Lipid interactions with in vitro development of mammalian zygotes

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  • Gamete Research 21:243-254 (1988)

    Lipid Interactions With In Vitro Development of Mammalian Zygotes R.A. Waterman and R.J. Wall

    Reproduction Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland

    Rabbit zygotes were tested for their ability to sequester radiolabeled acetate, oleate, and arachidonate in intracellular lipid. Radiolabeled arachidonic acid was concentrated 170 k 28-fold (mean 2 SEM) and oleic acid was concentrated 105 * 26-fold in zygotic lipids during 6 hr of culture when compared with the initial concentrations in culture medium. Acetate was not concentrated into lipids by cultured zygotes. Both long chain fatty acids were incorporated mainly as triglyceride. Polydimethylsiloxane fluid, used to cover the microdroplets of medium during culture, demonstrated lipophilic properties. This charac- teristic was utilized to indirectly transfer lipids to culture medium, permitting examination only of lipoidal properties of test extracts on embryonal development. For rabbit zygotes, blood plasma extract was detrimental and whole blood extract was beneficial for embryonal cleavage rates during the first 24 hr of culture. A higher proportion of mouse zygotes developed to blastocysts when cultured in modified Hams F-10 medium compared to BMOC medium, and this difference was negated by inclusion of a lipid extract prepared from rabbit oviductal fluid in the culture system. Comparison of fatty acid analyses of the lipid extracts with development rates of zygotes suggests that modified rates of embryo development may be associated with ratios of individual fatty acids presented to the culture medium rather than with the presence of any single fatty acid.

    Key words: rabbit, mouse, cultured zygote, lipid, fatty acid

    INTRODUCTION

    Despite the efforts of many workers using a variety of culture media and conditions, the uniform and predictable culture of mammalian zygotes to blastocysts has not been attained in many species. In nearly all culture media, protein, amino acid, carbohydrate, salt, vitamin, antibiotic, and other metabolite quantities and qualities have been carefully described. Almost all successful culture media contain

    Received January 22, 1988; accepted June 17, 1988.

    Address reprint requests to Dr. R.A. Waterman, Reproduction Laboratory, Beltsville Agricultural Research Center, USDA Agricultural Research Service, Beltsville, MD 20705.

    Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply approval to the exclusion of other products that may be suitable.

    0 1988 Alan R. Liss, Inc.

  • 244 Waterman and Wall

    either albumin or blood serum. However, descriptions of these additions to media regarding lipid class content or their respective fatty acid compositions are not usually reported. With the many attempts to identify the best albumin or serum source, it is likely that the lipid content or quality presented to zygotes by the culture medium may be important to the development of an optimal and predictable culture system.

    This study was conducted to examine the ability of rabbit zygotes to incorporate fatty acids during in vitro culture. Effects of lipid extracts, transferred to culture medium by lipophilic diffusion, on the development of rabbit and mouse zygotes during in vitro culture are also demonstrated. Preliminary reports have been made [Waterman and Wall, 1985; Waterman, 19861.

    MATERIALS AND METHODS Rabbit Zygotes

    New Zealand White does were induced to superovulate by subdermal admin- istration of 130 IU pregnant mares serum gonadotropin (PMSG; Intercontinental Biologic Corp., Millsboro, DE). Three days later, the does were mated twice and 300 IU human chorionic gonadotropin (hCG) was administered intravenously (Ly- phomed, Melrose Park, IL). Zygotes were recovered between 19 and 20 hr after breeding by flushing the oviducts with culture medium. A flared 1.14-mm ID polyethylene tube was inserted into the fimbriated end of each oviduct to a depth of about 1 cm and clamped into place. A blunt, thin-walled 22-gauge needle was inserted through the uterine wall near the uterotubal junction and threaded through the uterotubal junction. Six milliliters of medium was gently flushed toward the fimbria and collected via the polyethylene tube in a 60 X 15 mm sterile petri dish. Oocytes and zygotes collected from three rabbits were pooled (n = 40-60), washed free of oviductal fluids with culture medium, and microscopically examined for the presence of both pronuclei.

