The Jackson Laboratory, Bar Harbor, ME 04609, USA
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Abstract |
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Key words: in-vitro-grown oocyte/mouse oocytes/nuclear and cytoplasmic maturation/preimplantation embryo
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Introduction |
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Meiotically competent oocytes in most mammalian species undergo spontaneous maturation when released from their follicular environment and cultured (Pincus and Enzmann, 1935; Edwards, 1965
). The mechanisms involved in germinal vesicle breakdown (GVB) as well as the cell signalling pathways driving the oocyte into metaphase II in response to the preovulatory gonadotrophin surge are not fully understood. However, both epidermal growth factor (EGF) and follicle stimulating hormone (FSH) induce nuclear maturation in mouse oocytecumulus complexes in vitro through a mechanism mediated by cumulus cells (Downs et al., 1988
).
There are reports that EGF promotes nuclear maturation of human (Das et al., 1991), bovine (Kobayashi et al., 1994
; Lorenzo et al., 1994
; Lonergan et al., 1996
; Rieger et al., 1998
), and porcine (Singh et al., 1993
; Ding and Foxcroft, 1994
; Grupen et al., 1997
) oocytes, as well as cytoplasmic maturation of mouse (Das et al., 1991
), bovine (Kobayashi et al., 1994
; Lonergan et al., 1996
; Rieger et al., 1998
), porcine (Ding and Foxcroft, 1994
; Wang and Niwa, 1995
; Grupen et al., 1997
; Abeydeera et al., 1998a
) and human (Goud et al., 1998
) oocytes. However, effects of EGF are still not clear since there are other reports that EGF does not improve the cytoplasmic maturation of mouse oocytes (Merriman et al., 1998
).
Systems for oocyte development in vitro provide an opportunity to characterize critical somatic and germ cell interactions that confer the mammalian oocyte with full developmental competence (Eppig and Schroeder, 1989; Spears et al., 1994
; Cortvrindt et al., 1996
; Eppig and O'Brien, 1996
; Eppig et al., 1996
). Nevertheless, the maturation of in-vitro-grown (IVG) oocytes is deficient compared with that of oocytes grown in vivo. For example, the onset of GVB is delayed in oocytegranulosa cell complexes cultured from the preantral follicle stage (Eppig et al., 1996
). Furthermore, a lower percentage of IVG oocytes reach the blastocyst stage after in-vitro fertilization compared with oocytes grown in vivo (Eppig and O'Brien, 1998
).
In combination with gonadotrophins, EGF promotes nuclear maturation in oocytes obtained from intact preantral follicles cultured to the antral follicle stage (Boland and Gosden, 1994; Smitz et al., 1998
). However, effects of EGF on cytoplasmic maturation of IVG mouse oocytes have not been evaluated. The objective of the present study was to determine the effects of EGF, alone or in combination with FSH, during in-vitro-maturation (IVM) on nuclear and cytoplasmic maturation of mouse oocytes. The effects of EGF were evaluated using maturing oocytes from three different sources; namely, in-vivo-grown oocytes obtained from large antral follicles of gonadotrophin-primed (PR) or unprimed (UPR) females, as well as IVG oocytes.
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Materials and methods |
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In-vitro-grown (IVG) oocytes
Oocytegranulosa cell complexes of preantral follicles were isolated from 12-day-old mice and cultured as previously described (Eppig et al., 1992; Eppig and O'Brien, 1996
). Briefly, oocytegranulosa cell complexes were obtained from preantral follicles of 12-day-old females after collagenase digestion. Oocytegranulosa cell complexes were washed three times in collagenase-free Waymouth medium supplemented as described above, and with 3 mg/ml BSA (crystallized; ICN Biochemicals, Aurora, OH, USA), 5 µg/ml transferrin, 5 ng/ml selenium (ITS; Collaborative Research Inc., Bedford, MA, USA) and 5 µg/ml insulin (Collaborative Research Inc.). Approximately 300350 oocytegranulosa cell complexes were cultured per well in a 6-well dish on collagen-coated membranes (Biocoat Collagen I Inserts, Becton Dickinson, Bedford, MA, USA) in 4 ml of medium. IVG medium was supplemented with 1 mg/ml fetuin, to prevent hardening of the zona pellucida (Eppig et al., 1996
), but not with gonadotrophins or EGF. Cultures were maintained for 10 days at 37°C under a humidified atmosphere of 5% O2, 5% CO2 and 90% N2 in modular incubation chambers (Billups-Rothenberg, Del Mar, CA, USA). At the end of the culture period, oocytegranulosa cell complexes were detached from the collagen membranes and collected in BSA-free Waymouth medium supplemented with 1 mg/ml fetuin. ITS mixture was omitted from the maturation medium. Complexes were washed three times in 2.5 ml of fresh medium and groups of 150200 oocytegranulosa cell complexes were allocated randomly to different maturation conditions.
