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
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Abstract |
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Key words: fertilization/follicular fluid-meiosis-activating sterol (FF-MAS)/oocyte maturation/meiosis/preimplantation development
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Introduction |
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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, 1972; Sorensen and Wassarman, 1976
; Eppig and Schroeder, 1989
). 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, 1989
; Eppig et al., 1992
, 1994
). Our previous studies showed that 520 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., 2004
). 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.
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Materials and methods |
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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 1520 min before each experiment.
Isolation and culture of oocytes
Cumulusoocyte 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)- 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 0.110 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 4060 oocytes per group with 39 replicate studies (Grondahl et al., 1998
; Ruan et al., 1998
; Byskov et al., 2002
). 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 (19802310 total oocytes per experiment) were cultured for 1617 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, 1996
) 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 6070 oocytes per group within an experiment. A total of 19802310 oocytes obtained from 3645 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).
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Results |
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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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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, 2001), 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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Discussion |
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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., 1992; Pavlok et al., 1992
; Lonergan et al., 1994
; Crozet et al., 1995
; Cognie et al., 1998
). 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., 1976
; Bar-Ami and Tsafriri, 1981
). 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., 1990; Bergere et al., 2001
; Combelles et al., 2002
; Levran et al., 2002
; Neal et al., 2002
; Schmiady and Neitzel, 2002
). 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.
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Acknowledgements |
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Submitted on March 22, 2004; accepted on July 6, 2004.