THE JOURNAL OF EXPERIMENTAL ZOOLOGY 253:220-225 (1990)

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Preparation of Nucleate and Anucleate Fragments of Hamster and Mouse Eggs by Centrifugation R. YANAGIMACHI AND C.H. YANG Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822 ABSTRACT Nucleate and anucleate fragments of hamster and mouse eggs can be prepared by centrifuging zona-free eggs in a Percoll gradient. These fragments are fertilizable and can be used for various studies of nucleus-ooplasm interactions.

To study nucleus-ooplasm interactions in mammals, it is often desirable t o prepare eggs without nuclei. In the past, this has been done by removing nuclei from eggs using a micropipette (see, e.g., McGrath and Solter, '86) or microsurgically dissecting each egg into two (see, e.g., Tarkowski, '80).We report here that a large number of nucleate and anucleate fragments of mammalian (hamster and mouse) eggs can be prepared rapidly by centrifuging eggs after removal of their zonae pellucidae. Fragments thus obtained are fertilizable.

MATERIALS AND METHODS The medium used for handling eggs before and during centrifugation was a HEPES/NaHC03buffered medium, M2 (Quinn et al., '82), supplemented with 4 mg/ml bovine serum albumin (BSA) and 10 pgiml gentamicin sulfate. HEPES was neutralized and freeze-dried prior t o use. The pH of the medium was adjusted to 7.4 by adding a small quantity of 1 M NaOH. The medium was kept at 4°C and used within 3 days of preparation. Special reagents used in this study and their sources were as follows: BSA, Fr. V (Calbiochem, La Jolla, CAI; HEPES (Research Organics, Cleveland, OH); bovine testicular hyaluronidase (300 USP units/mg) and bovine pancreatic trypsin (100 TAME units/mg) (ICN Pharmaceutical, Cleveland, OH); bovine pancreatic u-chymotrypsin (43 BTEE unitdmg), cytochalasin D (CD), gentamicin sulfate, Hoechst 33342, and Percoll (Sigma Chemical Co., St. Louis, MO); and lysolecithin (Avanti Polar Lipids, Birmingham, AL). Mature unfertilized eggs of the golden hamster and Swiss albino mouse were collected from oviducts of superovulated females within 4-5 hr after ovulation (Yanagimachi, '69). Hamster eggs 0 1990 WILEY-LISS, INC.

were freed from cumulus cells by treating them for 5-10 min in M2 containing 0.1% hyaluronidase. After rinsing in M2, they were treated for 1-2 min with 0.1% trypsin in M2 to dissolve the zonae pellucidae. Zona-free eggs thus obtained were rinsed thoroughly in M2 before being used in experiments. Zona-free mouse eggs were similarly prepared except that zona were dissolved by 4-10 min of treatment of the eggs with 0.001% u-chymotrypsin in M2. The zonae of 20-30% of mouse eggs were not dissolved even if they were drawn in and pushed out of a small-bore pipette repeatedly in the chymotrypsin solution. These eggs were discarded. The method for centrifuging zona-free eggs has been described previously (Yang and Yanagimachi, '89). Briefly, 100 pl each of 45%, 30%, and 7.5% Percoll in M2 was placed sequentially in a 400 pl plastic microcentrifuge tube (5 x 45 mm, heparin-coated; Beckman Instruments, Palo Alto, CA), and a drop of M2 containing the zona-free eggs was placed in the 7.5% Percoll layer. The tube was put in a high-speed microcentrifuge (Model Z-230-M, Vangard International, Neptune, NJ) and centrifuged at 9,500g. During centrifugation, eggs migrated to the 30% Percoll layer, elongated, and then divided into fragments. After centrifugation for varying lengths of time, the contents of the tube were gently sucked into a large-bore pipette and transferred t o a watchglass containing M2 to search for eggs and egg fragments under a dissecting microscope. Occasionally, we fixed eggs and egg fragments with 2% glutaraldehyde in M2 immediately after their recovery from the centrifuge tube. This was necessary to determine the precise location of chromoReceived July 28, 1989; revision accepted September 21, 1989.

