Physiology of meiosis-activating sterol: endogenous formation and mode of action

Christian Grøndahl1,3, Jens Breinholt1, Philip Wahl1, Anthony Murray1, Thomas Høst Hansen1, Inger Færge1, Carsten E. Stidsen1, Kirsten Raun1 and Christa Hegele-Hartung2

1 Fertility Team, Research and Development, Novo Nordisk A/S, Copenhagen, Denmark and 2 Research Laboratories, Schering AG, Berlin, Germany 3 To whom correspondence should be addressed at: Fertility Team, Novo Nordisk A/S, Sauntesvej 13, DK-2820 Gentofte, Denmark. e-mail: chgr{at}novonordisk.com


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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BACKGROUND: In the context of mammalian oocyte maturation, it has been suggested that intermediates of cholesterol biosynthesis may represent the physiological signal that instructs the oocyte to reinitiate meiosis. METHODS: Endogenous levels of follicular fluid meiosis-activating sterol (FF-MAS) were monitored in rabbit ovarian tissue, and the influence of exogenous gonadotrophins on sterol formation was assessed. The involvement of cAMP in FF-MAS-induced versus spontaneous oocyte maturation in vitro in mice was also investigated, as was the direct microinjection of FF-MAS into mouse oocytes. RESULTS: Levels of FF-MAS in rabbit ovaries were significantly elevated 1 h after hCG/LH induction and remained so for 4 and 12 h after induction. In naked oocytes undergoing spontaneous maturation, a significant decrease in cAMP was detected after 30 min of culture. However, FF-MAS-mediated induction of oocyte maturation in hypoxanthine-arrested naked oocytes was not associated with any detectable decrease in intracellular cAMP levels. Microinjected FF-MAS failed to induce any noticeable meiosis. CONCLUSIONS: A rapid increase in FF-MAS level occurred in vivo in the rabbit ovary in response to LH, and clear differences were seen in the cAMP pattern during spontaneous and induced oocyte maturation in mice.

Key words: FF-MAS/follicle/gonadotrophins/IVM/oocyte


    Introduction
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 Abstract
 Introduction
 Materials and methods
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 References
 
Fertilization of the mammalian oocyte completes the meiotic process that is initiated at the time of oocyte formation in fetal life. Meiotic cell division is a prerequisite for sexual reproduction, and constitutes a unique cell division not only to produce gametes but also to control their successful interaction. The pituitary hormones FSH and LH are generally believed to control the overall regulation of ovarian physiology, and thereby also control the processes of follicular growth, oocyte maturation and ovulation.

It is well established that in a number of species (including humans), oocytes liberated from fully grown follicles are capable of undergoing spontaneous meiotic maturation in vitro (Pincus and Enzmann, 1935Go; Edwards, 1965Go; Cha et al., 1991Go; Trounson et al., 1994Go; Barnes et al., 1995Go, 1996; Russell et al., 1997Go). However, the subsequent embryonic development in vitro varies tremendously among species, and so far the human embryonic competence of in vitro maturation has been poor (Trounson et al., 2001Go).

It has been shown by many groups that cAMP is essential for meiotic arrest inside the follicle, and that a fall in intracellular levels of cAMP leads to the initiation of resumption of meiosis in mammalian oocytes (Eppig, 1989Go). Spontaneous maturation in vitro can be blocked or delayed by the addition of membrane-permeable derivatives of cAMP, or by phosphodiesterase inhibitors such as hypoxanthine (Hx) or isobutylmethylxanthine (IBMX); this either prevents the cAMP inside the oocytes from being degraded (Cho et al., 1974Go; Eppig and Downs, 1984Go; Downs, 1997Go), or it increases the level of cAMP by virtue of activators of the adenylate cyclase such as forskolin (Dekel et al., 1984Go).