    Rabbit Zygote Cultures

    Five to ten zygotes were cultured in 100-p1 droplets of medium in 60 X 15 mm culture dishes under 7.0 ml of polydimethylsiloxane fluid (Dow Corning 360 medical fluid, 20 centistokes, Dow Corning Medical Products, Midland, MI). Zygotes were cultured in a humidified incubator at 37.5C with an atmosphere of 5% carbon dioxide to 95% air. Culture medium was based on a modified [Kane and Foote, 19701 Hams F-10 formulation (GIBCO formula 83-5181, lot 61P4140, GIBCO Laborato- ries, Grand Island, NY). The medium also contained 1 % (wiv) bovine serum albumin (Fraction V, A-9647, Sigma Chemical Co., St. Louis, MO), 14.3 mM sodium bicarbonate, and 1.0 mM glucose. The medium was adjusted to pH 7.3 by gassing with 5% carbon dioxide and to 290 mOsM by addition of sodium chloride. This medium permitted in vitro development of zygotes which was comparable to in vivo rates of development.

    Fatty Acid Incorporations Into Rabbit Zygotes

    Experiment 1 was conducted to verify utilization of exogenous fatty acids by rabbit zygotes during in vitro culture. Radiolabeled medium was prepared by transferring an aliquot of radiolabel source, about 4 pCi [1-14C] acetate (714 pCi/mg), 1 pCi [1-I4C] oleate (199 pCi/mg) in ethanol, or [ 1-I4C] arachidonate (196

  • Lipids and In Vitro Development of Zygotes 245

    pCi/mg) in ethanol, to a glass vial and, for oleate and arachidonate, evaporating the carrier solvent with nitrogen. Two milliliters of medium was then added to the vial, and equilibration was allowed to proceed at 4C for 18 hr. Medium was sampled after equilibration to determine radioactivity presence per unit volume medium.

    Microdrops were formed with the equilibrated medium and zygotes were cultured for 2, 4, or 6 hr. After culture, embryos were recovered from each culture droplet, rinsed twice in about 8 ml of fresh unlabeled medium to remove contami- nating surface radioactivity, and transferred to an extraction tube. Lipids were extracted from each pool of five to ten embryos by the described micro-Folch extraction. Lipid classes were isolated by thin-layer chromatography [Waterman, 19801, and individual classes were analyzed for radiolabel incorporations by liquid scintillation counting corrected for quench. Label enrichment values were calculated as follows: For each group of zygotes cultured in a single droplet, the level of radioactivity found in each lipid fraction was divided by the level of radioactivity present in an equivalent volume of medium sampled at initiation of culture. The equivalent volume was calculated assuming that each zygote represented a metabolic, or biochemically active, fluid volume of 0.5 nl.

    Lipid Microextractions Lipids were extracted from zygotes and fluids by a micromodification of the

    procedure described by Folch et al. [1957]. One drop of acetic acid and 1 ml of methanol were added to each teflon-capped extraction tube containing embryos or 0.1-0.2 ml of fluid to be processed. After mixing, 2 ml of chloroform was added, the tube was vortexed, and the extraction was allowed to proceed overnight at 4C. Lipids were phased into the chloroform layer by adding 1.1 ml of aqueous 2% potassium dihydrogen phosphate followed by vigorous mixing. After 2 hr, the extraction tubes were centrifuged at 4C for 10 min at 1OOOg. The upper aqueous phase, containing nearly all hydrophilic compounds, was completely aspirated and discarded.

    Lipid extracts were examined for long chain fatty acid compositions by temperature-programmed gas-liquid chromatography [Waterman, 19801. Fatty acid weight percentages were calculated from the summation of peak areas of palmitic (C16:0), stearic (C18:0), oleic (CIS:l), linoleic (C18:2), linolenic (Cl8:3), and arachidonic (C20:4) acids.

    Lipid Extracts and Rabbit Zygote Development Experiment 2 was conducted to determine if lipid extract presentation to culture

    medium by a unique procedure could modify in vitro cleavage rates of cultured rabbit zygotes. Extracts to be tested were dried on coverslips, which were then placed in 60 X 15 mm culture dishes (Fig. 1). Two or three 100-p1 droplets of medium were placed about 1.5 cm from the coverslip and 7 ml of medical fluid was added. The prepared culture dishes were held for 2-3 hr in an incubation chamber while surgical recovery and microscopic evaluation of zygotes was accomplished. Zygotes were added to the quilibrated culture dishes, five to ten zygotes per microdroplet.

