Permeability of the mouse zona pellucida: a structure-staining-correlation model using coloured probes K. Turner and R. W. Horobin Department of Biomedicai Science, Western Bank, University of Sheffield, Sheffield SlO 2TN, UK

Coloured molecular probes of diverse molecular structure were used to define the permeability of the zona pellucida of preimplantation mouse embryos. The molecular characteristics examined were: electric charge, the size of conjugated aromatic groups and the hydrophilic\p=n-\lipophiliccharacter, all of which can be described numerically. It was hoped that definition of the types of compound able to cross the zona pellucida would contribute to identification and understanding of potential maternal\p=n-\embryonicsignals. The staining pattern of 51 dyes in unfertilized and fertilized one-cell eggs was recorded and 16 dyes were subsequently used to test two-, four- and eight-cell embryos further, to establish whether the permeability of the zona pellucida altered with developmental age. Four different staining patterns were observed that directly correlated with the numerical parameters of the probe but were independent of developmental age. The size of the conjugated system and the hydrophilic\p=n-\lipophilicbalance of the probe were important in determining its interaction with the zona pellucida and embryo whereas electric charge appeared to have little influence. The resulting model is useful for predicting likely interactions between chemically defined molecules and biological entities and suggests that most biologically active molecules and metabolites can pass through the zona pellucida unhindered and enter the embryo with ease.

Introduction some bacteria and fungi may also be excluded from the zona pellucida (Eaglesome et al, 1980). The is surrounded the zona developing embryo by pellucida To date studies have focussed on the relationship between the This is fhroughout preimplantation period. layer respon¬ the molecular weight of a substance and its ability to cross the sible for of fertilization and is the site of species specificity zona pellucida. Little or no attention has been given to other the secondary block to polyspermy (Yanagimachi, 1994; molecular properties that affect the interaction of molecules Wassarman and Mortillo, 1991). In addition, it defines a with biological structures. micro-environment within which cleavage and blastocyst The penetration and accumulation patterns of molecular formation take place, and presents the only physical barrier to probes of the dye and fluorochrome type into living cells has the movement of molecules between and maternal embryonic shown that there are several physicochemical properties of tissue. significance (Horobin and Rashid-Doubell, 1993). In addition to The mouse zona is of three acidic pellucida composed unique molecular size, electric charge, the size of the conjugated or with about 80% of its mass glycoproteins: being protein and aromatic system of a molecule and its hydrophilic-lipophilic 20% almost all the remaining carbohydrate (Wassarman, 1988). character are often important. These properties can all be The zona is a structure since apparently highly porous very defined or modelled numerically, and applied to a large number large molecules, like horseradish peroxidase, ferritin, immuno- of probes with diverse combinations of physicochemical globulins and even viruses are capable of passing through characteristics (Horobin and Rashid, 1990). it (Hastings et al, 1972; Sellens and Jenkinson, 1975; Thus, the electric charge (Z) of a molecule may be assessed Wassarmann, 1988). Furthermore, zona permeability appears to from its structural formula, plus the pK values of its acidic and be independent of developmental age. However, there is some basic substituents. The size of the molecule's conjugated evidence zona act as a that the pellucida may protective barrier system is an indication of the significance of Van der Waals' For by excluding harmful compounds. example, embryos attractions. These are non-directed, short-range polar forces cultured in the absence of their zona pellucida appear to be that are often associated with aromatic groups. An estimate of more sensitive to toxic compounds than if they are cultured their strength is given by the conjugated bond number (CBN) with their zonae intact (Fleming et al, 1987). Large viruses, and relates to the number of conjugated bonds within the molecule involved in electron delocalization. *Present address: Sheffield Fertility Centre, 26 Glen Rd, Nether Edge, Sheffield Hydrophilicity— S7 IRA, UK. lipophilicity may be modelled using the logarithm of the Received 3 April 1997. octanol—water partition coefficient (log P). A positive value

