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a Health Care Discovery, Pharmacology, Novo Nordisk A/S, Copenhagen, Denmark
b Research Laboratories, Schering AG, Berlin, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 µg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls.
In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis.
CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action.
In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocytes liberated from follicles have been observed in the case of a number of species to spontaneously mature in vitro without any stimulatory treatment [13]. It has been shown by many groups that cAMP is essential in the meiotic arrest and that a drop in the intracellular level of cAMP leads to the initiation of resumption of meiosis in mammalian oocytes [14–18]. The spontaneous maturation of liberated mammalian oocytes can be blocked or delayed by addition of membrane-permeable derivatives of cAMP or phosphodiesterase inhibitors, preventing cAMP inside the oocytes from degrading [19–21], or by increasing the level of cAMP by activators of the adenylate cyclase such as forskolin [22]. In contrast, several groups [17, 23] have presented evidence from follicle-enclosed oocytes that cAMP may have a dual role, being involved both in the arresting mechanisms and in mediating resumption of meiosis. Treatment of follicles with either gonadotropins or forskolin mediates a transient rise in cAMP levels inside the oocyte that stimulates oocyte maturation.
There is growing evidence, however, that two counteracting mechanisms exist in vivo: an inhibitory stimulus constantly preventing the oocyte from resuming meiosis and an activating stimulus downstream from the gonadotropic surge that is capable of overriding the inhibitory mechanism in vivo as the follicle approaches ovulation. The nature and origin of the inhibitory mechanism are not yet fully elucidated, although several observations point toward soluble factors originating from the follicular wall [24], the granulosa cells [25, 26] in the follicular fluid [27], and even the cumulus cells [28].
With regard to the activating principle, Byskov and coworkers [29] isolated a class of C-29 sterols from human follicular fluid and bull testicles that they designated meiosis-activating sterols (MAS) because of its maturational effect on hypoxanthine-arrested mouse oocytes cultured in vitro. The origin of these stimulating components also remains unclear but has been reported to be the cumulus or follicle cells [30–32]. The synthesized sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3ß-ol (FF-MAS), identical to that purified from human follicular fluid, has since been reported to stimulate the resumption of meiosis in a dose-dependent manner in mouse oocytes arrested with either hypoxanthine, isobutylmethylxanthine (IBMX), or dibutyryl cAMP [33]. The Mangelsdorf group [34] recently observed FF-MAS to be a ligand for the orphan nuclear receptor LXR-. Therefore, a possible hypothesis is that a putative nuclear MAS receptor has similarities at least in the ligand-binding domain to LXR-, since our previous findings made it unlikely that LXR- was the receptor [33].
The classical mechanism of all known mammalian nuclear receptors involves transcription [35]. Therefore, to explore some of the molecular mechanisms of MAS and possibly characteristics of a putative receptor, the aim of the present study was to elucidate whether the action of MAS involves transcription of DNA to RNA or translation of existing maternally derived RNA to protein.
Guanyl nucleotide-binding proteins (G proteins), because of their close coupling to cAMP turnover, have previously been investigated for their role in meiotic maturation [36, 37]. We therefore tested the effect of a G protein-activating toxin from Vibrio cholera (cholera toxin [CT]) on MAS-induced and spontaneous maturation in order to further characterize the MAS signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The in vitro culture assay for resumption of meiosis in mouse oocytes was conducted as previously reported [33]. Briefly, oocytes were obtained from immature (21–24 day old) female mice (C57Bl/6J x DBA/2J F1-hybrids; M&B, Ry, Denmark) weighing 13–16 g, that were kept under controlled lighting and temperature. The mice received an i.p. injection of 0.2 ml gonadotropins (Gonal-F; Serono, Randolph, MA) containing 20 IU FSH, and 48 h later the animals were killed by cervical dislocation. The ovaries were dissected out, and the oocytes were isolated in Hx-medium (see below) under a stereomicroscope by manual rupture of the follicles using a pair of 27-gauge needles. Spherical naked oocytes (NkO) or cumulus-enclosed oocytes (CEO) displaying an intact germinal vesicle (GV) were selected and placed in -minimum essential medium (-MEM without ribonucleosides; Gibco BRL, Gaithersburg, MD; cat. no. 22561) supplemented with 3 mM hypoxanthine (Sigma Chemical Co., St. Louis, MO; cat. no. H-9377), 8 mg/ml human serum albumin (State Serum Institute, Copenhagen, Denmark), 0.23 mM pyruvate (Sigma; cat. no. S-8636), 2 mM glutamine (Flow Sciences, Wilmington, NC; cat. no. 16–801), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Flow Sciences; cat. no. 16–700). This medium was designated Hx-medium.