With regard to oocyte maturation, it has recently been suggested that certain intermediates of cholesterol biosynthesis may represent the physiological signal that originates in the somatic compartment of the follicle and instructs the oocyte to reinitiate meiosis. A lipophilic molecule (4,4-dimethyl-5{alpha}-cholest-8,14,24-trien-3ß-ol) has been identified in human follicular fluid, and designated as follicular fluid meiosis-activating sterol (FF-MAS) (Byskov et al., 1995Go). Since this original observation, many studies using follicle-derived or synthetic FF-MAS have been carried out to observe the efficacy of FF-MAS in inducing oocyte maturation. Thus, FF-MAS has been shown to stimulate germinal vesicle breakdown (GVBD) in meiotically arrested rodent oocytes (Grøndahl et al., 1998Go, 2000a; Hegele-Hartung et al., 1999Go, 2001;Go Downs et al., 2001Go). These studies have shown that FF-MAS, in a dose-dependent manner, can induce resumption of meiosis in culture in the presence of different meiosis-inhibiting principles (e.g. Hx, IBMX, dbcAMP), and this is consistent with the physiological role of FF-MAS in oocyte maturation.

In vivo, oocytes will resume meiosis as a consequence of the mid-cycle rise in gonadotrophin levels (Adashi, 1994Go). In particular, the LH rise/peak will trigger a timed resumption of meiosis, leading to synchronous nuclear maturation and cytoplasmic maturation that is seen as nuclear membrane breakdown and extrusion of the first polar body. At the time of follicle ovulation this leads to the production of a fully mature metaphase II oocyte which is ready for fertilization.

Thus, under in-vivo circumstances the resumption of meiosis requires gonadotrophin stimulation, and this is mediated by the somatic compartment which is thought to generate a positive stimulus that triggers GVBD. However, if FF-MAS is able to trigger the resumption of meiosis and initiate both nuclear and cytoplasmic maturation, it might be clinically important in the treatment of infertile couples undergoing IVF. Recently, FF-MAS has been seen positively to influence the survival rate of human oocytes during in-vitro maturation (Cavilla et al., 2001Go), and also to act as a meiotic signal in human oocytes, thus inducing nuclear maturation in vitro (Grøndahl et al., 2000bGo).

It has been shown that arrested cumulus-enclosed oocytes, but not naked oocytes, can be induced to resume meiosis by treatment with either FSH or epidermal growth factor (EGF) (Downs et al., 1988Go; Morishige et al., 1993Go; Singh et al., 1993Go; Merriman et al., 1998Go). In contrast, LH/hCG has little (if any) effect on the isolated cumulus–oocyte complex (Downs et al., 1988Go); rather, a fully functional follicle is required to signal, perhaps because the oocyte down-regulates LH-receptors in the cumulus cells adjacent to the oocyte (Eppig et al., 1997Go). It has also been shown that the signalling pathways of spontaneous and induced meiotic maturation are different (Grøndahl et al., 2000aGo; Leonardsen et al., 2000bGo; Færge et al., 2001Go); for example, the mos/MAP kinase pathway is involved in both FF-MAS and FSH signalling in vitro, in contrast to spontaneous maturation.

Whether MAS is involved in the physiology of oocyte maturation in vivo remains to be fully determined, and this point has been the subject of debate in recent reports (Downs et al., 2001Go; Vaknin et al., 2001Go).

The purpose of the present study was to observe the in-vivo generation of FF-MAS in the ovary, to observe the influence of gonadotrophin stimulation, and to determine if either FSH or LH is a major director of FF-MAS production in vivo. Furthermore, the ability of FF-MAS to signal upon oocyte injection was observed in order to elucidate further the potential signalling pathways of FF-MAS.

The involvement of cAMP in these clearly different resumptions of meiosis—actively induced meiotic maturation and spontaneous in-vitro maturation—have been studied. Finally, the kinetics of action of FF-MAS in vitro was studied in cumulus-enclosed and naked oocytes, while the dependency of FF-MAS on the presence of a chaperone protein in order to exact its action as an inducer of meiosis.


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Preparations of rabbits
Thirty female New Zealand White rabbits (aged 14–16 weeks) were housed under controlled lighting and temperature. The rabbits were either killed without any hormone stimulation (control group; n = 6) or after two different priming protocols. In the first group (n = 6), FSH priming was induced by s.c. injection of 200 IU human recombinant FSH (Gonal-F®; Serono, Geneva, Switzerland) 48 h before tissue collection. In the second group (n = 6), FSH and LH priming was induced by s.c. injection of 200 IU Gonal-F®, followed after 48 h by a single s.c. injection of hCG (50 IU) (Physex®; Leo, Ballerup, Denmark). The rabbits were killed at 1, 4 or 12 h after hCG. The ovaries were removed immediately after euthanasia, frozen in Eppendorf tubes and stored at –80°C until analysed by high-performance liquid chromatography (HPLC). All animal procedures were carried out in accordance with the guidelines and regulations of the Danish Animal Protection law and under permission for animal studies permit No. 1996-101-74.