Downloaded from Bioscientifica.com at 10/02/2021 11:02:21AM via free access indicates a lipophilic molecule, whereas a negative value used. Four-cell embryos were obtained in the early afternoon of indicates a hydrophilic molecule. the second day following plug identification. We, therefore, set out to define at the light microscope level, Mice on a normal light cycle provided two-cell and eight- the types of molecule permitted access to, or excluded from, cell embryos. Two-cell embryos were obtained during the the mouse embryo by the zona pellucida, before and after morning of the day following plug identification, and eight-cell fertilization. A set of coloured molecular probes, varying embryos were obtained during the morning of the third day widely in values of Z, CBN and log P, were selected. Although after plug identification. Blastocyst stage embryos were not molecular size is related to the CBN, since large molecules have examined since the zona pellucida cannot be guaranteed to be the potential to have many aromatic groups within them, it completely intact at this stage (Ljungkvist and Nilsson, 1974). was not examined in the work presented here since this had been done this is irrelevant, previously. Fortunately, probably Calculation numerical molecular since earlier work has shown size to have no significant effect of parameters of probes on penetration of the zona pellucida (Hastings et al, 1972; The numerical parameters of the molecular probes were Sellens and Jenkinson, 1975). The embryo was viewed simply calculated using procedures described by Horobin and Rashid of the zona as being composed two compartments, pellucida (1990). A large number of dyes and fluorochromes were used. and the cell or blastomeres comprising the embryo itself. The perivitelline space was not considered as a separate compart¬ ment because of its small size and the inherent difficulties of Selection of molecular probes due to it to determining any staining patterns being necessary Probes were chosen that varied widely over the three observe the zona it through pellucida. parameters of Z, CBN and log P, and hence the physico- The of the model was then tested validity resulting by chemical characteristics of electric charge, ability to bind by applying it to a number of biologically relevant molecules: Van der Waals' forces, and hydrophilicity-lipophilicity. platelet activating factor, produced by preimplantation Unfertilized and fertilized were to 51 of of which culture medium eggs exposed dyes embryos and the presence in appears diverse physicochemical character. A selection of 16 of these to influence favourably (O'Neill, 1985; embryo growth Ryan dyes was further tested on two-, four-and eight-cell embryos et al, 1990); oestrogen and progesterone, detected in the early (Table 1). embryo in a number of species but may also be of maternal origin (Kennedy, 1983); and histamine and prostaglandin E2, present in or produced by preimplantation mouse embryos Preparation of molecular probe working solutions (Dey and Johnson, 1980; Niimura and Ishida, 1987). The The dyes were usually made as 0.1% (w/v) solutions in metabolites glucose, pyruvate and lactate and amino acids, up PBS. Celestin Blue and Thionin, however, were dissolved in treated as a single group, were also studied. More general 0.9% (w/v) saline since they precipitated in PBS. The pH values consideration was given to other classes of biological of all dye solutions were checked and adjusted to lie within the molecule and to drugs. normal physiological range of pH 7.0—7.4. The purity of each dye was also checked using a thin-layer Chromatographie (Proctor and Horobin, 1985). Materials and Methods procedure

Animals Assessment of uptake of the molecular probe of the molecular was examined Randomly bred female Swiss albino mice aged 6—10 weeks Uptake probes by placing or in a small of solution in the centre were used to provide unfertilized eggs and embryos. Mice eggs embryos drop dye of welled slide over which then were allowed to ovulate and mate naturally. In order to obtain a a coverslip was applied. 10 at each eggs and embryos at the correct stage of development, most Approximately eggs or embryos developmental were examined for a and were mice were kept in a room where the light—dark period was stage given dye. Eggs embryos viewed an BHS intervals reversed. Males were introduced and female mice were checked using Olympus microscope at of 5, 30 and 60 min. was scored in both the zona and at regular intervals for the presence of a vaginal plug, which Staining pellucida the as weak or absent. indicated that mating had taken place. Unfertilized eggs were embryo cytoplasm being strong, obtained within 2 h of mating. In naturally ovulating mice, penetration of eggs by spermatozoa usually occurs between 3 Calculation of numerical parameters of biologically active and 5 h after ovulation. molecules Cumulus cells were removed by incubation in 0.1% (w/v) hyaluronidase (Sigma, Poole, Dorset) in PBS for 3—5 min at The permeability of the zona pellucida and uptake, by the room temperature. When the cumulus cells were dispersed, the embryo, of biologically relevant molecules were tested using eggs were removed and washed thoroughly in two changes of the resultant model. Calculations of , CBN and log were PBS. All of the oocytes used had only one polar body and no performed for the following: platelet activating factor, oestro¬ pronuclei. gen, progesterone, histamine, prostaglandin E2; and the One-cell embryos were obtained early in the morning on the metabolites: glucose, pyruvate, lactate, and amino acids, treated day after plug identification. Only those embryos with two as a single group. Calculations are only possible on non-protein distinct pronuclei, identified with phase contrast optics, were molecules using current procedures, since the three-dimensional