Inhibition of Protein Synthesis
In order to evaluate the effect of the protein inhibitor cycloheximide on MAS-induced oocyte maturation, 50 µg/ml cycloheximide (Sigma; cat. no. C-7698) was added either 4 h before or simultaneously with the addition of 10 µM FF-MAS (synthesized at Novo Nordisk A/S). Thus, NkO and CEO, respectively, were cultured in 4-well multidishes (Nunclon; Nunc, Roskilde, Denmark) in which each well contained 0.4 ml of Hx-medium and 35–45 oocytes. Two different control wells (i.e., 35–45 oocytes cultured in identical medium) were always cultured simultaneously with two cycloheximide wells: an Hx control with no addition to the Hx-medium and a positive control with addition of 10 µM FF-MAS exclusively. The oocytes were cultured for 22 h after FF-MAS addition. By the end of the culture period, the numbers of oocytes with GV, germinal vesicle breakdown (GVB), and polar bodies (PB), respectively, were counted using a stereomicroscope (Wildt, Leica MZ 12; Milton Keynes, UK). The resumption of meiosis, defined as percentage of oocytes undergoing GVB per total number of oocytes in that well, was calculated as: %GVB = (number of GVB + number PB/total number oocytes) x 100. The experiment described was performed 3 times and the results presented as mean ± SD.
Inhibition of Transcription
To evaluate the involvement of transcription in MAS-induced oocyte maturation, NkO or CEO were cultured as described above with the same controls and treated either 4 h before or simultaneously with the addition of 10 µM FF-MAS with either 50 or 100 µg/ml -amanitin (a potent polymerase II inhibitor; Sigma; cat. no. A-2263) or 10 or 50 µg/ml actinomycin D (a potent transcription inhibitor; Sigma; cat. no. A-1410). The experiment described was performed 3 times and the results presented as mean ± SD.
Inhibition of G Proteins by CT
To evaluate the effect of CT (Calbiochem, San Diego, CA; cat. no. 227035-S) on MAS-induced maturation as well as spontaneous maturation, NkO were cultured with the addition of CT in the range from 0.1 to 10 µg/ml to either Hx-medium with 10 µM MAS or Hx-free medium. Control groups were cultured simultaneously in identical medium without CT. The oocytes were cultured and assessed as described above. The experiment described was performed 3 times and results presented as mean ± SD.
Statistics
In the study of protein synthesis and transcription inhibition, the percentages of oocytes showing GVB were examined by a one-way ANOVA. When ANOVA yielded significance, pairwise comparison (Student's t-test) between treatment groups and controls was conducted. A P value of 0.05 or less was considered to be significant.
In the study of CT inhibition of G proteins, data from three experiments for each treatment were pooled; and because of higher variance in oocyte numbers, the percentage of oocytes reaching GVB (mean ± SD) was tested for statistically significant difference for each CT concentration by chi-square test. A P value of 0.05 or less was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Four hours of cycloheximide pretreatment significantly influenced the NkO responsiveness to MAS. Simultaneous treatment with MAS and the translation inhibitor likewise reduced the NkO response to MAS by around 50% (Fig. 1). The level of spontaneously maturing oocytes in the hypoxanthine-arrested control oocytes was observed to be 15.9 ± 3.1 %GVB (mean ± SD), and the MAS-induced oocytes were observed to respond at the level of 81.9 ± 2.8 %GVB (mean ± SD). The oocytes pretreated for 4 h with cycloheximide and then challenged with MAS were observed to achieve 14.6 ± 9.9 %GVB (mean ± SD); this value was significantly lower than that for the MAS-treated positive controls (Fig. 1) and not significantly different from the unstimulated Hx control level. Simultaneous treatment with cycloheximide and MAS resulted in 43.9 ± 1.4 %GVB (mean ± SD), which was also significantly lower than in positive MAS-treated controls but significantly higher than in Hx controls. Spontaneous maturation in Hx-free medium was observed to be 84 ± 6.1 %GVB (mean ± SD); this value was not significantly affected by cycloheximide treatment (68.5 ± 2.5 %GVB) (Fig. 1).
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In the CEO there was a tendency, although nonsignificant, for 4-h pretreatment with the protein inhibitor cycloheximide to lower the %GVB. Thus, treatment of CEO with cycloheximide and FF-MAS yielded a less striking effect than in NkO (Fig. 2).
