1 Department of Reproductive Biology, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 Jiao Chang Dong Lu, Kunming, Yunnan 650223, China, 2 Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, WI 53706, USA and 3 China-US Primate Biology Laboratory, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming 650223, China
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Abstract |
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Key words: blastocysts/in-vitro maturation/primate oocytes/protein-free medium
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Introduction |
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Well-defined culture conditions that support successful oocyte maturation resulting in embryo development will facilitate examination of the inter- and intracellular mechanisms responsible for initiating and completing normal maturation. In most reports, IVM of mammalian oocytes was performed in undefined systems containing co-cultured somatic cells, blood serum, bovine serum albumin (BSA) or cell-conditioned medium. Besides being a potential source of infectious agents, these undefined components make it difficult to undertake proper quality control and to evaluate the basic requirements for metabolic substrates, nutritional and growth factors, and hormones during IVM (Bavister, 1995).
Establishing a chemically defined IVM system for primate oocytes could also benefit efforts to alleviate human infertility by providing consistent results. Chemically defined IVM media that support successful oocyte maturation have been established in cattle (Keskintepe and Brackett, 1996; Rose-Hellekant et al., 1998
) and pigs (Abeydeera et al., 1998
). But in primates, including humans, little is known about the meiotic and developmental abilities of oocytes matured in a protein-free medium. The most widely used primate model for preimplantation studies is the rhesus monkey (Macaca mulatta). In the present study, two IVM media, either with serum supplementation or without any commercial protein source (i.e. chemically defined), were tested for their competence to support rhesus monkey oocyte meiotic maturation. Success was evaluated by the ability of IVM metaphase II (MII) ova to undergo IVF and subsequent embryo development to the blastocyst stage.
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Materials and methods |
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Culture media
The protein-free media consisted of: (1) CMRL-1066 (Connaught Medical Research Laboratories) medium (Gibco, Grand Island, NY, USA) modified as previously described (Boatman, 1987) (mCMRL-1066), with no macromolecular supplement; (2) a modification of hamster embryo culture medium version 9 (HECM-9), containing 11 amino acids and pantothenic acid, with 2.0 mmol/l Mg2+ and 1.0 mmol/l Ca2+, and 0.1 mg/ml polyvinyl alcohol (PVA) as the macromolecule (Lane et al., 1998
; McKiernan and Bavister, 2000
) (HECM-10). The HECM-10 was used for IVM because it has a relatively simple, chemically defined formulation and because an earlier variant (HECM-3) supported IVM of bovine oocytes (Rose-Hellekant et al., 1998
). These media were sometimes supplemented with 20% heat- inactivated bovine calf serum (BCS; Hyclone Laboratories Inc., Logan, UT, USA) to produce IVM treatments 3 and 4 respectively. All IVM media were supplemented with 5 µg/ml ovine FSH [oFSH-NIADDK-NIH (National Institutes of Health, Bethesda, MD, USA), AFP55518] and 10 µg/ml ovine LH (oLH-NIADDK-NIH, AFP4117A). These concentrations of gonadotrophins were chosen as they were previously used in a study that successfully matured rhesus oocytes in vitro (Schramm and Bavister, 1996
). Treatment 3 (mCMRL-1066 + BCS) was regarded as the control or baseline for this study because this medium was previously used for rhesus monkey IVM (Schramm and Bavister, 1994
).
Oocyte culture for IVM
Oocytes collected from each female monkey that appeared normal (non-vesiculated, round and medium to lightly pigmented and surrounded by several layers of cumulus cells) and contained an intact germinal vesicle (GV), as revealed by observation under Nomarski optics, were retrieved and randomly divided among the four IVM media treatments. Oocytes that appeared shrunken or dark were discarded. Within one replicate (female), each treatment group usually consisted of three or four oocytes, but occasionally two or five oocytes, depending on availability. Each group was washed three times in its respective treatment medium, then placed into 50µl drops of the same medium covered with equilibrated silicone oil contained in 35 mm plastic Petri dishes and matured for 3640 h at 37°C in a humidified atmosphere of 5% CO2 in air.
Evaluation of cumulus expansion and meiotic maturation
At the end of IVM culture, the degree of cumulus expansion was assessed subjectively under a stereomicroscope and classified into five categories: 0, no expansion; 1, very slight expansion observed only in the outermost layer; 2, slight expansion observed in two or three layers from the periphery; 3, moderate expansion, observed in ~50% of the cumulus cell layers; and 4, full expansion except in the corona radiata layer. Then oocytes were examined under a microscope using Nomarski optics for the morphological status of nuclear maturation: GV breakdown (GVB) was taken as the initiation of nuclear maturation and extrusion of the first polar body (metaphase II, MII) as the criterion for completion of nuclear maturation. Detection of these morphological details was facilitated by drawing the oocytes in and out of a Pasteur pipette to partially remove the cumulus cell layer prior to examination.