Downloaded from Bioscientifica.com at 10/02/2021 11:02:21AM via free access Table 1. Structural parameters of dyes and their respective staining pattern of the mouse embryo

Structure Staining Structure Staining parameters" intensities parameters2 intensities Dye name Dye name (CI No.) CBN log Zona Embryo (CI No.) CBN log Zona Embryo

Region A Region Alizarin Brilliant Sky (62045) 1 20 0.04 Alcian Blue 2GX (0) + 2 48 2.00 + + + Alizarin Cyanin 2R (58550) - 1 23 - 4.62 + + Alcian Blue 8GX (74240)* + 4 48 - 9.66 + + +

- - Alizarin Cyanin Green G (61590) 2 32 + 2.75 + + Astra Blue 6GLL (0) +3 57 -10.39- ++ +

- Biebrich Scarlet (26905) -2 30 -3.17 + + Chicago Blue 6B (24410)* -4 47 -11.76 + + Bixin (75120) -1 22 +3.46 + + Eosin (45400)* 2 36 + 0.91 + + Brilliant Green (42040)* + 1 23 + 2.26 + + Gallein (45445) -2- 36 -3.83 + + Carbolan Green G (61580) 2 32 + 2.88 + + Geranine G (14930) 2 32 2.71 + +

- Celestin Blue (51050)* +1 21 -3.46 + + Indulin (50400) +1- 51 +4.44- + + Crystal Violet (42555)* + 1 24 + 1.20 + + + Monosol Fast Blue (74200) 3 48 -10.15 + + + Eriochrome Black (14645) 1 30 0.00 + + + Supranol Bordeaux G(24810)* - 2 46 + 1.61 + + - Iodine Green (42556) +2 23 -2.11 + + Thiazine Red (14780) - 2 32 2.05 + +

- Janus Black (11825) +1 33 +2.71 + + Trypan Blue (23850)* -4 45 -11.42- + + Malachite Green (42000) +1 23 +0.10 + + Metanil Yellow 1 22 + 0.85 + + Region C - Methyl Red (13020) -1 19 -0.14 + + Janus Blue (12211)* +1 38 +5.12 ++ + + Méthylène Blue (52015) + 1 18 0.91 + + Janus Green (11050)* +1 34 +4.02 ++ + + New Méthylène Blue (52030) + 1 18 +- 1.95 + + + Nile Blue (51180)* +1 23 -3.37 + + + Region D Palatine Fast Orange (18745) 1 20 + 0.62 + + + Cuprolinic Blue (0)* +4 44 -17.91 + + - Patent Blue A (42080) 1 35 4.86 + + Fast Green FCF (42053) -2 36 -10.13 +- + +

- Primulin (49000) -1 29 +0.18- + + Indigo Carmine (73015) 2 23 5.90 Solochrome Azurine BS (43830) -3 31 -4.40 Light Green SF (42095) - 2 34 - 6.62 - - Solochrome Dark Blue (15705)* -1 27 +0.26 New Coccine (16255) 3 26 8.59 - Solway Purple (60730)* 1 26 1.20 Orange G (16230) -2- 21 -5.01- - - Thionin (52000) +1 18 -2.12- + + Patent Blue V (42051) 1 24 7.10 Zapon Fast Blue (51004) + 1 18 + 1.84 + + Ponceau 4R (16255)* - 3 26 - 8.60 - Sirius Red (35780) -6- 62 -17.14- - Solochrome Cyanin R (43820)* -4 29 -11.28 - Solochrome Red (0) 3 24 8.97 -

