MOLECULAR REPRODUCTION AND DEVELOPMENT 31:ll4-121 (1992)

Cyanide-Resistant Reduction of Nitroblue Tetrazolium and Hydrogen Peroxide Production by the Rabbit Blastocyst COLE MANES Department of Biology, University of Sun Diego, S a n Diego, California ported here is a cytochemical demonstration of cell surface oxidase activity in the trophoblast layer of the rabbit blastocyst, indicating yet another nonmitochondrial site of oxygen utilization. The quantitative contribution of nonmitochondrial oxidases to total oxygen superoxide radical. A cytochemical method initially develutilization by the blastocyst is not yet known, but their oped for the detection and localization of hydrogen peroxpresence is significant in that they are sources of oxyide production at the ultrastructural level in phagocytosing gen free radicals and hydrogen peroxide (Fridovich, leukocytes (Briggs et al.: J Cell Biol 67:566, 1975)was also 1978). The superoxide anion (02-), produced by the applied to the blastocyst. The results demonstratethat the one-electron reduction of molecular oxygen, is the parrabbit blastocyst acquires the ability to reduce NBT by a ent radical; through its dismutation to hydrogen peroxcyanide-insensitive process and to generate hydrogen peride and its further reaction with hydrogen peroxide to oxide between the fourth and fifth days postcoitum. The form hydroxyl radicals, highly reactive tissue-damagenzymatic activity responsible is apparently ar; NAD(P)Hing products are generated that must be used or dependent oxidase in the outer, microvillous plasma mempromptly scavenged (Halliwell and Gutteridge, 1989). brane of the trophoblast. Cytochemical methods for detecting and localizing oxygen radicals a t the cellular level were developed Key Words: Trophoblast, NAD(P)H oxidase, NBT primarily to study phagocytic leukocytes (Karnovsky et al., 1981). About half of the oxygen consumption in INTRODUCTION leukocytes during phagocytosis is not inhibited by cyaDefining the roles of oxygen in the metabolism of the nide and is due to the activation of a plasma membrane mammalian blastocyst is of importance not only for oxidase requiring NAD(P)H (Curnutte and Babior, understanding the full metabolic repertoire of the em- 1987). The activity of this cell surface oxidase can be bryo but also for designing optimum conditions for its readily detected by the ability of leukocytes to reduce maintenance in vitro. It has been recognized for over 30 nitroblue tetrazolium (NBT) to formazan in the presyears that on a cellular basis the blastocyst consumes ence of cyanide (Baehner and Nathan, 1967). More refar more oxygen than the cleaving embryo (Fridhand- cently, cytochemical methods for localizing sites of suler et al., 1957). When mouse and rabbit embryos un- peroxide and hydrogen peroxide production a t the dergo the transition from morulae to blastocysts, their ultrastructural level in phagocytosing leukocytes have Qo, rises about fivefold (Brinster, 1974). This high been developed (Briggs et al., 1975,1986).The findings level of oxygen uptake by blastocysts has been mea- described below are the result of applying these techsured under conditions in which glucose and pyruvate niques to the rabbit blastocyst. To the best of my knowlare the principal substrates (Fridhandler, 1961; Mills edge, this is the first cytochemical demonstration of and Brinster, 1967; Benos and Balaban, 1980). In this oxygen free radical production by the preimplanted situation, oxygen uptake is totally inhibited by cyanide mammal. (Fridhandler et al., 1957; Benos and Balaban, 19801, MATERIALS AND METHODS which suggests that the sole function of oxygen in blasEmbryo Recovery tocyst metabolism is to serve as the terminal electron acceptor for the mitochondrial electron transport sysEmbryos used in this work were recovered from New tem (ETS). Zealand white rabbits on days 4-6 postcoitum (p.c.1.On There is, however, evidence for nonmitochondrial ox- day 4 or 5 p.c. (approximately 84 and 108 h of embryo ygen utilization in the blastocyst. Filler and Lew (1981) demonstrated a mixed-function oxidase in the mouse blastocyst, which would appear to be closely related, if not identical, to the cytochrome P-450 system of the Received August 12, 1991; accepted October 4,1991. endoplasmic reticulum. Balling et al. (1985) detected a Address reprint requests to Cole Manes, Department of Biology, Unisimilar activity in the rabbit blastocyst. The work re- versity of San Diego. Alcala Park, San Diego, CA 92110.

