Department of Animal Health and Biomedical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA
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Abstract |
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Key words: development/embryo/pantothenate/vitamins
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Introduction |
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In contrast to the extensive studies with substrates and amino acids, the role of vitamins for normal embryo development in vitro has not been thoroughly examined, although as with somatic cells, these important nutrients may be expected to play a key role in the embryo's metabolic balance and general nutrition. Vitamins are organic nutrients required in small amounts by mammalian cells; however, most mammals have lost the ability to manufacture them and so vitamins must, therefore, be supplied as nutrients (Baum, 1978; Stryer, 1981
). Many water-soluble vitamins are precursors of co-enzymes that are necessary for numerous biochemical reactions in the cell. Vitamins involved in metabolic reactions include riboflavin and niacin as precursors of FAD and NAD respectively, pantothenate as a component of co-enzyme A and thiamine which is required for the conversion of pyruvate to acetyl CoA. The co-enzyme pyridoxal phosphate is an essential catalyst for the transamination and deamination of amino acids. Biotin is a co-enzyme in carboxylation reactions for the biosynthesis of purines and fatty acids. Ascorbic acid is an antioxidant, while choline and inositol are components of phosphoglycerides found in most membranes of higher organisms. Since these processes are so essential for normal cell functions and maintenance, it would appear that vitamins are indispensable nutrients for cultured cells including embryos.
Previous work from this laboratory showed that although particular vitamins, especially inositol, choline and pantothenate, stimulated zona escape or `hatching' by hamster blastocysts, they had no demonstrable effects on embryo development per se starting from the 8-cell or morula stages (Kane et al., 1986; Kane and Bavister, 1988a
,b
). In these studies, no information was obtained on possible vitamin effects on development of earlier stages. The apparent lack of effect of vitamins on advanced stages of embryo development may have been due in part to the use of inferior culture media, as shown by the relatively low incidence of blastocyst development in these studies. Only ~5070% of (in-vivo produced) 8-cell embryos reached the blastocyst stage in vitro using medium TLP-PVA (Kane and Bavister, 1988a
,b
), doubtless due to the presence of glucose and phosphate; when these compounds were eliminated from the medium, 8090% of 8-cell embryos reached the blastocyst stage (Seshagiri and Bavister, 1989
). In addition, because the earliest stages of embryo development (zygote and early cleavage stages) have markedly different nutritional needs to those of late cleavage and differentiating (morula/blastocyst) stages (Brinster and Troike, 1979
; Gardner and Lane, 1996
; Pinyopummintr and Bavister, 1996
), a need to re-examine the role of vitamins on development from the 1-cell stage is indicated. The present work was undertaken with a more refined culture medium, HECM-6 (McKiernan et al., 1995
), to examine the effect of 10 water-soluble vitamins either singly or in combination on development of hamster 1-cell embryos to the blastocyst stage and on their subsequent fetal development after embryo transfer.
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Materials and methods |
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Embryos were cultured in medium HECM-6 containing no vitamins (control) and in HECM-6 containing one or more of the following vitamins (Sigma: 10 µmol/l L-ascorbic acid (A-4034), 5µmol/l D-biotin (B-4639), 5µmol/l choline chloride (C-1879), 3µmol/l folic acid (F-7876), 3µmol/l myo-inositol (I-5125), 5µmol/l niacinamide (N-3376), 3µmol/l D-pantothenate (P-5155), 1 µmol/l pyridoxal HCl (P-9130), 1 µmol/l riboflavin (R-4500), and/or 3 µmol/l thiamine (T-4625) (Table I). The concentrations of vitamins used in these experiments were based on those in Ham's F10 medium (Ham, 1963
; Kane and Bavister, 1988b
).
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Embryo transfer experiments were conducted to determine post-implantation viability of blastocysts that had developed in the control medium (HECM-6) and in HECM-6 plus pantothenate. Recipient females were mated to vasectomized males the day 1-cell embryos were collected from donor females. The female hamster has two uteri, each with its own cervix opening separately into the vagina (Magalhaes, 1968), therefore, no embryos will migrate from one uterine horn to the other. Taking advantage of these separate uteri, 810 blastocysts cultured in control medium were transferred to one uterine horn and an equal number of morphologically similar blastocysts cultured in HECM-6 plus pantothenate were transferred to the other uterine horn as previously described (McKiernan and Bavister, 1994
). Embryos were transferred into 18 females. Recipient females were examined for fetuses 11 days post-transfer (day 14 of gestation, 2 days before term).
