'Blebbing' of the nuclear envelope of mouse zygotes, early embryos and hybrid cells MARIA S. SZOLLOSI* and DANIEL SZOLLOSI Vniti de Biologie de la Fe'coiidation, Station de Physiologie animale, INRA, 78350 Jouy-en-Josas, Fiance * Previous papers of the author have been published under the name "Soitynska" Summary In the mouse zygote and in two-cell stage embryos and embryonic (blastomere of 8-cell stage embryo) the inner leaflet of the nuclear envelope of pronuclei nuclei following cell hybridization with activated and that of blastomere and polar body II nuclei oocytes. Their response to the oocyte cytoplasm by evaginate, forming multiple blebs within the peri- initiating blebbing depends on: (1) the position of nuclear space, which contains a granular material. the host cell in its cell cycle at the moment of Blebbing exists only in oocytes activated by sperm hybridization, and (2) the time spent by the foreign in vivo or in vitro, or parthenogenetically by treat- nuclei in the host cytoplasm following cell fusion. If ment with ethanol or puromycin. The germinal donor nuclei are introduced close to the time of vesicle and an interphase nucleus formed after activation, they start to produce blebs at the time treatment of the oocyte at metaphase I by puromy- corresponding to the initiation of blebbing by the cin do not form blebs. Formation of blebs is specifi- female pronucleus in the first cell cycle. If foreign cally located in the cell cycle. The burst of the nuclei are introduced a few hours after activation blebbing activity occurs during the first half of the they must be incubated in the host cytoplasm for cell cycle in one-cell embryos and in the earliest some time before initiation of bleb formation, interphase period in the second cell cycle. Blebbing provided that the host pronucleus has initiated ceases from the beginning of the third cell cycle. blebbing by that time. The occurrence in the cytoplasm of 'double- The existence of blebbing in nuclei formed only layered' vesicles containing granular material re- after oocyte activation, and the timing and the sembling bleb contents and the disappearance of general occurrence of this event during the earliest blebs from the nuclear envelope by the end of the cleavage stages of almost every mammalian em- cell cycle provide evidence that blebs represent a bryo, suggest that this special nucleocytoplasmic step in the transport of some material from the transport plays a specific role at the beginning of nucleus to the cytoplasm. Ethanolic phosphotung- development. stic acid does not stain blebs, suggesting the ab- sence of basic protein in their contents. Key words: blebs, nuclear envelope, early mouse embryo, Blebbing can be induced in somatic (thymocyte) cell hybrids. Introduction used term in the literature. Nuclear envelope bleb formation was described for the In almost all mammalian zygotes studied by electron first time in rat pronuclei by Szollosi (1965) and later in microscopy, small evaginations of the inner leaflet of the rabbit zygotes by Gulyas (19716). In the rat bleb nuclear envelope (NE) have been observed in both formation ceases at the end of the first cell cycle, but in pronuclei, which project into the perinuclear space. the rabbit the blebbing process has been observed not Evaginations of various sizes containing an electron- only in pronucleate eggs (Zamboni & Mastroianni, 1966; dense, non-homogeneous material have been referred to Gondos & Bhiraleus, 1970; Gulyas, 1971a) but also in in the literature as tertiary nucleoli (Szollbsi, 1965), blebs two-cell (Gulyas, 19716; Longo, 1978) and four-cell (Gondos & Bhiraleus, 1970; Gulyas, 1971a; Longo, embryos (D. Szollosi, unpublished data). Blebs have 1978) and nuclear extrusions (Gulyas, 19716). In the been described also in rabbit blastocysts by Hadek & present work we will use the term 'bleb' and 'blebbing' as Swift (1962). Among other mammals in which nuclear the most descriptive, most neutral and most commonly envelope blebs have been detected in zygotes are: sheep Journal of Cell Science 91, 257-267 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 257 (Crozet et al. 1987), cattle (Crozet, 1984), chimpanzees Whittingham's medium for fertilization in vitro (FIV) (Fraser (Maul, 1977), golden hamsters, two species of wild & Drury, 1975). Puromycin was used at a concentration of mouse (Mus cervicolor, M. spretus) and all the examined 100/(gml~ of culture medium. All culture was carried out strains of house mouse (M. musculus) (D. Szollosi, under paraffin oil. unpublished). In the laboratory mouse, blebbing has a Ovarian oocytes were collected from ovaries 48 h after injec- tion of PMSG. They were cultured in medium M16 (Whitt- limited duration because blebs have never been seen in ingham, 1971) for 9-5 h. Oocytes in metaphase I stage were morulae or in blastocysts (M. Szollosi, unpublished). In transferred to medium M16 containing puromycin for 13 h of the human, blebs have been described in pronucleate culture. Oocytes that developed nuclei and extruded PB1 were eggs (Zamboni et al. 1986; Santhananthan & Trounson, fixed for EM. 1985), and in two-cell (Pereda & Coppo, 1987), four-cell Ovulated oocytes were collected 16 h after hCG injection, and (Sundstrome^a/. 