP while the levels of cAMP increase (Ratner 1976). Similar changes occur after the LH surge (Ratner & Sanborn 1980) in parallel with the resumption of meiosis. The possible role of cGMP in the regulation of meiosis is poorly understood. High concentrations of cGMP derivatives inhibit spontaneous maturation of hamster oocyte-cumulus complexes (Hubbard & Terranova 1982) and denuded rat oocytes (Tornell et al. 1984). Recently, we have shown that elevation of endogenous cGMP levels, by stimulation of the membranebound guanylate cyclase by atrial natriuretic peptide (ANP; Leitman et al. 1987), inhibits spontaneous rat oocyte maturation (Tornell et al. 1990). Stimulation of the soluble guanylate cyclase by sodium nitroprusside (SNP ; Leitman et al. 1987) also results in inhibition of oocyte maturation (Tornell et al. 1990). On the other hand, data from experiments with hamster oocyte-cumulus complexes suggest that ANP, via cGMP, actually stimulates meiosis at certain incubation times and concentrations (Hubbard & Price 1988). T o further evaluate the role of cGMP and cAMP in the regulation of meiosis in the rat we have measured endogenous cGMP and cAMP levels in spontaneously maturing oocytes. Furthermore, we have microinjected cGMP, cAMP or deoxy-CAMP (a cAMP derivative which does not activate PKA; de Wit et al. 1982) into rat oocytes and examined their effect on meiosis.
MATERIALS AND METHODS Animals. Female, 26-day-old, immature, Spraguee Dawley rats (Alab, Stockholm, Sweden) were injected S.C.with 10 IU of pregnant mare’s serum gonadotropin (Sigma Chemicals, St Louis, MO, USA) to induce synchronized follicular development. The animals were housed under standardized environmental conditions with lights on between 05.00 and 19.00 h. Water and food were provided ad libitum. Tissue preparation and incubation procedures. The rats, 28 days old, were killed by cervical dislocation in the morning before the L H surge. The ovaries were dissected out and cleaned of adherent tissue and transferred to M, culture medium buffered with Hepes (pH 7.4; Hogan et al. 1986). Large follicles were punctured with a hypodermic needle under a stereomicroscope to release oocyte-cumulus complexes. Oocyte-cumulus complexes were subsequently cultured for 1 4 h in 1.0 ml of medium M,, (Hogan et al. 1986) buffered with 25 mM HCO, in Falcon organ culture dishes (Becton Dickinson Labware, Iincoln Park, NJ, USA) under a humidified atmosphere of 5y0 CO, in air to study the spontaneous maturation. For microinjection experiments oocytecumulus complexes were collected and denuded in M, supplemented with 0.2 mM 3-isobutyl-1-methylxanthine (IBMX). The oocytes were denuded by removing the cumulus cells mechanically by a series of mouth-controlled micropipettes with successively smaller diameter (Magnusson et al. 1977). Oocytes with a germinal vesicle (GV) were considered arrested in prophase I and used for microinjection (see below) with vehicle or indicated substance, or cultured immediately. Oocytes were cultured in the medium M,, buffered with 25 mM HCO, under a humidified atmosphere of 5 yo CO, in air for 3 4 h and examined for the presence of GV at the indicated incubation times. I n experiments where cyclic nucleotides were measured, the oocyte-cumulus complexes were collected as described, except that no IBMX was present. Groups of 5G200 oocyte-cumulus complexes were either denuded immediately and the oocytes placed in 6G0/, ethanol within 15 min for cyclic nucleotide analysis with radioimmunoassay (RIA), or cultured for 1 or 2 h in MI, under a humidified atmosphere of 5 % CO, in air before being denuded and placed in 60% ethanol within 15 min for cyclic nucleotide analysis. Microinjectian of nocytes. Groups of 10 denuded oocytes were placed in droplets of M, supplemented with 0.2 mM IBMX covered with light paraffin oil (BDH Ltd, Poole, UK) on a depression slide at room temperature. T h e oocyte was sucked onto the end of a glass pipette held by a Narishigi hydraulic micromanipulator (Nikon, Tokyo, Japan). The injection pipette (tip less than 1 p m diameter) was made from
Meiosis and oocyte cGMP levels a glass capillary with an internal filament (Clark Electromedical Instruments, Pangbourne, UK) and also held by a Narishigi hydraulic micromanipulator. The injections were done with compressed air under a Nikon Diaphot inverted microscope with Nomarski optics under 400 x magnification. The injected volume was estimated to be approximately 10-20 pl. The substances injected were all in a concentration of 10 mM dissolved in water. After injection, the oocytes were collected, rinsed once in M, without IBMX and then transferred to 1.0 ml MI, medium in Falcon organ culture dishes for culture. Chemicals. Guanosine 3’,5’-cyclic monophosphate (cGMP), adenosine 3’,5’-cyclic monophosphate (CAMP), 2’-deoxyadenosine 3’,5’-cyclic monophosphate (deoxy-CAMP) and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma. Oocyte examination. In the microinjection experiments, the denuded oocytes were examined repeatedly at the indicated incubation times in the culture dishes with an inverted microscope to reveal the stage of meiosis. Microinjected oocytes showing signs of degeneration were not included in the data (see below). In experiments performed to evaluate the spontaneous maturation of oocyte-cumulus complexes, these were placed on microscope slides after the incubations to reveal the stage of meiosis with the help of Nomarski optics. Oocytes showing a germinal vesicle (GV) were considered to be still arrested in prophase I and those with GV breakdown (GVB) to have resumed meiosis. Determination of cGMP and C A M P . The cGMP and cAMP analyses were performed with RIA kits (Amersham, UK). The oocytes were placed in 60% ice-cold ethanol immediately or after 1 or 2 h of incubation as described’ above. The samples were centrifuged and the pellets rinsed with 750,d 60% ethanol and centrifuged. The supernatants were completely evaporated. The dried extracts were acetylated according to the manufacturer to improve sensitivity. Each sample contained 8&200 (cGMP) or 50-200 (CAMP) denuded oocytes, and the nucleotide content ranged between 12 and 38 or 6 and 52 fmol per tube respectively. The sensitivity was 0.5 and 1.0 fmol per tube respectively. Cross-reactivity for the cAMP RIA to cGMP was less than 0.0004% and for the cGMP RIA to cAMP less than 0.02% according to the manufacturer. Statistics. Each experiment was repeated three times with similar results and the data shown were pooled from these experiments. The values are given as mean fSEM. Statistical differences were calculated by analysis of variance followed by the StudentNewman-Keuls test. In the presented data on oocyte maturation n refers to the number of oocytes, and in data on the levels of cyclic nucleotides n refers to the number of samples. In the experiments regarding resumption of meiosis the data were treated as a
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normal approximation of the binomial distribution to obtain the standard error of the mean. Differences between different groups were calculated by two-way analysis of variance. Values of P < 0.05 were considered statistically significant.
