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Gamete Biology |
The Jackson Laboratory,4
Bar Harbor, Maine 04609 Novo Nordisk A/S,5
Discovery and Development, Novo Allé, DK-2880 Copenhagen, Denmark Schering AG,6
Corporate Research, Research Center Europe, Medicinal Chemistry, D-13342 Berlin, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gamete biology, in vitro fertilization, meiosis, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cholesta-8,14,24-trien-3ß-ol) originally isolated from follicular fluid (FF-MAS) could be such a signal [4, 5]. However, this idea has been controversial [6–8] because inhibitors of 14-delta-demethylase fail to block spontaneous maturation; however, it is not clear whether these inhibitors prevent accumulation of FF-MAS. Among the arguments against FF-MAS as a signal inducing the resumption of meiosis is the observation that concentrations of this sterol in mouse ovaries do not reach detectable levels until after germinal vesicle breakdown (GVB) has already commenced [9]. Instead, FF-MAS concentrations increase during the progression from metaphase I (MI) to metaphase II (MII), the stage of meiosis reached immediately before ovulation. In addition to preovulatory progression to MII (nuclear maturation), oocytes also undergo "cytoplasmic maturation"; this entails biochemical and molecular changes, separate from those involving chromosomal events, which give rise to oocytes competent to support fertilization and embryonic development [10]. Although nuclear and cytoplasmic maturation usually occur in synchrony, they can occur out of synchrony and can be experimentally separated. For example, in oocytes isolated from early antral follicles and matured in vitro, cytoplasmic maturation can occur even when the progress of nuclear maturation becomes arrested at MI [11, 12]; however, even though these eggs support fertilization and development to the blastocyst stage, the embryos are usually triploid, and thus further development is curtailed [11].
Because it is clear that both nuclear and cytoplasmic maturation are completed in a follicular environment of increasing FF-MAS concentration [9], this study focused on the hypothesis that FF-MAS promotes the MI to MII transition and cytoplasmic maturation in mouse oocytes. Because FF-MAS effects might be most readily detected when oocyte maturation is compromised, several experimental mouse models were selected to test this hypothesis. These were 1) oocytes from 22-day-old (C57BL/6J x SJL/J)F1, hereafter B6SJLF1, hybrid mice, which readily complete meiotic maturation to MII in vitro and have a high frequency of successful in vitro fertilization and preimplantation embryonic development [11, 13]; 2) oocytes from 18-day-old B6SJLF1 mice, which have a relatively low frequency of completing either nuclear or cytoplasmic maturation [14, 15]; 3) oocytes from 22-day-old C57BL/6J inbred mice, whose competence to complete both nuclear and cytoplasmic maturation in vitro is less than that of the hybrid oocytes described above (our unpublished data and results presented here); 4) oocytes from four recombinant inbred (RI) strains of mice, LTXBO and its related congenic strains (CX8-2, CX8-3, CX8-4), which have meiotic defects arising from either arrest or delay in the progression from MI to MII [16, 17]; and 5) oocytes from mouse strain I/LnJ, which exhibit slowed kinetics of GVB and low incidence of progression to MII [18]. The overall strategy was to mature cumulus cell-enclosed oocytes in medium supplemented with FF-MAS and to assess the progression of nuclear maturation to MII and competence to complete preimplantation embryonic development to the blastocyst stage after in vitro fertilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocytes were obtained from female mice of several different strains, including C57BL/6J, I/LnJ, LTXBO and its related strains (CX8-2, CX8- 3, CX8-4), and of the B6SJLF1 hybrid. The mouse strain LTXBO is a recombinant inbred strain derived from strains LT/Sv and C57BL/6J. Furthermore, the LT/Sv strain is a recombinant inbred strain derived from BALB/cJ and C58/J, which are also the progenitor strains of the CX8 recombinant inbred strains [16]. All mouse strains were maintained in the research colony of the investigators at The Jackson Laboratory. All experiments were conducted according to established ethical guidelines for the care and use of laboratory animals.
Preparation of FF-MAS
FF-MAS was synthesized and purified at Novo Nordisk A/S (Copenhagen, Denmark) and Schering AG (Berlin, Germany) [19, 20]. Stocks of crystalline FF-MAS (10 mM) 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, Milford, MA) at –80° C. An aliquot was diluted to 1, 5, 10, or 20 µM in culture media 15–20 min before each experiment.
Isolation and Culture of Oocytes
Oocyte-cumulus cell complexes were isolated from ovaries using 30- gauge needles by puncturing the largest antral follicles 44 h after priming with 2 IU (inbred mouse stains) or 5 IU (hybrid mice) eCG. Only completely cumulus-enclosed oocytes (CEOs) were used. To obtain denuded oocytes, the cumulus cells of CEOs were removed by repetitively drawing and expelling the cumulus cell-enclosed oocytes using a 0.5-ml pipet.
