Blastocoel Cavity Formation by Preimplantation Rat Embryos in the Presence of Cyanide and Other Inhibitors of Oxidative Phosphorylation D
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Blastocoel cavity formation by preimplantation rat embryos in the presence of cyanide and other inhibitors of oxidative phosphorylation D. R. Brison and H. J. Leese Department of Biology, University of York, Heslington, York YOl 5DD, UK The role of oxidative phosphorylation in blastocoel development in rats was determined by culturing morula stage embryos for 24 h in the presence of three inhibitors of ATP generation: cyanide, antimycin-A and 2,4-dinitrophenol (DNP). Rat morulae could form blastocysts in concentrations of cyanide that are toxic to the embryos of other mammals. Similar results were obtained with antimycin-A and DNP, although DNP reduced the number of blastocysts that formed. A non-invasive ultramicrofluorometric assay was used on single blastocysts and the glycolytic pathway was shown to be stimulated in the presence of these inhibitors. These results suggest that, uniquely among preimplantation embryos studied, the developing rat blastocyst does not have an absolute requirement for oxidative phosphorylation but may be able to compensate by increasing the amount of glucose consumed and metabolized by glycolysis. This pattern of metabolism may be related to the changing maternal environment during development, with blastocoel cavity formation and implantation taking place in increasingly anoxic conditions. Introduction the TCA cycle (Dufrasnes et ah, 1993). The question therefore arises as to what extent rat embryos depend on oxidative The preimplantation period of development in the rat lasts for phosphorylation to generate the ATP required for develop¬ 5 days after fertilization; during this time the embryo is a ment. We have addressed this question by examining the effect free-living structure with a high requirement for energy, of inhibitors of oxidative phosphorylation on blastocyst particularly during the critical differentiative event of blasto¬ formation and metabolism. coel cavity formation (Leese, 1991). The embryos of the most widely studied species, the mouse, are thought to generate ATP the during preimplantation period via two major meta¬ Materials and Methods bolic pathways: the oxidation of substrates via the tricarboxylic acid (TCA) cycle/oxidative phosphorylation, and the break¬ and culture down of glucose via glycolysis to lactate (Brinster, 1967a; Embryo generation In mouse Wales, 1969; Biggers and Stern, 1973; Leese, 1991). Rat embryos were generated and cultured as described by blastocysts freshly flushed from the reproductive tract, only Brison and Leese (1991, 1993). Immature (28-30 days old) 33—44% of the consumed is accounted for lactate glucose by random-bred female rats of the Wistar strain were given single production (Gardner and Leese, 1990; Gardner and Sakkas, injections of non-superovulatory doses of 5 iu pregnant mares' 1993). As the yields of ATP from oxidative phosphorylation serum gonadotrophin (PMSG: Folligon; Intervet, Cambridge, and glycolysis are, respectively, 3& and 2 molecules per UK), followed 45-50 h later by 5 iu hCG (Chorulon; Intervet) molecule of glucose, it may be concluded that in the pre¬ to synchronize the timing of ovulation. They were immediately implantation mouse embryo, as in most adult mammalian cells, placed singly with males overnight to mate. Female mice, ATP generation from oxidative phosphorylation is quantitat¬ 6-8 weeks of age, of the inbred strain CBA/Ca C5 7BL/6 ively much more significant than that from glycolysis. Mouse were superovulated with 5 iu PMSG, followed 48 h later by embryos, in common with most mammalian cells, are therefore 5 iu hCG and also mated singly with males. highly sensitive to inhibitors of oxidative phosphorylation Rat embryos were recovered at the two- to four-cell or such as cyanide (Thomson, 1967). morula stage by flushing the oviducts with H6, a Hepes- However, there is evidence that developing rat blastocysts buffered T6 medium (Wood and Whittingham, 1981), on day 2 different of may exhibit a pattern energy metabolism, as they or mid-day 4 after mating, respectively. Blastocysts were lactate and convert glucose quantitatively to (Brison Leese, recovered by flushing the uterus on day 5. Embryos were 1991), and do not oxidize significant amounts of glucose via cultured in a modification of T6 medium (Wood and 1981; Brison and *Present address and correspondence: Department of Biology, University of Whittingham, Leese, 1991) containing mmol + 1_I Pennsylvania, Philadelphia, PA 19104-6018, USA. 1.0 glucose 1 , 2.0 mmol (D L) lactate and Received 27 September 1993. 