RESULTS T h e endogenous levels of cGMP (0 h : 0.41 fmol per oocyte, corresponding to approximately 1.5 ,UM, assuming an oocyte radius of 40,um) in the oocyte decreased when the oocyte-cumulus complex was cultured in nitro for 1 h (0.25 fmol per oocyte, approximately 0.9 ,UM) but no further decline was seen when the oocyte-cumulus complex was cultured for 2 h (Fig. 1). Compared with cGMP, the concentration of cAMP was approximately 50% higher in the oocyte (0 h : 0.64 fmol per oocyte, approximately 2.4 ,UM) and decreased after 1 h of incubation (0.42 fmol per oocyte, approximately 1.6 /AM). No further decline was seen after 2 h of incubation (Fig. 1). The percentage decrease of cGMP was in the same range as for cAMP (Fig. 2 ) . The initial fall in cyclic nucleotide levels coincided with the resumption of meiosis (Fig. Microinjection of vehicle slightly delayed the spontaneous maturation compared with un0.71
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.,
Fig. 1. The oocyte levels of CAMP and cGMP after 0, 1 and 2 h of incubation. The median number of oocytes in each group was 745. The data were pooled from three experiments. +* = significantly lower ( P < 0.01) compared with 0 h of incubation. cGMP; 0, CAMP.
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d 0)
4
d
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?9
rD
?- P3)
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a)*
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.,
Fig. 2. The levels of cAMP and cGMP at 1 and 2 h expressed as a percentage of the levels at 0 h of incubation (left axis) and the percentage of immature oocyteecumulus complexes after 0, 1 and 2 h of incubation (right axis). The data were pooled from three experimepts. cGMP; 0, CAMP; A,immature oocytes.
injected oocytes (Fig. 3). Both cGMP and cAMP retarded the spontaneous maturation more than vehicle when microinjected into oocytes, with cGMP being somewhat more effective than
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Hours Fig. 3. The effect ofinjection of vehicle on resumption of meiosis compared with uninjected oocytes after 1,2,3 and 4 h of incubation. The median number of oocytes in each group was 55. The data were pooled from three experiments. The two groups differ from each other when analysed by two-way analysis of vehicle; 0, control. variance (P < 0.01).
.,
cAMP (Fig. 4). T h e retardation was transient, since the maturation rate was approximately the same after 4 h of incubation whether the oocytes were injected with vehicle or cyclic nucleotides. When deoxy-CAMP was injected into the oocyte, the spontaneous maturation was not retarded compared with vehicle-injected oocytes. An average of 11yo of the oocytes degenerated after injection and were excluded from the calcu-
DISCUSSION I n this study it was demonstrated that the levels of cGMP in rat oocytes decline during spontaneous maturation. It was also shown that microinjection of cGMP could transiently delay the spontaneous maturation of denuded oocytes. These results suggest that the decrease in cGMP levels may be of functional importance. A decrease in oocyte cAMP levels during maturation as previously demonstrated (Schultz et al. 1983, Vivarelli er al. 1983, Dekel 1987) was confirmed in this study. ~h~ finding that the inhibition was temporary may indicate that the oocyte has a capacity for degrading cGMP and diffuse Or that these through the membrane into the surrounding medium.
Meiosis and oocyte cGMP levels
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a
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Hours Fig. 4. The effect of injection of vehicle, CAMP, cGMP or deoxy-CAMP into oocytes. The median number of oocytes in each group was 48. The data were pooled from three experiments. When analysed by two-way analysis of variance the groups of vehicleand deoxy-CAMP-injected oocytes do not differ. Both the CAMP- and cGMP-injected oocytes differ from vehicle-injected ones ( P < 0.01) and cGMP-injected oocytes also differ from CAMP-injected ones (P< 0.01). 0, CAMP; vehicle; 0, cGMP; x , deoxyCAMP.