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In all cycloheximide-treated groups, the frequency of PB formation was zero, which was also observed in spontaneously maturing oocytes cultured with cycloheximide in medium without hypoxanthine.
Inhibition of Transcription
We observed no effect of MAS-induced or spontaneous resumption of meiosis using inhibitors of transcription, regardless of whether the inhibitory agent was -amanitin or actinomycin D and regardless of the protocol (0-, 4-, or 24-h pretreatment), when culturing NkO in vitro (Figs. 3 and 4). Furthermore, no significant difference from MAS-positive controls in the frequency of PB formation was observed in any treatment group.
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CEO were likewise cultured as described above with -amanitin pretreatment for 4 h at concentrations of 50 µg/ml and 100 µg/ml before addition of MAS, together with positive and negative controls. In the negative controls (Hx control), the resumption of meiosis was observed to be 13.4 ± 5.2 %GVB (mean ± SD). The positive control level of 10 µM MAS was observed to 55.8 ± 4.2 %GVB (mean ± SD). Four hours of pretreatment with -amanitin before MAS addition significantly increased the MAS-mediated resumption of meiosis to 86.4 ± 6.5 %GVB (mean ± SD) and 83.4 ± 9.4 with addition of 50 and 100 µg /ml -amanitin, respectively (Fig. 5).
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Effect of CT on MAS-Induced and Spontaneous Maturation
When culturing NkO in Hx-medium, it was observed that CT (2.5–10.0 µg/ml) was able to inhibit MAS-induced %GVB significantly in comparison to Hx control (Fig. 6). This was in contrast to spontaneously matured oocytes cultured in Hx-free medium, where it was observed that CT was not able to inhibit the spontaneous maturation (91.4 ± 2.8 vs. 86.1 ± 1.0 %GVB mean ± SD, Fig. 6). These findings were influenced neither by timing of CT supplementation in relation to onset of culture (4-h CT preincubation or simultaneously with MAS addition) nor by the dose of CT.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Full progression of the meiotic process to the second metaphase stage was never observed in the cycloheximide-treated MAS-mediated oocytes. Spontaneously maturing oocytes treated with cycloheximide did resume meiosis; however, they were also observed to arrest in the first metaphase, in accordance with previous reports [38–40]. The susceptibility of spontaneous oocyte maturation to protein synthesis inhibition has been observed to be very species specific. Protein synthesis has been observed to be essential for spontaneous maturation in large domestic species such as pigs and cattle, whereas in mice, GVB does occur in the presence of cycloheximide or other protein synthesis inhibitors [39–43]. Yet Downs [40] observed in oocytes from mice that if protein synthesis was blocked from 2 to 6 h while the CEO were arrested by IBMX, the IBMX could be washed away and cycloheximide alone was sufficient to prevent spontaneous maturation from occurring. Likewise, FSH- or epidermal growth factor-mediated resumption of meiosis was completely blocked by protein synthesis inhibitors. These findings suggest that the mechanism of MAS-mediated GVB is somewhat similar to the mechanism of hormonally induced resumption of meiosis in requiring de novo protein synthesis in mouse oocytes. This is, however, in contrast to spontaneous maturation that does not require protein synthesis. The interpretation of our data could be either that a certain level of protein synthesis is essential for the signaling pathway of MAS, or that a rapid receptor turnover makes it essential with an ongoing de novo protein synthesis inside the oocyte. It is possible that MAS-mediated maturation and spontaneous maturation are quite similar in the requirement for de novo protein synthesis in order to progress to second metaphases, since cycloheximide effectively prevents PB formation both in hypoxanthine-arrested, spontaneously maturing and in FF-MAS-induced oocytes. In fish, the induction of oocyte maturation by 17,20ß-dihydroxy-4-pregnen-3-one has been reported to be similarly inhibited by protein synthesis inhibition with cycloheximide and unaffected by transcription inhibition with actinomycin D [44].
In cultured intact follicles, both LH and FSH have been observed to induce maturation of the oocytes; however, in cumulus-enclosed hypoxanthine-arrested oocytes, only FSH, in contrast to LH, has activity to induce resumption of meiosis [45]. Also in line with our finding, the first 2 h of the LH-induced resumption of meiosis in follicle-enclosed oocytes has been reported to be sensitive to protein synthesis inhibitors but not to transcription inhibitors [45].