ABSTRACT

The ability of the rabbit blastocyst

to reduce nitroblue tetrazolium (NBT) to formazan in the presence of cyanide was assayed as an indicator of extramitochondrial oxidase activity capable of generating the

0 1992 WILEY-LISS, INC.

HYDROGEN PEROXIDE FROM RABBIT BLASTOCYSTS TABLE 1. Rabbit Blastocyst Culture Medium Weight Component (g/liter) Molarity Amino acids 1.24 x 10-3 L-alanine 0.1105 L-arginine HCl 0.0358 1.70 x 10-4 Lasparagine 1.00 x 10-4 0.0150 Gaspartic acid 1.40 x 10-4 0.0186 Gcysteine 1.00 x 10-3 0.1212 Gglutamic acid 5.90 x 10-4 0.0868 2.00 x 10-3 L-glutamine 0.2930 1.93 X 1.4498 Glycine 3.40 x 10-4 L-histidine 0.0527 1.10x 10-4 L-isoleucine 0.0144 2.00 x 10-4 L-leucine 0.0262 2.30 x 10-4 Llvsine HC1 0.0419 1.00 x 10-4 L-methionine 0.0149 8.00 x 10-5 0.0132 Lphenylalanine 3.00 x 10-4 L-proline 0.0345 8.00 x 10-4 0.0840 Lserine 5.60 x 10-4 L-threonine 0.0666 1.00 x 10-5 0.0021 L-tryptophan 9.00 x 10-5 L-tyrosine 0.0163 1.80 x 10-4 0.0211 L-valine Vitamins 3.00 X 0.000007 Biotin 1.00 x 10-6 D-Ca pantothenate 0.00048 1.00 x 10-4 Choline chloride 0.0140 3.00 X lop6 0.00132 Folic acid LOO x 10-4 0.0180 i-Inositol 3.00 x 10-7 Niacinamide 0.00004 3.00 x 10-7 0.00006 Pyridoxine HC1 1.00 x 10-7 Riboflavin 0.00004 1.00 x 10-6 Thiamine HCl 0.00034 Inorganic salts 1.70 x 10-3 0.2500 CaClz-2HzO 1.00 x 10-8 0.000002 CuS04-5H20 6.00 x 10-3 KCla 0.2237 6.00 x 10-4 0.1220 MgC12-6H20 5.00 x 10-7 MnC12-4H20 0.0001 1.30 X lo-' 7.6000 NaCl 3.50 x 10-3 0.2960 NaHC03 1.00 x 10-3 0.2681 Na2HP04-7HaO 3.00 X lop6 0.00086 ZnS04-7H20 . Other components 5.50 x 10-3 1.0000 D-glucose 2.00 x 10-2 4.7640 HEPES 3.00 x 10-5 0.0041 Hypoxanthine 1.00 x 10-6 0.0002 Lipoic acid 100,000 u K penicillin 1.00 x 10-6 0.0002 Putrescine diHCl 1.00 x 10-2 1.1000 Na pyruvate 0.0050 Soybean trypsin inhibitor 0.0500 Streptomycin 1.38 x 10-3 Taurine 0.1726 aThefinal 6 mM concentration of Kf is achieved by adding 2.80 ml of 1N KOH. The pH is finally brought to 7.38-7.40 by titration with concentrated NH40H.

development, respectively), blastocysts were flushed from the uterine horns with 5-10 ml of culture medium (Table 1) t h a t had been warmed to 37°C. The blastocysts were collected in sterile watch glasses maintained a t 37°C on a slide warmer. At day 6 p.c. (approximately 132 h of embryo development), the uterine horns were opened with bent-tipped forceps and the exposed blastocysts were transferred with the same forceps to pre-

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warmed culture medium on the slide warmer. As required, the "neozona" (Denker and Gerdes, 1979) was removed with needles a t this point. Blastocysts were subjected to cytochemical assay within 1 h of being removed from the reproductive tract. Some blastocysts recovered a t 4 days p.c. were cultured 24 h in the medium described (Table 11, under a gas phase of 10%02, 5% CO,, and 85% N, at 39°C with gentle rotation throughout the culture period.