.,
The role of cGMP in oocyte maturation has only been indirectly indicated : cyclic G M P derivatives can inhibit spontaneous maturation (Hubbard & Terranova, 1982, Tornell et al. 1984), and ANP and' SNP can inhibit spontaneous maturation via elevated cGMP levels (Tornell et al. 1990). In this study we more directly indicated a function for cGMP in regulating oocyte maturation by demonstrating changes in oocyte levels of cGMP during this process and by direct injection of cGMP into the oocyte. The importance of cAMP in the regulation of oocyte meiosis has been illustrated by several approaches, as discussed above. The estimated levels of cAMP found in the present study were in the same range as previously described for the mouse (Schultz et al. 1983, Vivarelli et al. 1984) and the rat (Dekel 1987). Microinjection of cAMP into rat oocytes delayed the maturation, as earlier described in mouse by Bornslaeger et al. (1986). Deoxy-CAMP did not affect the maturation rate, suggesting that the delay does involve activation of PKA. The demonstrated decrease in endogenous levels of oocyte cGMP and cAMP coinciding
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with the resumption of meiosis and the delay of this process by micro-injection of cGMP or cAMP into the oocyte suggest that not only cAMP but also cAMP may have an important role in controlling meiosis. Based on the presented data we can only speculate about possible interactions between these two systems. In other systems, PKA and the cGMP-dependent protein kinase (PKG) are able to phosphorylate the same proteins (Lincoln & Corbin 1983). When cGMP binds to PKG, or when cAMP binds to PKA, a phosphorylation of putative proteins important for arresting the oocyte in meiotic prophase may occur. In high concentrations, as obtained after microinjections, cGMP and cAMP might interact in an unspecific way with PKA and P K G respectively (Khoo & Gill 1979). However, the cGMP levels produced in the oocyte-cumulus complex in response to ANP and SNP were in a range not likely to interact directly with the PKA (Tornell et al. 1990). Hundred- to thousand-fold higher concentrations of cGMP are required for activation of PKA, and 25- to 1000-fold (depending on the substrate) higher concentrations of cAMP are necessary to activate P K G (Khoo & Gill 1979). Levels of cAMP sufficient to inhibit meiosis can be maintained by stimulating adenylate cyclase or inhibiting phosphodiesterase (PDE). Several different forms of PDE exist (Beavo 1988). Among them, a cGMP-inhibited PDE has been described (MacPhee et al. 1988). The presence of a cGMP-inhibited PDE in extracts from mouse oocytes has been reported. At a concentration of 0.1 ,UM cGMP inhibited PDE activity in these extracts by 50 % (Bornslaeger et al. 1984). Our results show that the levels of cGMP in the rat oocyte are of the order of 1-2 ,UM, suggesting that cGMP may effectively inhibit this form of PDE. The activity of cGMPinhibited PDE may be increased when the cGMP levels decrease in the oocyte. An increased PDE activity might be a part of the mechanism for decreasing cAMP levels during resumption of meiosis. The mechanisms for the regulation of cGMP levels in the oocyte are at present not known. Further studies will be necessary to explain in detail the interaction of cAMP and cGMP in the oocyte. Our present studies on rat oocytes confirm reports on the mouse and on the rat showing that cAMP is important in regulating meiosis and, furthermore, our results suggest
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that cGMP may also have an important role in this respect.
Manual, pp. 245-267. Cold Spring Harbour Laboratory, MO. HUBBARD, C.J. & PRICE,J. 1988. The effects of follicle-stimulating hormone and cyclic guanosine This study was supported by grants from the Swedish 3’,5’-monophosphate on cyclic adenosine 3’3’Medical Research Council (nos. B89-04x-27 and B89monophosphate-phosphodiesterase and resumption 04x-05650), the Swedish Society for Medical Reof meiosis in hamster cumulus-oocyte complexes. search, Goteborg Medical Society, Swedish Medical Biol Reprod 39, 829-838. Society, the Faculty of Medicine, University of P.F. 1982. Inhibitory C.J. & TERRANOVA, Goteborg, and Handl. Hjalmar Svensson’s Research HUBBARD, action of cyclic guanosine 5’-phosphoric acid Funds. We would like to thank Dr Bjorn Carlsson for (GMP) on oocyte maturation: Dependence on an valuable discussions and suggestions. intact cumulus. Biol Reprod 26, 628-632. KHOO,J. & GILL, G. 1979. Comparison of cyclic nucleotide specificity of guanosine 3’,5’-monoREFERENCES phosphate-dependent protein kinase and adenosine 3’,5’-monophosphate-dependent protein kinase. BEAVO,J.A. 1988. Multiple isoenzymes of cyclic Biochim Biophys Acta 584, 21-32. nucleotide phosphodiesterase. In : P. Greengard & D.C., AGNOST, V.L., TUAN,J.J., ANDRESEN, G A. Robison (eds.) Advances in Second Messenger LEITMAN, J.W. & MURAD, F. 1987. Atrial natriuretic factor and Phosphoprotein Research, vol. 22, pp. 1-38. and sodium nitroprusside increase cGMP in culRaven Press, New York. tured rat lung fibroblasts by activating different BORNSIAEGER, E.A., WILDE,M.W. & SCHULTZ, R.M. forms of guanylate cyclase. Biochem 3 244, 69-74. 1984. Regulation of mouse oocyte maturation : Involvement of cyclic AMP phosphodiesterase and LINCOLN,T.M. & CORBIN,J.D. 1983. Characterization and biological role of the cGMP-dependent calmodulin. Dev Bid 105, 488499. protein kinase. In: P. Greengard & G. Robison R.M. BORNSLAEGER, E.A., MATTEI,P. & SCHULTZ, (eds.) Advances in Cyclic Nucleotide Research, vol. 1986. Involvement of CAMP-dependent protein 15, pp. 139-192. Raven Press, New York. kinase and protein phosphorylation in regulation of T., LEREA, K. C., REIFSNYDER, D., MOORE, mouse oocyte maturation. Dev B i d 114, 453462. MACPHEE, CHARI,S.,HILLENSJO, T., MAGNUSSON, C., STURM, & BEAVO,J. 1988. Phosphorylation results in activation of a cAMP phosphodiesterase in human G. & DAUME,E. 1983. In vitro inhibition of rat platelets. 