In-vitro oocyte maturation (IVM)
Oocytecumulus complexes obtained from PR and UPR females as well as IVG complexes at the germinal vesicle (GV) stage were cultured in BSA-free Waymouth medium supplemented with either 10 ng/ml EGF (Collaborative Research Inc.), 10 ng/ml EGF plus 100 ng/ml FSH, or 100 ng/ml FSH. Control oocytecumulus and/or granulosa cell complexes were cultured in BSA-free Waymouth medium without any growth factor or hormonal supplementation. FSH (oFSH-20) was generously provided by The National Hormone and Pituitary Program of the National Institute of Diabetes and Diseases of the Kidney (NIDDK). At the end of 1718 h culture, the stage of nuclear maturation was assessed after removing surrounding granulosa cells by continuous pipetting of oocytegranulosa cell complexes in IVM medium. The percentage of oocytes undergoing GVB and polar body extrusion (metaphase II oocytes) in all groups was determined by observation under a dissecting microscope.
In-vitro fertilization and embryo culture
Oocytes at metaphase I or metaphase II were washed three times in Minimum Essential Medium (MEM; GIBCO, Life Technologies) supplemented with 3 mg/ml BSA. In-vitro fertilization and culture were performed as described previously (Ho et al., 1995; Eppig, 1999
). Eggs were removed from fertilization drops after 46 h, rinsed twice in 2.5 ml MEM and cultured overnight in 500 µl droplets of fresh medium under washed mineral oil in modular incubation chambers as described above. At 30 h post fertilization, cleavage-stage embryos were rinsed twice with KSOM medium supplemented with essential and non-essential amino acids (KSOM/AA) and cultured to the blastocyst stage at 37°C in 1 ml KSOM/AA medium in borosilicate tubes (Ho et al., 1995
; Eppig, 1999
). Blastocyst-stage embryos obtained on day 5 post fertilization were fixed for cell number determination as described previously (Van de Sandt et al., 1990
).
Statistical analysis
Data are presented as the mean percentage of at least three independent experiments, variation among replicates is presented as the standard error of the mean. The percentage of oocytes undergoing GVB and polar body extrusion as well as the proportion of 2-cell and blastocyst-stage embryos obtained after different maturation conditions were analysed using arcsin-transformed data and compared by analysis of variance (ANOVA) using StatView (SAS Institute Inc., Cary, NC, USA). When a significant F-ratio was defined by ANOVA, groups were compared using the Fisher's Protected Least Significant Difference (PLSD) post-hoc test using StatView software; when P 0.05, the difference was considered significant. The mean number of nuclei at the blastocyst stage on day 5 post fertilization is presented and groups compared using notched box and whisker plots (StatView). Non-overlapping notches between box plots indicates significant differences (P < 0.05) (Kafadar, 1985
).
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Results |
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Effect of maturation conditions on the cytoplasmic maturation of in-vivo grown oocytecumulus complexes
EGF alone, or in combination with FSH, increased the proportion of oocytes that cleaved to the 2-cell stage after in-vitro fertilization (72%; P = 0.05) and (74%; P < 0.05) respectively (Figure 2A) compared with untreated oocytes. Maturation with FSH alone had no effect (P = 0.16) on the proportion of oocytes that cleaved to the 2-cell stage (65%) compared with controls (52%). Furthermore, EGF as a sole stimulus, or EGF + FSH, during oocyte maturation increased the proportion of oocytes reaching the blastocyst stage (64%; P < 0.05) and (60%; P = 0.05) respectively. No significant differences were observed in the frequency of blastocyst formation between control (38%) or FSH-treated oocytes (46%; P = 0.3).
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Comparison of the number of cell nuclei per blastocyst on day 5 after fertilization of oocytes from PR females (Figure 3A), demonstrated significant differences after treatment with EGF + FSH (mean = 121.6 ± 4.1) and FSH (121.6 ± 5.0) compared with controls (97.2 ± 5; P < 0.05). Furthermore, EGF significantly increased the total number of nuclei (145.2 ± 5.0) compared with oocytes exposed to EGF + FSH (P < 0.0001) or FSH as an only stimulus (P = 0.0004). The number of nuclei in blastocysts obtained from UPR females (Figure 3B
) was higher after oocyte maturation with EGF (103.2 ± 4.0) or FSH alone (95.1 ± 3.5) or in combination (98.2 ± 3.7) compared with controls (83.6 ± 4.0; P < 0.05). However, no differences were observed among the different treatment groups.