NBT Reduction Assay Blastocysts were placed in 1 ml of culture medium containing NBT a t 300 pgiml and maintained a t 37°C. They were observed at 10-min intervals under a dissecting microscope to monitor the appearance of blue formazan. Ultrastructural Cytochemistry A minor modification of the cytochemical protocol for detecting hydrogen peroxide production in human leukocytes (Briggs et al., 1975) was used with rabbit blastocysts. The blastocysts were rinsed in 0.1 M Tris-maleate buffer, pH 7.5, containing 7% sucrose and 0.0002% Triton X-100 (Robinson, 1985) (TMST buffer) a t 37"C, then placed in the same buffer containing 1 mM 3-aminotriazole (3-AT) a t 37°C for 10 min. The basic cytochemical reaction mixture was TMST buffer containing 10 mM 3-AT and 2 mM CeCl,. The mixture was passed through a 0.45 pm pore size filter immediately before use. Blastocysts were incubated in this mixture for 20 min a t 37"C, then rinsed briefly in TMST buffer at room temperature. Fixation was accomplished in 4% paraformaldehyde, 5% glutaraldehyde, 4 mM CaC1, in 0.1 M sodium cacodylate buffer, pH 7.4 (Karnovsky's fixative), for 1h a t room temperature. Blastocysts were then rinsed in several changes of 0.1 M cacodylate buffer, pH 6.0, on ice over a 1-h period to remove nonspecific cerium hydroxide precipitate (Briggs et al., 1975). They were rinsed briefly in 0.1 M cacodylate buffer, pH 7.4, and postfixed in 1%osmium tetroxide in the same buffer in ice for 1h. Following osmication, the blastocysts were rinsed in water and stained en bloc with 0.5% uranyl acetate in water a t 4°C for 1 h. After a final rinse in water, the specimens were dehydrated through graded ethanols and propylene oxide and embedded in PolyBed 812 (Polysciences). Sections 70-80 nm in thickness were cut with a diamond knife and examined at 50 kV in a Zeiss EM900 electron microscope without further staining. Inhibitors and Prior Fixation The effects of potassium cyanide and N-ethylmaleimide (NEM) on NBT reduction and cerium perhydroxide formation were assayed by allowing a 10-min preexposure to the inhibitor at 1 mM concentration in either embryo medium or TMST buffer a s appropriate. Inhibitor a t the same concentration was also present in the cytochemical medium. The effect of catalase on cerium perhydroxide formation was tested by omitting 3-AT and adding bovine

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Fig. 1. NBT reduction in the presence of cyanide by rabbit blastocysts a t 5 days p.c. Both blastocysts were incubated for 60 min in medium containing NBT a t 300 Fgiml and 1mM KCN. The blastocyst to the left was fixed in 4% paraformaldehyde in PBS for 1 h prior to NBT exposure. The living blastocyst to the right shows formazan production in contrast to the blastocyst undergoing prior fixation. Early collapse of the living blastocyst a s a result of the 60-min exposure to KCN is evident.

catalase (Sigma) at 17.5 pg (364 UYml to both the TMST buffer and the cytochemical medium. Fixation of embryos prior to cytochemical assay was carried out in freshly prepared 4% paraformaldehyde in phosphatebuffered saline (PBS), pH 7.5, for 1 h at room temperature.

RESULTS AND DISCUSSION Trophoblast in the rabbit blastocyst is a highly attenuated, distinctly polarized, simple epithelium with a n outer surface densely populated with microvilli and a smoother inner surface adjoining the blastocoelic cavity (see Fig. 2). Among its many functions, it carries out active transport of sodium and chloride ions from the external environment into the blastocoelic cavity (Cross, 1973; Biggers et al., 1988), which, in the rabbit, leads to rapid expansion of the blastocyst from a diameter of 300 km on day 4 p.c. to 3 mm on day 6 P.c., resulting in a greater than 700-fold increase in volume during this 48-h period (Daniel, 1964). The studies reported here were carried out on rabbit blastocysts during this period of rapid expansion. By day 6 P.c., migration of parietal endoderm from the inner cell mass (ICM) results in a second cell layer on the blastocoelic surface of the trophoblast (see Fig. 2). When the early rabbit blastocyst at day 4 p.c. is incubated in the presence of NBT, blue color due to formazan deposition begins to appear between 90 and 120 min and is initially more intense in the region of the ICM. In contrast, 5- and 6-day rabbit blastocysts produce detectable formazan beginning a t 20 min, and the color is uniform over the entire surface of the blastocyst. Cyanide, which inhibits mitochondrial cytochrome oxidase, prevents the reduction of NBT to formazan in the 4-day blastocyst, whereas it has no effect on the rapid production of formazan in 5- and 6-day blastocysts (Fig. 1). NBT is a cation that is water soluble but does not readily cross cell membranes, while formazan is insoluble (Oberley and Spitz, 1985). It seems probable, therefore, that the rapid, cyanideinsensitive NBT reduction seen in the older blastocysts