CENTRIFUGATION OF MAMMALIAN EGGS

somes within the eggs and fragments before the eggs or fragments "round up." The eggs and fragments were examined with a phase-contrast microscope after mounting them between a slide and coverslip (Yang and Yanagimachi, '89). Some zona-free eggs were centrifuged without treatment. Other eggs were centrifuged after treating them for 30 min with M2 containing Hoechst 33342 (10 pg/ml) and/or CD (10 pM). Hoechst 33342 facilitated later examination of chromosomes. CD accelerated the fragmentation of eggs by centrifugation. All the procedures mentioned above were performed at 25-27°C in air. Some fragments were inseminated in vitro. Hamster spermatozoa were induced to undergo the acrosome reaction by incubating them for 1.5-2.0 hr in m-TALP-2 medium and then exposing them to 50 pg/ml lysolecithin for 15 min (Ohzu and Y anagimachi, '82). Mouse spermatozoa were incubated for 2 hr in TYH medium (Toyoda et al., '71) before being used for insemination. Both hamster and mouse eggs were inseminated (final sperm concentration 104/ml) and incubated in TYH medium. After 1-2 hr of incubation at 38°C under 5% COz in air, eggs were examined for evidence of fertilization. An egg was recorded fertilized when at least one decondensing sperm nucleus or sperm pronucleus was seen within the ooplasm.

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RESULTS Zona-free hamster eggs were separated into two or more fragments by 10-13 min of centrifugation at 9,500g (Yang and Yanagimachi, '89; Yang et al., '89; Fig. 1). When 20 eggs were examined shortly before they separated into two or more fragments, chromosomes (meiotic spindles) were found at various locations in elongated eggs. Apparently, chromosomes did not move from their original position in the egg cortex during centrifugation (Yang et al., '89). This explains why we found both light and heavy fragments, some with and some without chromosomes. The time needed for the separation of hamster eggs was reduced greatly when the eggs were treated with 10 p M CD for 30 min. Centrifugation for 35 sec at 9,500g was enough to separate all of the eggs into fragments. When the fragments were sorted into three groups according t o their size and examined for the presence or absence of chromosomes, none of 44 large fragments (larger than 64 pm in diameter) had chromosomes, whereas 68% of 74 smaller fragments (44-62 pm in diameter) and 17% of the smallest fragments

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Fig. 1. Diagrams showing the probable manner of separation of a hamster egg into two or more fragments by centrifugation. The midregion of an elongated egg is "pinched off" a t one place (a) or two or more places (b) during centrifugation, resulting in the production or two or more fragments. Fragments round up after centrifugation.

(smaller than 40 pm in diameter) contained chromosomes (Figs. 3, 9A). All the large fragments were believed t o be derived from the centrifugal (heavier) portion of the egg because chromosomes moved t o the centripetal pole of CDtreated eggs during centrifugation (Fig. 4).When CD-treated eggs were centrifuged for 60 sec, all eggs were separated into small fragments (39 2 8.3 pm in diameter, range 23-52 pm), some with and most without chromosomes. Fragments were even smaller (29 L 5.3 pm in diameter, range 1741 pm) when centrifuged for 10 min. Zona-free mouse eggs were also separated into fragments by centrifugation at 9,500g. It took 1520 rnin before intact eggs (without CD treatment) began t o separate. Chromosomes were found in the midregion of the egg before separation (Fig. 5b, c), and were in all of 41 light fragments after separation (Fig. 5d). As was the case in the hamster, CD treatment accelerated fragmentation of eggs by centrifugation. All the eggs were separated into two or more fragments by 5 min (not 2 min) of centrifugation at 9,500g. Chromosomes were absent in all but one of 40 large fragments (larger than 58 pm in diameter) (Fig. 7). About half of 37 small fragments (smaller than 52 pm in diameter) contained chromosomes (Figs. 8, 9B).