HPLC measurement of FF-MAS in rabbit ovarian tissue
Concentrations of FF-MAS in rabbit ovary tissue were measured using HPLC. The method involved the preparation of a total lipid extract (TLE) of minced ovarian tissue, followed by normal phase (NP) HPLC fractionation and quantification based on an internal reference substance (NNC90-1029) by reversed-phase (RP) HPLC. All solvents were either analytical or HPLC grade (Merck, Darmstadt, Germany).

One set of rabbit ovaries was placed in a 10 ml capped tube, and the tissue chopped and minced in the tube using a scalpel and forceps. The pulp was soaked in a mixture of chloroform:methanol (2:1, v/v; 5 ml), followed by addition of the reference material for quantification (250 ng; 50 µl of a 5 µg/ml stock solution in ethanol). After shaking (45 oscillations per min) for 16 h the tube was centrifuged at 45 rpm; the clear, lower chloroform layer was carefully transferred to a clean 10 ml tube and the solvent evaporated to dryness. The dried residue (TLE) was weighed and then dissolved/suspended in n-heptane:2-propanol (9:1, v/v; 2 ml). After centrifugation at 45 rpm, the clear supernatant was transferred to a 3 ml tapered tube and the solvent evaporated. To the TLE was added n-heptane:2-propanol (100:1, v/v; 150 µl), followed by a few drops of 2-propanol until a clear solution was obtained. The sample was subjected to NP-HPLC fractionation using the following system: column: YMC SIL (5 µ), 250x10 mm (YMC Europe, Weselerwald, Germany); solvent: n-heptane:2-propanol (100:1, v/v; flow rate 5 ml/min, using dual UV-detection at 210/250 nm). Using this set-up, the retention times of FF-MAS and NNC90-1029 were identical (~9 min). The column eluate between 8.2 and 9.7min (7.5 ml), containing both FF-MAS and NNC90-1029, was collected and the solvent evaporated. The fraction containing FF-MAS/NNC90-1029 was dissolved in ethanol (15 µl), and an aliquot (2 µl) analysed by RP-HPLC using the following conditions: column: Nucleosil 100 (3 µm) 60x1 mm (Grom Analytik, Herrenberg, Germany); solvent: 95% aqueous acetonitrile, flow rate 150 µl/min, 50°C, DAD-detection at 210–450 nm. Under these conditions, FF-MAS and NNC90-1029 were separated and exhibited retention times of 3.0 and 4.2 min respectively. The FF-MAS content of the ovaries was calculated as the area ratio of the FF-MAS and NNC90-1029 peaks monitored at 250 nm.

Culture of mouse oocytes
The in-vitro culture assay for resumption of meiosis in mouse oocytes was conducted as previously reported (Grøndahl et al., 1998Go). Briefly, oocytes were obtained from immature (21- to 24-day-old) female mice (C57Bl/6JxDBA/2J F1-hybrids; M&B, Denmark) of body weight 13–16 g that were maintained under controlled lighting and temperature. The mice were given an i.p. injection of 0.2 ml gonadotrophins (Gonal-F®) containing 20 IU FSH, and 48 h later were killed by cervical dislocation. The ovaries were dissected out, and the oocytes isolated in Hx-medium (see below) under a stereo microscope 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 {alpha}-minimum essential medium ({alpha}-MEM without ribonucleosides; Gibco-BRL) supplemented with 3 mmol/l Hx (Sigma Chemical Co.), 8 mg/ml human serum albumin (HSA; State Serum Institute, Denmark), 0.23 mmol/l pyruvate (Sigma), 2 mmol/l glutamine (Flow Laboratories), 100 IU/ml penicillin and 100 µg/ml streptomycin (Flow Laboratories). This medium was designated Hx-medium.

Source of FF-MAS
FF-MAS (4,4-dimethyl-5-{alpha}-cholest-8,14,24-trien-3ß-ol) was synthesized by Novo Nordisk, Department of Medicinal Chemistry, and purified to >99% as described previously (Murray et al., 2000Go).