    Lipid extracts were prepared from several sources. Rabbit oviductal flushings were collected from does subjected to the injection schedule as previously described and mated to vasectomized bucks. A 14 cm length of 1.14-mm ID polyvinyl tubing was inserted in the fimbriated end of each oviduct and clamped in place. Sterile saline was slowly injected through the uterotubal junction until the tubing was filled. The

  • 246 Waterman and Wall

    TEST RABBIT EXTRACT ZYGOTES

    OIFFUSION /I THROUGH OIL rn0IUIl.s COVERSLIP ZYGOTES 6 EXTRACT

    Fig. 1. Preparations for culture of zygotes in the presence of lipids extracted from various tissues. The test extract was dried on a coverslip, which was then placed in a culture dish. Droplets of medium were placed about 1.5 cm from the coverslip and 7 ml of medical fluid (oil) was added. After about 2 hr of equilibration in the incubator, zygotes were transferred to the medium. Exposure of zygotes to the lipid extract was moderated by the distributional equilibria of lipoidal compounds between the lipophilic medical fluid and the medium.

    tubing was removed from the infundibulum and backflushed with air into an extraction tube, expelling two to three drops of first-drawn oviductal fluids. Bovine luteal extracts, previously prepared from corpora lutea of cows at day 19 of gestations, were tested directly. Graded levels of this stock extract (10-160 pg lipid) were examined to see if beneficial or toxic exposures might be detected by this culture system. Lipids of plasma (about 30, 50, or 100 pg) or whole blood (about 50 pg), obtained from does subjected to the superovulation schedule, were extracted by the described micro-Folch procedure. During preliminary studies, transfers of dried, redissolved whole blood extracts to the coverslip were made with hexane, chloro- form, or methanol.

    Embryo development was estimated by counting nuclei. Embryos were stained with Hoescht dye 33342 (Sigma Chemical Co, St. Louis, MO), a DNA-specific fluorescence stain, according to the technique of Purse1 et al. [1985]. Nuclei were counted at 400 X using fluorescence microscopy. Data for in vitro embryo develop- ment are expressed as a ratio between embryos cultured in the absence and presence of lipid extract. Within each day, the nuclear count for each embryo was divided by the mean nuclear count for control embryos, resulting in a ratio greater than 1 .O if the lipid extract accelerated the cleavage rate relative to control embryos within culture replicate. In order to compare in vitro cleavage rates of control zygotes to the physiological rates, embryos were surgically recovered from three does 44 hr after mating, 24 hr after the normal recovery time, and immediately stained for DNA.

    Mouse Zygote Cultures

    Experiment 3 was conducted to determine whether blastocyst development within cultured mouse ova could be modified by medium and/or lipid extract exposure. Superovulation and breeding schedules for mice (B6SJL strain) and collection and treatment procedures for collected ova have been described [Biggers et

  • Lipids and In Vitro Development of Zygotes 247

    a c al L :SO 0

    2 40 -+

    m

    Arachidonate t r a n s f e r -- ~ Oleate t rans fer

    Acetate: No t r a n s f e r

    1 2 3 4 5 6 Hours of incubat ion

    Fig. 2. Distribution equilibria of radiolabeled fatty acids between medium and medical fluid (oil). Arachidonate loss from medium was 99.53 - 33.64 * e ** (- 1.68/t) and oleate loss from medium was 99.58 minus 25.50 * e **(- 1.68k).

    al., 19711. Physical orientation of the culture system was as described for rabbit zygotes (Fig. 1) . Culture media included modified Hams F-10 medium and BMOC medium [Brinster, 19721, which contained 0.5% defatted bovine serum albumin (crystallized bovine albumin, 8 1-001-4, Pentex, Miles Scientific, Naperville, IL). Rabbit oviduct fluid was obtained from an unmated, superovulated doe 48 hr after ligature of the uterotubal and fimbriated ends of the oviducts. Lipids were extracted from oviduct fluid (1.1 ml) according to Folch et al. [ 19571, and this stock extract preparation was used for the entire experiment at an equivalent volume of 0.08 ml of oviduct fluid per culture. Four experimental treatments (12-20 ova-which were presumed to be fertilized-per treatment and replicated five times) included BMOC medium with and without extract and modified Hams F-10 with and without extract. Data are expressed as the percentage of zygotes that were two-cell embryos by 24 hr which developed to blastocysts by 96 hr within each treatment.

