A synthetic analogue of meiosis-activating sterol (FF-MAS) is a potent agonist promoting meiotic maturation and preimplantation development of mouse oocytes maturing in vitro

C.L. Marín Bivens1, B. Lindenthal2, M.J. O'Brien1, K. Wigglesworth1, T. Blume2, C. Grøndahl3 and J.J. Eppig1,4

1 The Jackson Laboratory, Bar Harbor, Maine, USA, 2 Schering AG, Research Laboratories, D-13342 Berlin, Germany and 3 Novo Nordisk A/S, Discovery & Development, Novo Allé, DK-2880, Copenhagen, Denmark

4 To whom correspondence should be addressed at: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA. Email: jje{at}jax.org


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Follicular fluid-meiosis-activating sterol (FF-MAS) is a factor present in the pre-ovulatory follicle during the time of oocyte maturation. In mouse oocytes maturing in vitro, FF-MAS promotes the completion of meiotic maturation to metaphase II (MII) and improves competence to complete the 2-cell stage to blastocyst transition. We produced analogues of FF-MAS and selected three on the basis of potency to promote the resumption of meiosis by mouse oocytes maintained in meiotic arrest by hypoxanthine. The objective of this study was to determine whether these FF-MAS analogues also affect the quality of oocytes maturing in vitro with respect to the completion of meiotic maturation and augmenting the frequency of development to the blastocyst stage after fertilization in vitro. METHODS: Cumulus cell-enclosed oocytes were isolated from the small antral follicles of 18 or 20 day post-natal mice. These oocytes normally have a reduced competence to complete meiotic maturation and preimplantation embryo development. Oocytes were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0.1% ethanol, 1 µmol/l FF-MAS, or 0.1–10 µmol/l FF-MAS analogues ZK255884 (884), ZK255933 (933) and ZK255991 (991). Oocytes that progressed to MII were fertilized in vitro and the percentage developing to the 2-cell and blastocyst stages was determined. RESULTS: At 1 µmol/l, 991 and 933 increased the portion of oocytes progressing to MII, whereas the lowest dose of 991 and 884 was ineffective. Treatment of maturing oocytes with either 0.1 or 1 µmol/l 933 dramatically increased oocyte competence to complete preimplantation development. CONCLUSIONS: The synthetic analogue of FF-MAS, ZK255933, is a potent agonist that improves the quality of mouse oocytes matured in vitro. This compound may therefore have therapeutic value for treatment of oocytes from women undergoing therapy for infertility owing to poor oocyte quality.

Key words: fertilization/follicular fluid-meiosis-activating sterol (FF-MAS)/oocyte maturation/meiosis/preimplantation development


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Oocytes are maintained in meiotic arrest by somatic follicular cells until the pre-ovulatory surge of LH from the pituitary (Lindner et al., 1974Go). LH is thought to stimulate the resumption of meiosis by disassembling the meiotic arrest system and inducing the production of a positive signal to reinitiate meiosis (for reviews see Downs, 1995Go; Eppig et al., 2004Go). While the nature of this positive signal remains unknown, it has been proposed that a meiosis-activating sterol present in follicular fluid (known as FF-MAS: 4,4-dimethyl-5{alpha}-cholesta-8,14,24-trien-3{beta}-ol) may function as such a signal (Grondahl et al., 1998Go; Byskov et al., 2002Go). Controversy surrounds this idea (Downs et al., 2001Go; Vaknin et al., 2001Go; Tsafriri et al., 2002Go) because concentrations of FF-MAS in mouse ovaries do not reach detectable levels until after germinal vesicle breakdown (GVB) and peak during the progression from metaphase I (MI) to metaphase II (MII) (Baltsen, 2001Go). Furthermore, recent data reveal that this sterol does not accelerate the kinetics of GVB; instead, it dramatically promotes the later stages of meiotic maturation, namely the progression of MI to MII (Marin Bivens et al., 2004Go). Unexpectedly, FF-MAS treatment of oocytes during meiotic maturation also increased their subsequent competence to complete preimplantation development (Marin Bivens et al., 2004Go). In spite of these profound effects in vitro, possible shortcomings in the use of FF-MAS in a clinical setting might include its low solubility in media and rapid metabolization into cholesterol.

We have produced analogues of FF-MAS with higher solubility in media and greater stability than FF-MAS by introducing hydrophilic variations in the sterol backbone or side chain. This study focused on the hypothesis that these analogues might maintain the physiological properties of FF-MAS, and improve oocyte quality by the MI to MII transition and competence to complete preimplantation development.