The observation in NkO of the involvement of transcription of DNA to RNA in progression of final maturation revealed that transcription was not an essential requirement for MAS-mediated GVB and furthermore that PB formation was also unaffected.
Interestingly, in CEO, in contrast to NkO, -amanitin did have an effect on MAS-mediated GVB, since it boosted the percentage of oocytes resuming meiosis significantly. Noticeably, MAS does not have the same efficacy on naked and cumulus-enclosed oocytes in our laboratory, which has been reported previously [33]. This effect of -amanitin could be interpreted as an involvement of active transcription in the putative inhibitory mechanism that is communicated from the cumulus cells to the oocytes.
After removing the cumulus cells from oocytes and inhibiting heteronuclear RNA synthesis with -amanitin, it has been observed that the oocytes spontaneously mature and reach at least the first metaphase stage in cattle and pigs [46, 47]. However, in the cumulus-enclosed pig and cattle oocytes, inhibition of transcription early in the culture period markedly reduced the spontaneous maturation, suggesting the requirement for RNA synthesis in the cumulus cells [43, 46–49]. It could therefore be speculated that the formation of a meiosis-active substance made in the cumulus cells, hypothetically MAS, requires RNA transcription in the cumulus cells.
CT was observed to significantly inhibit MAS-mediated but not spontaneous oocyte maturation in mouse oocytes. CT catalyzes ADP-ribosylation of the subunit of Gs, one of several families of heterotrimer G proteins, resulting in the constitutive activation of adenylate cyclase [50, 51]. In naked rodent oocytes, CT has been observed to partly prevent the drop in intracellular cAMP associated with spontaneous maturation [16], whereas spontaneous maturation is not inhibited [15, 16, 52]. Our finding suggests that the G protein-binding receptor mechanism is involved in MAS signaling. In Xenopus laevis oocytes, the progesterone-mediated maturation and accompanying decrease in intracellular levels of cAMP [53] are mediated via modulation of membrane-bound adenylate cyclase and can likewise be inhibited by CT [54, 55].
In conclusion, an active protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to active transcription. The signaling pathway is still speculative; however, these data could be interpreted to suggest either that a certain level of protein synthesis is essential for the signaling pathway of MAS or that a rapid receptor turnover makes it essential with an ongoing de novo protein synthesis inside the oocyte. In either case it is unlikely that the MAS signaling mechanism involves a nuclear receptor dependent on active transcription. Finally, the CT results could be interpreted as suggesting that this signaling mechanism involves G protein-coupled receptor mechanisms. Further investigations need to be conducted in order to reveal the details regarding the signaling pathway of MAS.
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ACKNOWLEDGMENTS |
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-amanitin and cycloheximide cultures. |
FOOTNOTES |
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1 Correspondence: Christian Grøndahl, Health Care Discovery, Novo Nordisk A/S, Novo Nordisk Park, G 8.1.06. Må, DK-2760 Maaloev, Denmark. FAX: 45 44 43 45 37; chgr{at}novo.dk 
Accepted: October 15, 1999.
Received: January 14, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Nature 1996; 383:728–731.[CrossRef][Medline],20-ß-dihydroxy-4-pregnen-3-one-induced in-vitro maturation in oocytes of the catfish, clarias-batrachus. J Biosci 1995; 20:151–156.This article has been cited by other articles:
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L. M Mehlmann Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation Reproduction, December 1, 2005; 130(6): 791 - 799. [Abstract] [Full Text] [PDF]
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I. Faerge, B. Terry, J. Kalous, P. Wahl, M. Lessl, J.L. Ottesen, P. Hyttel, and C. Grondahl Resumption of Meiosis Induced by Meiosis-Activating Sterol Has a Different Signal Transduction Pathway than Spontaneous Resumption of Meiosis in Denuded Mouse Oocytes Cultured In Vitro Biol Reprod, December 1, 2001; 65(6): 1751 - 1758. [Abstract] [Full Text] [PDF]
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I. Faerge, C. Grøndahl, J.L. Ottesen, and P. Hyttel Autoradiographic Localization of Specific Binding of Meiosis-Activating Sterol to Cumulus-Oocyte Complexes from Marmoset, Cow, and Mouse Biol Reprod, February 1, 2001; 64(2): 527 - 536. [Abstract] [Full Text]
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S. M. Downs, B. Ruan, and G. J. Schroepfer Jr Meiosis-Activating Sterol and the Maturation of Isolated Mouse Oocytes Biol Reprod, January 1, 2001; 64(1): 80 - 89. [Abstract] [Full Text]
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