Microinjection of mouse oocytes
Injection of compounds was performed on GV oocytes loaded into a droplet of {alpha}-MEM supplemented with 0.8% HSA and 3 mmol/l Hx under mineral oil in a 35 mm Petri dish on the stage of an inverted microscope. Oocytes were sucked onto a holding pipette (120 µm outer diameter, 20 µm inner diameter) and an injection pipette (Eppendorf, Hamburg, Germany) was fitted to a pressure microinjector (Eppendorf). The pipette holder was attached to a piezoelectric positioning system (Burleigh, NY, USA) mounted on a motorized micromanipulator (Luigs and Neumann, Ratingen, Germany). The injection pipette was pushed against the zona pellucida, after which a piezoelectric pulse was given that moved the injection pipette forward by 20 µm. With this movement, the pipette penetrated the zona pellucida and oolemma; a brief pressure pulse was then applied to release a volume of ~10 pl into the oocyte cytoplasm. Resumption of meiosis was triggered by removal of the injected oocytes from the Hx-medium, or by addition of FF-MAS to the Hx-containing medium, and allowing meiosis to occur during a subsequent culture period (as described earlier) of 20–22 h.

Measurement of cAMP in mouse oocytes
Oocytes were taken from female mice, denuded and matured with or without FF-MAS as described above. At 0, 0.5, 1, 2, 3, 4 and 24 h during maturation, groups of 90–100 oocytes were placed in 50 µl cAMP buffer (50 mmol/l sodium acetate, pH 5.8, 0.01% sodium azide and 1 mmol/l IBMX (Sigma) and frozen at –80°C until analysed.

The oocytes were thawed and lysed by addition of 1 µl 1 mol/l NaOH for 5 min followed by neutralization with 1 µl 1 mol/l HCl. cAMP buffer was added to a final volume of 100 µl. The cAMP content was measured in duplicate using the acetylation protocol of the cAMP kit (Amersham Pharmacia Biotech, Uppsala, Sweden).

Kinetic studies
In order to evaluate the kinetics of MAS-induced oocyte maturation, NkO and CEO were cultured in 4-well multidishes (Nunclon, Denmark) in which each well contained 0.4 ml of Hx-medium and 35–45 oocytes. The oocytes were allowed to interact with FF-MAS for 1, 2, 4, 8 or 22 h before being thoroughly washed three times and subsequently cultured in Hx-medium without FF-MAS such that the total culture period was 22 h. On completion of the culture period the number of oocytes with a GV, GVBD and polar bodies (PB) were each counted using a stereomicroscope (Wildt, Leica MZ 12). The resumption of meiosis (defined as percentage of oocytes undergoing GVBD per total number of oocytes in the well), was calculated as: % GVBD = (number of GVBD + number of PB/ total number oocytes) x 100. All experiments were performed in triplicate, and results were presented as mean ± SEM.

FF-MAS dose–response studies and chaperone protein studies
The oocyte culture experiments were conducted in two laboratories (in Copenhagen and Berlin), with a range of FF-MAS concentrations and of HSA levels in CEO as well as NkO. The oocytes were cultured and evaluated as described above.

Statistical analysis
In observations of FF-MAS concentrations in ovarian tissues, mean levels were compared using Fisher’s exact test; a P-value <= 0.05 was considered to be statistically significant. In the kinetic study of FF-MAS and in the albumin dependency investigations, percentages of oocytes showing GVBD were analysed using a one-way analysis of variance (ANOVA). When the ANOVA indicated significance, this was followed by pair-wise comparison (Student’s t-test) between time points. A P-value <= 0.05 was considered to be statistically significant. ANOVA and Student’s t-test were also used in the cAMP studies, with a P-value <= 0.05 considered to be statistically significant.


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 Abstract
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 Materials and methods
 Results
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 References
 
Dynamics of FF-MAS in rabbit ovaries
The mean (±SEM) basic level of FF-MAS in ovarian tissue of prepubertal rabbits was 3.2 ± 0.8 ng per mg dry weight ovarian tissue, and was not significantly increased after FSH priming. In contrast, a rapid and significant increase in endogenous FF-MAS was seen after FSH stimulation followed by hCG (LH activity) as early as 1 h after hCG; subsequently, FF-MAS levels were continuously raised between 1 and 12 h after hCG exposure (Figure 1).