Competence of mammalian oocytes to complete meiotic maturation and preimplantation development is acquired in a stepwise manner. Oocytes isolated from small antral follicles of neonatal mice are only partially competent to undergo meiotic maturation. Albeit they are able to undergo GVB, the progression of meiosis arrests at MI instead of MII (Szybek, 1972Go; Sorensen and Wassarman, 1976Go; Eppig and Schroeder, 1989Go). Moreover, even if they do progress to MII, such oocytes are rarely able to complete the 2-cell stage to blastocyst transition after fertilization (Eppig and Schroeder, 1989Go; Eppig et al., 1992Go, 1994Go). Our previous studies showed that 5–20 µmol/l FF-MAS treatment of these partially competent oocytes during maturation in vitro after isolation from the small antral follicles of 18-day-old mice promoted the completion of both meiotic maturation to MII and the 2-cell stage to blastocyst transition after fertilization (Marin Bivens et al., 2004Go). Here we determined the ability of 1 µmol/l FF-MAS and three of its analogues to promote the completion of meiotic maturation and the 2-cell to blastocyst transition using partially competent oocytes retrieved at the germinal vesicle (GV) stage from hormonally primed 18- and 20-day-old mice.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals
Five IU of equine chorionic gonadotrophin (eCG) were injected into 16- and 18-day-old (C57BL/6J x SJL)F1 (i.e. B6SJLF1) mice, and GV stage cumulus cell-enclosed oocytes (CEOs) were obtained from these mice 44 h later at 18 and 20 days of age, hereafter referred to as P18 and P20. Sperm was collected from 2- to 4-month-old, singly housed B6SJLF1 mice for use. All mice were maintained in the research colony of the investigators at The Jackson Laboratory. All experiments were conducted with prior approval from the institutional Animal Care and Use Committee and according to established ethical guidelines for the care and use of laboratory animals.

Preparation of FF-MAS and agonists
FF-MAS, ZK255844, ZK255933 and ZK255991 were synthesized and purified at Schering AG (Berlin, Germany). Stocks of crystalline FF-MAS, ZK255844, ZK255933 and ZK255991 (10 mmol/l) were dissolved in absolute ethanol in a light-attenuated room. To prevent oxygenation and light-induced degradation of FF-MAS, aliquots were immediately stored under argon in gas-impermeable, deactivated, amber glass microvials (Waters, USA) at –80°C. An aliquot was diluted to 0.1, 1 or 10 µmol/l in culture medium 15–20 min before each experiment.

Isolation and culture of oocytes
Cumulus–oocyte complexes were isolated from ovaries using 30 gauge needles by puncturing the largest antral follicles. Only completely enclosed CEOs were used. To assess the effects of the sterols on nuclear competence and ability to complete preimplantation development, the CEOs were matured in minimal essential medium (MEM)-{alpha} with Earle's salts, and supplemented with 10 µg/ml streptomycin sulphate, 75 µg/ml penicillin G and 5% fetal bovine serum (FBS) in 35 mm culture dishes (Falcon brand, Fisher Scientific, USA). FF-MAS at 1 µmol/l, ZK255884, ZK255933 and ZK255991 at 0.1–10 µmol/l or 0.1% ethanol (control) were added to the medium. For initial experiments to test the activity of the analogues, the ability of the sterol to reverse the meiotic arrest imposed on cumulus cell-denuded oocytes by 4 mmol/l hypoxanthine (Sigma, St Louis, MO) was evaluated as described previously using 40–60 oocytes per group with 3–9 replicate studies (Grondahl et al., 1998Go; Ruan et al., 1998Go; Byskov et al., 2002Go). For all subsequent experiments on meiotic and cytoplasmic maturation, there was no hypoxanthine in the oocyte maturation medium. Sixty to 70 CEOs per group (11 experimental groups) in triplicate studies (1980–2310 total oocytes per experiment) were cultured for 16–17 h at 37°C in modular incubation chambers (Billups Rothenberg, Del Mar, CA) infused with an atmosphere of 5% CO2, 5% O2, 90% N2. After in vitro maturation, the cumulus cells were removed and the oocytes were examined and classified using a stereomicroscope according to meiotic stage (GV, MI or MII). MII stage oocytes were rinsed three times to remove sterol and ethanol, and inseminated with B6SJLF1 sperm in a 0.5 ml droplet of medium under oil. Preimplantation embryo development was carried out as described previously (Eppig and O'Brien, 1996Go) in the absence of FF-MAS or analogues. The percentages of embryos that developed to the blastocyst stage within 5 days were determined.