is occurring a t the cell surface, whereas the delayed, cyanide-sensitive reaction in the 4-day blastocyst is the result of mitochondrial cytochrome oxidase activity. Cyanide-insensitive reduction of NBT in leukocytes is associated with the production of 0,- by a cell surface NAD(P)H oxidase (Curnutte and Babior, 1987). If this were true of the rabbit blastocyst, dismutation of the 0, would lead to hydrogen peroxide formation on the cell surface. This possibility was investigated by applying to the blastocyst the cytochemical method devised by Briggs et al. (1975) for the detection and localization of hydrogen peroxide formation in leukocytes. The method depends on the reaction of hydrogen peroxide with Ce3' ions to form electron-dense deposits of cerium perhydroxides that remain well-localized a t the reaction site. To inhibit any endogenous catalase that would rapidly destroy hydrogen peroxide, 3-aminotriazole (3-AT)is included in the cytochemical reaction medium. Triton X-100 is also included at 0.0002% to permeabilize cell membranes and promote access of the reaction components to cytoplasmic sites (Robinson, 1985). When the 6-day blastocyst is reacted with the basic cytochemical medium lacking additional substrate, a thin electron-dense precipitate forms along the microvillous surface of the trophoblast (Fig. 3). Although this result is presumptive evidence for hydrogen peroxide formation a t the microvillous surface, it must be noted that cerium has also been used a s a capture reagent to detect sites ofphosphatase activity (Blok et al., 1982; Robinson, 1985). Preliminary studies of rabbit blastocysts with the azo dye technique described by Ziomek et al. (1990) have shown abundant alkaline phosphatase activity (Manes, unpublished), a n enzyme characteristically associated with the plasma membrane. It was therefore necessary to carry out controls to rule out alkaline phosphatase a s the source of the precipitate. Stimulation of the reaction by the provision of exogenous substrates was attempted. The addition of 2 mM P-glycerophosphate, a standard substrate used in the identification of phosphatases (Robinson, 1985),did not alter the baseline reaction seen in the absence of substrate (Fig. 4). In contrast, addition of NADH or NADPH at 0.88 mM produced a marked increase in reaction product (Fig. 5). Precise quantitation of reaction product is not possible, but it is my distinct impression from viewing sections of nearly 20 blastocysts that NADPH yields more extensive precipitate than does NADH. Based on these findings, the surface activity appears to be due to a n NAD(P)H-dependent oxidase, not a phosphatase. Further evidence that the reaction product was not due to phosphatase activity is the effect of prior fixation of blastocysts. Fixation of embryos in 4% paraformaldehyde is the standard protocol prior to assay for phosphatase (Ziomek et al., 1990). Prior fixation, however, totally abolishes precipitate formation in the assay used here. Furthermore, the omission of 3-AT from the cytochemical medium while adding exogenous catalase

Fig. 2. Rabbit blastocyst wall at 6 days P.c., showing a trophoblast cell (TI with the typical outer microvillous surface (mv)and a parietal endoderm cell (El adjacent to the blastocoel (B). The specimen was fixed shortly after recovery from the reproductive tract and without exposure to the cytochemical medium. X 6,800. Fig. 3. Trophoblast cell of 6-day rabbit blastocyst exposed to the basic cytochemical medium without added substrate. A thin deposit of cerium perhydroxide (arrows) is visible along the microvillous border. The 20-min exposure to the cytochemical medium typically results in an increase in cytoplasmic vacuoles (compare with Fig. 2) and varying degrees of distortion of cytoplasmic structures. N, nucleus. x 5,700.

Fig. 4. Trophoblast cell as in Figure 3. 6-Glycerophosphate was present a t 2 mM during the cytochemical reaction. There is no increase in cerium perhydroxide precipitation along the microvillous border beyond that seen in Figure 3. C , crystalline inclusion typical of rabbit trophoblast. ~ 6 , 8 0 0 . Fig. 5. Trophoblast cell as in Figure 3. NADPH was present at 0.88 mM during the cytochemical reaction. Note the large increase in cerium perhydroxide precipitation along the microvillous border in comparison with that seen in Figures 3 and 4. NADH at 0.88 mm resulted in a similar increase in reaction product. ~ 6 , 8 0 0 .