Data collection and analysis
Embryos were examined at 48 and 72 h post-egg activation for morphological development to 8-cell stage and to morula and blastocyst stages respectively. Percentages of 8-cell embryos, morulae/blastocysts and blastocysts were calculated from the total number of embryos cultured. After culture, embryos were either fixed and Hoechst-stained for determining cell numbers (Boatman et al., 1988) or embryos were transferred to pseudopregnant recipient females to determine post-implantation viability.
In all statistical analyses, proportions were transformed using an arc sine square root transformation and mean nuclear number data were transformed using a square root transformation. These transformations were used to control for possible heterogeneity of variance (Snedecor and Cochran, 1980). Treatment differences were determined using a two-way analysis of variance (ANOVA) using the GLM procedure of SAS (SAS Institute, 1989). Treatments were compared using Fisher's least significant difference test (LSD); significance was set at P < 0.05. Embryo transfer data were analysed using a paired t-test.
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Results |
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Discussion |
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The constructive method of media development has, in our laboratory, produced the chemically-defined, protein-free `hamster embryo culture medium' (HECM) series. Culturing embryos in a protein-free, completely defined medium provides an opportunity to examine effects of any compound on in-vitro development without confounding factors from serum components such as bovine serum albumin (BSA), which are notoriously variable in composition and properties (Kane, 1983; McKiernan and Bavister, 1992
; Bavister, 1995
). Incremental modifications of HECM during the past 10 years, incorporating changes in energy substrates and amino acids, have dramatically increased developmental responses, from only 26% of hamster 2-cell embryos developing through one cleavage division with HECM-1 (Schini and Bavister, 1988
), to 27% of 1-cell embryos reaching blastocyst with HECM-3 (McKiernan et al., 1991
), to 59% blastocysts from 1-cell embryos with HECM-6 (McKiernan et al., 1995
). Each increment in development, with a concomitant reduction in the variability of responses, indicates an improvement in the medium, which forms the basis of the next series of experiments. The present study on the effects of vitamins on hamster 1-cell embryo development in vitro represents another logical step in this process.
Pantothenate was the single most stimulatory vitamin affecting hamster 1-cell embryo development in vitro. Pantothenate significantly increased blastocyst development compared with the control and compared with every other single vitamin, except for thiamine. Development in medium containing combinations of vitamins (including pantothenate) was never significantly greater than when embryos were cultured in pantothenate alone. There appeared to be no synergistic interaction between pantothenate and any of the vitamins that were tested. It is not known at what stage pantothenate exerts its action on cultured embryos. Experiments carried out in our laboratory in which pantothenate was added at 0 h (1-cell stage), at 24 h (2-cell stage) or at 48 h (8-cell stage) of culture were inconclusive, and there was no significant difference between the three treatments (data not shown).
Embryo viability was significantly enhanced in the presence of pantothenate. The number of live fetuses produced per 100 1-cell embryos cultured in HECM-6 containing pantothenate was >2-fold greater than the number of live fetuses produced per 100 embryos cultured in HECM-6 alone.
The mechanism of the effect of pantothenate on blastocyst formation may be 2-fold. Pantothenate is an essential vitamin for the biosynthesis of co-enzyme A, which is a co-factor for a multitude of enzymatic reactions including the oxidation of fatty acids, carbohydrates, pyruvate, lactate and amino acids (Tahiliani and Beinlich, 1991). Satisfying the embryo's specific energy substrate needs is critical to successful development in vitro (discussed by Barnett and Bavister, 1996). In several species, glucose (at normal plasma concentrations) inhibits embryo development in vitro while pyruvate and/or lactate are the preferred energy substrates (Leese and Barton, 1984
; Takahashi and First, 1992
; Conaghan et al., 1993
; Bavister, 1995
). Increased glycolysis and decreased tricarboxylic acid (TCA) cycle activity are indicators of perturbed energy metabolism in cultured embryos (Seshagiri and Bavister, 1991
; Gardner and Lane, 1993
; Barnett and Bavister, 1996
). Addition of vitamins and amino acids to the culture medium helps maintain in-vivo rates of glycolysis and oxidation, indicating that these compounds are key regulators of embryo metabolism (Lane and Gardner, 1998
). Thus, the role of pantothenate in embryo development may be to encourage formation of acetyl-CoA, and hence to stimulate oxidative metabolism.
