* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download PDF
Survey
Document related concepts
SNARE (protein) wikipedia , lookup
Extracellular matrix wikipedia , lookup
Cell growth wikipedia , lookup
Cellular differentiation wikipedia , lookup
Signal transduction wikipedia , lookup
Cell encapsulation wikipedia , lookup
Cell culture wikipedia , lookup
Ethanol-induced non-lamellar phases in phospholipids wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Cytokinesis wikipedia , lookup
Cell membrane wikipedia , lookup
Transcript
/ . Emhryol. exp. Morph. Vol. 60, pp. 303-319, 1980 Printed in Great Britain © Company of Biologists Limited 1980 303 Membrane sterols and the development of the preimplantation mouse embryo HESTER P.M. PRATT,1 JO KEITH AND JYOTSNA CHAKRABORTY2 SUMMARY The role of membrane sterols in the compaction and subsequent development of the preimplantation mouse embryo was studied by incubating embryos in 7-ketocholesterol and other oxygenated sterols. These sterols have been shown to inhibit sterol synthesis and deplete membranes of cholesterol in a variety of other cell types. Compaction and subsequent blastocyst formation were normal when embryos were incubated in physiological sterols but were inhibited by oxygenated sterols to a degree which depended upon the concentration of sterol, duration of incubation and developmental age of the embryos. Precompaction 8-cell embryos were most susceptible to the action of these sterols and failed to compact (as assessed by cell flattening and increased intercellular adhesion) but continued to divide, whilst later stage embryos developed normally. 7-ketocholesterol had a specific effect on the ultrastructure of the smooth endoplasmic reticulum of treated embryos. The developmental and ultrastructural effects induced by the oxygenated sterols could be reversed or prevented by the use of products of the blocked reaction (i.e. mevalonate, desmosterol or cholesterol). These results substantiate the evidence that preimplantation mammalian embryos are capable of synthesizing membrane sterols from the 8-cell stage onwards and emphasize the importance of the sterol composition of membranes for normal cytokinesis and compaction of the mouse embryo. INTRODUCTION The presence of sterols in the cell membranes of all eukaryotes and some prokaryotes (Nes, 1974) implies a fundamental role in membrane physiology for these molecules. Studies of artificial bilayers of defined lipid composition have demonstrated that sterols interact with phospholipids to counteract phase transition effects and thereby create an intermediate fluidity state which stabilizes the bilayer and regulates its permeability (Lee, 1975). The variation in sterol content amongst different intracellular membranes, the enrichment of cholesterol in the normal plasma membrane (Bretscher, 1973) and the well documented examples of pathological conditions associated with cells of abnormal sterol composition (Papahadjopoulos, 1974; Cooper, 1977) all suggest 1 Author's address: Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2 3DY, U.K. 8 Author's address: Department of Physiology, Medical College of Ohio, C.S. No. 10008, Toledo, Ohio 43699, U.S.A. 304 H. P. M. PRATT, J. KEITH, J. CHAKRABORTY that the properties of membranes can be influenced by their component sterols. Experimental modifications of the cholesterol content of membranes in intact cells have confirmed that cholesterol can regulate the fluidity and permeability of the lipid bilayer in natural as well as artificial membranes (Papahadjopoulos, 1974; Demel & de Kruyff, 1976). Sterol modification experiments have also demonstrated that the activity of membrane transport enzymes can be influenced and may be regulated by the cholesterol composition of their immediate lipid microenvironment (Kimelberg, 1977; Warren, Houslay, Metcalfe & Birdsall, 1975). Other functions of cell membranes, namely the ability to undergo cytokinesis (Pratt, Fitzgerald & Saxon, 1977) and natural or experimentally induced cell fusion (Hope, Bruckdorfer, Hart & Lucy, 1977; Horwitz, Wight, Ludwig & Cornell, 1978) as well as the capacity to bind and transmit proliferative and differentiative stimuli (Alderson & Green, 1975; Pratt et al. 1977) are all radically and specifically altered by modifications in sterol composition. With these observations in mind, it is clear that many of the crucial reorganizations of membranes, particularly