    Statistics

    A general procedure of least-squares analysis for data with unequal subclass numbers was used for the analysis of variance (see Tables 1-3, Figs. 3 , 5 , 6) and generation of least-squares means [Barr et al., 19761. For each significant F statistic, probability values for the hypothesis Ho: [least squares mean (i) = least squares mean (i)] were generated for the tested means. Reported data are arithmetic means and standard errors unless otherwise noted. Significance was tested at P < .05. Smoothed, fitted curves (Fig. 2) and incorporation kinetic curves (Fig. 4) were calculated by an iterative least-squares approach on a personal computer. Source programs in FORTRAN and curve-fitting algorithms are available from the authors.

    RESULTS Experiment 1

    The medical fluid used to cover the microdrops of media was found to be lipophilic. Several blank incubations without zygotes were conducted to determine to what extent the radiolabeled substrates would be lost from the culture medium (Fig. 2). Arachidonic acid loss from the medium to the medical fluid approached 33%

  • 248 Waterman and Wall

    2o01 I80 1 A,A I

    2 4 6

    Hours o f culture

    Fig. 3. Concentration of radiolabeled substrates in the lipids of rabbit zygotes during the first 6 hr of culture. Values represent means 2 SEM for acetate (Ac; n = 5 ) , oleate (OA; n = 5 ) , and arachidonate (AA; n = 4). Enrichment values were calculated as (dpm in zygotes/dpm in equivalent volume of medium).

    within 6 hr, whereas oleic acid loss approached 25%. Acetate was not transferred from the medium to the medical fluid. Data reported in this paper are not adjusted for this time-dependent loss. The length of time between preparation of culture plates and initiation of the culture period varied, as a result of variation in timing of surgical and zygote evaluation procedures, by as much as 1 hr between days, resulting in uncertainty about what the actual radiolabel distribution might have been during the culture period. Thus, enrichment values presented herein are conservative estimates of the abilities of zygotes to concentrate lipids during in vitro culture.

    Rabbit zygotes were able to sequester and to actively concentrate exogenous oleate and arachidonate but not acetate (Fig. 3 ) into cellular lipids. Contents of radiolabeled arachidonic acid were 170 ? 28 times (17,000%) higher in embryos than in an equivalent volume of medium after 6 hr. Likewise, radiolabeled oleic acid was concentrated 105 k 26-fold within the first 6 hr of in vitro culture. Radiolabeled acetate was minimally concentrated into lipid classes during in vitro culture at this stage of embryo development. Acetate accumulation in rabbit zygotes was 4.9 2 2.2-fold above medium activity during the first 6 hr of in vitro culture.

    Analyses of the various lipid fractions revealed that most of the radiolabeled fatty acids were accumulated as intraceliular triglyceride and not as free fatty acid. Distributional patterns of radiolabeled lipids were similar after 4 hr (Table 1) and 6 hr of culture. Concentration of radiolabeled fatty acids in triglycerides was between 12-24 times greater than in free fatty acids, cholesterol esters, or phospholipids. Kinetic analyses of lipid fraction radioactivities (Fig. 4) reveal that flux rates of arachidonic acid (1.17 pCi, 5.90 pg/ml medium) through the free fatty acid pool were about two to three times greater than those of oleic acid (0.90 pCi, 4.58 pg/ml medium).

    Experiment 2 During the vitro culture, zygotes were not in direct contact with the extracts, but

    rather were exposed to the extracts through the diffusion of lipophilic compounds through the medical fluid as shown in Figure 1. By utilizing this approach, hydrophilic compounds that might have accidently been transferred and concentrated

  • Lipids and In Vitro Development of Zygotes 249

    TABLE 1. Enrichment of Radiolabeled Substrates in Lipid Fractions of Rabbit Zygotes After 4 Hr of Culture*

    LiDid Class

    Lipid enrichment from labeled:

    Acetate Oleate Arachidonate

    Phospholipid 0.9 t 0.1" 6.4 ? 1.2' 10.3 t I.2b Free fatty acid 0.7 ? 0.3" 5.6 2 1.7' 7.6 ? 0.9b Trig1 yceride 0.7 t 0.1" 73.0 f 24.1' 128.5 t 17.6' Cholesterol ester 0.6 f 0.2" 5.6 2 1.6' 5.3 ? 0.7b

    *Label enrichment was calculated as (dpm in zygotes/dpm in equivalent volume of medium). Values represent means t SEM; n = 5 for acetate and oleate and n = 4 for arachidonate. "Columns and rows with differing superscript letters are significantly different (P < .05).