IVF of IVM ova
Ova exhibiting a first polar body (MII stage) were inseminated. Semen was collected by penile electroejaculation as previously described (Gould and Mann, 1988). Sperm capacitation and IVF were performed as described previously (Bavister et al., 1983
). Briefly, washed motile spermatozoa were resuspended at 20x106/ml in 2 ml Tyrode's albumin-lactate-pyruvate (TALP) medium (containing 3 mg/ml fraction V BSA) overlaid with 2 ml silicone oil that had been equilibrated with 5% CO2 in air, and incubated for 47 h at 37°C in a humidified atmosphere of 5% CO2 in air in plastic Petri dishes. Spermatozoa were then incubated for an additional 11.5 h with 1.0 mmol/l each of caffeine and dibutyryl cyclic AMP (dbcAMP) to induce hyperactivation (Boatman and Bavister, 1984
). The hyperactivated spermatozoa were diluted to 1x105/ml in 100 µl drops of equilibrated TALP medium containing 2% BCS and co-incubated with the MII ova for 1216 h at 37°C in a humidified atmosphere of 5% CO2 in air, keeping the four IVM treatment groups separate. After co-incubation, spermatozoa were removed from ova manually by aspirating through a pulled glass pipette. Ova were examined with an inverted microscope using Nomarski optics for evidence of activation (containing two polar bodies and/or
1 pronucleus). Any ova containing three or more pronuclei were excluded from the fertilization data as indicating polyspermic fertilization.
Embryo culture
Keeping the treatment groups separate, putative embryos (with two polar bodies and/or 1 pronucleus) were washed and cultured for 2436 h at 37°C in 50µl drops of equilibrated mCMRL-1066 containing 20% BCS, in a humidified atmosphere of 5% CO2, 5% O2 and balance N2. Then, embryos with two or more cells were transferred to fresh 50 µl drops of the same medium and cultured until development was arrested or until `hatching' (escape from the zona pellucida) occurred. The embryo culture medium was changed every other day. Embryos were examined daily using Nomarski optics (x200400 magnification) on a Nikon Diaphot TMD microscope.
Experimental design and data analyses
The entire procedure (oocyte collection, IVM, IVF and embryo culture) was replicated eight times with oocytes from different females (each female = one replicate). Values for GVB and MII were analysed as percentages of total oocytes, and values for activated ova and all embryonic stages were analysed as percentages of MII ova. All percentage data were subjected to arcsin (square root) transformation. Transformed data were analysed by analysis of variance (ANOVA) and Fisher's protected least significant difference (LSD) test. A probability of P 0.05 was considered statistically significant.
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Results |
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Effect of different media on cumulus expansion and meiotic maturation
All COC showed cumulus expansion of category 4 after IVM in mCMRL-1066 + BCS or in HECM-10 + BCS (IVM treatments 3 and 4, respectively). Oocytes matured in mCMRL-1066 alone showed category 3 cumulus expansion, whereas those cultured in HECM-10 showed only slight cumulus expansion (category 1; Figure 1). In the four IVM treatments, similar proportions of oocytes initiated meiosis (GVB: 23/35, 66%; 24/36, 67%; 27/33, 82%; and 24/35, 69% respectively; data not shown) and completed meiosis (MII: 60, 50, 76 and 57% respectively; Table I
).
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The ability of ova to cleave and to undergo embryo development was not significantly different among the four IVM treatments (P > 0.05). The time intervals for blastocyst formation and hatching ranged from 166180 h and 204220 h after insemination respectively, among the four groups. Blastocysts derived from each medium had visible inner cell masses and the four IVM treatments could not be distinguished from each other by blastocyst sizes or by the timing of development. Among the blastocysts, four out of four, one out of three, one out of four, and three out of six hatched from their zonae pellucidae in IVM treatment groups 1 to 4 respectively (data not shown).