3 Biol Chem 263, 10353-10358. oocyte meiosis by human follicular fluid fractions. MAGNUSSON, C. & HILLENSJO, T. 1977. Inhibition of Arch Gynecol 233, 155-164. maturation and metabolism in rat oocytes by cyclic CHO, W.K., STERN, s. & BIGGERS, J.D. 1974. ‘ AMP. 3 E X P Zoo1 201, 139-147. Inhibitory effect of dibuturyl cAMP on mouse C., HILLENSJO, T., TSAFRIRI,, A., HULToocyte maturation in vitro.3Exp Zoo1 187,383-386. MAGNUSSON, BORN,R. & AHREN,K. 1977. Oxygen consumption DEKEL,N. 1987. Interaction between the oocyte and of maturing rat oocytes. Bzol Reprod 17, 9-15. the granulosa cells in the preovulatory follicle. In : G. & ENZMANN, E.V. 1935. The comparative P. Leung, D. Armstrong, K. Ruf, W. Moger & PINCUS, behavior of mammalian eggs in vivo and in vitro. I. H. Friesen (eds.) Endocrinology and Physiology of The activation of ovarian eggs. 3 Exp Med 62, Reproduction, pp. 197-209. Plenum Press, New York. 665-675. DOWNS,S.M. & EppiG, J.J. 1984. Cyclic adenosine RATNER,A. 1976. Effects of follicle stimulating monophosphate and ovarian follicular fluid act hormone and luteinizing hormone upon cyclic AMP and cyclic GMP levels in rat ovaries in vitro. synergistically to inhibit mouse oocyte maturation. Endocrinology 114, 418426. Endocrinology 99, 1496-1500. C.R. 1980. Effect of DOWNS,S.M., COLEMAN, D.L., WARD-BAILEY, P.F. & RATNER,A. & SANBORN, endogenous L H secretion on ovarian cyclic AMP EPPIG, J.J. 1985. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low and cyclic GMP levels in the rat. Lzfe Sci 26, molecular weight fraction of porcine follicular fluid. 439445. SCHULTZ, R.M., MONTGOMERY, R.R. & BELANOFF, Proc N a t l Acad Sci USA 82, 454458. HILLENSJO, T., EKHOLM, C. & AHREN,K. 1978. Role J.R. 1983. Regulation of mouse oocyte meiotic maturation: Implication of a decrease in oocyte of cyclic AMP in oocyte maturation and glycolysis in the pre-ovulatory rat follicle. Acta Endocrinol cAMP and protein dephosphorylation in com(Copenh) 87, 377-388. mitment to resume meiosis. Dev Biol 97, 264273. F. & LACY, E. 1986. In vitro TORNELL, J., BRANNSTROM, M. & HILLENSJO, T . 1984. HOGAN, B., COSTANTINI, Different effects of cyclic nucleotide derivatives culture of eggs, embryos and teratocarcinoma cells. In: B. Hogan, F. Costantini & E. Lacy (eds.) upon the rat oocyte-cumulus complex in vitro. Acta Physiol Scand 122, 507-513. Manipulating the Mouse Embryo. A Laboratory a
Meiosis and oocyte cGMP levels TORNELL, J , , CARLSSON, B. & BILLIG,€1. 1990. Atrial natriuretic peptide inhibits spontaneous rat oocyte maturation. Endocrinology 126, 1504-1508. TSAFRIRI, A. & CHANNING, C.P. 1975. An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro. Endocrinology 96, 922-927. TSAFRIRI, A,, POMERANTZ, S.H. & CHANNING, C.P. 1976. Inhibition of oocyte maturation by porcine follicular fluid : Partial characterization of the inhibitor. Biol Reprod 14, 51 1-516. A,, CHANNING, C.P., POMERANTZ, S.H. & TSAFRIRI, LINDNER, H.R. 1977. Inhibition of maturation of
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isolated rat oocytes by porcine follicular fluid. 3 Endocrinol 75, 285-291. 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 Daferentiation 12, 271-276. DE WIT, R.J.W., HOPPE,J., STEC,W.J., BARANIAK, J. & JASTOROFF, B. 1982. Interaction of cAMP derivatives with the ‘stable’ cAMP binding site in the CAMP-dependent protein kinase type I. Eur 3 Biochem 122, 95-99.
Resumption of rat oocyte meiosis is paralleled by a decrease in guanosine 3’,5’-cyclic monophosphate (cGMP) and is inhibited by microinjection of cGMP J. T O R N E L L , H. B I L L I G and T. H I L L E N S J O * Departments of Physiology and *Obstetrics and Gynecology, University of Goteborg, Sweden TORNELL, J., BILLIG,H. & HILLENSJO, T. 1990. Resumption of rat oocyte meiosis is paralleled by a decrease in guanosine 3’,5‘-cyclic monophosphate (cGMP) and is inhibited by microinjection of cGMP. Acta Physiol Scund 139, 511-517. Received 24 January 1990, accepted 6 March 1990. ISSN 0001-6772. Departments of Physiology and Obstetrics and Gynecology, University of Goteborg, Sweden.
The aim of the present study was to measure the level of cyclic GMP (cGMP) compared with the level of cyclic AMP (CAMP) in rat oocytes during resumption of meiosis I (oocyte maturation) and to microinject these cyclic nucleotides into the oocyte to study their effects on oocyte maturation. Immature oocytes were obtajned from prepubertal rats primed with pregnant mare’s serum gonadotropin. Oocytes were isolated and shortterm cultured under conditions enabling spontaneous maturation. The levels of cGMP and cAMP decreased in the oocyte during spontaneous maturation (from 0.41 to 0.25 and from 0.64 to 0.42 fmol per oocyte respectively). The decrease was observed during the first hour of culture, and no further decline was seen after 2 h. Microinjection of cGMP or cAMP into isolated immature oocytes delayed the spontaneous maturation, cGMP being slightly more effective than CAMP, whereas 2-deoxy-CAMP (which does not stimulate protein kinase A) did not. These results demonstrate for the first time that the level of cGMP decreases in the oocyte parallel to spontaneous meiosis, as already shown for CAMP. This suggests that cGMP, as well as CAMP, may be of importance for regulating this process. This assumption is further supported by data demonstrating a delay in the maturation of oocytes injected with cGMP. Key words: CAMP, cGMP, meiosis, oocyte, rat.
