Lipid interactions with in vitro development of mammalian zygotes

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<ul><li><p>Gamete Research 21:243-254 (1988) </p><p>Lipid Interactions With In Vitro Development of Mammalian Zygotes R.A. Waterman and R.J. Wall </p><p>Reproduction Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland </p><p>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. </p><p>Key words: rabbit, mouse, cultured zygote, lipid, fatty acid </p><p>INTRODUCTION </p><p>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 </p><p>Received January 22, 1988; accepted June 17, 1988. </p><p>Address reprint requests to Dr. R.A. Waterman, Reproduction Laboratory, Beltsville Agricultural Research Center, USDA Agricultural Research Service, Beltsville, MD 20705. </p><p>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. </p><p>0 1988 Alan R. Liss, Inc. </p></li><li><p>244 Waterman and Wall </p><p>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. </p><p>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. </p><p>MATERIALS AND METHODS Rabbit Zygotes </p><p>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. </p><p>Rabbit Zygote Cultures </p><p>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. </p><p>Fatty Acid Incorporations Into Rabbit Zygotes </p><p>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 </p></li><li><p>Lipids and In Vitro Development of Zygotes 245 </p><p>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. </p><p>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. </p><p>Lipid Microextractions Lipids were extracted from zygotes and fluids by a micromodification of the </p><p>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. </p><p>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. </p><p>Lipid Extracts and Rabbit Zygote Development Experiment 2 was conducted to determine if lipid extract presentation to culture </p><p>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. </p><p>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 </p></li><li><p>246 Waterman and Wall </p><p>TEST RABBIT EXTRACT ZYGOTES </p><p>OIFFUSION /I THROUGH OIL rn0IUIl.s COVERSLIP ZYGOTES 6 EXTRACT </p><p>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. </p><p>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. </p><p>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. </p><p>Mouse Zygote Cultures </p><p>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 </p></li><li><p>Lipids and In Vitro Development of Zygotes 247 </p><p>a c al L :SO 0 </p><p>2 40 -+ </p><p>m </p><p>Arachidonate t r a n s f e r -- ~ Oleate t rans fer </p><p>Acetate: No t r a n s f e r </p><p>1 2 3 4 5 6 Hours of incubat ion </p><p>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). </p><p>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. </p><p>Statistics </p><p>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 &lt; .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. </p><p>RESULTS Experiment 1 </p><p>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% </p></li><li><p>248 Waterman and Wall </p><p>2o01 I80 1 A,A I </p><p>2 4 6 </p><p>Hours o f culture </p><p>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...</p></li></ul>


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