plasma membranes, that occur during the differentiation and proliferation of the preimplantation mouse embryo could be influenced by, or possibly even mediated by, changes in their sterol composition. For example, the redistribution of plasma membrane components (both pre-existing and newly synthesized) and the membrane fusion that accompanies each cleavage or mitotic division must require a degree of fluidity in the lipid matrix. Furthermore, the sequence of cell flattening and intercellular adhesion that occurs during compaction of the morula and is associated with changes in membrane transport (Biggers, Borland & Powers, 1977) together with the development of intercellular junctions and a polarized epithelial configuration (Ducibella, Ukena, Karnovsky & Anderson, 1977; Johnson, Pratt & Handy side, 1980) could also be critically dependent on the sterol composition of embryo plasma membranes. In order to study the possible influence of membrane sterol composition on these processes, we have investigated the effects of incubating mouse morulae with synthetic sterols which have been shown in a variety of other systems to inhibit the rate-limiting enzyme in sterol synthesis, HMG Co A reductase (hydroxymethylglutaryl Co A reductase), and thus to deplete cellular membranes of cholesterol (Brown & Golstein, 1974; reviewed, Kandutsch, Chen & Heiniger, 1978). The morphological and developmental consequences of this treatment reinforce the biosynthetic evidence for sterol synthesis in preimplantation embryos (Pratt, 1978) and suggest a role for sterols in the reorganization of the embryo that occurs during compaction. Membrane sterols andpreimplantation mouse embryos 305 MATERIALS AND METHODS Animals CFLP female mice were obtained from Anglia Labs., Carworth, Essex, U.K., superovulated using 5 i.u. PMS (pregnant mare's serum) (Folligon, lntervet) and 5 i.u. hCG (human Chorionic Gonadotrophin) (Chorulon, lntervet) 44-48 h later and were then mated with CFLP males. Detection of a vaginal plug the following morning was taken to indicate successful mating and the embryos were staged chronologically by defining the time of the hCG injection as 0 h. Mice were killed by cervical dislocation and the embryos recovered by flushing the oviducts and uteri with culture media. When zona-free embryos were used, the zona was removed with acid Tyrode's solution + 0-4 % polyvinylpyrrolidone pH 2-5. Embryo culture The conventional embryological technique of incubating embryos in microdrops of culture medium covered with paraffin oil could not be used without modification since the solubility of the sterols in the oil would lead to uncertainty about the effective concentration of sterol in the culture medium. Embryos were therefore incubated in groups of five to ten embryos in the wells (0-1 ml medium per well) of the microtitre plates (Gibco Bio-cult Ltd, Scotland) which are conventionally used for immunological assays. The plates were covered with adhesive plate covers (Gibco Bio-cult Ltd, Scotland) which permit gas exchange, and incubated at 37 °C in 95 % air, 5 % CO2. Embryos were scored morphologically either by direct observation in the wells of the microtitre plates or else by removing them to Petri dishes and examining them under an inverted phase microscope. Qualitatively similar results were obtained by using the conventional micro-drop technique and pre-equilibrating the paraffin oil with the sterol-containing media though in this case the minimal effective dose was substantially higher due to partitioning of the sterol into the oil. In some experiments 2 % foetal calf serum was added to the medium. Chromatographically pure sterols were obtained from Steraloids Ltd, Croyden, U.K. Stock solutions were made up in benzene at 50 mg/ml and stored under N 2 at 4 °C for a maximum of 2 weeks before use. Samples of these stock solutions were assessed periodically for their chromatographic purity and the absence of autooxidation products by thin layer chromatography on silica gel plates using the solvent petroleum ether:ether:acetic acid (40:60: 0-1 vol./vol.) (Bowyer & Davies, 1976). Immediately before use, these stock solutions were diluted into 5 % bovine serum albumin (BSA) 0-14MNaCl pH 7-0, vortexed repeatedly and cleared by centrifugation as described by Kandutsch & Chen (1973) to give a sterol solution of 500 ju,g/ml. This solution was then diluted into culture medium containing 4 mg/ml BSA (Biggers, Whitten & Whittingham, 1971) to give the appropriate sterol concentration for embryo 306 H. P. M. PRATT, J. KEITH, J. CHAKRABORTY culture. Benzene from the original sterol stock solution had no effect on normal embryo development at these dilutions. The type of BSA (and associated fatty acids) used for the original suspension of sterols was not an important factor in the developmental inhibition observed since sterols diluted into delipidated BSA had the same effect. However, for the experiments described here one single batch of BSA was used throughout. Autooxidation of sterols during long incubation periods was a potential problem and a-tocopherol was included in some culture media as suggested by Kandutsch & Chen (1977). However since this did not result in enhanced embryonic growth or development, autooxidation was not considered to be a major contribution to the results obtained and a-tocopherol was not included in routine cultures. In experiments involving the addition of mevalonic acid a stock solution of 100 mM mevalonic acid was made up in culture medium and then diluted appropriately for embryo culture. Cell counting Assessments of cell numbers were made by counting cell nuclei prepared using the technique of Tarkowski (1966). Electron microscopy Embryos were collected in culture medium number 16 containing 4mg/ml BSA and fixed for 1 h at room temperature in 2-5 % glutaraldehyde, 0-1 Mcacodylate buffer pH 7-2, 10% sucrose. Embryos were washed in 0-1 Mcacodylate buffer, post fixed in 1 % osmium tetroxide 0-1 M cacodylate buffer, dehydrated in ethyl alcohol and embedded in Epon. Thin sections were stained with lead citrate and viewed with a Phillips EM300. RESULTS Table 1 demonstrates that precompacted 8-cell embryos compacted (as assessed by cell flattening at the light microscope level) normally and formed blastocysts when cultured continuously in natural precursors of cholesterol and in cholesterol itself at concentrations up to 100yMg/ml (the highest concentration tested). However when embryos were treated with a variety of sterol synthesis inhibitors (Brown & Goldstein, 1974) a dose-related inhibition of compaction and blastocyst formation was observed. 7-ketocholesterol and 6-ketocholestanol were the most potent inhibitors but 20-a-hydroxycholesterol and 25-hydroxycholesterol were also effective. Embryos treated with 5 /xg/ml of 7-ketocholesterol continued to divide through one to two cycles of cell division during 48 h of culture in a simple medium containing BSA (Table 2) before eventually lysing. The rate of proliferation was significantly reduced compared to either control or cholesterol-treated embryos which did not differ Membrane sterols and preimplantation mouse embryos 307 Table 1. Growth of 8-cell embryos (60-64 h post hCG) in sterols and sterol synthesis inhibitors Desmosterol Lanosterol 7-dehydrocholesterol Squalene Mevalonic acid (5 m.M) (10 mM) Cholesterol synthesis inhibitors 7-ketocholesterol 6-ketocholestanol 25-hydroxycholesterol 20-a-hydroxycholesterol — ooo Control Cholesterol precursors Cholesterol 10 10 10 10 — 1 5 10 20 50 1 5 10 50 5 10 25 5 10 25 % compacting;* % forming blastocysts No. of embryos 98 82 (49) 70 96 89 100 93 80 100 100 100 65 81 89 80 60 60 100 100 100 (57) (81) (19) (10) (15) (15) (15) (9) (9) 80 19 0 3 6 80 .12 0 0 80 6 0 0 0 80 0 0 0 (5) (69) (57) (75) (72) 19 0 0 47 0 0 13 0 0 ooo /tg/ml (5) (17) (14) (4) (12) (24) (16) (15) (15) (11) * Compaction was assessed by cell flattening using an inverted phase microscope and an embryo was considered to be compacted if it was indistinguishable from an untreated control. significantly from one another (Table 2). The viability and morphology of 7-ketocholesterol treated embryos was substantially improved by inclusion of 2 % foetal calf serum (FCS) in the medium though the rate of cell division still remained reduced compared to untreated controls. In the presence of 2 % FCS the minimum dose of 7-ketocholesterol sufficient to inhibit compaction was increased to 20-50 /*g/ml. The inclusion of 2 % FCS thus provided a means of analysing the ultrastructural consequences of long term (24-48 h) sub-lethal inhibition. Compacting embryos treated with 20-50/*g/ml 7-ketocholesterol (in the absence of FCS) were demonstrably less compact than controls within 6 h of 308 H. P. M. PRATT, J. KEITH, J. CHAKRABORTY Table 2. Cell numbers of control and sterol-treated 8-cell embryos after 24-48 h in culture* Sterol None Cholesterol 7-ketocholesterol Concentration Otg/ml) Hrs in culture Mean cell numbert No. of embryos — — 50 50 5 5 24 48 24 48 24 48 14-2 ± 2-8 42-6 ±160 2 19-7+ 60 49-6±15-52 10 3± 20 23-4 ± 4-81 (39) (27) (14) (10) (17) (10) * Embryos placed in culture medium+4 mg/ml BSA at 60-64 h post hCG. Cell number 8±1. f Mean ± standard deviation of the mean (4-10 embryos) at end of incubation period. 