T h e mammalian oocyte matures, i.e. resumes meiosis I, spontaneously when liberated from the follicle and cultured in vitro (Pincus & Enzmann 1935). I t has therefore been suggested that the oocyte is under constant inhibition within the follicle, preventing it from undergoing meiosis prematurely. A number of substances have been proposed to be intrafollicular inhibitors of oocyte maturation. Spontaneous maturation of isolated porcine, rat and mouse oocytes is prevented by addition of follicular Correspondence : Jan Tornell, Department of Physiology, University of Goteborg, PO Box 33031, S-400 33 Goteborg, Sweden.
fluid (FFl) or by co-culture with granulosa cells (Tsafriri & Channing 1975, Tsafriri et al. 1977, Chari et al. 1983, Downs & Eppig 1984). A partially purified inhibitor of oocyte maturation (OMI) has been isolated from porcine FF1 (Tsafriri et al. 1976), but its chemical nature is still unknown. Hypoxanthine in concentrations found in mouse FF1 is inhibitory to spontaneous mouse oocyte maturation (Downs et al. 1985). Moreover, cyclic AMP (CAMP) can inhibit spontaneous oocyte maturation in several species (Cho et al. 1974, Magnusson & Hillensjo 1977). Experimental studies on the effects of cAMP on meiosis are complicated by the fact that there is
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a compartmentalization of cAMP levels within the follicle. In the mouse the levels of cAMP decrease in the oocyte and increase in granulosa cells prior to the resumption of meiosis in response to the luteinizing hormone (LH) surge (Schultz et al. 1983). Under certain conditions cAMP derivatives actually stimulate meiosis in follicle-enclosed rat oocytes (Hillensjo et al. 1978). However, microinjection of cAMP or the catalytic subunit of protein kinase A (PKA) into mouse oocytes inhibits their spontaneous maturation. Oocytes injected with a PKA inhibitor undergo spontaneous maturation in spite of the presence of dibuturyl-CAMP or the phosphodiesterase inhibitor isobutyl-metb.ylxanthine (IBMX; Bornslaeger et al. 1986). Therefore, the direct role of cAMP in the oocyte is generally believed to be inhibitory to meiosis. Ovarian cyclic G M P (cGMP) levels are also influenced by gonadotropins. Whole rat ovaries exposed to gonadotropins in vitro respond with decreased levels of cGMP while the levels of cAMP increase (Ratner 1976). Similar changes occur after the LH surge (Ratner & Sanborn 1980) in parallel with the resumption of meiosis. The possible role of cGMP in the regulation of meiosis is poorly understood. High concentrations of cGMP derivatives inhibit spontaneous maturation of hamster oocyte-cumulus complexes (Hubbard & Terranova 1982) and denuded rat oocytes (Tornell et al. 1984). Recently, we have shown that elevation of endogenous cGMP levels, by stimulation of the membranebound guanylate cyclase by atrial natriuretic peptide (ANP; Leitman et al. 1987), inhibits spontaneous rat oocyte maturation (Tornell et al. 1990). Stimulation of the soluble guanylate cyclase by sodium nitroprusside (SNP ; Leitman et al. 1987) also results in inhibition of oocyte maturation (Tornell et al. 1990). On the other hand, data from experiments with hamster oocyte-cumulus complexes suggest that ANP, via cGMP, actually stimulates meiosis at certain incubation times and concentrations (Hubbard & Price 1988). T o further evaluate the role of cGMP and cAMP in the regulation of meiosis in the rat we have measured endogenous cGMP and cAMP levels in spontaneously maturing oocytes. Furthermore, we have microinjected cGMP, cAMP or deoxy-CAMP (a cAMP derivative which does not activate PKA; de Wit et al. 1982) into rat oocytes and examined their effect on meiosis.
MATERIALS AND METHODS Animals. Female, 26-day-old, immature, Spraguee Dawley rats (Alab, Stockholm, Sweden) were injected S.C.with 10 IU of pregnant mare’s serum gonadotropin (Sigma Chemicals, St Louis, MO, USA) to induce synchronized follicular development. The animals were housed under standardized environmental conditions with lights on between 05.00 and 19.00 h. Water and food were provided ad libitum. Tissue preparation and incubation procedures. The rats, 28 days old, were killed by cervical dislocation in the morning before the L H surge. The ovaries were dissected out and cleaned of adherent tissue and transferred to M, culture medium buffered with Hepes (pH 7.4; Hogan et al. 1986). Large follicles were punctured with a hypodermic needle under a stereomicroscope to release oocyte-cumulus complexes. Oocyte-cumulus complexes were subsequently cultured for 1 4 h in 1.0 ml of medium M,, (Hogan et al. 1986) buffered with 25 mM HCO, in Falcon organ culture dishes (Becton Dickinson Labware, Iincoln Park, NJ, USA) under a humidified atmosphere of 5y0 CO, in air to study the spontaneous maturation. For microinjection experiments oocytecumulus complexes were collected and denuded in M, supplemented with 0.2 mM 3-isobutyl-1-methylxanthine (IBMX). The oocytes were denuded by removing the cumulus cells mechanically by a series of mouth-controlled micropipettes with successively smaller diameter (Magnusson et al. 1977). Oocytes with a germinal vesicle (GV) were considered arrested in prophase I and used for microinjection (see below) with vehicle or indicated substance, or cultured immediately. Oocytes were cultured in the medium M,, buffered with 25 mM HCO, under a humidified atmosphere of 5 yo CO, in air for 3 4 h and examined for the presence of GV at the indicated incubation times. I n experiments where cyclic nucleotides were measured, the oocyte-cumulus complexes were collected as described, except that no IBMX was present. Groups of 5G200 oocyte-cumulus complexes were either denuded immediately and the oocytes placed in 6G0/, ethanol within 15 min for cyclic nucleotide analysis with radioimmunoassay (RIA), or cultured for 1 or 2 h in MI, under a humidified atmosphere of 5 % CO, in air before being denuded and placed in 60% ethanol within 15 min for cyclic nucleotide analysis. Microinjectian of nocytes. Groups of 10 denuded oocytes were placed in droplets of M, supplemented with 0.2 mM IBMX covered with light paraffin oil (BDH Ltd, Poole, UK) on a depression slide at room temperature. T h e oocyte was sucked onto the end of a glass pipette held by a Narishigi hydraulic micromanipulator (Nikon, Tokyo, Japan). The injection pipette (tip less than 1 p m diameter) was made from