Enrichment of the embryo culture medium with pantothenate may also have a secondary role as a defence against free oxygen radicals. Free oxygen radicals can be generated in aerobic organisms, particularly in metabolically active mitochondria (Boveris et al., 1972). Cells preincubated with pantothenate contained less lipid peroxides and their plasma membranes were more resistant to permeabilization when exposed to UV radiation or hydroxyl radicals (Slyshenkov et al., 1996
). Further work has shown that the protective effect of culture with pantothenate was not due to this vitamin scavenging oxygen free radicals but rather because it greatly increased the intracellular level of co-enzyme A. Co-enzyme A may facilitate removal of lipid peroxides by increasing metabolism of fatty acids and promote repair of the plasma membrane by activating phospholipid biosynthesis (Slyshenkov et al., 1996
). Increased energy production coupled with structurally sound cell membranes should facilitate blastocele formation. In addition, by stimulating TCA cycle activity, pantothenate may indirectly increase the cellular supply of glutamate, a glutathione precursor (Slyshenkov et al., 1996
). Supplementing culture medium with glutathione, which scavenges H2O2 and oxygen free radicals, relieved the 2-cell block in a mouse strain whose embryos were resistant to culture (Legge and Sellens, 1991
), while synthesis of glutathione during oocyte maturation in vitro in the pig (Abeydeera et al., 1998
) and cow (Luvoni et al., 1996
; de Matos et al., 1996a,b) significantly improves subsequent blastocyst development.
In contrast to the significant stimulatory effect of pantothenate, the remaining nine vitamins tested had no demonstrable effects on hamster 1-cell embryo development and responses were never significantly different from control values. Even though these vitamins showed no overt negative effects, simply supplying combinations of vitamins, such as the Ham's F-10 or minimum essential medium (MEM) vitamins requires caution. Mouse embryos showed reduced morula/blastocyst development and cell number in the presence of F-10 vitamins, and although MEM vitamins had a less drastic effect on morphological development, blastocyst cell number was also reduced (Tsai and Gardner, 1994). Specific vitamin requirements for preimplantation stage embryos, especially those grown in vitro, must be determined empirically. Few investigators have examined the effects of individual vitamins on preimplantation embryo development in vitro (Daniel, 1967
; Kane, 1988
; Kane and Bavister, 1988b
; Shirley, 1989
; Tsai and Gardner, 1994
). In these studies, three species were used: rabbit, mouse and hamster. Common to all these studies, investigators found a majority of `neutral' vitamins, some that were required and some that inhibited development, while no vitamin had the same effect in all three species. Rabbit blastocysts required inositol, pyridoxal, riboflavin and niacinamide for expansion and growth (Daniel, 1967
; Kane, 1988
). Hamster 8-cell embryos required inositol, pantothenate and choline for blastocyst hatching (Kane and Bavister, 1988b
). Mouse embryos do not appear to have any specific vitamin requirement; however, blastocyst cell numbers are reduced by physiological levels of niacinamide (Tsai and Gardner, 1994
) and high levels of niacinamide, biotin, riboflavin, and ascorbic acid arrest development (Shirley, 1989
). Perhaps general vitamin requirements of cultured embryos are met, to a large extent, by endogenous sources. A study by O'Neill (1998) indicated that mouse embryos had an absolute requirement for folate, shown by decreased development in the presence of methotrexate, an inhibitor of dihydrofolate reductase, although folic acid added to the medium had no beneficial effect on development.
When selected combinations of vitamins (each containing pantothenate) were tested in the present study, embryo development was unchanged or significantly greater than control (Tables VI and VII). Indeed, the blastocyst-stimulating effect of pantothenate was the same regardless of the presence or absence of other vitamins. In general, any observed stimulation of embryo development with combinations of vitamins could be attributed entirely to the action of pantothenate because none of the responses were greater than with pantothenate alone. In keeping with our strategy for improving embryo culture media design, we now routinely incorporate pantothenate into the medium, and HECM-6 + pantothenate is designated HECM-9. In summary, different vitamins, individually or in combination, have neutral or stimulatory effects on blastocyst development of cultured 1-cell hamster embryos. The most striking finding in this study was that 3µmol/l pantothenate can account for virtually all of the embryo stimulatory effects observed. This is the first report of a single vitamin significantly stimulating blastocyst formation from cultured embryos in any mammalian species.