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Discussion |
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Culture conditions during meiotic maturation can also have a significant impact on the progression to metaphase II (Downs and Mastropolo, 1997) and developmental potential (Van de Sandt et al., 1990
). For example, several culture media support the completion of meiosis to a different extent according to the concentration and type of energy substrates contained. Moreover, variations in the culture environment (pH) may limit the availability of substrates required for proper metabolism in the oocytecumulus complex (Downs and Mastropolo, 1997
). The frequency of polar body extrusion after maturation in Waymouth medium is lower compared with other oocyte maturation media (Downs and Mastropolo, 1997
). However, maximal rates of blastocyst formation were found after in-vitro maturation of mouse oocytes in Waymouth medium (Van de Sandt et al., 1990
). Culture conditions may explain the differences in the rates of polar body extrusion observed after EGF and/or FSH stimulation of mouse oocytes in the present study compared with those of others (Merriman et al., 1998
). In the latter study, 8496% of oocytes extruded a polar body when cultured in serum-supplemented MEM medium, albeit with a lower preimplantation development in vitro (Merriman et al., 1998
).
In combination with gonadotrophins, EGF increased cumulus expansion and the rates of polar body extrusion in oocytes obtained using a whole follicle culture system (Boland and Gosden, 1994; Cortvrindt et al., 1998
; Smitz et al., 1998
). Maturation medium supplemented with EGF plus FSH and 5% serum supported polar body formation in 90% of oocytes (Boland and Gosden, 1994
), with between 9% and 41% of oocytes reaching the blastocyst stage after in-vitro fertilization (Spears et al., 1994
). Although recombinant meiosis-stimulating ligands were used in these experiments, oocyte maturation was conducted in the presence of serum, which could confound interpretation. Culture and maturation of oocytegranulosa cell complexes in the presence of serum results in similar rates of cleavage and blastocyst formation after in-vitro fertilization compared with in-vivo-grown oocytes (Eppig and O'Brien, 1998
). However, serum- or BSA-supplementation during meiotic maturation remain a source of undefined components that may `mask' the effects of growth factor or hormonal supplementation on cumulus expansion and meiotic progression. For example, stimulation with EGF alone had a comparable effect on nuclear maturation of pig oocytes as treatment with porcine follicular fluid (PFF) and supplementation of PFF-containing medium with EGF affords no additional improvements in the rates of oocyte maturation (Reed et al., 1993
). Moreover, EGF supplementation to medium containing fetal calf serum or PFF had no effect on meiotic maturation or cleavage rates in bovine (Lonergan et al., 1996
) or porcine oocytes (Abeydeera et al., 1998b
) respectively.
EGF treatment of maturing oocytes from UPR females did not affect the percentage of embryos that completed preimplantation development, in contrast to the beneficial effect of EGF on the development of embryos derived from oocytes of PR females. EGF promoted higher cell numbers in the blastocysts of both groups. Thus, effects of EGF during oocyte maturation on blastocyst quality are separable from effects on frequency of blastocyst development. In addition, these results suggest that gonadotrophins in vivo increase the sensitivity or responsiveness of complexes to EGF, thereby promoting both nuclear and cytoplasmic maturation of in-vivo-grown cumulus cell-enclosed oocytes cultured with EGF. However, IVG oocytes behaved more similarly to oocytes from PR females than UPR females with regard to their response to the maturation-stimulating ligands; the frequency of completion of preimplantation development was increased, as well as the number of cells per blastocyst. This common response to these ligands during maturation occurred despite the development of oocytegranulosa cell complexes in vitro in the absence of gonadotrophins. The implications of this observation are not clear, but it has also been noted that more blastocysts are produced per ovary after IVG than development in vivo when calculated on a per animal basis (Eppig and O'Brien, 1998). Perhaps both the responses of maturing oocytes in vitro to EGF noted above and the greater production of blastocysts per animal when oocytes were grown in vitro reflect suppressive or selective follicular regulatory processes occurring in vivo that are not functional under the conditions used in vitro.
In our previous studies on the preimplantation developmental competence of in-vivo-grown and in-vitro-matured oocytes, or oocytes both grown and matured in vitro, FSH has been used as the standard promoter of maturation. It is worthwhile, therefore, to consider whether EGF alone or EGF plus FSH offer benefits compared with FSH alone for promoting nuclear and cytoplasmic maturation in vitro. These comparisons are summarized in Table II. EGF or EGF plus FSH are always at least the equal of FSH alone for promoting nuclear and cytoplasmic maturation. There were no benefits of EGF, compared with FSH, detected for oocytes from UPR females. However, there were significant benefits noted for using EGF alone, or EGF and FSH together, for the nuclear and cytoplasmic maturation of oocytes from PR females and IVG oocytes. In fact, EGF treatment of IVG oocytes resulted in the production of blastocysts having a cell number equivalent to that of oocytes from UPR females. However, the frequency at which these IVG oocytes are able to complete preimplantation development is still below that for both groups of in-vivo-grown oocytes. Nevertheless, the use of EGF for the maturation of IVG oocytes still represents a significant advance in the technology for producing oocytes in vitro that are competent of undergoing preimplantation development.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 27, 1999; accepted on September 20, 1999.