    Oleic a c i d

    0 FFA

    \ U- $ Arach idon ic a c i d

    T o t a l

    0 2 4 6

    Hours o f cu l tu re

    Fig. 4. Calculated incorporations of oleate or arachidonate into lipids of cultured rabbit zygotes. Kinetic analyses indicate that substrates were rapidly conveyed through free fatty acids (FFA) to triglycerides (TG).

    in the organic extraction of lipids were not transferred to the culture medium. No significant differences in numbers of nuclei were observed between control embryos cultured for 24 hr (9.64 2 0.35, n = 78) and embryos of the same chronological age recovered from rabbit oviducts (10.7 2 0.44, n = 33).

    Extracts from rabbit oviductal flushings, presented qualitatively to the culture medium by diffusion through the medical fluid, did not affect rabbit zygote development (Fig. 5) . Likewise, extracts from bovine luteal tissue at day 19 of pregnancy at any of six concentrations tested did not affect zygote development (pooled data are reported). Extracts of whole blood accelerated ( P < .05), while those of blood plasma significantly slowed ( P < .05) cleavage rates of treated embryos compared to control embryos obtained from the same does and cultured at the same time. Attempts to partially isolate a lipid fraction from whole blood which would be beneficial to embryo development were unsuccessful. All attempted extract transfers using methanol (favored polar lipid transfer such as phospholipid), chloroform (all lipid classes transferred), or hexane (favored neutral lipids such as cholesterol ester and triglyceride) accelerated embryo cleavage rates (pooled data are reported).

  • 250 Waterman and Wall

    +J

    a! 1.2 U - 1 . 1 - 0 L +J 1.0 t 0 0.9.

    a! 0 0.8 L

    k 1 0.7 1

    CT OV C L WE BP

    Fig. 5 . Indirect effects of lipid extracts on development of rabbit zygotes during 24 hr of culture. Values represent means k SEM for (treatedkontrol) ratios (see text) of control zygotes (CT; n = 78) and zygotes exposed to extracts from oviduct fluid (OV; n = 102), corpora lutea (CL; n = 86), whole blood (WB; n = 44), and blood plasma (BP; n = 26). Blood plasma slowed ( P < .05) and whole blood lipid extract accelerated ( P < .05) zygote development compared to control zygotes.

    Plasma extract proved to be detrimental to zygote development at lipid concentrations that were about 60% lower than, equal to, and 200% greater than whole blood exposure levels (pooled data are reported).

    Fatty acid analyses of the various extract sources are presented in Table 2. Oviductal flushings were significantly higher in proportions of palmitic and stearic acids. Bovine luteal lipids were significantly higher in proportions of oleic and arachidonic acids. Both blood preparations contained significantly higher proportions of linoleic acid. The calculated oleic to arachidonic acid ratio, as presented to this test system in the original extract, was significantly elevated for blood plasma compared to other extract sources.

    Experiment 3 Mouse ova were cultured in either modified Hams F-10 or BMOC medium

    and, as with Experiment 2, media were not in direct contact with the lipid extract prepared from rabbit oviductal fluid. There was a significant difference between media in the proportion of ova that were two-cell embryos within the first 24 hr which developed to blastocyts by 96 hr (Fig. 6). Lipid extract from rabbit oviductal fluid, presented separately and qualitatively to each culture medium by diffusion through the medical fluid, did not significantly affect the development of 2-cell mouse embryos to blastocysts within either medium, yet it did remove developmental differences between media.

