THE JOURNAL OF EXPERIMENTAL ZOOLOGY 255~355-358 (1990)

Nuclear Transplantation in the Pig Embryo: Nuclear Swelling R.S. PRATHER, M.M. SIMS, AND N.L. FIRST Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211 (R.S.P.), and Department of Meat and Animal Science (M.M.S., N.L.F.), University of Wisconsin-Madison, Madison, Wisconsin 53706 ABSTRACT The transfer of nuclei from cleavage stage embryos to enucleated activated meiotic metaphase I1 oocytes results in a reprogramming of the transferred nucleus such that it behaves as a zygotic nucleus. One estimator of nuclear reprogramming is nuclear swelling after nuclear transfer. The diameter of nuclei after nuclear transfer was not found to be dependent upon the amount of cytoplasm transferred with the donor cell or the amount of cytoplasm in the recipient cell. Nuclei from 4-, 8-, and 16-cell stage embryos swelled to a similar diameter after nuclear transfer (26.9,27.3, and 27.2 pm, respectively) and this was significantly different from the diameter of contemporary donor embryos (18.3, 14.3,and 13.0 km, respectively).This is a swelling of 47,91, and 10910, respectively. Since the degree of nuclear swelling does not appear to be related to cytoplasmic volume it is concluded that the components mediating nuclear swelling are not in a limiting supply.

Nuclear transfer for the production of cloned individuals has recently been developed for the sheep, cow, rabbit, and pig (see review by Prather and First, '89). The ability t o produce large numbers of identical individuals is based upon the assumption that all of the nuclei in the donor embryo are genetically identical, that the recipient cells supplying cytoplasm are genetically identical (see review by Seidel, '831, and the transferred nucleus is reprogrammed to behave as a zygotic nucleus. The procedures for nuclear transplantation have been developed for amphibians and the resulting nuclear reprogramming has been described (see reviews by Gurdon, '86; DiBerardino, '89; Prather and First, '90a). The nuclear reprogramming observed in amphibian nuclei after transfer is gene specific (Wakefield and Gurdon, '83; Gurdon et al., '84).The morphological nuclear modifications that occur after nuclear transfer include swelling of the nucleus and disappearance of nucleoli, while biochemical changes include an exchange of proteins between the cytoplasm and the transferred nucleus and precise regulation of DNA synthesis. The nuclear transfer procedures presently employed for sheep (Willadsen, '86; Smith and Wilmut, '89), cattle (Prather et al., '871, rabbits (Stice and Robl, '88), and pigs (Prather et al., '89a) transfer a nucleus (blastomere) to an oocyte from which half of its cyto0 1990 WILEY-LISS, INC.

plasm was removed. In a n effort to begin t o describe changes in nuclear morphology that occur in mammals after nuclear transfer, pig embryonic nuclei were transferred to activated, enucleated metaphase I1 oocytes and the effect of the amount of cytoplasm in the recipient cell and donor cell on the degree of nuclear swelling was evaluated.

MATERIALS AND METHODS Embryos, micromanipulation, and membrane fusion Unfertilized oocytes and embryos were collected and manipulated as previously described (Prather et al., '89a). Briefly, the cells were treated with cytoskeletal inhibitors (cytochalasin B and colchicine) and the arrested metaphase I1 chromosomes were localized by using UV fluorescence (Prather and First, 'gob), and removed by aspiration into a micropipette. A donor blastomere or karyoplast was then aspirated from a donor embryo into the pipette and then deposited into the perivitelline space of the enucleated recipient oocyte. The bore of the transfer pipette was sufficiently small such that the donor nucleus could easily be visualized. Electrofusion was conducted Received November 13, 1989; revision accepted February 21, 1990. Address reprint requests to R.S. Prather, Department of Animal Sciences, 164 ASRC,University of Missouri, Columbia, MO 65211.

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as previously described (Prather et al., '89a) using a Zimmermann cell fusion unit or a BTX Electrocell Manipulator 200 (BTX, San Diego, CA) was used. No differences in the fusion rates or nuclear swelling were observed between the two cell fusion instruments. Electrofusion was monitored at 30 min intervals for 2 hr. Individual nuclear transfer embryos were rolled in the 50 pl drop of culture medium and visually assessed for fusion. Those embryos that had fused were cultured in vitro for 20 hr.

transferred only t o one-half oocytes. Each of these cell stage transfers was conducted on two separate occasions. Statistical analysis Nuclear diameter measurements were analyzed using the Student's t test (Snedecor and Cochran, '80).

RESULTS To assay the importance of cytoplasmic volume on the degree of swelling after nuclear transfer a In vitro culture 2 x 2 factorial design incorporated the transfer of Nuclear transfer embryos were cultured in an intact four-cell stage blastomere or a karyo50 p1 drops of N-2-hydroxyethylpiperazine-N1-2-plast (equivalent in cytoplasmic volume to an ethane sulfonic acid (HEPESI-buffered Tyrode's eight-cell blastomere) t o either a one-half enumedia (HbT; Bavister et al., '83) supplemented cleated oocyte or an intact enucleated oocyte (an with bovine serum albumin (3 mgiml, Sigma, St. oocyte from which a volume of cytoplasm equivaLouis, MO) and gentamicin sulfate (25 pg/ml, lent to, or slightly larger than, the first polar body Sigma) under paraffin oil, in a humidified in- was removed). There was no significant difference cubator at 37°C. This culture system allows one- in nuclear swelling between the treatments (Fig. and two- cell stage pig embryos t o develop to the 1). This experiment was repeated for 8- and 16morulahlastocyst stage (Hagen et al., '89). cell stage nuclei, except the entire blastomere was transferred t o either a one-half oocyte or an intact Nuclear diameter enucleated oocyte. Again there was no significant After culture, embryos were fixed in 70% meth- difference in the degree of swelling among the two anol:30% acetic acid, then rehydrated in phos- oocyte types (Fig. 2). phate-buffered saline containing 2 pg/ml DAPI. Since there were no differences between oocyte Embryos were stained for 4 h r at 4°C. A scale bar or karyoplast type within cell stages the data was projected on a monitor attached t o the micro- were grouped. Four-, 8-, 16-, and 32-cell stage nuscope and measured. Stained embryos were then clei all swelled significantly ( P < 0.05) after nuexcited through a UV 1A filter cube on a Nikon inverted Diaphot. Two diameter measurements NUCLEAR SWELLING IN EARLY PIG EMBRYOS were taken directly from the monitor, averaged, and converted to micrometers. A single nucleus, 30 -, I selected at random, was measured in control nonmanipulated 4-,8-, 16-, or 32-cell stage embryos 4CELL derived from the same flush as the donor embryos.