- -

*Z, is nominal electric charge; CBN, conjugated bond number; log P, logarithm of the octanol-water partiti) coefficient. b( ) represents no staining; ( + ) weak staining; ( + + ) strong staining. *Dyes- that were tested further on two-, four- and eight-cell embryos. shape of a protein and thus, the exposed chemical groups Region A of Fig. 1 contains probes to which the zona contributing to overall molecular behaviour, are unknown. pellucida offered no barrier, and which could accumulate in the embryo (compare with Fig. 2a). Almost all the probes (25/26) in this region showed this behaviour. These probes are Results relatively small (CBN < 35); moderately to weakly hydrophilic or lipophilic (log > 5); and either anionic or cationic. Individual molecular probes showed differences in their relative Nearly all the probes —in Region (11/12) accumulated in uptake by the zona pellucida and the embryo (Table 1). For substantial amounts in the zona pellucida, but did not accumu¬ those probes able to gain access, passage across the zona late in the embryo (compare with Fig. 2b). The common feature pellucida was rapid with visible staining of the embryo of these compounds was that they all had large conjugated occurring within minutes. No changes in staining pattern were systems (CBN > 30) irrespective of whether they were ani¬ observed after 5 min, although staining intensities continued to onic, cationic, hydrophilic or lipophilic. Region C contains only increase for up to 30 min. two probes; these accumulated in the embryo as well as in the There were several links between probe structure and sites zona pellucida (compare with Fig. 2c). Both probes are strongly of probe accumulation. These links may be seen if patterns of lipophilic (log > +4), cationic, with large conjugated sys¬ probe accumulation are plotted on a CBN-log diagram, in a tems (CBN > 34). Most of the probes in Region D (9/11) slightly simplified form with probe uptakes plotted either as failed to accumulate in either the zona pellucida or the embryo 'present' or 'absent' (Fig. 1). The staining patterns observed, (compare with Fig. 2d). The characteristic feature of these latter with respect to charge, CBN and log P, could be summarized compounds is their strongly hydrophilic nature (log < 5); — by four pictographs. and wide range of sizes of conjugated system. These

Downloaded from Bioscientifica.com at 10/02/2021 11:02:21AM via free access Fig. 1. The relationship between physicochemical structure, as defined by charge, conjugated bond number (CBN) and the logarithm of the octanol-water partition coefficient (log P), and staining patterns observed in mouse preimplantation embryos. O: oestrogen; P: progesterone; H: histamine; PAF: platelet-activating factor; Pg: prostaglandin E2; Gl: glucose; Pt: pyruvate; L: lactate. Individual amino acids are not shown. compounds, with one exception, are anionic; small hydrophilic model used here, the first possible barrier to probe entry is the cationic probes are not commercially available. zona pellucida. Although permeable to certain large com¬ pounds, probes with high affinity for biopolymers in the zona pellucida would nevertheless be excluded. Accumulation of Effect of developmental age probes within the zona pellucida reflects binding of the to somewhat anionic, Sixteen, selected were tested on two-, four- and dyes highly hydrated, glycoproteins dyes favoured Van der Waals' eight-cell embryos, in addition to unfertilized and fertilized (Wassarman, 1988). Such binding, by has been in one-cell eggs. No marked in the or intensity of forces, long reported analogous systems (Schetty, changes pattern Brook and Horobin and Bennion, was observed for of the at of the 1955; Munday, 1970; 1973; probe uptake any dyes any will be stages. Rashid et al, 1991). Van der Waals' attractions most significant in probes with large conjugated systems and, hence, large CBN values. Numerical parameters for the biological molecules The second barrier is the plasmalemma of the embryonic cells. such membranes either active The calculated values for charge, CBN and log for the Passage through requires transport or for the probe to be lipophilic. Probe accumulation chosen biological molecules are shown (Table 2). Their within embryonic cells can arise from into various physicochemical characteristics relative to the molecular probes uptake organelles and by a variety of mechanisms (Rashid and are indicated their on Fig. I. by positions Horobin, 1990a, b; Rashid et al, 1991; Rashid-Doubell and Horobin, 1993). The four regions of Fig. 1 can now be as as the bases of Discussion explained well probable physicochemical entry and accumulation. The permeability of the mouse embryo zona pellucida has been The Region A probes fail to adsorb onto the zona pellucida, explored by observing the penetration of coloured probes. Of reflecting their low CBN values and consequent minor Van der the three physicochemical properties examined, the size of the Waals' binding. Passive diffusion through the lipid-rich plas¬ conjugated system and the hydrophilic—lipophilic character of malemma is possible for the lipophilic members of this set. the molecule were the main determinants of staining pattern. Moreover, most hydrophilic members are bases, possessing There was little dependence on its electric charge. The staining lipophilic (and hence membrane permeant) free-base forms. pattern was also independent of embryo developmental stage Region probes, all with large CBN values, will bind to the and duration of exposure to the probe. zona pellucida because of Van der Waals' attractions. Region C The various staining patterns illustrated in Fig. 1 can be probes, having large CBN values and being lipophilic, could divided into those favouring and those limiting probe accumu¬ accumulate both in the zona pellucida and in the embryonic lation within the embryo. In the simple two-compartment compartment.