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Figure 1. Endogenous formation of FF-MAS in rabbit ovaries. FF-MAS was measured in ovarian tissue in pre-pubertal rabbits, in non-stimulated animals (controls), after FSH stimulation and at 1, 4 and 12 h after FSH and hCG stimulation. FSH alone had no significant influence on FF-MAS levels; however, addition of LH significantly increased (*, P < 0.01) FF-MAS levels after only 1 h of stimulation.

 
Microinjection of mouse oocytes to investigate the signalling nature of FF-MAS.
In order to address the question of whether FF-MAS-induced GVBD in mouse oocytes was mediated by cell-surface or intracellular receptors, FF-MAS was injected directly into the cytoplasm of oocytes. Injection of FF-MAS plus 20% HSA (such that the intracellular concentration of FF-MAS was 1.5 and 3 µmol/l) did not cause any induction of GVBD (Table I). In contrast, application of 3 or 10 µmol/l FF-MAS to the extracellular medium effectively induced GVBD (Table I). In other experiments, 22-R-OH-cholesterol (a potent inhibitor of FF-MAS-induced meiotic resumption) was injected into the cytoplasm (at estimated final concentrations of 2.5–3.5 µmol/l); subsequent addition of FF-MAS to the extracellular medium showed that, under these conditions, 22-R-OH-cholesterol did not induce inhibition from an intracellular site.


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Table I. Effect on GVBD of microinjecting FF-MAS, 22-R-OH-chol and GTP modulators into mouse oocytes
 
Subsequent investigations were made to determine whether FF-MAS-induced GVBD involved the activation of GTP binding proteins. Injection of the non-hydrolysable GTP analogue GTP{gamma}S (which would be expected irreversibly to bind and activate all G{alpha} subunits; see Table I) had no stimulatory effect on GVBD (as would have been expected by G-protein activation). Moreover, only a minimal effect was seen on FF-MAS-induced GVBD (87% in controls versus 73% for injected) (Table I). In contrast, when GDPßS was injected, the FF-MAS-induced meiosis activation was significantly blunted (87% controls versus 65% injected). Hence, FF-MAS-induced GVBD may require the activation of a G-protein.

Measurement of cAMP during spontaneous and FF-MAS-induced maturation
The cAMP content of naked and denuded oocytes was measured during spontaneous and FF-MAS-induced maturation. A transient and significant decrease in cAMP was seen within 1 h, but this returned immediately to baseline levels in spontaneously maturing mouse oocytes. Any such fluctuations were absent from FF-MAS-induced maturation over the observation period. Otherwise, the two curves were parallel, though with values for the spontaneously matured oocytes always slightly lower (Figure 2).



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Figure 2. cAMP levels (mean ± SD) in in-vitro cultured mouse NkO during spontaneous maturation in hypoxanthine (Hx)-free medium or in FF-MAS-induced maturation in Hx-medium. A significant fall in cAMP level occurred after 1 h for spontaneous maturation (**, P < 0.005), but there was no significant fall in cAMP level for FF-MAS-induced maturation at any time-point.

 
Kinetic investigation of FF-MAS in mouse NkO and CEO
The culture of FF-MAS for specific periods, followed by FF-MAS wash out and subsequent culture for 22 h, revealed a marked difference in the response of mouse NkO and CEO. A significant effect of FF-MAS was noted after 2 h pulse-chase in NkO, but no effect was seen in CEO. After FF-MAS culture for 4 h, an almost full effect (75% GVBD) was seen in NkO, in contrast to that in CEO, where only slight—though significant—activation was noted (Figure 3).



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Figure 3. Kinetic observation of effects of FF-MAS exposure during culture of mouse oocytes. A significant effect of FF-MAS (***, P < 0.001) was seen after 2h in naked oocytes (NkO) (A), but not in cumulus-enclosed oocytes (CEO) (B). After 4 h of culture, an almost full effect (75% GVBD) was seen in NkO, but only slight (though significant) activation was seen in CEO (GVBD <40%). Thus, CEO required >4 h exposure for FF-MAS to signal to full effect. There was also an apparent difference in Emax for NkO and CEO at 30 µmol/l FF-MAS concentration (~80% versus 60% GVBD respectively). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Influence of protein co-culture on efficacy of FF-MAS-induced meiosis in mouse NkO arrested by Hx
The culture of oocytes in a serum and protein-free medium showed FF-MAS to have no effect, irrespective of the dose used (range 1–50 µmol/l; data not shown). When oocytes and FF-MAS (10 µmol/l) were cultured with a protein source [bovine serum albumin (BSA) or HSA in this case], a dose-dependent effect of HSA was noted (Figure 4). FF-MAS activity was not seen at very low albumin levels (<=1 mg/ml), but was dose-dependent at albumin levels >=2 mg/ml. It is possible that albumin might simply influence solubility of the highly lipophilic C-29 sterol, but it may also act as a complex carrier or chaperone protein that binds FF-MAS, thereby presenting or transporting the sterol to the active site in the oocyte.



