Action of phorbol myristate acetate (PMA) at fertilization of mouse oocytes in vitro

Cytochalasin B, the drug affecting microfilaments (Godman & Miranda, 1978), blocks cytokinesis of the second maturation division when present during fertilization of mouse oocytes (Niemierko, 1975; Niemierko & Komar, 1976).

There are reports that phorbol myristate acetate (PMA) blocks cytokinesis without impairing karyokinesis as well, which results in binucleation of sea urchin (Bresh & Arendt, 1978), frog (Ellinger, 1982) and mouse (Sawicki & Mystkowska, 1981) blastomeres.

The aim of the present work was the application of PMA to block cytokinesis of the second maturation division in mouse oocytes fertilized in vitro.

Oocytes were inseminated according to the method of Toyoda, Yokoyama & Hosi (1971) as described in detail on our earlier work (Niemierko & Komar, 1976). The oocytes were obtained from outbred Swiss albino females induced to superovulate by i.p. injections of 5i.u. PMSG (Gestyl, Organon) and hCG (Chorulon, Intervet) given 43–48 h apart. Females were killed 13 h after the second injection. Sperm was collected from F₁ (CBA/H×C57B1) males.

Phorbol 12-myristate 13-acetate (PMA; Sigma, lot no. 99c-0131) was used in concentrations 5ng ml^-1 and 10 ng ml^-1, which were obtained by dissolving stock solution containing 0 ·1 mg PMA per 1 ml DMSO in fertilization medium. Three experiments were carried out.

1 –1 ·5 h after mixing the gametes, oocytes were treated with 0·25 % hyaluronidase in 0 ·9% NaCl to remove the cumulus oophorus. Oocytes showing the presence of cytoplasmic bulge heralding the second polar body and freshly penetrated oocytes with a spermatozoon under the zona pellucida were placed in a drop of medium containing 5 ng ml^-1 PMA under paraffin oil. After 1 h of observation under dissecting microscope (warm stage), the eggs were cultured for 3 –4 h in 37°C and 5 % CO₂ in air.

The eggs were fertilized in vitro in the presence of 5 ng ml^-1 or 10 ng ml^-1 PMA. Control eggs were fertilized in PMA-free medium. All eggs were incubated for 6h. In order to determine whether PMA-induced triploids cleaved to 2-cell stage, a few tripronucleate eggs from this experiment were cultured in the medium without PMA for 20 h after mixing the gametes.

Oocytes were preincubated for 1 ·5 h in fertilization medium containing 5 ng ml^-1 PMA before they were fertilized in vitro in the presence of 5 ng ml^-1 PMA. The double control experiments were carried out. In Control 1 eggs preincubated 1-5 h in vitro were fertilized. In Control 2 eggs obtained from females 14 ·5 h post hCG were fertilized. Each experiment lasted 5 h and then the eggs were fixed. Whole-mount preparations were made from all the eggs according to Tarkowski & Wróblewska (1967). Eggs were examined with light microscope. The results were treated statistically with χ² test.

PMA at a concentration 5 and 10 ng ml^-1 prevents cytokinesis of the second maturation division of the mouse oocytes, so that digynic triploids arise (Figs 1,2). When oocytes were subjected to PMA at the onset of the second maturation division, yield of triploids, reached only 14 ·4% (Table 1). The eggs used in this experiment were at different stages of the second maturation division, which correlated with the size of the cytoplasmic bulge heralding the second polar body. PMA did not cause retraction of this bulge, the nascent polar body. Cytokinesis was effectively blocked only in freshly penetrated eggs. In such eggs the spindle of metaphase II has not rotated.

The percent of triploid eggs increased to about 40 % in Experiment II (Table 2), when oocytes were fertilized in the presence of PMA. Because spermatozoa penetrated the oocytes at the end of the first hour, PMA acted for about one hour on unfertilized oocytes. Neither concentration of PMA impaired the fertilization process (in control eggs the rate of fertilization was insignificantly higher). However, at both concentrations the yield of triploids was similar. As shown in Table 3 half of triploids obtained with PMA enters (Fig. 3) or finishes the first cleavage division.