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CENTRIFUGATION OF MAMMALIAN EGGS

When 43 small (lighter) and 57 large (heavier) fragments of CD-treated hamster eggs were inseminated in vitro and examined 1-2 hr later, all of them were fertilized (Fig. 10). Similarly, 85%of 20 small (lighter) and 92% of 36 large (heavier) fragments of CD-treated mouse eggs were fertilized when examined 1-2 hr after insemination (Fig. 11).

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0 0 0 o o DISCUSSION 00 0 0 o 00 ao 00 I I I , 1 so We have previously reported that centrifugation of zona-free hamster eggs results in the production of nucleate and anucleate fragments that are fertilizable (Yang et al., '89). We report here B: Mouse that nucleate and anucleate fragments obtained from CD-treated eggs are also fertilizable. Al-63t3.0 0 a though CD treatment is not essential for obtain0 2 : ing egg fragments, it certainly accelerates fragmentation. It is interesting that hamster and mouse eggs respond to centrifugation differently when they are not treated with CD. For hamster eggs, chroD i a m e t e r I p m l o f egg f r a g m e n t mosomes do not move from their original position in the egg cortex during centrifugation at Fig. 9. Relationship between the size (diameter) of egg 9,500g. Consequently, chromosomes go to either fragment and the presenceiabsence of chromosomes in the light or heavy fragments (Yang et al., '89; this fragment. A: Hamster. B: Mouse. Arrows indicate the diamestudy). For the mouse egg, on the contrary, chro- ters of hamster and mouse eggs before centrifugation. mosomes move t o the midregion of the egg during centrifugation and are almost always incorporated into the lighter fragments. The heavier ently, the metaphase I1 chromosomes (spindles) of fragments seldom contain chromosomes. Appar- the mouse egg are much less tightly anchored to the egg cortex than those of the hamster egg. Acceleration of egg fragmentation by CD treatFigs. 2-8. Hoechst fluorescence (Figs. 2-4, 6-8) and ment was impressive, particularly in the hamphase-contrast (Fig. 5) micrographs of hamster and mouse ster. Centrifugation for 35 sec at 9,5009 was eggs before and after centrifugation. All eggs, except for those in Figure 5, were treated with CD prior to centrifugation. enough to separate all of the CD-treated hamster Magnification of all figures x 300. eggs into fragments. Neither the light nor the heavy fragments of CD-treated hamster egg Fig. 2. Hamster eggs before centrifugation. showed distinct stratification of their cytoplasmic Fig. 3. Two large hamster egg fragments without chromo- inclusions after 35 sec of centrifugation. somes, and eight small fragments with or without chromoIn both the hamster and the mouse, it is the somes. heavy (large) fragments that are consistently Fig. 4. a, b: Hamster eggs shortly before fragmentation; (hamster) or almost consistently (mouse) devoid note that chromosomes move to the centripetal pole of the of chromosomes. If we are interested in obtaining egg. An arrow indicates the direction of centrifugation. a pure population of large fragments from the Fig. 5. Mouse eggs before centrifugation (a) and at vari- mixed population of egg fragments, placing one or ous stages of separation into two fragments (b-e). ch, two glass beads of known size (e.g., 60 pm; Duke Chromosomes. Scientific Corp., Palo Alto, CA) in the dish as a standard reference would facilitate their sepaFig. 6. Mouse eggs before centrifugation ration. Fig. 7. Four large mouse egg fragments without chroIt was somewhat disappointing that we could mosomes. not separate a pure population of nucleated (light Fig. 8. Seven small mouse egg fragments with or without and small) fragments under the dissecting microchromosomes. scope. In some of our experiments, all small frag0