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Figure 4. Effect of albumin level in culture medium on efficacy of FF-MAS to induce meiotic resumption. No significant inductive effect was observed at low albumin levels (<=1 mg/ml), but a dose-dependent significant increase in FF-MAS (10 µmol/l) efficacy occurred at albumin levels >=2 mg/ml (*, P < 0.05).

 

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In the present study, hCG (LH) was clearly seen as the key gonadotrophin influencing FF-MAS activity in the ovary, where a rapid, 3- to 4-fold increase in FF-MAS levels was observed after hCG treatment. In the rabbit ovary, elevated FF-MAS levels were seen after only 1 h, but were sustained during the investigation period (12 h). These findings contrasted with recently reported data regarding the formation of MAS sterols in mice (Baltsen, 2002Go), whereby a slower onset of gonadotrophin induction of MAS sterols was found that peaked post-meiotically. The timing of meiosis resumption in vivo is species-dependent, and in rodents GVBD has been reported to occur 1–2 h after hCG injection. Rabbits are non-rodents however, and the exact meiotic timing is not identical to that in mice and rats; however, 1 h post LH is considered to be pre- or peri-meiotic in this species.

As the mid-cycle LH rise triggers oocyte maturation to occur in vivo, it could be postulated that the timing of the FF-MAS rise is consistent with a physiological role in the meiosis-triggering signal in vivo to influence the meiotic process. The effect of LH on the isolated cumulus–oocyte complex is difficult to investigate as the LH-receptors are not expressed at the cumulus cells. However, CEO can be induced to resume meiosis by stimulation with FSH or EGF only if cellular contact between the somatic cellular network and the oocyte, mediated by numerous gap-junctions, is intact (Downs et al., 1988Go; Byskov et al., 1997Go; Leonardsen et al., 2000aGo). In contrast to the in-vivo findings of the present study, it has been shown recently that FSH stimulation of CEO in vitro results in increased formation of FF-MAS (Leonardsen et al., 2000aGo). However, based on these results, the rise in FF-MAS levels to signalling concentrations is predominantly under LH regulation and is not dramatically affected by single FSH administration.

Although several groups have identified an essential role for LH in achieving full developmental competence of oocytes, the mediation of this role remains the subject of controversy. Experiments with in-vitro follicle culture have shown that both meiotic maturation rate and developmental competence are enhanced by the addition of LH to in-vitro cultured follicles (Weston et al., 1996Go; Cortvrindt et al., 1998Go). When culturing isolated human cumulus–oocyte complexes, positive effects of both FSH and LH in the culturing media on development competence have been observed (Zhang et al., 1993Go; Wynn et al., 1998Go; Anderiesz et al., 2000Go). Others (Shimada and Terada, 2002Go) have recently reported that the essential role of FSH and LH is to induce progesterone production and receptor expression in cumulus cells; this might be crucial for normal oocyte cumulus physiology.

In order to determine the cellular location of the target for FF-MAS—i.e., plasma membrane versus intracellular—FF-MAS was microinjected into the cytoplasm of mouse oocytes. The intracellular application of FF-MAS failed to induce GVBD. Furthermore, the microinjection of 22-R-OH-cholesterol—a potent inhibitor of FF-MAS-induced GVBD (Grøndahl et al., 1998Go; C. Grøndahl et al., unpublished results)—had no such inhibitory effect. These experimental approaches suggest that the effect of FF-MAS requires an initial interaction with a putative target located in the plasma membrane, and is consistent with the hypothesis that FF-MAS signalling putatively is mediated by cell-surface receptors. It cannot be ruled out however, that the injected FF-MAS or 22-R-OH-cholesterol leaks out or is metabolized before the induction of GVBD. Interestingly, Xenopus oocytes injected with progesterone do not undergo GVBD in most cases (Cork and Robinson, 1994Go), and steroids attached to cell-impermeable polymers still mediate maturation (Godeau et al., 1978Go).