To test the activity of FF-MAS, the ability of the sterol to reverse the meiotic arrest imposed on cumulus cell-denuded oocytes by 4 mM hypoxanthine (Sigma, St. Louis, MO) was evaluated as described previously [4, 5, 21]. Minimum essential medium (MEM), supplemented with 5 mg/ ml of crystalline BSA (Sigma) as is required for the activity of FF-MAS [22], was used with either 0.2% ethyl alcohol or 5, 10, or 20 µM FF- MAS for 22 h.
To assess the effects of FF-MAS on nuclear and cytoplasmic maturation in the absence of hypoxanthine and not on the reversal of meiotic arrest in vitro, the CEOs were matured in MEM-alpha supplemented with Earle salts, 10 µg/ml streptomycin sulfate, 75 µg/ml penicillin G, and 5% fetal bovine serum (FBS). FF-MAS was added to the hypoxanthine-free medium at concentrations ranging from 1 to 20 µM with control groups receiving 0.2% ethanol. Forty to 60 oocytes per group (720–1000 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, and 90% N2. After in vitro maturation, the cumulus cells were removed and the oocytes were examined and classified using a stereomicroscope according to developmental stage (GV, MI, or MII). MII stage oocytes of all genotypes were inseminated with B6SJLF1 sperm, and preimplantation embryo development was carried out as described previously without FF-MAS [23]. The proportions of embryos that developed to the blastocyst stage within 5 days were determined.
Statistical Analysis
The data are presented as the mean percent (± SEM) of six experiments with each model. There were 40–60 oocytes per group within an experiment; therefore, 720–1000 oocytes were used in each experiment. Oocytes were isolated from 10–12 animals per experiment. These frequency data were transformed by arc-sin computation to comply with the assumptions of analysis of the variance using the statistical software package JMP version 5.1 (SAS Institute, Inc., Cary, NC). ANOVA with Tukey highly significant differences post hoc analyses was conducted to determine whether FF-MAS had statistically significant effects on meiotic maturation or preimplantation embryo development (P < 0.05 was considered significant).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This model was used to assess the effects of FF-MAS on nuclear and cytoplasmic development of oocytes for which the success rate in vitro is high, using only control medium for maturation. To verify the biological activity of synthetic FF-MAS, preparations were tested by assessing the reversal of hypoxanthine-maintained meiotic arrest as described by others [4, 5, 21]. The peak FF-MAS concentration required to induce GVB by cumulus cell-enclosed oocytes was 10 µM (Fig. 1A). In the absence of hypoxanthine, FF-MAS did not accelerate the time at which spontaneous GVB occurred in cumulus cell-enclosed oocytes (Fig. 1B). However, 5 µM FF-MAS increased the percentage of oocytes in which meiosis progressed to MII (Fig. 2A). Although FF-MAS had no effect on the percentage of MII oocytes that cleaved to the two-cell stage after in vitro fertilization (in control medium almost 90% cleaved to the two-cell stage), there was a significant increase in the percentage of two-cell stage embryos that developed to the blastocyst stage resulting from treatment with 10 and 20 µM FF-MAS (Fig. 2B). As a consequence of increased frequencies of maturation to MII and completion of the two- cell to blastocyst transition, the percentage GV-stage oocytes present at the start of culture that developed to the blastocyst stage increased dramatically, from 42–75%, when maturation was promoted by FF-MAS (Fig. 2B). Thus, both nuclear and cytoplasmic maturation were improved in oocytes from 22-day-old B6SJLF1 hybrid mice by treatment with FF-MAS.
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Oocytes from 18-Day-Old B6SJLF1 Mice
Oocytes from 18-day-old B6SJLF1 mice (primed with eCG at 16 days of age) have a lower frequency of maturation to MII and are less competent to complete the two- cell to blastocyst transition than oocytes isolated from 22- day-old mice [14]. Thus, these oocytes, which are isolated from relatively small antral follicles, are deficient in their competence to complete both nuclear and cytoplasmic maturation. Upon maturation in control medium, only 17% progressed to MII (Fig. 3A). However, treatment with FF-MAS during maturation in vitro increased the frequency to 56%. Although only 44% of inseminated MII oocytes cleaved to the two-cell stage in the control group, this proportion increased to 73% when oocytes were treated with 20 µM FF- MAS (Fig. 3B). Thus, there was a dramatic increase, almost 10-fold, in the percentage of the initial number of GV-stage oocytes that produced blastocysts (from 3% to 28%) when treated with FF-MAS during maturation in vitro (Fig. 3B).