The incidence of triploidy was highest (72 %) in Experiment III (Table 4), when oocytes, preincubated with PMA, were fertilized in the presence of 5 ng ml^-1 PMA. However, preincubation in vitro decreased the rate of fertilization itself (compare Control 1 and Control 2). In Experiment III PMA acted for 1 ·5 h on untreated eggs (pre-incubation) and about Ih on unfertilized eggs (as in Experiment II). Since PMA is ineffective after cytokinesis starts (cf. Exp. I), it might be suggested that the high yield of triploidy achieved in Experiment III is due to prolongation of PMA treatment to about 2 h.

Among oocytes spontaneously activated in the presence PMA, 2-pronuclear eggs without second polar body preveil. Because up to 20 h after HCG injection the majority of activated eggs remains haploid with second polar body (Kaufman & Surani, 1974, controls to experiments II and III), it is concluded that diploid, two pronuclear, eggs are the result of PMA action.

The rate of polyspermie (3n+2nd p.b; 4n; 4n+2nd p.b.; 5n) eggs in Experiment II and III was similar to that in the Control. The majority of the polyspermie eggs were dispermic. From these results it would appear that PMA does not affect the block to polyspermy at the plasmalemma level.

Phorbol ester (PMA) prevents cytokinesis of the second maturation division of the mouse oocytes. The action of PMA is most effective when it precedes cytokinesis for about two hours. Karyokinesis of the second maturation division was not impaired and, as in the presence of cytochalasin B, digynic triploid oocytes were obtained. In contrast to the rapidly acting cytochalasin B, which caused retraction of large bulges of cytoplasm heralding the second polar body (Niemierko, 1975; Niemierko & Komar, 1976), PMA did not inhibit cytokinesis of the second maturation division once it had started. PMA does not inhibit initiated cytokinesis of the first cleavage division of mouse blastomeres and immediate cleavage of spontaneously activated eggs (Niemierko & Komar, unpublished data).

Induction of triploidy with PMA is procedurally more complicated, but it is as effective as the method using cytochalasin B. Half of triploids obtained with the application of either drug undergoes first cleavage division.

The mechanism of blocking cytokinesis by PMA is unrecognized. PMA causes many cellular effects. Binding of PMA to a receptor, protein kinase C, which is the integral enzyme of cell membrane, mediates rapid changes in membrane structure (Kraft & Anderson, 1983; Parker, Stabel & Whaterfield, 1984). Scarce study on effects of PMA on the cytoskeleton of differentiated cells suggest selective action of the drug on intermediate filaments. PMA induces translocation of these filaments and the redistribution of cytoplasmic action and microtubules (Croop, Toyama, Dlugosz & Holtzer, 1980; Phaire-Washington, Silverstein & Wang, 1980).

The cytoskeleton of unfertilized eggs and one-cell zygotes comprises tubulin, actin and α-actinin (Wassarman & Fujiwara, 1978; Lehtonen & Badley, 1980). Intermediate filaments appear as non-filamentous cytokeratin and scarce cyto-keratin filaments appear until cleavage-stage embryos (Lehtonen et al. 1983). Unfortunately, myosin in mouse oocytes and one-cell zygotes has not been investigated, although it is one of the major components of the embryo cytoskeleton already in 2-cell stage (Sobel, 1983). Recently, studies have been made on the organization and dynamics of actin microfilaments (Maro, Johnson, Pickering & Flach, 1984) and microtubules (Schatten, Simerly & Schatten, 1985) during fertilization events. Despite extensive studies of the cytoskeleton of early mouse embryos, little is known at present about the functional cooperation of cytoskeletal elements, or about the interaction of the cytoskeleton with plasma membrane.

It seems probable that PMA blocks cytokinesis of the second maturation division indirectly. Because karyokinesis of this division is not disturbed by PMA this excludes the action of the drug on the spindle microtubules. The present study reveals that PMA does not interfere with organized and functioning actin filaments during cytokinesis. Maybe PMA affects precytokinetic organization of actin and/or the connections of this cytoskeletal system with plasma membrane. An important initial event during extrusion of second polar body is the attachment of the equator of the meiotic spindle to the membrane. Thus the connection of the actin equatorial part of the meiotic spindle with the overlying egg plasma membrane could be a target site for PMA action.

The authors are indebted to Professor J. C. Czyba for supplying the PMA. This investigation was supported by the Academy of Agriculture (grant MR II10) and the Small Supplies Programme of the WHO.