    Fatty acid percentages of the media and oviductal fluid extract used for this experiment are presented in Table 3. The BMOC medium, which contained defatted bovine serum albumin, had a subjectively lower yet measurable fatty acid profile than did the modified Hams F-10 medium, which contained normal bovine serum albumin. Percentages of stearate and of linoleate were similar for both media, which were, in turn, significantly lower than those of oviductal fluid extract. Percentages of palmitate and oleate were similar for modified Hams F-10 medium and oviductal fluid, and differed ( P < .05) between BMOC medium and oviductal fluid extract. The percentage of arachidonic acid did not differ between media and oviductal fluid

  • Lipids and In Vitro Development of Zygotes 251

    TABLE 2. Major Fatty Acid Weight Percentages in Lipids Isolated From Extract Source Tissues*

    Fatty acid weight percentage

    Extract source C16:O C18:O C18:l C18:2 C20:4 Ratio"

    Oviduct fluid 34.4 & l.Sb 27.4 * 1.1' 18.4 IT 1.2d 13.2 t 0.9d 5.34 f 0.6" 3.88 t 0.36d Whole blood 26.1 t 0.4d 17.0 f 0.5b 17.4 f 0.6d 32.5 f 0.4b 5.01 2 0.3' 3.80 & 0.39d Corpora lutea 28.2 t 0.5" 15.5 f O.le 33.9 f 0.2b 11.2 f 0.2d 8.79 5 0.7b 3.95 & 0.33d Blood plasma 28.4 t 0.5" 12.9 f 0.2d 22.0 L 0.3e 30.8 L 0.2" 3.55 k 0.3d 6.41 t 0.62'

    *Values represent means 2 SEM; n = 13 for oviductal flushings, n = 16 for whole blood, and n = 5 for bovine luteal tissue and rabbit blood plasma. aRatio = C18:l weight percentage to C20:4 weight percentage. &Columns with differing superscript letters are significantly different (P < .05).

    100

    80 PI 0) 2 60 C PI 2 40 PI a

    20

    0

    CT ov

    Fig. 6. Comparison of mouse embryo development as affected by fatty acid exposure and culture medium. Vertical displacements represent means t SEM (n = 5 replicates of 10-20 zygotes each per medium X treatment cell) for the percentage of two-cell embryos at 24 hr of culture which became blastocysts by 96 hr. The percentage of control zygotes (CT) which became blastocysts during culture in modified Ham's F-10 medium (shaded bars) was greater (P < .05) than that during culture in BMOC medium (open bars). Inclusion of oviduct fluid extract (OV) in the culture system removed the difference in zygotic developmental efficacy between media.

    TABLE 3. Major Fatty Acid Weight Percentages in Lipids Isolated From Modified Ham's F-10 and BMOC Media and Oviduct Fluid*

    Fatty acid weight percentage

    Extract source C16:O c 18:O C18:l C18:2 C20:4 Ratioa BMOC medium 45.1 IT 0.4b 16.5 t 0.1" 17.6 f 0.2' 10.4 L 0.2' 6.7 -C 0.5 2.7 t 0.2' F-10medium 28.2 k 1.4' 16.1 IT 0.4' 31.8 * 0.2d 13.0 t 1.3' 8.8 t 1.8 3.9 f 0.7b.C Oviduct fluid 24.5 & 1.0' 20.2 t 0.4b 30.0 t 0.2b 19.3 f 0.7b 4.8 k 0.8 6.7 f l.Ob

    *Values represent means * SEM; n = 4 for BMOC medium and oviductal fluid and n = 3 for modified Ham's F-10 medium. "Ratio = C18:l weight percentage to C20:4 weight percentage. b-dColumns with differing superscript letters are significantly different (P < .05).

    extracts. The calculated oleic to arachidonic acid ratio, as presented to this test system in the original extract, was significantly elevated for oviductal fluid compared to either medium.

  • 252 Waterman and Wall

    DISCUSSION

    Successful and uniform culture of zygotes currently depends on our ability to repeatably formulate and prepare an optimal culture medium. Unfortunately, lipid characteristics of various culture media have not been defined [see Eppig and Schroeder, 1986; Kane, 19871. Usually, various sources of serum or serum albumin are tested until a successful and uniform result is obtained in embryo culture, and then this one source of serum or lot of albumin is used for continued research efforts. More important, our understanding of the lipid requirements for successful culture of zygotes is, at best, inadequate.