Statistical analysis
The data are presented as the mean percentage (±SEM) of three independent experiments. There were 60–70 oocytes per group within an experiment. A total of 1980–2310 oocytes obtained from 36–45 animals were used in each experiment. These frequency data were transformed by arc-sin computation to comply with the assumptions of analysis of variance using the statistical software package JMP version 5.1 (SAS Institute, Cary, NC). ANOVA with Tukey's HSD post hoc analyses was conducted to determine whether any sterol compound had statistically significant effects on meiotic maturation or preimplantation embryo development compared with control (P<0.05 was considered significant).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The sterol FF-MAS promotes the completion of meiotic maturation and the competence of oocytes to complete the 2-cell stage to blastocyst transition (Marin Bivens et al., 2004Go). The objective of this study was to determine whether analogues of FF-MAS, initially identified on the basis of their greater potency to induce the resumption of meiosis by oocytes cultured in medium containing hypoxanthine, could also promote the completion of meiotic maturation and improve competence to complete preimplantation development in medium not containing hypoxanthine.

Production and characterization of agonists
ZK255884 (884) was produced by substituting the sterol side chain of FF-MAS with amines (Figure 1A and B). Using 884 as the source structure, 100 new analogues were generated by parallel synthesis in carousel reactors by inducing variations in one of three positions: side chain amine, double bonds in the sterol backbone structure, or dimethyl substitution in position 4. Two compounds, ZK255933 (933) and ZK255991 (991) (Figure 1C and D, respectively), were found to be more potent and efficient than FF-MAS in promoting the resumption of meiosis in the presence of hypoxanthine (Figure 2).



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Figure 1. Chemical structure and water solubility of FF-MAS (A), and its related compounds ZK255884 (884, B), ZK255933 (933, C) and ZK255991 (991, D).

 


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Figure 2. Assay of synthetic analogues and FF-MAS: reversal of inhibition of germinal vesicle breakdown (GVB) maintained by 4 mmol/l hypoxanthine in cumulus-enclosed oocytes. The bars indicate the percentage of GV stage oocytes that progressed to either metaphase I (MI) or metaphase II (MII) stage after 24 h culture. (*) Indicates significant difference from the control, P<0.05.

 
Effect of FF-MAS analogues on oocyte maturation
Oocytes isolated from the antral follicles of mice increasing in age from P16 to P26 show an increase in the percentages of oocytes competent to complete meiotic maturation to MII and the 2-cell stage to blastocyst transition (Eppig and Schroeder, 1989Go). Here, GV stage CEOs were isolated from P18 and P20 mice 44 h after eCG injection. Therefore, the frequency of oocyte maturation to MII and development of embryos to the blastocyst stage was relatively low in the P18 group when compared with P20, although higher doses of FF-MAS enhance meiotic maturation even in the P20 oocytes (Marin Bivens et al., 2004Go). Thus the suboptimal maturational potential of P18 oocytes provided an ideal model system to assess the effects of the FF-MAS analogues on meiotic maturation and embryonic developmental competence.

Upon maturation in control medium, only 38 and 74% of oocytes from P18 and P20 mice, respectively, progressed to MII (Figures 3 and 5). Near a physiological concentration of 1 µmol/l (Baltsen, 2001Go), FF-MAS did not affect the progression of meiotic maturation (Figures 3 and 5). Among the analogues, 1 µmol/l 991 had the most robust effect on meiotic maturation in vitro, increasing the frequency of oocyte progression to MII to 71 and 93% in P18 and P20, respectively, compared with controls (Figures 3 and 5). Analogue 933 also promoted progression to MII in oocytes from P18, from 38 to 77% (Figure 5). The highest concentration of all the analogues, 10 µmol/l, was toxic, resulting in either cell death or retardation of maturation (data not shown).



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Figure 3. Effect of increasing doses of FF-MAS analogues and FF-MAS on nuclear maturation of oocytes isolated from eCG-primed 18-day-old B6SJLF1 mice. White bars indicate the percentage of oocytes at the germinal vesicle (GV) stage, striped bars indicate the percentage of oocytes at metaphase I (MI) and black bars indicate the percentage of oocytes at metaphase II (MII) after 16–17 h culture. (*) Indicates significant difference from the control, P<0.05.

 


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Figure 5. Effect of increasing doses of FF-MAS analogues and FF-MAS on nuclear maturation of oocytes isolated from eCG-primed 20-day-old B6SJLF1 mice. White bars indicate the percentage of oocytes at the germinal vesicle (GV) stage, striped bars indicate the percentage of oocytes at metaphase I (MI) and black bars indicate the percentage of oocytes at metaphase II (MII) after 16–17 h culture. (*) Indicates significant difference from the control, P<0.05.

