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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.

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