12 P < 0001 Student's t test. 22 - P > 005 Student's t test. treatment. Embryos incubated for longer periods in 20-50 /xg/ml + 2 % FCS remained as a cluster of poorly compacted cells with their plasma membranes deformed and aligned in regions of incipient intercellular contact but without the generalized intercellular adhesion that occurs during normal compaction (Figs. 1, 2). At the electron microscope level, embryos cultured for 2 4 h i n 20-50 /tg/ml 7-ketocholesterol + 2 % FCS formed fewer cell contacts and junctional complexes than controls and had the general appearance of poorly compacted morulae with occasional, small blastocoelic cavities (Fig. 3). Numerous whorls were present in the cells of these embryos (Figs. 3, 6c). The control and cholesterol-treated embryos on the other hand developed many intercellular contacts with normal junctional complexes and compacted and formed normal blastocysts (Fig. 4). Microvilli were localized to the basal and apical regions of cells of 7-ketocholesterol-treated embryos and there was no apparent effect of the inhibitor on the membranes of microvilli, mitochondria, Golgi and membranous lamellae (Fig. 5). However, smooth endoplasmic reticulum (ER) was always disrupted by the inhibitor although rough ER remained morphologically undamaged even in regions where the two membrane Fig. 1. (a) Phase-contrast micrograph of mouse embryos recovered at the 8-cell stage (60-64 h post hCG) and cultured for 12 h in medium 16 + 2% FCS containing 20 /ig/ml cholesterol. The embryos are fully compacted as judged by cell flattening, x 250. (b) As (a) but the medium contained 20 /tg/ml 7-ketocholesterol. Note that the degree of cell flattening is substantially reduced compared with (a), x 250. Fig. 2. (a) Phase-contrast micrograph of embryos shown in Fig. (1 a) after 36 h of culture in 20/ig/ml cholesterol. The embryos have formed normal blastocysts. x 250. (b) As (a) but after 36 h culture in 20 /ig/ml 7-ketocholesterol. Note the absence of fluid accumulation and persistent reduction in cell flattening, x 650. Membrane sterols and preimplantation mouse embryos £7i 1a 309 310 H. P. M. PRATT, J. KEITH, J. CHAKRABORTY 1 ). Fig. 3. 8-cell embryo treated with 50/fg/ml of 7-ketocholesterol + 2 % FCS for 24 h. Small blastocoele with portions of extruded cells is identifiable ( *)• Two cells (Ci and Q) are dividing. Numerous membranous whorls (arrows) are clearly seen in several cells of this embryo, x 2000. systems were continuous (Fig. 6 a). Rough and smooth ER were frequently found in close contact with membranous whorls (Fig. 6 c) and membranebound dense bodies which were morphologically similar to lipid vesicles (Fig. 6b, c). Partial disruption of localized areas of nuclear membrane was also observed in 7-ketocholesterol-treated embryos. The damaging effects of the inhibitor were more marked at higher concentrations. The smooth ER and nuclear membranes of cholesterol-treated embryos were unaffected and indistinguishable from untreated controls (Fig. 4 b). Membrane whorls were absent though cytoplasmic membrane-bound vesicles apparently containing lipid were abundant (Fig. 4a). The timing of 7-ketocholesterol action was examined by culturing 8-cell Membrane sterols and preimplantation mouse embryos i f Fig. 4. (a) 8-cell embryo treated with 50/ig/ml of cholesterol + 2 % FCS for 24 h. Note the normal blastocyst morphology, x 2000. (b) Higher magnification of the portions of two cells (Q and C2) from the embryo shown in Fig. 4 (a), showing unaffected rough (arrow) and smooth (arrow heads) endoplasmic reticulum. Cellular contact areas are normal (double arrows), x 54378. 311 312 H. P. M. PRATT, J. KEITH, J. CHAKRABORTY >K Fig. 5. 