In order to investigate the roles of GTP-binding proteins in FF-MAS signalling, the effects of microinjecting the non-hydrolysable guanine nucleotides GTP{gamma}S and GDPßS were investigated. Microinjection of GTP{gamma}S, which is expected to bind and maintain all G{alpha} subunits in an active state, had a minimal effect on FF-MAS-induced GVBD. By contrast, the microinjection of GDPßS, which maintains the G{alpha} subunit in an inactive state, clearly blunted the meiotic response to a maximal concentration of FF-MAS. The results thus demonstrate the involvement of G-proteins in FF-MAS-mediated signalling. A similar finding has been observed for progesterone-mediated signalling in Xenopus oocytes, although GTP{gamma}S was found to inhibit progesterone-induced GVBD (Cork et al., 1990Go; Lutz et al., 2000Go; P.Wahl, unpublished observations). Progesterone-induced maturation of Xenopus oocytes is a well-known example of non-genomic signalling by steroids; however, little is known about the early signalling events involved in this process. At present, we cannot explain the observed difference between the effects of guanidine analogues on Xenopus versus mouse oocytes, but it is clear that progesterone- and FF-MAS-induced GVBD occur over a time frame of several hours. This allows the activation or inhibition of additional G-protein pathways to occur, and this in turn may interfere with the interpretation of results. It should also be noted that the inhibition produced by GDPßS was somewhat variable, as was also reported for other G-protein-coupled responses (Yatani et al., 1987Go; Kozlowski et al., 1994Go). The present results are compatible with the previous observation that the effects of FF-MAS may involve interaction with cholera toxin-susceptible G-protein-coupled receptors (Grøndahl et al., 2000bGo).

Endogenous meiotic signalling has been identified in fish and amphibians, and a firm paradigm of the signalling pathway is currently under construction. Recently, a general model of maturation-promoting factor (MPF) formation during oocyte maturation in vertebrates has been proposed (Yamashita et al., 2000Go). In this model, progesterone (which is synthesized in the supporting cells in the oocyte microenvironment upon gonadotrophin regulation) mediates a membrane receptor-induced signalling that, via mos, MEK and MAP-kinase, converts pre-MPF to MPF in Xenopus laevis oocytes. In fish and amphibians other than Xenopus, the initiation of meiosis is mediated by 17{alpha},20ß-dihydroxy-4-pregnen-3-one (17,20-DP), and this involves activation of the same pathway. It was observed recently (Schmitt and Nebreda, 2002Go) that in Xenopus oocytes, progesterone-induced oocyte maturation can be inhibited by PKA (cAMP-dependent kinase), even if the kinase is catalytically inactive and does not bind to the PKA regulatory subunits. The catalytically inactive PKA also inhibits mos-induced maturation. This points toward cAMP and PKA involvement that is very different in spontaneous and induced maturation in Xenopus and may be a well-preserved mechanism. In mammals, a putative MAS receptor is hypothesized to involve three pathways: (i) the mos/MEK/MAP-kinase pathways leading to MPF formation and stabilization; (ii) direct translational activation of masked cyclin B mRNA translation to cyclin B; and (iii) a pathway of decreasing cAMP and A-kinase activation (Yamashita, 2000Go; Yamashita et al., 2000Go). It is also hypothesized that FF-MAS and related endogenous sterols could form this gonadotrophin-induced signal in mammals that is responsible for many important aspects of meiosis signalling and oocyte maturation, both of nuclear and cytoplasmic character, chromatin condensation and spindle assembly.

In considering the mode of action of FF-MAS, one approach was to measure cAMP levels during maturation. The cAMP level in mouse oocytes decreased significantly during the first hour of spontaneous maturation (from 17.3 ± 1.1 to 12.5 ± 0.4 fmol per 50 oocytes, P < 0.005), but during the following 2 h returned to baseline. This finding was in agreement with previously reported data (Schultz et al., 1983Go; Moss et al., 1993Go; Aktas et al., 1995Go). Thus, a fall in cAMP level appears to be an initiator of spontaneous maturation in mouse oocytes.