1981) and nine-cell embryos (Lopatae? after removal of follicular cells were divided into four groups: al. 1983). However, blebbing was never observed either (1) oocytes cultured for 9h in M16 containing puromycin; in zygotes or in any early embryonic stages of the oocytes that developed pronuclei, i.e. were activated by drugs, domestic pig (Szollosi & Hunter, 1973; D. Szollosi, were fixed for EM; (2) zona-free oocytes inseminated in me- unpublished). Nuclear blebbing has also been described dium for 2 h containing puromycin and then transferred to M16 in oocytes from foetal and prepubertal ovaries in cattle, with puromycin for 7 h of culture; (3) zona-free oocytes insemi- nated and cultured as for group (2), but both media contained rhesus monkeys and humans (Baker & Franchi, 1969), tritiated methionine at a final concentration of 20juCiml~ ) in but they have never been seen in nuclei (germinal addition to puromycin; (4) zona-free oocytes inseminated and vesicles) of ovarian oocytes from the large antral follicles cultured as for group (2) but both media contained tritiated of adult females of rabbit, mouse, pig, sheep and humans methionine and no puromycin, in order to control the influence (D. Szollosi, unpublished). of puromycin on methionine incorporation. Fertilized oocytes The above information strongly suggests that blebbing were fixed for EM. of the nuclear envelope is a common event during early Autoradiography mammalian development. Surprisingly, this phenom- Semithin sections of oocytes from groups (3) and (4) were enon is known from fragmentary studies only, and its placed on slides and dried. They were coated with emulsion biological significance is at present far from being under- (Ilford K-5, stored for 6 days in a light-proof box at 4°C) then stood. Because fertilization and early embryonic develop- developed in D-19 (Eastman Kodak) and stained with Richard- ment of the laboratory mouse can be easily controlled and son's Methyl Blue-Azur B (Richardson et al. 1960). experimentally investigated we carried out a detailed study on the dynamics of blebbing shown by the nuclear Oocyte-thymocyte and oocyte-blastomere hybrids envelope in this species. Ovulated oocytes were obtained from the oviducts of Swiss albino mice (superovulated with PMSG and hCG as above) 14-16h after hCG administration. After dispersing the cumu- 1 Materials and methods lus cells with hyaluronidase (300i.u. ml" ) and digestion of zona pellucida with 05 % Pronase, zona-free oocytes were Ovarian oocytes placed in drops of Whitten's medium (Whitten, 1971) or in medium 16 under liquid paraffin and cultured for 0-5-2-5h Swiss albino female mice (8-10 weeks old) were treated by an (37°C, 5 % CO in air) before handling. intraperitoneal injection of 5i.u. of pregnant mare's serum 2 Thymocytes were obtained from thymus of 1- to 5-day-old gonadotrophin (PMSG) for development of ovarian follicles. Oocytes were removed, 48 h after PMSG injection, from newborn mice as described by Czolowska et al. (1984). ovarian follicles with a sharp sterile needle in medium 2 (Quinn Blastomeres were isolated from eight-cell mouse embryos of 1 Swiss albino females mated with Fl (C57 BL/10 X CBA) males et al. 1982) containing dibutyryl cyclic AMP (100/igmr ) and on the third day of pregnancy. A few embryos with less than fixed for electron microscopy (EM). eight cells were always present among the uncompacted eight- cell embryos. Thus they were considered to be embryos from Zygote and embryos the first half of the fourth cell cycle. Ovulation was induced in female mice of CBA-T6T6 and CBA- Oocytes, either before or after cell hybridization, were H strains by intraperitoneal injections of PMSG and human activated with 7-8 % ethanol in culture medium for 5 min at chorionic gonadotrophin (hCG; 5-10i.u. of each), at 48-h room temperature under liquid paraffin, according to the intervals, and females were mated with males of their own method of Cuthbertson (Cuthbertson et al. 1981; Cuthbertson, strain. Fertilized eggs were flushed from the oviduct at almost 1983). hourly intervals between 16 and 34 h after hCG administration, Cell fusion was achieved by agglutinating thymocytes or a and two-cell stage embryos were removed at 32, 34, 39, 40, 50, blastomere to an oocyte with phytohaemagglutinin and treating 56 h after hCG, three- to four-cell embryos at 50, 56 h, and six- agglutinated cells with polyethylene glycol (PEG: Loba-Che- to eight-cell embryos at 66h after hCG. Embryos were fixed mie Mr1000 50% (w/v)) and Fluka (/Wr2000 45% (w/v)).