8-cell embryo treated with 50 /*g/ml of 7-ketocholesterol + 2 % FCS for 24 h. Note the undamaged mitochondria (M), golgi (G), membranous lamellae (arrow head) and plasma membrane (arrows) of two cells ( Q and C2). Damaged smooth ER (double arrow heads) and membranous whorls (double arrows) are scattered in the cytoplasm, x 27000. Membrane sterols and preimplantation mouse embryos 313 precompaction embryos with 7-ketocholesterol for varying periods, removing them to control media and observing their subsequent development (Table 3). Developmental inhibition became irreversible within 6-12 h of exposure to 5 /xg/ml 7-ketocholesterol, the period during which untreated embryos became fully compacted. Higher concentrations had irreversible effects within the first 6 h of culture (Table 3). Ultrastructural damage to the SER was detectable within 4 h of treatment (Fig. 6) but could be overcome if cholesterol or serum cholesterol (as FCS) was added to the culture within this period. The influence of the age of the embryo on its susceptibility to treatment with 7-ketocholesterol was then assessed by taking embryos at 8-cell precompaction stage (60-65 h post hCG), as well as compacted morulae (74-76 h post hCG) and fluid-accumulating morulae (85-88 h post hCG) and culturing them continuously in 5 /<g/ml 7-ketocholesterol. The precompaction 8-cell embryo is more sensitive to the inhibitor than either of the later stage embryos (Table 4) which proliferated at the same rate in the presence or absence of the inhibitor. The average cell numbers of morulae cultured from 88 h post hCG for 48 h in the presence of absence of 7-ketocholesterol were 39-8 ±4-9 and 35-5 ±6-8 for control and treated embryos respectively. An attempt was made to investigate the possible mode of action of 7-ketocholesterol by incubating inhibited precompaction 8-cell embryos with the immediate (mevalonic acid) or end (demosterol and cholesterol) products of the putative blocked reaction (i.e. the conversion of HMG Co A to mevalonic acid). The ability of embryos to compact (assessed by cell flattening) and form blastocysts was at least partially recovered in media containing any of these compounds in addition to 7-ketocholesterol (Table 5), and their rate of cellular proliferation increased though it did not reach control levels. DISCUSSION The results presented in this paper demonstrate that incubation of preimplantation mouse embryos with sterols can inhibit compaction and blastocyst formation to varying degrees depending upon the nature and dose of the sterol (Table 1), the length of exposure (Table 3) and the age of the embryo (Table 4). 7-ketocholesterol and other oxygenated sterols (e.g. 20-a-hydroxycholesterol, 25-hydroxycholesterol and 6-ketocholestanol) have been used to block sterol synthesis in a variety of cell types by their inhibition of the rate limiting enzyme, hydroxymethylglutaryl Coenzyme A (HMG Co A) reductase (Brown & Goldstein, 1974; Kandutsch & Chen, 1977; Kandutsch et al 1978). Preimplantation mouse embryos treated with these agents undergo specific and irreversible changes in ultrastructure within 4 h of treatment (Fig. 6) and exhibit subsequent reductions in cell proliferation., intercellular apposition, junction formation and fluid accumulation (Figs. Ib, 2b, 3). These ultrastructural and developmental consequences of 7-ketocholesterol treatment reinforce 314 H. P. M. PRATT, J. KEITH, J. CHAKRABORTY Membrane sterols and preimplantation mouse embryos 315 Table 3. Effect of exposure time on development inhibition by 7-ketocholesterol Hrs in inhibitors* 1-6 12 18-24 Concentration (/ig/ml) No. of % compacting % forming blastocysts embryos 97 1 73 10 7 182 5 10 25 50 5 5 1 4 90 1 13 10 0 0 42 2 (76) (15) (10) (30) (ID (28) * 8-cell embryos (60-64 h post hCG) were cultured in media (+4 mg/ml BSA) containing the indicated concentrations of 7-ketocholesterol for the stated duration, then washed and replaced in control medium for the remainder of the 48 h incubation period. ^ P < 0001 x2 test. 2fi P > 005 x2 test. Table 4. Effect of developmental age on susceptibility to inhibition by 7-ketocholesterol (5 ftg/ml) Hrs post hCG* 7-ketocholesterol 60-65 74-76 85-88 Control 60-65 Cellnumberf % compactingJ % forming blastocystsJ No. of embryos 7 ±0-7 (7) 18 ±3-2 (7) 28 ±5-9 (4) 191 662 792 51 51 2 632 (84) (53) (24) 7-5 ±1 (4) 962 892 (45) * Developmental age of the embryos when placed in control media (containing 4 mg/ml BSA) or media containing 5 /*g/ml 7-ketocholesterol. t Mean number of cells in embryos at start of incubation ± standard deviation. Number of embryos analysed in parentheses. X % of embryos which compacted (or remained compacted) and formed blastocysts during a 48 h incubation period. **P < 0001 x2 test. 2 2 - P > 005 x2 test. Fig. 6 (a) 8-cell embryo treated with 20/*g/ml of 7-ketocholesterol + 2 % FCS for 24 h. Continuity between the damaged smooth ER (arrows) and intact rough ER (arrow heads) is clearly visible, x 31920. (b, c) 8-cell embryo treated with 50/*g/ml of 7-ketocholesterol+ 4 mg/ml BSA for 4 h. Selective damage in the smooth ER (arrows Fig. 6b) and membranous whorls (arrows Fig. 6 c) is clearly seen. Lipid-like dense bodies surrounded by membrane remained undamaged (arrow heads) 6b x 36700, 6c x 27100. 