Downloaded from Bioscientifica.com at 10/02/2021 11:02:21AM via free access Fig. 2. (a) A two-cell mouse embryo after exposure to Crystal Violet (Region A). Probes from Region A accumulate in the embryo but not in the zona pellucida, (b) An eight-cell mouse embryo after exposure to Supranol Bordeaux G (Region B). Probes from Region accumulate in the zona pellucida but not in the embryo, (c) A one-cell mouse embryo after exposure to Janus Green (Region C). Probes from this Region C accumulate in the embryo as well as in the zona pellucida, (d) A four-cell mouse embryo after exposure to New Coccine (Region D). Probes from this Region D failed to accumulate in either the embryo or the zona pellucida. Scale bars represent 20 µ .

Region D probes, being hydrophilic and consequently A number of probes characteristic of Region appeared to membrane impermeant, do not enter cells by passive diffusion stain the perivitelline space more intensely than either the zona and have no affinity for the constituent molecules of the zona pellucida or the embryonic cells. The composition of the pellucida. The influences of the CBN and lipophilic—hydrophilic perivitelline space is unknown, but owing to the porous nature character of a probe on staining patterns are summarized of the zona pellucida, it is generally assumed to be the same as (Fig. 3). that of the surrounding medium. The findings of the present

Downloaded from Bioscientifica.com at 10/02/2021 11:02:21AM via free access Table 2. The numerical parameters of selective biological previous studies (Calarco and Brown, 1969; Hastings and molecules and metabolites Enders, 1974). Four probes show behaviour uncharacteristic of the regions Name Charge CBNa Log Pb into which they fall. Cuprolinic Blue and Fast Green FCF (Region D), both extremely hydrophilic and hence membrane the molecules impermeant, stained embryonic compartment. Fluid-phase Biological similar Oestrogen 0 7 +3.66 pinocytosis is unlikely, since probes failed to enter. Progesterone 0 3 +5.04 Cuprolinic Blue probably forms lipophilic, membrane permeant Histamine +1 4 4.47 pseudo-bases (Bunting, 1980). Fast Green FCF, an industrial Platelet-activating factor 0 0 +2.16- polysulphonated dye, probably contains weakly lipophilic, (and Prostaglandin E2 1 0 2.14 hence membrane permeant) - impuri¬ - incompletely sulphonated Metabolites ties. No explanations can be offered for the staining patterns of Glucose 0 0 2.98 Monosol Fast Blue and Solway Purple. Pyruvate 1 0 - 1.43 This discussion will now be applied to biological molecules

- - Lactate 1 0 2.12 relevant to the developing embryo. A molecule acting as - - Amino acids 1 to + 1 0 to 9 1.0 to 3.0 a messenger between maternal tissues and embryo must - - - penetrate the zona pellucida and plasmalemma quickly and aCBN, conjugated bond number; Log logarithm of the octanol—water without obstruction. Such a molecule should therefore fall into coefficient. partition Region A of Fig. 1. Thus, messengers need to have low CBN values and, if basic, positive or only slightly negative log values (log > — 5). The five biologically active molecules considered here all meet these requirements and are expected to cross the zona pellucida and pass into the embryo without difficulty. Although the embryo probably has specific mem¬ brane receptors for these molecules, they can to some extent pass through the membrane passively. Glucose, pyruvate, lactate and amino acids are important metabolites for early embryonic development (Leese, 1991). These molecules would also fall into Region A of Fig. 1 and have ready access to the embryonic cell contents. This access is confirmed by autoradiographical and immunofluorescence studies (Glass, 1963; Biggers, 1971), although amino acid transport across the plasmalemma appears to be by a combi¬ nation of facilitated and passive diffusion (Van Winkle et al 1985). Regarding other larger biological molecules, the model predicts that lipids and most lipid-containing molecules will gain access to the embryo with relative ease, since these molecules are unlikely to have any conjugated bonds and are Fig. 3. The influence of the conjugated bond number (CBN) and obviously lipophilic. However, one limitation of the model is hydrophilic—lipophilic character of a molecule on its ability to that these calculations do not take into account the complex mouse permeate embryos. physiological situation where many such molecules are bound by high molecular weight carriers, such as albumin, which will then influence their ability to interact with the embryo. study suggest that this may not be the case and there are two Large polysaccharide molecules, like glycogen and heparin, possible explanations. The probes may bind to substances are extremely hydrophilic and will be surrounded by large within this space and so accumulate. Alternatively, the zona hydration shells in aqueous solution. Heparin is anionic and pellucida may be acting as a one-way gate, allowing molecules will, therefore, be expected to behave similarly to probes in to reach the embryonic cell contents from the surrounding Region D and neither interact nor accumulate within the zona medium but selectively restricting their exit. Such a concept has pellucida since there are no strong binding forces. This analysis been proposed previously by Kapur and Johnson (1986). may explain the observation of Austin and Lovelock (1958) Evidently, physicochemical factors can predict entry and that heparin (molecular weight 1200) is unable to cross the accumulation of probes in the embryo, without invoking cell zona pellucida, leading them to believe that there was an upper physiology. This view is supported by observations that limit to the size of molecule permitted access to the embryo. hydrophilic probes with high CBN values (that is, Region Accessibility of the zona pellucida to proteins and nucleic probes with log < 0) or low CBN values (that is, Region D acids cannot be predicted using the present model. These large probes) are respectively typical markers of adsorptive and fluid polymers fold in a complex manner, so their tertiary structures phase pinocytosis (Rashid et al, 1991). Failure of such probes to control which groups are exposed and able to undergo accumulate in the embryo suggests that little pinocytotic intermolecular interactions. Although their CBN values will uptake occurred in the present case and is consistent with be high, this will not accurately reflect the occurrence of