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R. YANAGIMACHI AND C.H. YANG

ments of CD-treated hamster eggs had chromosomes; in other experiments, only about 50% of small fragments contained chromosomes. This seems to be due to the variation in the manner of fragmentation of eggs during centrifugation. If all the eggs separate into two fragments, all the small fragments will have chromosomes. On the other hand, if only some of the eggs separate into two and the rest separate into three or more fragments, then there will be small fragments with and without chromosomes. If we are able to prevent multiple fragmentation of the egg, we should be able to obtain a pure populations of small, nucleate fragments. Various biological and physical conditions (e.g., “aging” of the eggs before and after collecting them from the oviduct and the temperature within the centrifuge) seem t o influence the speed and manner of fragmentation. As we learn more about these conditions, we may be able t o avoid multiple fragmentation of the egg. Both nucleate and anucleate egg fragments will provide an interesting experimental system. For example, we should be able to obtain valuable information about the nucleus-ooplasm relationship by inseminating nucleated fragments containing various amounts of ooplasm. Anucleate fragments can be used as the “ooplasm-carrying liposomes” in introducing the ooplasm of one species into the egg of another species. These fragments will also be very useful in studying the biology and chemistry of sperm nuclei in the ooplasm without intervention from the egg nuclei. Karyotyping of human spermatozoa using zona-free hamster eggs (Rudak et al., ’78; Martin, ’SS), for example, could be done more easily by using anucleate fragments instead of intact eggs with maternal (hamster) chromosomes. All the chromosomes appearing in these anucleate fragments are undoubtedly of sperm (human) origin.

ACKNOWLEDGMENTS This study was supported by a grant from the National Institute of Health (HD-03402). We thank Mr. T. Timothy Smith and Mrs. Hiroko Yanagimachi for their assistance in the preparation of the manuscript. Fig. 10. Small (a) and large (b) fragments of CD-treated hamster eggs, penetrated by spermatozoa, photographed at 1 hr after insemination. Arrows indicate decondensed sperm nuclei in the ooplasm. Fig. 11. Small (a) and large (b) fragments of CD-treated mouse eggs, penetrated by spermatozoa, photographed at 1hr after insemination. Arrows indicate decondensed sperm nuclei in the ooplasm.

LITERATURE CITED Martin, R.H. (1988) Human sperm karyotyping: A tool for the study of aneuploidy. In: Aneuploidy: Part B. B.K. Vig and A.A. Sandberg, eds. Alan R. Liss, Inc., New York, pp. 297316. McGrath, J., and D. Solter (1986) Nuclear and cytoplasmic transfer in mammalian embryos. In: Developmental Biol-

CENTRIFUGATION OF MAMMALIAN EGGS ogy. L.W. Browder, ed.; R.B.L. Gwatkin, vol. ed. Plenum Press, New York, Vol. 4, pp. 37-55.

Ohzu, E., and R. Yanagimachi (1982) Acceleration of acrosome reaction in hamster spermatozoa by lysolecithin. J. Exp. Zool., 224:259-263. Quinn, P., C. Barros, and D.G. Whittingham (1982) Preservation of hamster oocytes t o assay the fertilizing capacity of human spermatozoa. J. Reprod. Fertil., 66:161-168. Rudak, E., P.A. Jacobs, and R. Yanagimachi (1978) Direct analysis of the chromosome constitution of human spermatozoa. Nature, 274:911-912. Tarkowski, A.K. (1980) Fertilization of nucleate and anucleate egg fragments in the mouse. Exp. Cell Res., 128:7377.

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Toyoda, Y., M. Yokoyama, and T. Hoshi (1971) Studies on the fertilization of mouse eggs in vitro. I. In vitro fertilization of eggs by epididymal sperm. Jpn. J. Anim. Reprod., 14:147151. Yanagimachi, R. (1969) In vitro capacitation of hamster spermatozoa by follicular fluid. J. Reprod. Fertil., 18:275-286. Yang, C.H., and R. Yanagimachi (1989) Differences between mature ovarian and oviductal oocytes: A study using the golden hamster. Hum. Reprod., 4:63-71. Yang, C.H., R. Yanagimachi, and H. Yanagimachi (1989) Morphology and fertilizability of zona-free hamster eggs separated into halves and quarters by centrifugation. Biol. Reprod., in press.

Preparation of nucleate and anucleate fragments of hamster and mouse eggs by centrifugation.

Nucleate and anucleate fragments of hamster and mouse eggs can be prepared by centrifuging zona-free eggs in a Percoll gradient. These fragments are f...
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