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also prevents precipitate formation (Fig. 61, strengthening the conclusion that hydrogen peroxide is one of the reactants. The enzymatic basis for hydrogen peroxide formation a t the trophoblast surface is indicated by its sensitivity to aldehyde fixation and by the inhibitory effect (not shown) of the sulfhydryl-blocking reagent N-ethylmaleimide (Smyth et al., 1960). However, there is something of a paradox in these results. Although hydrogen peroxide can be formed directly by some oxidases through the two-electron reduction of molecular oxygen, its usual mode of formation is by the “dismutation” of 0,- (Halliwell and Gutteridge, 1989).The enzymatic dismutation of 0,- by the CuiZn superoxide dismutase found in the cytoplasm of animal cells would be inhibited by cyanide (Weisiger and Fridovich, 1973). As is shown in Figure 7, cerium perhydroxide precipitation on the trophoblast surface is not inhibited by cyanide. The remaining possibilities would appear to be that the hydrogen peroxide is being formed directly without the intervention of 0,- or by nonenzymatic dismutation of 0,- or by a novel membrane-bound superoxide dismutase that is not inhibited by cyanide. It should be noted that hydrogen peroxide formation on the surface of leukocytes by the well-characterized NAD(P)H oxidase is also insensitive to cyanide (Briggs et al., 1975). The apparent localization of hydrogen peroxide formation in the rabbit blastocyst to the outer, microvillous surface of the trophoblast was seen initially in intact 6-day blastocysts “permeabilized” with 0.0002% Triton X-100. Higher magnification of the microvilli (Fig. 8 ) shows the discretely localized character of the reaction product, as opposed to occasional amorphous small collections of precipitate found elsewhere, apparently not entirely removed by the postreaction wash in acidic cacodylate. To verify that the cell surface enzymatic activity is confined to the outer face of the trophoblast, 6-day blastocysts were removed from the neozona and opened widely to ensure equal exposure of outer and inner surfaces of the trophoblast to the cytochemical medium. As is shown in Figure 9, in these opened blastocysts there is no specific reaction on the blastocoelic surface of the trophoblast, nor on the attached parietal endoderm. ICM cells (not shown) were also devoid of surface reaction product. Access of the cytochemical reactants to intracellular sites was evidenced by the appearance of precipitate within mitochondria when NADH was added to the cytochemical medium (not shown). The production of superoxide and hydrogen peroxide in mitochondria by “leakage” of electrons to molecular oxygen from intermediates of the ETS has been well documented (Boveris, 1977). The developmental appearance of the trophoblast oxidase was determined by assaying 4- and 5-day p.c. blastocysts. As can be seen in Figure 10, there is abundant activity in the 5-day blastocyst. Again, quantitation of oxidase activity is not possible, but the impression is that the activity is even more vigorous in the 5-day than in the 6-day blastocyst. In contrast, very minimal activity is seen in the 4-day blastocyst (Fig.

11);occasional microvilli can be found that are outlined by reaction product, but the majority of the 4-day trophoblast surface shows virtually no reaction. If these 4-day blastocysts are incubated for 24 h in the medium described (Table l ) , intense activity of the hydrogen peroxide-generating system is evident at the end of the incubation period (Fig. 12). Four-day blastocysts cultured for 24 h also acquire the ability to reduce NBT in a cyanide-insensitive reaction. The most concise interpretation of the cyanide-insensitive reduction of NBT and hydrogen peroxide production by older blastocysts is that both cytochemical methods are demonstrating the same enzymatic activity. Both phenomena appear simultaneously during early blastocyst expansion in the rabbit. Both are suppressed by prior fixation of the blastocyst and by the sulfhydryl-inactivating agent NEM. Furthermore, as can be seen in Figure 13, the addition of NBT to the cerium cytochemical medium greatly reduces hydrogen peroxide formation. This reduction would be predicted if NBT competes with molecular oxygen for electrons generated in the cell surface reaction. Collectively, these findings suggest that cyanide-insensitive reduction of NBT to formazan will be a simple, useful test for trophoblast oxidase activity in other mammalian blastocysts. Ultrastructural cytochemistry using cerium can be reserved for those situations in which the results with NBT are equivocal. The findings presented here raise the obvious question of the significance of the production of reactive oxygen species by the uterus-facing surface of the trophoblast. The question can be phrased in terms of whether the generation of superoxide anion andior hydrogen peroxide on the trophoblast surface is a n unfortunate byproduct of a n essential oxidation process or whether these molecules are useful in their own right (Halliwell and Gutteridge, 1989). In biological systems where hydrogen peroxide is known to be “useful,” such a s the hardening of the fertilization membrane of the sea urchin egg (Foerder and Shapiro, 1977) and the generation of chloramines for bacterial killing by leukocytes (Weiss et al., 19831, a peroxidase is present. To date, assays of soluble and particulate fractions of both rabbit blastocysts and the adjacent endometrium have failed to detect any peroxidase activity (Manes, unpublished). However, hydrogen peroxide is known to mimic the action of insulin in increasing glucose uptake by adipocytes (Czech et al., 19741, and a n NADH oxidasecytochrome system capable of generating hydrogen peroxide has been identified in renal brush border membranes (Gimenez-Gallego et al., 1980). Thus hydrogen peroxide may conceivably be a n autocrine factor promoting trophoblast transport functions. An intriguing possibility is that hydrogen peroxide may be serving a paracrine function to increase guanylate cyclase activity in the endometrium, a s it has been shown to function in pulmonary arterioles (White et al., 1976; Bruke and Wolin, 1987). It may be a n important embryonic “signal” in the initiation of implantation (Kennedy, 1983). Pierce e t al. (1991) recently reported