    Two findings are reported in this study. Rabbit zygotes can actively sequester long chain fatty acids from the culture medium, but they cannot utilize acetate for de novo lipid synthesis or fatty acid chain elongation. Cleavage rates of cultured rabbit and mouse zygotes can be affected by exogenous lipid presentations and exposures within the culture medium. Also contained within these two findings is the observation that oil used to cover the microdrops of medium can variably affect lipophilic substrate and metabolite concentrations within the culture medium.

    During the conduct of preliminary studies, it was observed that radiolabel recovery of added arachidonic acid from the culture medium was less than 70% whereas recovery of added acetate usually exceeded 97%. The divergence in radiolabel recoveries was easily traced to the lipophilic/hydrophobic nature of the medical fluid used to cover the medium droplets during culture. Cultures with radiolabeled substrate and no zygotes verified a lipophilic equilibrium between the medium and the medical fluid oil which was sensitive to the comparative lack of molecular dipole moment, or fatty acid chain equivalent, of the tested fatty acid. Miller and Purse1 [1987] have reported transfer rates for steroids which reflect a similar effect.

    With a low partition coefficient, the medical fluid can both supply lipophilic compounds to and remove lipophilic compounds from culture medium. Conse- quently, the physical culture volume and associated metabolite distribution for lipophilic compounds become greater than that associated with just the medium microdroplet in which the embryos are contained. In other words, any lipophilic compound that is added to the medium at a stated concentration will change in concentration once the medical fluid is placed over the medium. Also, as the lipophilic compound is utilized from the medium by the cultured embryos, the compound will slowly diffuse back from the oil into the medium. Thus, studies with lipophilic compounds [e.g., Plante et al., 19871 should be conducted with regard not only for the medium, but also for the medical fluid volume and isolation within and between treatments.

    This study is a qualitative rather than a quantitative examination of fatty acid incorporation into rabbit zygotes. Rabbit zygotes were cultured in pools of five to ten per microdrop of medium. The small tissue mass precluded any direct analytical measurement of fatty acid content within individual embryo pools. Therefore, the ability of the embryos to concentrate the radiolabeled substrate above the level present in the medium was examined in order to estimate any active capacity of the zygote to incorporate and retain exogenous fatty acids.

    Rabbit zygotes actively concentrated oleic and, especially, arachidonic acids into cellular lipids. There was, however, a lack of acetate incorporation into lipids

  • Lipids and In Vitro Development of Zygotes 253

    within the cultured zygotes, indicating that fatty acid synthesis and chain elongation were not active at this stage of development. Palmitic acid incorporation into the lipids of eight-celled mouse embryos was reported by Flynn and Hillman [1980]. They also reported that carbon dioxide production from palmitate was comparatively low until after the embryos had developed beyond the eight-cell stage, indicating that long chain fatty acids are not readily utilized for @-oxidation and probably do not serve as a normal source of energy production during the initial stages of develop- ment.

    Experiments 2 and 3 demonstrate that, in a qualitative manner, lipid extracts presented only to the polydimethylsiloxane fluid can diffuse to the culture medium and affect subsequent zygote development. The effects of varying fatty acid compositions on membrane structure and function are complex, although transitory substitutions of available rather than ideal fatty acids into the membrane bilayer usually do not adversely affect most cellular functionality [Spector and Yorek, 19851. A combination of palmitic and oleic acids, fatty acids most prevalent in the pig oocyte [Homa et al., 19861, was better than either single fatty acid in promoting the formation and hatching of mouse blastocysts from the one-cell stage [Quinn and Whittingham, 19821. Indirect presentation of the extract prepared from oviduct fluid to mouse zygotes cultured in the presence of defatted albumin (BMOC medium) could have increased the availability of oleate relative to palmitate, permitting a slightly enhanced ability for blastocyst formation. Data reported for experiment 2 also suggest that, rather than one fatty acid, the indirect presentation of a lipid extract containing two or more fatty acids in an undefined proportion can affect embryo development during in vitro culture beyond a simple survival concept.

    The zygote, then, is apparently capable of sequestering any exogenous fatty acid present in its microenvironment, but is not capable of modifying or metabolizing any long chain fatty acid sequestered at this stage of development. The ability of the zygote to sequester exogenous fatty acids is indirectly shown by this study to have carry-over effects, either beneficial or detrimental, on subsequent developmental potential.

    ACKNOWLEDGMENTS

    The authors would like to thank P. Lazik for her technical assistance.

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