 
Compared with the results on meiotic maturation, there was a different pattern of agonist efficacy upon competence to complete preimplantation embryonic development. Treatment with analogue 933 caused the most robust increase in the percentage of oocytes cleaving to 2-cell embryos since the percentage of embryos developing to blastocysts in both P18 and P20 was doubled (Figures 4 and 6). However, analogue 991 did not affect, or was detrimental to, the ability of oocytes to develop to the 2-cell and blastocyst stages (Figures 4 and 6). Interestingly, FF-MAS (1 µmol/l) also increased the percentage of oocytes that developed to the 2-cell stage, and doubled the percentage of blastocysts per MII oocytes of P18 (Figure 6). Analogue 884 did not affect oocyte competence to complete blastocyst development. Analogue 991 was highly effective in promoting the completion of meiotic maturation to MII (Figures 3 and 5). Yet, this analogue had no apparent ability to promote the acquisition of competence to complete preimplantation development (Figures 4 and 6).



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Figure 4. Effect of increasing doses of FF-MAS analogues and FF-MAS on preimplantation development of oocytes isolated from eCG-primed 18-day-old B6SJLF1 mice. White bars indicate the percentage of metaphase II (MII) oocytes that cleaved to the 2-cell stage after insemination. Striped bars indicate the percentage of 2-cell stage embryos that developed to the blastocyst stage. Black bars indicate the percentage of the total number of MII oocytes present at the start of culture that developed to the blastocyst stage. (*) Indicates significant difference from the control, P<0.05.

 


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Figure 6. Effect of increasing doses of FF-MAS analogues and FF-MAS on preimplantation development of oocytes isolated from eCG-primed 20-day-old B6SJLF1 mice. White bars indicate the percentage of metaphase II (MII) oocytes that cleaved to the 2-cell stage after insemination. Striped bars indicate the percentage of 2-cell stage embryos that developed to the blastocyst stage. Black bars indicate the percentage of the total number of MII oocytes present at the start of culture that developed to the blastocyst stage. (*) Indicates significant difference from the control, P<0.05.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Little is known about the mechanisms that underlie the activity of FF-MAS or its analogues on either meiotic maturation or competence to complete preimplantation development. The results with analogue 991, which strongly promoted meiotic maturation, and analogue 933, that both promoted meiotic maturation and increased the competence to complete preimplantation development, suggest that different mechanisms or sensitivities promote these two processes. These analogues might, therefore, be useful to dissect the biochemical and physiological pathways underlying the effects of the endogenous ligand, FF-MAS. Furthermore, these analogues may help reveal the pathways involved in establishing the normal synchrony between meiotic and cytoplasmic maturation (Eppig et al., 1994Go).

It is well established for several species that oocytes obtained from small antral follicles are less competent than those from large antral follicles to complete either meiotic maturation or preimplantation embryonic development (Eppig et al., 1992Go; Pavlok et al., 1992Go; Lonergan et al., 1994Go; Crozet et al., 1995Go; Cognie et al., 1998Go). Clearly, oocytes from small antral follicles require further development before they become developmentally equivalent to oocytes from large antral follicles. It is unlikely that endocrine factors such as gonadotrophins or steroids, which affect somatic follicular development, have any direct effect on the acquisition of maturational competence of oocytes, although indirect effects are possible (Hunter et al., 1976Go; Bar-Ami and Tsafriri, 1981Go). Nevertheless, in this study, treatment of both P18 and P20 oocytes with analogue 933 during their maturation in vitro promoted their competence to complete meiotic maturation and preimplantation development. Treatment with either FF-MAS or analogue 933 promoted the completion of the developmental or physiological processes in these oocytes isolated from small antral follicles that would have been acquired by the time the oocytes would be present in large antral follicles. Treatment of maturing oocytes with the sterols in vitro had succeeded in promoting oocyte development where follicular stimulation with gonadotrophins (eCG) in vivo had failed.

Many cases have been reported of the failure of human oocytes to complete meiotic maturation during clinical IVF protocols (Rudak et al., 1990Go; Bergere et al., 2001Go; Combelles et al., 2002Go; Levran et al., 2002Go; Neal et al., 2002Go; Schmiady and Neitzel, 2002Go). Undoubtedly multiple aetiologies may underlie these failures. However, the developmental deficiencies producing at least some of these arresting defects of oocyte maturation may be amenable to correction by treatment of oocytes in vitro with FF-MAS or analogue 933. Moreover, considering the low frequency of successful fertilization and development following in vitro maturation of human oocytes, the addition of FF-MAS or analogue 933 to the maturation medium may prove highly beneficial.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Dr Mary Ann Handel for her helpful suggestions in the preparation of this manuscript. This research was supported by contracts from Novo Nordisk A/S and Schering AG (C.L.M.B. and J.J.E.) and a grant (HD21970) (J.J.E., M.J.O. and K.W.) from the National Institutes for Child Health and Human Development. Scientific Resources at The Jackson Laboratory are supported in part by a Cancer Centre Core Grant (CA34194) from the National Cancer Institute.


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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 22, 2004; accepted on July 6, 2004.