Meiosis and oocyte cGMP levels a glass capillary with an internal filament (Clark Electromedical Instruments, Pangbourne, UK) and also held by a Narishigi hydraulic micromanipulator. The injections were done with compressed air under a Nikon Diaphot inverted microscope with Nomarski optics under 400 x magnification. The injected volume was estimated to be approximately 10-20 pl. The substances injected were all in a concentration of 10 mM dissolved in water. After injection, the oocytes were collected, rinsed once in M, without IBMX and then transferred to 1.0 ml MI, medium in Falcon organ culture dishes for culture. Chemicals. Guanosine 3’,5’-cyclic monophosphate (cGMP), adenosine 3’,5’-cyclic monophosphate (CAMP), 2’-deoxyadenosine 3’,5’-cyclic monophosphate (deoxy-CAMP) and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma. Oocyte examination. In the microinjection experiments, the denuded oocytes were examined repeatedly at the indicated incubation times in the culture dishes with an inverted microscope to reveal the stage of meiosis. Microinjected oocytes showing signs of degeneration were not included in the data (see below). In experiments performed to evaluate the spontaneous maturation of oocyte-cumulus complexes, these were placed on microscope slides after the incubations to reveal the stage of meiosis with the help of Nomarski optics. Oocytes showing a germinal vesicle (GV) were considered to be still arrested in prophase I and those with GV breakdown (GVB) to have resumed meiosis. Determination of cGMP and C A M P . The cGMP and cAMP analyses were performed with RIA kits (Amersham, UK). The oocytes were placed in 60% ice-cold ethanol immediately or after 1 or 2 h of incubation as described’ above. The samples were centrifuged and the pellets rinsed with 750,d 60% ethanol and centrifuged. The supernatants were completely evaporated. The dried extracts were acetylated according to the manufacturer to improve sensitivity. Each sample contained 8&200 (cGMP) or 50-200 (CAMP) denuded oocytes, and the nucleotide content ranged between 12 and 38 or 6 and 52 fmol per tube respectively. The sensitivity was 0.5 and 1.0 fmol per tube respectively. Cross-reactivity for the cAMP RIA to cGMP was less than 0.0004% and for the cGMP RIA to cAMP less than 0.02% according to the manufacturer. Statistics. Each experiment was repeated three times with similar results and the data shown were pooled from these experiments. The values are given as mean fSEM. Statistical differences were calculated by analysis of variance followed by the StudentNewman-Keuls test. In the presented data on oocyte maturation n refers to the number of oocytes, and in data on the levels of cyclic nucleotides n refers to the number of samples. In the experiments regarding resumption of meiosis the data were treated as a
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normal approximation of the binomial distribution to obtain the standard error of the mean. Differences between different groups were calculated by two-way analysis of variance. Values of P < 0.05 were considered statistically significant.
RESULTS T h e endogenous levels of cGMP (0 h : 0.41 fmol per oocyte, corresponding to approximately 1.5 ,UM, assuming an oocyte radius of 40,um) in the oocyte decreased when the oocyte-cumulus complex was cultured in nitro for 1 h (0.25 fmol per oocyte, approximately 0.9 ,UM) but no further decline was seen when the oocyte-cumulus complex was cultured for 2 h (Fig. 1). Compared with cGMP, the concentration of cAMP was approximately 50% higher in the oocyte (0 h : 0.64 fmol per oocyte, approximately 2.4 ,UM) and decreased after 1 h of incubation (0.42 fmol per oocyte, approximately 1.6 /AM). No further decline was seen after 2 h of incubation (Fig. 1). The percentage decrease of cGMP was in the same range as for cAMP (Fig. 2 ) . The initial fall in cyclic nucleotide levels coincided with the resumption of meiosis (Fig. Microinjection of vehicle slightly delayed the spontaneous maturation compared with un0.71
1 I
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0
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Hours
.,
Fig. 1. The oocyte levels of CAMP and cGMP after 0, 1 and 2 h of incubation. The median number of oocytes in each group was 745. The data were pooled from three experiments. +* = significantly lower ( P < 0.01) compared with 0 h of incubation. cGMP; 0, CAMP.
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d 0)
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?9
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Fig. 2. The levels of cAMP and cGMP at 1 and 2 h expressed as a percentage of the levels at 0 h of incubation (left axis) and the percentage of immature oocyteecumulus complexes after 0, 1 and 2 h of incubation (right axis). The data were pooled from three experimepts. cGMP; 0, CAMP; A,immature oocytes.
injected oocytes (Fig. 3). Both cGMP and cAMP retarded the spontaneous maturation more than vehicle when microinjected into oocytes, with cGMP being somewhat more effective than
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iJ
Hours Fig. 3. The effect ofinjection of vehicle on resumption of meiosis compared with uninjected oocytes after 1,2,3 and 4 h of incubation. The median number of oocytes in each group was 55. The data were pooled from three experiments. The two groups differ from each other when analysed by two-way analysis of vehicle; 0, control. variance (P < 0.01).