Pantothenate had a specific effect on blastocyst development, and therefore more transferable embryos were produced. Addition of this vitamin could have a significant effect on human embryo development in vitro, especially now that there is greater emphasis on blastocyst transfer. It remains to be seen if pantothenate can stimulate embryo development in species other than the hamster, especially in primates including human. However, a medium essentially the same as HECM-9 (Bavister's defined medium, BDM) has been used to support development of human IVF embryos from the zygote to 4-cell stage, with birth of healthy babies following embryo transfer (Rinehart et al., 1998 and unpublished data).
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Acknowledgments |
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Notes |
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References |
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Barnett, D.K. and Bavister, B.D. (1996) What is the relationship between the metabolism of preimplantation embryos and their developmental competence? Mol. Reprod. Dev., 43, 105133.[ISI][Medline]
Bavister, B.D. (1995) Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update, 1, 91148.[ISI]
Bavister, B.D. and Andrews, J.C. (1988) A rapid sperm motility bioassay procedure for quality-control testing of water and culture media. J. In Vitro Fertil. Embryo Transfer, 5, 6775.[ISI][Medline]
Bavister, B.D., Leibfried, M.L. and Lieberman, G. (1983) Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol. Reprod., 28, 235247.[Abstract]
Baum, S.J. (ed.) (1978) Introduction to Organic and Biological Chemistry. Macmillan, New York.
Boatman, D.E., Andrew, J.C. and Bavister, B.D. (1988) A quantitative assay for capacitation: evaluation of multiple sperm penetration through the zona pellucida of salt-stored hamster eggs. Gamete Res., 19, 1929.[ISI][Medline]
Boveris, A., Oshimo, N. and Chance, B. (1972) The cellular production of hydrogen peroxide. Biochem. J., 128, 617630.[ISI][Medline]
Brinster, R.L. and Troike, D.E. (1979) Requirements for blastocyst development in vitro. J. Anim. Sci., 49, 2634.[ISI][Medline]
Conaghan, J., Handyside, A.H., Winston, R.M.L. and Leese, H.J. (1993) Effect of pyruvate and glucose on the development of human preimplantation embryos in vitro. J. Reprod. Fertil., 99, 8795.[Abstract]
Daniel, J.C. (1967) Vitamins and growth factors in the nutrition of rabbit blastocysts in vitro. Growth, 31, 7177.[ISI][Medline]
Gardner, D.K. and Lane, M. (1993) Amino acids and ammonium regulate mouse embryo development in culture. Biol. Reprod., 48, 377385.[Abstract]
Gardner, D.K. and Lane, M. (1996) Alleviation of the `2-cell block' and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum. Reprod., 11, 27032712.[Abstract]
Ham, R.G. (1963) An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp. Cell Res., 29, 515526.[ISI]
Kane, M.T. (1983) Variability in different lots of commercial bovine serum albumin affects cell multiplication and hatching of rabbit blastocysts in culture. J. Reprod. Fertil., 69, 555558.[Abstract]
Kane, M.T. (1988) The effects of water-soluble vitamins on the expansion of rabbit blastocysts in vitro. J. Exp. Zool., 245, 220223.[ISI][Medline]
Kane, M.T. and Foote, R.H. (1970) Culture of two- and four-cell rabbit embryos to the expanding blastocyst stage in synthetic media. Proc. Soc. Exp. Biol. Med., 133, 921925.
Kane, M.T. and Bavister, B.D. (1988a) Protein-free culture medium containing polyvinyl alcohol, vitamins, and amino acids supports development of eight-cell hamster embryos to hatching blastocysts. J. Exp. Zool., 247, 183187.[ISI][Medline]
Kane, M.T. and Bavister, B.D. (1988b) Vitamin requirements for development of eight-cell hamster embryos to hatching blastocysts in vitro. Biol. Reprod., 39, 11371143.[Abstract]
Kane, M.T., Carney, E.W. and Bavister, B.D. (1986) Vitamins and amino acids stimulate hamster blastocysts to hatch in vitro. J. Exp. Zool., 239, 429432.[ISI][Medline]
Lane, M. and Gardner, D.K. (1998) Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts. Hum. Reprod., 13, 991997.[Abstract]
Leese, H.J. and Barton, A.M (1984) Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J. Reprod. Fertil., 72, 913.[Abstract]
Legge, M. and Sellens, M.H. (1991) Free radical scavengers ameliorate the 2-cell block in mouse embryo culture. Hum. Reprod., 6, 867871.[Abstract]
Luvoni, G.C., Keskintepe, L. and Brackett, B.G. (1996) Improvement in bovine embryo production in vitro by glutathione-containing culture media. Mol. Reprod. Dev., 43, 437443.[ISI][Medline]
Magalhaes, H. (1968) Gross anatomy. In Hoffman, R.A., Robinson, P.F. and Magalhaes, H. (eds), The Golden Hamster: Its Biology and Use in Medical Research. Iowa State University Press, Ames, Iowa, pp. 91118.