The cAMP level during FF-MAS-induced oocyte maturation did not change significantly, but remained higher at all time points compared with that in the spontaneously maturing oocytes. This is most likely due to the presence of the phosphodiesterase inhibitory effect of hypoxanthine during culture stabilizing the cAMP level. Therefore, the maturation exerted by FF-MAS appears not to depend upon a fall in cAMP levels, which is suggestive of the hypothesis that FF-MAS signalling occurs via a different intracellular route. This result was in close accordance with previous reports from our laboratory detailing the signalling pathway of FF-MAS and observing that PKA was not involved in the FF-MAS-induced signalling (Færge et al., 2001Go)

Somewhat controversially, mouse oocytes can be inhibited with supraphysiological doses of Hx or IBMX to a state where FF-MAS can no longer induce maturation (>5 mmol/l Hx; >200 mmol/l IBMX, C.Grøndahl, unpublished observation). Therefore, subtle unobservable fluctuations in cAMP might be important for normal oocyte physiology. In Xenopus oocytes, maturation is believed to be governed by progesterone. As described previously for FF-MAS, the induction of maturation in mammalian oocytes is sensitive to cholera toxin, precisely as observed for progesterone-mediated meiotic maturation in Xenopus oocytes (Schorderet-Slatkine et al., 1978Go). In Xenopus oocytes it has been seen that, by inducing very high cAMP levels, the formation of MPF was abolished and maturation did not occur; however, if cAMP was only slightly elevated, then progesterone could induce maturation. A similar effect in mammalian oocytes could explain the controversy about cAMP seen in the present study, that whilst a significant fall in cAMP is not observed in FF-MAS-induced maturation, cAMP does play a role in meiosis inhibition and perhaps cAMP levels may be too high to allow meiotic maturation to occur. Noticeably, a small but non-significant fall in MAS-induced cAMP level was noted, though whether this has any importance and whether it would be abolished with higher artificial cAMP levels requires further investigation.

In the present study, it was observed that the microinjection of FF-MAS did not lead to any induction of meiosis. This corresponds well with observations in Xenopus oocytes, where the microinjection of progesterone did not induce maturation, and this was suggestive of a membrane-bound receptor mechanism. Indeed, a membrane-bound progesterone receptor has recently been cloned (Bayaa et al., 2000Go; Tian et al., 2000Go). However, albumin clearly has a pronounced effect on FF-MAS efficacy, either as a chaperone protein presenting the oocyte to the sterol, by facilitating transport, or simply because protein must be present in the medium in order for FF-MAS to remain available in solution.

In conclusion, LH was observed to be the key gonadotrophin influencing FF-MAS activity in the rabbit ovary, thereby providing evidence for the hypothesis that FF-MAS is part of ovarian physiology and plays a role in oocyte maturation in vivo. These findings support the hypothesis that two counteracting mechanisms exists in vivo: (i) an inhibitory stimulus that constantly prevents oocytes from resuming meiosis; and (ii) an activating stimulus downstream from the gonadotrophic surge, especially LH, that is capable of overriding the inhibitory mechanism in vivo as the follicle approaches ovulation. A further consequence of this hypothesis is that spontaneous maturation in vitro is far from physiological, and this is also reflected in the poor developmental capabilities observed clinically when transferring in-vitro-derived embryos, at least in humans. Further studies are required to elucidate fully the complex difference between in-vivo maturation, induced in-vitro maturation and spontaneous maturation in vitro.

Finally, based on the available data, it is hypothesized that the sterol family of FF-MAS represents a physiological signal downstream of the LH surge that is the mammalian counterpart to steroid (progesterone or 17{alpha},20ß-dihydroxy-pregnen-3-one) -induced and plasma membrane receptor-mediated oocyte maturation in amphibians and fish. However, further studies are required to reveal the full physiological importance of the MAS sterol family. Moreover, it remains to be investigated whether there is a clinical role for this molecule—in other words, whether FF-MAS-induced maturation qualitatively improves human oocyte maturation and leads to embryos with improved embryonic competence by inducing a signalling pathway of meiotic resumption that is closer to the in-vivo maturational process.


    Acknowledgements
 
The authors thank Ms Elene J.Carlsen, Tina Olesen, Anne Mette Jacobsen and Dorthe Andersen for skilful technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 24, 2002; resubmitted on August 29, 2002. accepted October 3, 2002