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Discussion |
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In the control group (Table I; mCMRL-1066 + serum), the nuclear maturation frequency of COC was similar to a previous report on rhesus monkey IVM, while the incidence of blastocyst formation was higher (16 versus 4%; Schramm and Bavister, 1994
). However, there was no significant difference among the IVM treatments: oocytes matured in the four IVM media exhibited a similar potential to mature, become fertilized and to reach various `benchmark' stages of embryo development after IVF. Blastocyst development, which ranged from 16 to 30% of inseminated IVM ova, did not differ significantly whether serum was present in the IVM medium or not. Furthermore, there was no significant difference in the time intervals for blastocyst formation and hatching, or in the morphology and size of blastocysts across all groups. Thus, this study demonstrates that serum factors are not obligatory for IVM of primate oocytes.
In contrast, previous studies with rodents showed that maturation of mammalian oocytes in serum-free medium resulted in `zona hardening', leading to poor sperm penetration and failure of fertilization (Downs et al., 1986; Kito and Bavister, 1996
). Zona hardening is thought to result from changes of zona pellucida (ZP) structure catalysed by a protease released by precocious cortical granule exocytosis during IVM (Ducibella et al., 1990
). Fetuin, an alpha-trypsin inhibitor in serum, can inhibit this precocious hardening of the ZP in a dose-dependent manner (Schroeder et al., 1990
). However, our IVF data provide evidence that, unlike in rodents, zona hardening does not occur during IVM of rhesus monkey oocytes in protein-free medium. The lack of need for serum during rhesus monkey oocyte IVM in the present study is consistent with previous reports with oocytes of cattle (Keskintepe and Brackett, 1996
; Rose-Hellekant et al., 1998
) and pigs (Abeydeera et al., 1998
). Possibly in all these species, premature cortical granule exocytosis does not occur during IVM.
Blastocyst development also did not differ whether a complex (mCMRL-1066) or simple (HECM-10) medium was used for IVM. Medium mCMRL-1066, supplemented with 20% BCS, has been used successfully for rhesus monkey oocyte IVM and for embryo culture (Schramm and Bavister, 1994, 1996
). The formulation of mCMRL-1066, as modified by Boatman (1987), involves many components believed to be necessary for viability and function of somatic cells in vitro, such as 21 essential and non-essential amino acids, the energy substrate `triad' (pyruvate, glucose and lactate; Bavister, 1995
), various vitamins, nucleotides, inorganic salts, and so on. In contrast, HECM-10 is a relatively simple or `minimal' medium that was developed over many years to support development of 1-cell hamster embryos to the blastocyst stage, and to ascertain their nutrient requirements. Apart from the usual inorganic salts, HECM-10 contains only one member of the energy substrate triad (lactate), 11 amino acids, one vitamin (pantothenic acid) and PVA as a substitute for protein. Lactate combined with 11 amino acids as energy substrates and nutrients can effectively support hamster and rhesus monkey embryo development (McKiernan et al., 1991
, 1995
; McKiernan and Bavister, 1994
; Schramm and Bavister, 1996
) whereas lactate plus glutamine together can support bovine oocyte maturation in vitro (Rose-Hellekant et al., 1998
).
Glucose, pyruvate and lactate are the main substrates for energy metabolism in somatic cells. Additionally, glutamine can also serve as an energy substrate to improve in-vitro nuclear maturation of hamster (Gwatkin and Haidri, 1973) and rabbit (Bae and Foote, 1975
) oocytes. Among the substrate triad, pyruvate is considered to be essential for supporting oocyte meiotic events (Brinster, 1971
; Rieger and Loskutoff, 1994
). Oocyte utilization of pyruvate is closely dependent upon cumulus cells which can convert glucose or lactate into pyruvate for use by oocytes (Leese and Barton, 1985
). However, pyruvate alone is insufficient for oocyte cytoplasmic maturation. As used in the present study, mCMRL-1066 contains all four energy substrates (glutamine, glucose, lactate and pyruvate) whereas HECM-10 has only lactate and glutamine.
The data presented here indicate that the minimal but highly selected components of HECM-10, including energy substrates and amino acids, are sufficient for supporting oocyte nuclear and cytoplasmic events in the rhesus monkey and comparable in effectiveness to all those present in the much more complex medium mCMRL-1066. However, further improvement in the culture system is needed. Although the proportions of GV oocytes that reached MII and of MII oocytes that fertilized were similar to those in previous studies on rhesus oocyte IVM (summarized in Schramm and Bavister, 1999), the frequency of blastocyst development in all these studies was still much lower than with in-vivo matured oocytes (~5060%; Schramm and Bavister, 1996
). Further studies to examine constituents of the IVM culture medium that could enhance cytoplasmic maturation are in progress. In particular, addition of glucose to the `minimal' medium HECM-10 may improve IVM results, as indicated by other studies from our laboratory (unpublished data), and enhance cumulus expansion (see below).