0.25 mmol pyruvate 1~ , adjusted to an osmolarity of 1994 Journals of and Fertility Ltd Downloaded from Bioscientifica.com at 09/26/2021 08:34:40PM Reproduction via free access + 4 1 258 mosmol kg- , and supplemented with g polyvinyl blastocyst by comparing their concentrations in the spent alcohol 1~ Mouse morulae were recovered from the ovi¬ microdrops to those in non-embryo containing control drops ducts on day. 3, and cultured in a modified medium M16 in the same dish. The inhibitors used did not affect the assays (Whittingham, 1971) supplemented with 4 g BSA 1 (ICN and in any case were included in the control drops for each Immunobiologicals, High Wycombe, Bucks). Embryos of both species were cultured in 20 µ drops of medium under light paraffin oil (BDH, Poole, Dorset) at 37°C in a humidified atmosphere of 5% C02 in air. Expression of results and statistical analysis The development of morulae in culture was expressed as the Oxidative phosphorylation inhibitors percentage reaching each developmental stage. Differences between treatments were tested for statistical significance by All three oxidative inhibitors were phosphorylation pre¬ analysis. Differences in blastocyst cell numbers and glucose on the of the as 10 stock solutions in pared day experiment were tested for by T6 consumption/lactate production significance T6, and serially diluted in 20 µ culture drops of pre- Student's t test. equilibrated overnight. Cyanide was dissolved in T6 at a concentration of 10 mmol l~ . Antimycin-A was initially dissolved in ethanol, diluted with medium T6 to a concen¬ 20 1 , and to a clear solution. Results tration of mg ~ sonicated yield The maximum final concentration of ethanol was 0.02%. 2,4-Dinitrophenol (DNP) was dissolved in T6 at a concen¬ Embryo culture ~ experiments tration of 10 mmol 1 , and sonicated. None of the inhibitor The role of oxidative in blastocoel devel¬ concentrations used altered the osmolarity or pH of the phosphorylation in rats was determined from the medium significantly. Control drops of T6 were treated by opment by culturing embryos morula in various concentrations of three known inhibi¬ adding an equivalent amount of T6 medium, containing stage ethanol if appropriate. tors of oxidative phosphorylation: cyanide, antimycin-A and (Slater, 1963, 1967). Each culture Fresh stocks of inhibitors were used for each replicate 2,4-dinitrophenol experiment was carried out between three and five times, and similar of each experiment. Two sources of cyanide, NaCN and results were obtained for each which were then KCN, were used, with identical results, and two sources of replicate, Antimycin-A (Sigma A-2006 and A-8674), also with identical pooled. Cyanide had no significant effect on the proportions of results. morulae reaching each developmental stage (Table 1 and Fig. 1) and the blastocysts that formed were of normal morphology. Cyanide (1.0 mmol 1) caused a slight reduction in the number Morphological assessment of cells, suggesting a minor effect on cell division (Table 1). Antimycin-A had no significant effect on development, and Morulae were cultured for 24 h from 4 to 5 after day day no effect on final blastocyst cell number, in a concentration fertilization in the absence or of the inhibitors. At the presence range that inhibits respiration in rabbit blastocysts (Benos and end of this were scored as period they compacting morulae, Balaban, 1983) and cultured cells (Gauthier et ah, 1990). No blastocysts (if the blastocoel cavity was less than fully early effect on development was seen in concentrations up to formed), and blastocysts (if they were fully expanded). The 0.1 mmol DNP 1_1, which effectively uncouple electron total numbers of cells of were determined the blastocysts using transport from ATP production in rabbit blastocysts (Benos fluorochrome bis-benzamide to label polynucleotide-specific and Balaban, 1980) and other cells (Slater, 1963). However, in cell nuclei and were (Handyside Hunter, 1984). Embryos mmol 1~ was 38% 1.0 DNP , development reduced, although classed as morphologically normal if, in the case of blastocysts, of morulae still formed blastocysts, which contained fewer they remained fully expanded, or in the case of morula and cells than did control blastocysts (Table 1). In addition, earlier if the was clear and cell cleavage stages, cytoplasm freshly flushed day 5 blastocysts were cultured overnight membranes intact. were scored as if the Embryos degenerate in the maximum concentrations of all three inhibitors, or if blastomeres showed cell blastocysts collapsed, disrupted and remained fully expanded for at least 24 h (data not membranes and dark, condensed cytoplasm. shown). In view of the above results, additional controls were to ensure that the inhibitors could block oxidative Non-invasive assays performed phosphorylation in preimplantation embryos. Mouse embryos Glucose consumption and lactate production by the same cultured from the morula stage in 1.0 mmol KCN I-1, taken embryo were measured non-invasively using the ultramicro- from the same source and prepared in the same manner as for fluorometric technique described previously (Leese and Barton rat morulae, were completely degenerate after 24 h. In control 1984; Gardner and Leese, 1990; Brison and Leese, 1991). medium, 100% of mouse morulae formed normal blastocysts Day 5 blastocysts, of a similar size and degree of expansion, (data not shown). All three inhibitors were also tested for their freshly flushed from the uterus, were incubated individually for effect on earlier cleavage (two—four-cell) stage rat embryos. In 2 h in 48.1 nl drops of T6 ± KCN or DNP.