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Oocytes from 22-Day-Old C57BL/6J Inbred Mice
Oocytes and embryos from inbred mice are generally much less successful in undergoing in vitro fertilization and preimplantation development than those from hybrid or outbred mice [24, 25]. Although only 32% of the oocytes cultured in control medium matured to MII, 79% progressed to MII when treated with 10 µM FF-MAS (Fig. 4A). This is equivalent to the percentage observed for oocytes from 22-day-old B6SJLF1 mice matured in control medium (Fig. 2B). Maturation in 10 µM FF-MAS also promoted cytoplasmic maturation as the percentage of two-cell stage embryos increased from 38% in the control group to 60% in the FF-MAS–treated group, and there was also an increase in the percentage of embryos that completed the two-cell to blastocyst transition after oocyte maturation in medium containing FF-MAS (Fig. 4B). Because there was such a dramatic increase in the percentage of FF-MAS- treated oocytes that progressed to MII, the total number of oocytes that developed to the blastocyst stage was also greatly increased (4–17%).
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Oocytes from 22-Day-Old LTXBO and Related Strains
Strain LTXBO is a recombinant inbred strain derived from strains LT/Sv and C57BL/6J. Strain LT/Sv is a recombinant inbred strain derived from BALB/cJ and C58/J, which are also the progenitor strains of the CX8 strains [16]. Many of the oocytes from these strains exhibit a prolongation of MI or MI-arrest [16, 17], although the etiology of this defect is not necessarily identical in all strains. FF- MAS promoted the progression of meiotic maturation to MII of oocytes of all of these strains (Fig. 5, A, C, E, and G). FF-MAS treatment during maturation in vitro also promoted development to the two-cell stage of oocytes of all four RI strains (Fig. 5, B, D, F, and H). Moreover, FF-MAS treatment of maturing oocytes promoted competence to complete the two-cell stage to blastocyst transition by oocytes from all strains (LTXBO, CX8-2, CX8-3) except CX8-4 (Fig. 5, B, D, F, and H). Oocytes from CX8-4 females were notably poor in their competence to complete preimplantation development in vitro.
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Oocytes from 22-Day-Old I/LnJ Mice
Both nuclear and cytoplasmic maturation of I/LnJ oocytes in vitro are atypical. Many fully grown oocytes either exhibit retarded kinetics of GVB or simply fail to initiate GVB [18]. Furthermore, oocytes that complete GVB often arrest at MI and thus fail to progress to MII and form diploid embryos [18, 26]. Because of the limited number of I/ LnJ mice available, the effects of only one concentration of FF-MAS, 20 µM, were assessed. FF-MAS promoted nuclear maturation to MII: 55% of treated oocytes, compared with 28% of those in the control group, progressed to MII (Fig. 6A). Moreover, FF-MAS promoted cytoplasmic maturation as indicated by a significantly higher (P < 0.05) incidence of cleavage to the two-cell stage after fertilization in vitro (Fig. 6B). Although FF-MAS treatment of maturing I/LnJ oocytes did not improve the frequency of completing the two-cell stage to blastocyst transition, treatment dramatically and significantly (P < 0.05) improved the frequency of blastocysts (7–26%) due to the increases that were promoted in both the percentage of oocytes progressing to MII and of two-cell stage embryos (Fig. 6B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hybrid B6SJLF1 oocytes isolated from the large antral follicles of 22-day-old eCG-primed mice routinely yield high percentages of MII oocytes competent to undergo fertilization and development to the blastocyst stage after spontaneous maturation in vitro in our laboratory. Yet, FF- MAS treatment of maturing B6SJLF1 oocytes in vitro improves the quality even of these oocytes known to have a high incidence of successful development. It is well established that oocytes obtained from small antral follicles are less competent to mature to MII or to undergo preimplantation embryonic development [15, 27–30]. This is illustrated by comparison of the control groups of oocytes obtained from 22- versus 18-day-old mice (Figs. 2, A and B and 3, A and B). Oocytes from small antral follicles require further development before they become developmentally equivalent to oocytes from large antral follicles. Although gonadotropin treatments can accelerate follicular development, such treatments have limited ability to accelerate oocyte development [31]. Nevertheless, treatment of oocytes from 18-day-old mice with FF-MAS during their maturation dramatically improves their competence to complete preimplantation development to approximately the frequency of control oocytes obtained from 22-day-old mice (but not to FF-MAS-treated oocytes from these mice).