In a study of bovine IVM, there was some correlation between cumulus expansion and oocyte developmental capacity (Rose-Hellekant et al., 1998). In contrast, in the present study, maturation of oocytes in unsupplemented HECM-10 resulted in very poor cumulus expansion. Nevertheless, this IVM treatment supported meiosis and acquisition of developmental competence in oocytes equally as well as the other treatments in which cumulus expansion was superior. We have also noted poor developmental capability of rhesus oocytes exhibiting degrees of cumulus expansion (unpublished data). It is concluded from these studies that cumulus expansion does not correlate with the acquisition of meiotic and developmental competence by rhesus monkey COC matured in vitro. The same conclusion was drawn in a study in the pig (Abeydeera et al., 1998
). Thus, it may be inferred that cumulus expansion cannot be used as a reliable marker for oocyte maturation in the rhesus monkey.
The most cumulus expansion (category 4) was observed with COC matured in both serum-containing media (mCMRL-1066 + 20% BCS and HECM-10 + 20% BCS). Oocytes matured in the two serum-free media displayed quite different amounts of cumulus expansion (category 3 in mCMRL-1066 versus category 1 in HECM-10). These findings suggest that rhesus monkey cumulus expansion can be affected not only by serum but also by other components of the basic culture medium. The stimulatory action of serum on cumulus expansion observed in this study was also investigated in rodents (Eppig, 1980; Kito and Bavister, 1996
). It is clear that some components of serum, such as epidermal growth factor (EGF) (Downs, 1989
), insulin-like growth factor and other factors increasing intracellular cyclic AMP (Dekel and Kraicer, 1978
; Racowsky, 1985
) can induce cumulus expansion in vitro. As in pigs (Abeydeera et al., 1998
) and cattle (Rose-Hellekant et al., 1998
), rhesus monkey COC are capable of a certain degree of cumulus expansion in culture media that contain gonadotrophins without serum or follicular fluid supplementation. In contrast, no cumulus expansion was observed in mouse (Eppig, 1980
) or hamster (Kito and Bavister, 1996
) COC matured with gonadotrophins, BSA or PVA but without serum. This discrepancy may reflect species-specific differences in the cumulus expansion mechanism. Because cumulus expansion requires substrates, such as the combination of glucose with glutamine, for synthesis of mucopolysaccharides (Chen et al., 1990
), it is proposed that the absence of glucose contributes to the inferior cumulus expansion of COC matured in HECM-10 although this medium contains glutamine. This assumption has been confirmed by other work from our laboratory showing category 3 cumulus expansion of rhesus COC matured in glucose-supplemented HECM-10 (unpublished data). Thus, glucose is required for cumulus expansion of rhesus monkey COC matured in serum-free medium. In the present study, enhancement of cumulus expansion in serum-supplemented media can be at least partly explained by the presence of glucose in serum. Whether or not additional serum components such as EGF are required for maximum expansion of rhesus monkey cumulus oophorus remains to be established.
In summary, this study demonstrates that primate oocytes can be successfully matured in a protein-free, chemically defined culture medium with subsequent development to blastocysts after IVF. The presence of serum during IVM did not improve the capabilities of the oocytes to undergo fertilization and development, although it did stimulate expansion of the cumulus oophorus. Lactate and 11 amino acids provided in HECM-10 as energy substrates and nutrients can support the acquisition of meiotic and developmental competence of rhesus oocytes. It is inferred that, in order to improve the developmental competence of IVM monkey oocytes, future studies can use chemically defined HECM-10 as the base medium to assess the importance of specific factors such as hormones and growth factors in supporting primate oocyte maturation. Elucidation of these factors would be much more difficult in media containing blood serum or serum albumin preparations both because of the notorious variability of different batches of serum and their chemical complexity (Bavister, 1995). It is anticipated that the results of studies using rhesus monkey oocytes will be useful for developing strategies for oocyte maturation in other primates including humans, which could help to avoid the use of excessive gonadotrophin stimulation needed for collection of in-vivo matured ova. This strategy could be especially useful for women with polycystic ovarian syndrome (Barnes et al., 1996
).
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Acknowledgments |
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Notes |
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5 To whom correspondence should be addressed at: Department of Reproductive Biology, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 Jiao Chang Dong Lu, Kunming, Yunnan 650223, China. E-mail: wji{at}mail.kiz.ac.cn
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References |
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Submitted on June 7, 2000; accepted on October 13, 2000.