deMatos, D.G., Furnus, C.C. and Moses, D.F. (1996a) Glutathione synthesis during in vitro maturation of bovine oocytes: role of cumulus cells. Biol. Reprod., 57, 14201425.[Abstract]
deMatos, D.G., Furnus, C.C., Moses, D.F. et al. (1996b) Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability. Mol. Reprod. Dev., 45, 451457.[ISI][Medline]
McKiernan, S.H. and Bavister, B.D. (1992) Different lots of bovine serum albumin inhibit or stimulate in vitro development of hamster embryos. In Vitro Cell. Dev. Biol., 28a, 154156.
McKiernan, S.H. and Bavister, B.D. (1994) Timing of development is a critical parameter for predicting successful embryogenesis. Hum. Reprod., 11, 21232129.
McKiernan, S.H., Bavister, B.D. and Tasca, R.J. (1991) Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage. Hum. Reprod., 6, 6475.[Abstract]
McKiernan, S.H., Clayton, M.K. and Bavister, B.D. (1995) Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro. Mol. Reprod. Dev., 42, 188199.[ISI][Medline]
O'Neill, C. (1998) Endogenous folic acid is essential for normal development of preimplantation embryos. Hum. Reprod., 13, 13121316.[Abstract]
Pinyopummintr, T. and Bavister, B.D (1996) Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts. Reprod. Fertil. Dev., 8, 835841.[ISI][Medline]
Rinehart, J., Chapman, C., McKiernan, S. and Bavister, B. (1998) A protein-free chemically defined embryo culture medium produces pregnancy rates similar to human tubal fluid (HTF) supplemented with 10% synthetic serum substitute (SSS). Hum. Reprod., 13 (Abstract Book 1), 59.
SAS (1989) SAS User Guide. SAS Institute, Cary, NC.
Schini, S.A. and Bavister, B.D. (1988) Development of golden hamster embryos through the two-cell block in chemically defined medium. J. Exp. Zool., 245, 111115.[ISI][Medline]
Seshagiri, P.B. and Bavister, B.D. (1989) Glucose inhibits development of hamster 8-cell embryos in vitro. Biol. Reprod., 40, 599606.[Abstract]
Seshagiri, P.B. and Bavister, B.D. (1991) Glucose and phosphate inhibit respiration and oxidative metabolism in cultured hamster eight-cell embryos: evidence for the `Crabtree Effect'. Mol. Reprod. Dev., 30, 105111.[ISI][Medline]
Shirley, B.A. (1989) Inhibition of development of preimplantation mouse embryos in vitro by some water soluble vitamins. Med. Sci. Res., 17, 465466.
Slyshenkov, V.S., Moiseenok, A.G. and Wajtczak, L. (1996) Noxious effects of oxygen reactive species on energy-coupling processes in ehrlich ascites tumor mitochondria and the protection by pantothenic acid. Free Rad. Biol. Med., 20, 793800.[ISI][Medline]
Snedecor, G.W. and Cochran, W.G. (eds) (1980) Statistical Methods. Iowa State University Press, Ames, Iowa.
Stryer, L. (ed.) (1981) Biochemistry. W.H.Freeman, San Francisco, pp. 247249.
Tahiliani, A.G. and Beinlich, C.J. (1991) Pantothenic acid in health and disease. In Aurbach, G.D. and McCormick, D.B. (eds), Vitamins and Hormones: Advances in Research and Applications. Academic PressHarcourt Brace Javonovich, San Diego, pp. 165228.
Takahashi, Y. and First, N.L. (1992) In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology, 37, 963978.[ISI]
Tsai, F.C. and Gardner, D.K. (1994) Nicotinamide, a component of complex culture media, inhibits mouse embryo development in vitro and reduces subsequent developmental potential after transfer. Fertil. Steril., 61, 376382.[ISI][Medline]
Submitted on May 4, 1999; accepted on September 13, 1999.