316 H. P. M. PRATT, J. KEITH, J. CHAKRABORTY Table 5. Development of8-cell embryos (60-64 h post hCG) in 7-ketocholesterol and mevalonic acid or cholesterol* % forming % compacting blastocysts No. of embryos Cell numberf Mevalonic acid (10 ITLM) Cholesterol (50 /*g/ml) 7-ketocholesterol (5 /tg/ml) 7-ketocholesterol (5 /tg/ml) + Mevalonic acid (10 ITLM) + Cholesterol (50 /tg/ml) + Desmosterol (50/^g/ml) 1001 96 1 192 1001 81 1 62 (10) (47) (69) 53-7 ±13-3 (8) 580±13-5 (6) 21 -5 ± 6-4 (6) 733 23 683 703 (30) (19) (10) 270±3-8 (5) 3 5 0 ± 7 1 (18) 32-4 ±7-2 (5) 743 903 * Embryos were cultured for 60 h in media+ 4 mg/ml BSA. t Blastocysts or the most advanced embryos were counted at the end of the culture period. Mean ± S.D. Number of embryos counted in parentheses. X *P < 0001 x2 test. 2 3 - P < 005 x2 test. l »P > 005 x2 test. the biosynthetic evidence that mouse embryos are capable of synthesizing membrane sterols from the 8-cell stage onwards (Huff & Eik-Ness, 1966; Pratt, 1978). First, only those sterols known to inhibit HMG Co A reductase (Brown & Goldstein, 1974) induced abnormal development (Table 1), and all of these exerted their effects within short periods of time (Table 3) which are compatible with the half life (3^ hours) of the enzyme (Brown & Goldstein, 1974). Secondly the developmental abnormalities could be overcome by simultaneous exposure of the embryos to both the inhibitor and natural precursors of cholesterol or cholesterol itself (Table 5). This observation probably accounts for the improved viability and morphology of embryos treated with 7-ketocholesterol in the presence of FCS and its associated serum cholesterol, a fact which was exploited when analyzing the ultrastructure of embryos exposed to long term (24-28 h) sub-lethal treatment with 7-ketocholesterol. Finally the fact that the ultrastructural disruptions induced by 7-ketocholesterol were restricted to smooth endoplasmic reticulum (SER) (Figs. 5, 6) argues for a primary action on these membranes which are known to be the sites of sterol synthesis (Chesterton, 1968). We consider it unlikely that this SER is derived from rough ER as a secondary consequence of 7-ketocholesterol treatment since rough ER remains intact and two parameters of membrane-bound ribosome activity are unaffected, namely total protein synthesis and the assembly of membrane glycoproteins as assessed by the binding of concanavalin A (Pratt, unpublished). Furthermore, the membrane whorls associated with these lesions (Fig. 6 c) have been observed in other cell types where sterol synthesis has been inhibited (Dietert & Scallen, 1969) and are possible abnormal intermediates in membrane biosynthesis. The Membrane sterols and preimplantation mouse embryos 317 proliferation of SER observed here and in other systems has been suggested to be compensatory growth to replace damaged membranes (Yates, Arai & Rappoport, 1967). HMG Co A reductase activity (Pratt, 1978) of inhibited embryos is now being analyzed to provide a direct demonstration of the action of 7-ketocholesterol on sterol synthesis. The greater sensitivity of the precompaction 8-cell embryo to 7-ketocholesterol as compared with the compacting or fully compacted embryo could be explained by a reduced permeability to sterols at these later stages. However, this is not borne out by experiments using labelled cholesterol (Tarver & Pratt, unpublished observations), or by examination of the localization of sterols in embryos before or during compaction since sterols appear to be taken up from the medium to similar extents irrespective of embryonic age. On the other hand, synthesis of sterols is not detectable until the 8- to 16-cell stage (Pratt, 1978 and unpublished observations) and the increased susceptibility of the precompaction 8-cell embryos to 7-ketocholesterol may therefore be due to the immaturity of the sterol-synthesizing machinery and its inability to