.,
cAMP (Fig. 4). T h e retardation was transient, since the maturation rate was approximately the same after 4 h of incubation whether the oocytes were injected with vehicle or cyclic nucleotides. When deoxy-CAMP was injected into the oocyte, the spontaneous maturation was not retarded compared with vehicle-injected oocytes. An average of 11yo of the oocytes degenerated after injection and were excluded from the calcu-
DISCUSSION I n this study it was demonstrated that the levels of cGMP in rat oocytes decline during spontaneous maturation. It was also shown that microinjection of cGMP could transiently delay the spontaneous maturation of denuded oocytes. These results suggest that the decrease in cGMP levels may be of functional importance. A decrease in oocyte cAMP levels during maturation as previously demonstrated (Schultz et al. 1983, Vivarelli er al. 1983, Dekel 1987) was confirmed in this study. ~h~ finding that the inhibition was temporary may indicate that the oocyte has a capacity for degrading cGMP and diffuse Or that these through the membrane into the surrounding medium.
Meiosis and oocyte cGMP levels
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Hours Fig. 4. The effect of injection of vehicle, CAMP, cGMP or deoxy-CAMP into oocytes. The median number of oocytes in each group was 48. The data were pooled from three experiments. When analysed by two-way analysis of variance the groups of vehicleand deoxy-CAMP-injected oocytes do not differ. Both the CAMP- and cGMP-injected oocytes differ from vehicle-injected ones ( P < 0.01) and cGMP-injected oocytes also differ from CAMP-injected ones (P< 0.01). 0, CAMP; vehicle; 0, cGMP; x , deoxyCAMP.
.,
The role of cGMP in oocyte maturation has only been indirectly indicated : cyclic G M P derivatives can inhibit spontaneous maturation (Hubbard & Terranova, 1982, Tornell et al. 1984), and ANP and' SNP can inhibit spontaneous maturation via elevated cGMP levels (Tornell et al. 1990). In this study we more directly indicated a function for cGMP in regulating oocyte maturation by demonstrating changes in oocyte levels of cGMP during this process and by direct injection of cGMP into the oocyte. The importance of cAMP in the regulation of oocyte meiosis has been illustrated by several approaches, as discussed above. The estimated levels of cAMP found in the present study were in the same range as previously described for the mouse (Schultz et al. 1983, Vivarelli et al. 1984) and the rat (Dekel 1987). Microinjection of cAMP into rat oocytes delayed the maturation, as earlier described in mouse by Bornslaeger et al. (1986). Deoxy-CAMP did not affect the maturation rate, suggesting that the delay does involve activation of PKA. The demonstrated decrease in endogenous levels of oocyte cGMP and cAMP coinciding
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with the resumption of meiosis and the delay of this process by micro-injection of cGMP or cAMP into the oocyte suggest that not only cAMP but also cAMP may have an important role in controlling meiosis. Based on the presented data we can only speculate about possible interactions between these two systems. In other systems, PKA and the cGMP-dependent protein kinase (PKG) are able to phosphorylate the same proteins (Lincoln & Corbin 1983). When cGMP binds to PKG, or when cAMP binds to PKA, a phosphorylation of putative proteins important for arresting the oocyte in meiotic prophase may occur. In high concentrations, as obtained after microinjections, cGMP and cAMP might interact in an unspecific way with PKA and P K G respectively (Khoo & Gill 1979). However, the cGMP levels produced in the oocyte-cumulus complex in response to ANP and SNP were in a range not likely to interact directly with the PKA (Tornell et al. 1990). Hundred- to thousand-fold higher concentrations of cGMP are required for activation of PKA, and 25- to 1000-fold (depending on the substrate) higher concentrations of cAMP are necessary to activate P K G (Khoo & Gill 1979). Levels of cAMP sufficient to inhibit meiosis can be maintained by stimulating adenylate cyclase or inhibiting phosphodiesterase (PDE). Several different forms of PDE exist (Beavo 1988). Among them, a cGMP-inhibited PDE has been described (MacPhee et al. 1988). The presence of a cGMP-inhibited PDE in extracts from mouse oocytes has been reported. At a concentration of 0.1 ,UM cGMP inhibited PDE activity in these extracts by 50 % (Bornslaeger et al. 1984). Our results show that the levels of cGMP in the rat oocyte are of the order of 1-2 ,UM, suggesting that cGMP may effectively inhibit this form of PDE. The activity of cGMPinhibited PDE may be increased when the cGMP levels decrease in the oocyte. An increased PDE activity might be a part of the mechanism for decreasing cAMP levels during resumption of meiosis. The mechanisms for the regulation of cGMP levels in the oocyte are at present not known. Further studies will be necessary to explain in detail the interaction of cAMP and cGMP in the oocyte. Our present studies on rat oocytes confirm reports on the mouse and on the rat showing that cAMP is important in regulating meiosis and, furthermore, our results suggest
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that cGMP may also have an important role in this respect.