HYDROGEN PEROXIDE FROM RABBIT BLASTOCYSTS

Fig. 6. Trophoblast cell as in Figure 5. In this reaction, 0.88 mM NADPH was present, but 3-AT was omitted. Bovine catalase was added as described in Materials and Methods. Note the absence of precipitate on the microvilli. X 15,000. Fig. 7. Trophoblast cell as in Figure 5. KCN was present a t 1 mM during the cytochemical reaction. x 24,000. Fig. 8. Trophoblast cell as in Figure 3, showing the discrete, patchy

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deposition of reaction product along the microvilli seen in the absence of added NADPH. X22,500. Fig. 9. Trophoblast cell as in Figure 6 . This blastocyst was removed from the neozona and opened widely prior to the cytochemical reaction. Cerium perhydroxide precipitate is confined to the outer microvillous surface and is absent from the surface facing the blastocoel

(B). x5,700.

Fig. 10. Trophoblast cell of 5-day rabbit blastocyst. NADPH was present at 0.88 mM during the cytochemical reaction. This cell shows the large number of cytoplasmic vacuoles frequently seen following exposure to the cytochemical medium. There is also some reaction product in or adjacent to several of the vacuoles. x 12,000. Fig. 11. Trophoblast cell (T) of 4-day rabbit blastocyst. NADPH was present a t 0.88 mM during the cytochemical reaction. Note the close apposition of the trophoblast to the zona pellucida (ZP) and the absence of reaction product on the blunt microvilli. x 15,000. Fig. 12. Trophoblast cell (T) of rabbit blastocyst removed from the reproductive tract on day 4 p.c. and cultured 24 h in serum-free me-

dium (Table 1). Cytochemical conditions were as in Figure 11. Note the dense deposits of cerium perhydroxide between the trophoblast and the zona pellucida (ZP). x 12,000. Fig. 13. Trophoblast cell as in Figure 5. NBT (0.8 mgiml) was present during the cytochemical reaction. Note the greatly reduced formation of cerium perhydroxide along the microvillous border in comparison with that in Figure 5 and the flocculent deposits of formazan (arrows) resulting from the reduction of NBT. These blastocysts turned visibly blue during the 20-min cytochemical reaction. x9,ooo.

HYDROGEN PEROXIDE FROM RABBIT BLASTOCYSTS finding hydrogen peroxide in the blastocoelic fluid of the mouse blastocyst. The source of this hydrogen peroxide was not determined, but it was shown to exert a selective toxicity on totipotent cells within the ICM. Thus there are a number of possible roles for hydrogen peroxide itself in mammalian reproduction. It will be of great interest to investigate how general the production of hydrogen peroxide by blastocysts may be and how mammalian embryos make use of this potentially lethal molecule.

ACKNOWLEDGMENTS This work was begun while the author was a visitor in the laboratory of Dr. John Biggers. The author also thanks Drs. Betsey Williams and David Begg for their generous assistance with the initial electron microscopy and Drs. Manfred and Morris Karnovsky for their helpful suggestions regarding the cytochemistry. The work was supported as part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development through cooperative agreement HD22847, NICHHD.

NOTE ADDED IN PROOF After this manuscript was submitted, it was brought to the author’s attention that Nasr-Esfahani et al. (Development 109:501,1990),using a fluorimetric method, have demonstrated a transient rise in the intra-cellular concentration of hydrogen peroxide or lipid peroxides in the 2-cell mouse embryo growing in vitro.

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