Little is known about the mechanisms that regulate the MI-to-MII transition. The transition requires a decrease in cyclin-dependent kinase 1 activity brought about by ubiquitin-targeted, proteosome-mediated cyclin B degeneration [32, 33]. This proteosome-mediated system also appears required for the degradation of securin and consequent activation of separase to enable segregation of chromosomes at the MI to anaphase I transition [34, 35]. Both the multifunctional calcium/calmodulin-dependent protein kinase II (CaM KII) and protein kinase C (PKC) are also involved, but their functions are much less defined. Inhibition of CaM KII inhibits the MI-to-MII transition, suggesting that the activity of this kinase is required for this transition to occur [36, 37]. Conversely, inhibition of PKC promoted the MI- to-MII transition in LTXBO oocytes, suggesting that the activity of this kinase blocks, or delays, this transition [17]. Whether the modus operandi by which FF-MAS promotes the MI-to-MII transition involves the regulation of either of these kinases, or some other mechanism, remains to be determined. Nevertheless, different targets of FF-MAS action could mediate its effects on nuclear and cytoplasmic maturation because these maturational processes are not necessarily coupled [11, 12]. The results presented here demonstrate a quantitative effect of FF-MAS on nuclear maturation, but there also appears to be a qualitative improvement. Treatment of maturing mouse oocytes with FF-MAS reduced precocious chromatid separation in vitro and therefore may prevent aneuploidy [38]. Although it is unknown whether this would affect successful preimplantation development, a benefit to the long-term health of the fetus or offspring is clearly possible.
FF-MAS-treatment promoted both the nuclear and cytoplasmic maturation of oocytes from all of the experimental models used in this study. The concentration of FF-MAS increases in follicular fluid during oocyte maturation in vivo [9]. The studies presented here show that this sterol could function to promote complete oocyte maturation. The processes of cytoplasmic maturation are clearly affected by this sterol in ways manifested after fertilization during preimplantation development. Clearly, the conditions of oocyte maturation, such as those that may occur in preovulatory follicles, can dramatically affect not only the meiotic processes occurring during maturation but also the competence of the embryos derived from them to complete preimplantation development.
Although several of the models used in this study exhibit defects in the progression of nuclear maturation, frequently resulting in arrest or delay at MI, it is not likely that the etiology of the defects in all cases is identical. Moreover, the mechanism(s) by which FF-MAS functions to overcome the defect(s), at least in part, are unknown. Similarly, the oocytes of some women seeking clinical resolution of infertility problems exhibit defects in the progression of meiosis [39, 40]. The etiologies underlying these defects are undoubtedly also diverse. Whether FF-MAS or related compounds might assist in resolving these problems or enhance the maturation of all human oocytes in clinical settings is an exciting prospect.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 207 288 6073; jje{at}jax.org
3 Current address: Department of Obstetrics and Gynecology, Stanford Medical Center, Stanford, CA 94305
Received: 12 December 2003.
First decision: 1 January 2004.
Accepted: 12 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cholesta-8,14,24-trien-3ß-ol and intermediates obtained by said method. PCT Int. Application WO 9952930 (pending). Date of application: October 21, 1999 This article has been cited by other articles:
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 S. Cukurcam, I. Betzendahl, G. Michel, E. Vogt, C. Hegele-Hartung, B. Lindenthal, and U. Eichenlaub-Ritter Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes Hum. Reprod., March 1, 2007; 22(3): 815 - 828. [Abstract] [Full Text] [PDF]
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X. Cao, S. H. Pomerantz, M. Popliker, and A. Tsafriri Meiosis-Activating Sterol Synthesis in Rat Preovulatory Follicle: Is It Involved in Resumption of Meiosis? Biol Reprod, December 1, 2004; 71(6): 1807 - 1812. [Abstract] [Full Text] [PDF]
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M. M. Viveiros, M. O'Brien, and J. J. Eppig Protein Kinase C Activity Regulates the Onset of Anaphase I in Mouse Oocytes Biol Reprod, November 1, 2004; 71(5): 1525 - 1532. [Abstract] [Full Text] [PDF]
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C.L. Marin Bivens, B. Lindenthal, M.J. O'Brien, K. Wigglesworth, T. Blume, C. Grondahl, and J.J. Eppig 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 Hum. Reprod., October 1, 2004; 19(10): 2340 - 2344. [Abstract] [Full Text] [PDF]
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C. Bergh, A. Loft, K. Lundin, S. Ziebe, L. Nilsson, M. Wikland, C. Grondahl, J.-C. Arce, and for the CEMAS II Study Group Chromosomal abnormality rate in human pre-embryos derived from in vitro fertilization cycles cultured in the presence of Follicular-Fluid Meiosis Activating Sterol (FF-MAS) Hum. Reprod., September 1, 2004; 19(9): 2109 - 2117. [Abstract] [Full Text] [PDF]
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