Downloaded from Bioscientifica.com at 10/02/2021 11:02:21AM via free access intermolecular Van der Waals' forces. Thus the model cannot Horobin RW and Bennion PJ (1973) The interrelation of the size and substan- the role of van der Waals and be used to assess accessibility of the embryo to such molecules tivity of dyes: attractions hydrophobic bonding in biological staining Histochemie 33 191-204 as chorionic factors and interleukins. gonadotrophin, growth Horobin RW and Rashid F (1990) Interaction of molecular probes with living The model be used to the interaction may, however, predict cells and tissues. I. Some general mechanistic proposals, making use of a of drugs and toxins with the embryo. As an example, nicotine simplistic Chinese box model Histochemistry 94 205-209 has pK values of 6.2 and 11. Hence at physiological pH, it Horobin RW and Rashid-Doubell F (1993) Fluorescent probes: where do they in cells and In of Microinjection, Micro¬ will comprise three species: a lipophilic free base and two go why? Biotechnology Applications scopic Imaging and Fluorescence pp 61-66 Eds Bach, CH Reynolds, JM Clark, cations. Thus even in the absence of hydrophilic receptor- J Mottley and PL Poole. Plenum Press, New York mediated nicotine will enter the it uptake, embryo where may Kapur RP and Johnson LV (1986} Selective sequestration of an oviductal fluid localize in the lysosomes or on the DNA of the nuclei and glycoprotein in the perivitelline space of mouse oocytes Journal of potentially cause damage. Experimental Zoology 238 249—260 The model described here demonstrates the validity of Kennedy TG (1983) Embryonic signals and the initiation of blastocyst implan¬ tation Australian Journal of Biological Science 36 531—543 the of molecular a predicting uptake probes by combining Leese HJ (1991) Metabolism of the preimplantation mammalian embryo. In of their with that of the knowledge chemistry biological Oxford Reviews of Reproductive Biology Vol. 13 pp 35-72 Ed. SR Milligan. structure. This approach coupled with experimental evidence Oxford University Press, Oxford suggests that most molecules can gain entry to the embryo Ljungkvist I and Nilsson O (1974) Blastocyst—endometrial contact and blue reaction normal in the rat the zona and that it does not constitute a pontamine during implantation Journal of through pellucida 60 149-154 barrier to the of hazardous Endocrinology significant penetration potentially Niimura S and Ishida (1987) Immunohistochemical demonstration of prosta¬ compounds. glandin E2 in preimplantation mouse embryos Journal of Reproduction and Fertility 80 505-508 This work was supported by a grant from the Anatomical Society O'Neill C (1985) Partial characterization of the embryo-derived platelet- of Great Britain and Northern Ireland. We would like to thank activating-factor in mice Journal of Reproduction and Fertility 75 375—380 H. Leese for critically reviewing the manuscript and F. Rashid-Doubell Proctor GB and Horobin RW (1985) A widely applicable analytical system for stains: thin Stain for her assistance with the we wish to biological reverse-phase layer chromatography Technology chromatography. Finally, 60 1-6 the contribution to this work of A.W. who acknowledge Rogers, sadly Rashid F and Horobin RW Interaction of molecular with died before its (1990a) probes living completion. cells and tissues. 2. 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