Manual, pp. 245-267. Cold Spring Harbour Laboratory, MO. HUBBARD, C.J. & PRICE,J. 1988. The effects of follicle-stimulating hormone and cyclic guanosine This study was supported by grants from the Swedish 3’,5’-monophosphate on cyclic adenosine 3’3’Medical Research Council (nos. B89-04x-27 and B89monophosphate-phosphodiesterase and resumption 04x-05650), the Swedish Society for Medical Reof meiosis in hamster cumulus-oocyte complexes. search, Goteborg Medical Society, Swedish Medical Biol Reprod 39, 829-838. Society, the Faculty of Medicine, University of P.F. 1982. Inhibitory C.J. & TERRANOVA, Goteborg, and Handl. Hjalmar Svensson’s Research HUBBARD, action of cyclic guanosine 5’-phosphoric acid Funds. We would like to thank Dr Bjorn Carlsson for (GMP) on oocyte maturation: Dependence on an valuable discussions and suggestions. intact cumulus. Biol Reprod 26, 628-632. KHOO,J. & GILL, G. 1979. Comparison of cyclic nucleotide specificity of guanosine 3’,5’-monoREFERENCES phosphate-dependent protein kinase and adenosine 3’,5’-monophosphate-dependent protein kinase. BEAVO,J.A. 1988. Multiple isoenzymes of cyclic Biochim Biophys Acta 584, 21-32. nucleotide phosphodiesterase. In : P. Greengard & D.C., AGNOST, V.L., TUAN,J.J., ANDRESEN, G A. Robison (eds.) Advances in Second Messenger LEITMAN, J.W. & MURAD, F. 1987. Atrial natriuretic factor and Phosphoprotein Research, vol. 22, pp. 1-38. and sodium nitroprusside increase cGMP in culRaven Press, New York. tured rat lung fibroblasts by activating different BORNSIAEGER, E.A., WILDE,M.W. & SCHULTZ, R.M. forms of guanylate cyclase. Biochem 3 244, 69-74. 1984. Regulation of mouse oocyte maturation : Involvement of cyclic AMP phosphodiesterase and LINCOLN,T.M. & CORBIN,J.D. 1983. Characterization and biological role of the cGMP-dependent calmodulin. Dev Bid 105, 488499. protein kinase. In: P. Greengard & G. Robison R.M. BORNSLAEGER, E.A., MATTEI,P. & SCHULTZ, (eds.) Advances in Cyclic Nucleotide Research, vol. 1986. Involvement of CAMP-dependent protein 15, pp. 139-192. Raven Press, New York. kinase and protein phosphorylation in regulation of T., LEREA, K. C., REIFSNYDER, D., MOORE, mouse oocyte maturation. Dev B i d 114, 453462. MACPHEE, CHARI,S.,HILLENSJO, T., MAGNUSSON, C., STURM, & BEAVO,J. 1988. Phosphorylation results in activation of a cAMP phosphodiesterase in human G. & DAUME,E. 1983. In vitro inhibition of rat platelets. 3 Biol Chem 263, 10353-10358. oocyte meiosis by human follicular fluid fractions. MAGNUSSON, C. & HILLENSJO, T. 1977. Inhibition of Arch Gynecol 233, 155-164. maturation and metabolism in rat oocytes by cyclic CHO, W.K., STERN, s. & BIGGERS, J.D. 1974. ‘ AMP. 3 E X P Zoo1 201, 139-147. Inhibitory effect of dibuturyl cAMP on mouse C., HILLENSJO, T., TSAFRIRI,, A., HULToocyte maturation in vitro.3Exp Zoo1 187,383-386. MAGNUSSON, BORN,R. & AHREN,K. 1977. Oxygen consumption DEKEL,N. 1987. Interaction between the oocyte and of maturing rat oocytes. Bzol Reprod 17, 9-15. the granulosa cells in the preovulatory follicle. In : G. & ENZMANN, E.V. 1935. The comparative P. Leung, D. Armstrong, K. Ruf, W. Moger & PINCUS, behavior of mammalian eggs in vivo and in vitro. I. H. Friesen (eds.) Endocrinology and Physiology of The activation of ovarian eggs. 3 Exp Med 62, Reproduction, pp. 197-209. Plenum Press, New York. 665-675. DOWNS,S.M. & EppiG, J.J. 1984. Cyclic adenosine RATNER,A. 1976. Effects of follicle stimulating monophosphate and ovarian follicular fluid act hormone and luteinizing hormone upon cyclic AMP and cyclic GMP levels in rat ovaries in vitro. synergistically to inhibit mouse oocyte maturation. Endocrinology 114, 418426. Endocrinology 99, 1496-1500. C.R. 1980. Effect of DOWNS,S.M., COLEMAN, D.L., WARD-BAILEY, P.F. & RATNER,A. & SANBORN, endogenous L H secretion on ovarian cyclic AMP EPPIG, J.J. 1985. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low and cyclic GMP levels in the rat. Lzfe Sci 26, molecular weight fraction of porcine follicular fluid. 439445. SCHULTZ, R.M., MONTGOMERY, R.R. & BELANOFF, Proc N a t l Acad Sci USA 82, 454458. HILLENSJO, T., EKHOLM, C. & AHREN,K. 1978. Role J.R. 1983. Regulation of mouse oocyte meiotic maturation: Implication of a decrease in oocyte of cyclic AMP in oocyte maturation and glycolysis in the pre-ovulatory rat follicle. Acta Endocrinol cAMP and protein dephosphorylation in com(Copenh) 87, 377-388. mitment to resume meiosis. Dev Biol 97, 264273. F. & LACY, E. 1986. In vitro TORNELL, J., BRANNSTROM, M. & HILLENSJO, T . 1984. HOGAN, B., COSTANTINI, Different effects of cyclic nucleotide derivatives culture of eggs, embryos and teratocarcinoma cells. In: B. Hogan, F. Costantini & E. Lacy (eds.) upon the rat oocyte-cumulus complex in vitro. Acta Physiol Scand 122, 507-513. Manipulating the Mouse Embryo. A Laboratory a
Meiosis and oocyte cGMP levels TORNELL, J , , CARLSSON, B. & BILLIG,€1. 1990. Atrial natriuretic peptide inhibits spontaneous rat oocyte maturation. Endocrinology 126, 1504-1508. TSAFRIRI, A. & CHANNING, C.P. 1975. An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro. Endocrinology 96, 922-927. TSAFRIRI, A,, POMERANTZ, S.H. & CHANNING, C.P. 1976. Inhibition of oocyte maturation by porcine follicular fluid : Partial characterization of the inhibitor. Biol Reprod 14, 51 1-516. A,, CHANNING, C.P., POMERANTZ, S.H. & TSAFRIRI, LINDNER, H.R. 1977. Inhibition of maturation of
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isolated rat oocytes by porcine follicular fluid. 3 Endocrinol 75, 285-291. 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 Daferentiation 12, 271-276. DE WIT, R.J.W., HOPPE,J., STEC,W.J., BARANIAK, J. & JASTOROFF, B. 1982. Interaction of cAMP derivatives with the ‘stable’ cAMP binding site in the CAMP-dependent protein kinase type I. Eur 3 Biochem 122, 95-99.