compensate for damage. In cells exposed to 7-ketocholesterol a secondary consequence of blocked sterol synthesis is a depletion in the cholesterol content of cellular membranes, particularly the plasma membrane (Kandutsch & Chen, 1977). Some properties of 7-ketocholesterol-treated embryos could be attributed to cholesteroldepleted plasma membranes. For example, intercellular adhesion and fluid accumulation are reduced or absent, cell proliferation declines and methionine uptake and N a + / K + ATP ase activity (assessed by ouabain sensitivity) are reduced (Pratt, unpublished). Similar phenomena have been observed in cells with cholesterol-depleted membranes (Papahadjopoulos, 1974; Kimelberg, 1977; Horwitz et al. 1978). However 7-ketocholesterol is not having an indiscriminate effect on all cellular processes since total protein synthesis, binding of concanavalin A, presence of the glycolytic pathway and phospholipid synthesis (Pratt, 1980) are similar in sterol-treated and normal embryos. Compaction of the 8-cell embryo involves the coordinated reorganisation of the cytoskeleton which is vital for the subsequent differentiation of the embryo (Johnson et al. 1980). Important features of the compaction process are the deformation of the nearly spherical cleavage stage blastomeres to form an epithelial-type tissue and the ability of the component cells to spread across their neighbours and adhere to them (Ducibella et al. 1977). This process is known to be Ca 2 + dependent, requires the participation of intact microfilaments (Ducibella & Anderson, 1975) and can be inhibited by antibodies directed against cell surface determinants (Kemler, Babinet, Eisen & Jacob, 1977; Johnson et al. 1979). From the experiments described in this paper it seems that sterol-mediated properties of the membrane may be additional important features to be considered. These properties could include intercellular adhesion (possibly influenced by phase separation effects (Hope et al. 1977), activity of membrane-bound enzymes (Kimelberg, 1977) and cell motility 21 EMB 60 318 H. P. M. PRATT, J. KEITH, J. CHAKRABORTY (mediated through interactions between sterols and proteins associated with the cytoskeleton (discussed, Pratt, 1978)). In conclusion the results described here are consistent with evidence that the preimplantation mouse embryo is capable of synthesizing its own membrane sterols (Pratt, 1978) and emphasize the role that sterol synthesis plays in the normal metabolism of the embryo as well as its probable importance in facilitating the changes in surface properties that occur during development. Future studies will be aimed at analyzing the sterol composition of 7-ketocholesterol-treated embryos and investigating any consequent modifications to membrane properties by studying lateral mobility of membrane lipids and proteins (Johnson & Edidin, 1978) and the activities of membrane transport sites (Holmberg & Johnson, 1979). We wish to thank Mr Dave Tarver and Ms Gin Flach for assistance with these experiments and Dr David Bowyer and Dr Martin Johnson for helpful discussions. This work was supported by grants from the Ford Foundation and the Medical Research Council to H.P. M.P. J.C. was in receipt of Grant No: BRSG-507-RR-05700-09. REFERENCES J. C. E. & GREEN, C. (1975). Enrichment of lymphocytes with cholesterol and its effect on lymphocyte activation. FEBS Letters, 52, 208-211. BIGGERS, J. D., BORLAND, R. M. & POWERS, R. D. (1977). Transport mechanism in the preimplantation mammalian embryo. In The Freezing of Mammalian Embryos. Ciba Foundation Symposium 52 (New Series), pp. 129-146. Amsterdam, New York: Elsevier, North Holland. BIGGERS, J. D., WHITTEN, W. K. & WHITTINGHAM, D. G. (1971). The culture of mouse embryos in vitro. In Methods in Mammalian Embryology (ed. J. C. Daniel), pp. 86-116. Table 6-9. San Francisco: W. H. Freeman. BOWYER, D. E. & DAVIES, P. F. (1976). Effect of EPL on the metabolism of lipids in the arterial wall. In Phosphatidylcholine. Biochemical and Clinical Aspects of Essential Fhospholipids (ed. H. Peeters), pp. 160-186. Berlin, Heidelberg: Springer-Verlag. BRETSCHER, M. S. (1973). Membrane structure, some general principles. Science 181, 622-629. BROWN, M. S. & GOLDSTEIN, J. L. (1974). Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol. /. biol. Chem. 249, 7306-7314. CHESTERTON, C. J. (1968). Distribution of cholesterol precursors and other lipids among rat liver intracellular structures. /. biol. Chem. 243, 1147-1151. COOPER, R. A. (1977). Abnormalities of cell membrane fluidity in the pathogenesis of disease. New Engl. J. Med. 297, 371-377. DEMEL, R. A. & DE KRUYFF, B. (1976). The function of sterols in membranes. Biochim. Biophys. Ada. 457, 109-132. DIETERT. S. E. & SCALLEN, T. J. (1969). An ultrastructural and biochemical study of 3 inhibitors of cholesterol biosynthesis upon murine adrenal gland and testis. /. Cell Biol. 40, 44-60. DUCIBELLA, T. & ANDERSON, E. (1975). Cell shape and membrane changes in the 8-cell mouse embryo. Prerequisites for morphogenesis of the blastocyst. Devi Biol. 47, 45-48. DUCIBELLA, T., UKENA, T., KARNOVSKY, M. & ANDERSON, E. (1977). Changes in cell shape and cortical cytoplasmic organisation during embryogenesis of the preimplantation mouse embryo. /. Cell Biol. 74, 153-167. HOLMBERG, S. R. M. & JOHNSON, M. H. (1979). Amino acid transport in the unfertilised and fertilised mouse egg. /. Reprod. Fert. 56, 223-231. ALDERSON, Membrane sterols andpreimplantation mouse embryos 319 M. J., BRUCKDORFER, D. R., HART, C. A. & LUCY, J. A. (1977). Membrane cholesterol and cell fusion of hen and guinea-pig erythrocytes. Biochem. J. 166, 255-263. HORWITZ, A. F., WIGHT, A., LUDWIG, P. & CORNELL, R. (1978). Interrelated lipid alterations and their influence on the proliferation and fusion of cultured myogenic cells. /. Cell Biol. 77, 334-356. HUFF. R.. L. & EIK-NESS, K. B. (1966). Metabolism in vitro of acetate and certain steroids by six-day-old rabbit blastocysts. /. Reprod. Fert. 11, 57-63. JOHNSON, M. H., CHAKRABORTY, J., HANDYSIDE, A. H., WILLISON, K. & STERN, P. (1979). The effect of prolonged decompaction on the development of the preimplantation mouse embryo. J. Embryo!, exp. Morph. 54, 241-261. JOHNSON, M. H. & EDIDIN, M. (1978). Lateral diffusion in plasma membrane of mouse egg is restricted after fertilisation. Nature 272, 448-450. JOHNSON, M. H., PRATT, H. P. M. & HANDYSIDE, A. H. (1980). The generation and recognition of positional information in the preimplantation mouse embryo. In Cellular and Molecular Aspects of Implantation, (ed. S. R. Glasser and D. W. Bullock). Plenum Press. (In press.) KANDUTSCH, A. A. & CHEN, H. W. (1973). Inhibition of sterol synthesis in cultured mouse cells by 7-a-hydroxycholesterol, 7/tf-hydroxycholesterol and 7-ketocholesterol. /. biol. Cliem. 248; 8408-8417. KANDUTSCH, A. A. & CHEN, H. W. (1977). Consequences of blocked sterol synthesis in cultured cells. /. biol. Client. 252, 409-415. KANDUTSCH, A. A., CHEN, H. W. & HEINIGER, H. J. (1978). Biological activity of some oxygenated sterols. Science. 201, 498-501. KEMLER, R., BABrNET, C, EISEN. H. & JACOB, F. (1977). Surface antigen in early differentiation. Proc. natn. Acad. Sci., U.S.A. 74, 4449-4452. KIMELBERG, H. K. (1977). The influence of membrane fluidity on the activity of membrane bound enzymes. In Dynamic Aspects of Cell Surface Organization (ed. G. Poste & G. L. Nicolson), pp. 205-279. Elsevier, North Holland Biomedial Press. LEE, A. G. (1975). Functional properties of biological membranes, A physical chemical approach. Prog. Biophys. Mol. Biol. 29, 3-56. NES, W. R. (1974). Role of sterols in membranes. Lipids 9, 596-612. PAPAHADJOPOULOS, D. (1974). Cholesterol and cell membrane function. A hypothesis concerning the etiology of atherosclerosis. /. theor. Biol. 43, 329-337. PRATT, H. P., FITZGERALD, P. A. & SAXON, A. (1977). Synthesis of sterol and phospholipid induced by the interaction of phytohemagglutinin and other mitogens with human lymphocytes and their relation to blastogenesis and DNA synthesis. Cell Immunol. 32, 160-170. PRATT, H. P. (1978). Lipids and transitions in embryos. In Development in Mammals, vol. 3 (ed. M. H. Johnson), pp. 83-129. Elsevier, North-Holland. PRATT, H. P. (1980). Phospholipid synthesis in the preimplantation mouse embryo. /. Reprod. Fert. 58, 237-248. TARKOWSKI, A. K. (1966). An air drying method for chromosome preparations from mouse eggs. Cytogenetics. 5, 394-400. WARREN, G. B., HOUSLAY, M. D., METCALFE, J. C. & BIRDSALL, N. J. M. (1975). Cholesterol is excluded from the phospholipid annulus surrounding an active transport protein. Nature 255, 684-687. YATES, R. D., ARAI, K. & RAPPOPORT, D. A. (1967). Fine structure and chemical composition of opaque cytoplasmic bodies of triparanol treated Syrian hamsters. Expl Cell Res. 47, 459-478. HOPE, {Received 26 February 1980, revised 20 June 1980)