I

Experiments Four-cell stage blastomeres and four-cell karyoplasts (equivalent in volume to a n eight-cell stage blastomere) were transferred to enucleated onehalf oocytes or enucleated intact oocytes. Intact oocytes had their metaphase I1 chromosomes removed in a volume of cytoplasm equivalent t o the first polar body, whereas one-half oocytes had half of their cytoplasm removed. Thus the four-cell stage nuclear transfer experiments had a 2 x 2 factorial design and was conducted on four separate occasions. Eight- and 16-cell stage blastomeres were transferred to either one-half oocytes or intact oocytes, while 32-cell stage blastomeres were

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4K-112

40-1

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Fig. 1. Nuclear diameter after transfer of a n intact 4-cell stage blastomere or 4-cell stage karyoplast (equivalent in volume t o a n %cell stage blastomere) to either a one-half oocyte or a n intact oocyte (see text for full description).

NUCLEAR SWELLING AFTER NUCLEAR TRANSFER NUCLEAR DIAMETER AFTER NUCLEAR TRANSFER IN UQEMBRYOS

DIAMETER

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OF PIG EMBRYO NUCLEI

TT

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8

16

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64

CELL STAGE

Fig. 4. Nuclear diameter of 4- through 64-cell stage nuclei and a n appropriate regression equation

TVPE OF NUCLEUS

Fig. 2. Nuclear diameter after transfer of a n 8- or 16-cell stage blastomere to one-half oocyte or a n intact oocyte (see text for full description).

clear transfer and 20 h r of culture (Fig. 3). Fourcell stage nuclei swelled from 18.3 to 26.9 pm ( n = 13, n = 32, respectively), 8-cell stage nuclei from 14.3 to 27.3 pm ( n = 15, n = 11, respectively), 16-cell stage nuclei from 13.0 to 27.2 pm ( n = 13, n = 15, respectively), and 32-cell stage nuclei from 10.8 to 21.4 pm ( n = 10, n = 3, respectively). This represents a 47, 91, 109, and 9896, increase in nuclear diameter, respectively. Comparisons between cell stages can be done only retrospectively, as the experiments were conducted on separate days. A retrospective analysis suggests that the diameter of embryonic nuclei progressively decreases after the four-cell stage (Fig. 4). Four-cell

stages nuclei have a diameter of 18.3 pm. The diameter is significantly less (P< 0.05) for the 8(14.3 pm), 16- (13.0 pm), 32- (10.8 pm), and 64(9.2 pm) cell stage nuclei, although the 32- and 64-cell stage nuclei were not significantly different. The resulting equation best fit the data: y = 22.1 - 4 . 3 5 ~+ 0.348x2, with a n R2 of 0.98; where y = diameter (in pm) and x = cell stage.

DISCUSSION Nuclear modifications after nuclear transfer t o enucleated activated oocytes have been well defined for the amphibian (Gurdon, '86; DiBerardino, '89). However, nuclear transfer for cloning has only recently been successful in mammals (Prather and First, '89). Some nuclear modifications after nuclear transfer have been described in mammals, including nuclear swelling (Stice and Robl, ,891, a n exchange of lamin proteins NUCLEAR SWELLING AFTER NUCLEAR TRANSFER IN THE PIG (Prather et al., '89b), and a modification of nucleolar morphology (Czolowska et al., '84), which all I may be dependent upon the simultaneous transDOJORCBLGTME 30 T fer of the nuclei and activation of the oocyte (Szollosi et al., '88). Since nucler swelling was one of the first morphological changes described for "reprogrammed" nuclei (Gurdon, '64) i t was decided t o evaluate this characteristic in mammalian nuclear transfer embryos. The nuclear transfer procedures presently employed for the sheep (Willadsen, '86; Smith and Wilmut, ,891, cattle (Prather et al., '87), rabbits (Stice and Robl, '881, and pigs (Prather et al., '89a) transfer a nucleus (blastomere) t o an oocyte from CCELL WELL lCCELL 3bCELL Fig. 3. Nuclear diameter after transfer of 4-,8-, 16-, or 32- which half of its cytoplasm was removed. The macell stage nuclei to enucleated activated meiotic metaphase I1 jor question to be answered was: Does the amount oocytes. of cytoplasm in the donor cell or recipient cell afMKLEPRTRLNSRF

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fect reprogramming? This was indirectly assessed by observing nuclear swelling after nuclear transfer. If there was a direct competition for the DNA by cytoplasmic proteins from the donor and recipient cytoplasm differential degrees of nuclear swelling would have been observed between the different treatments. The least amount of nuclear swelling would be expected in the four-cell stage blastomere transferred t o a one-half oocyte group. The greatest degree of nuclear swelling would have been expected in the four-cell stage karyoplast transferred to an intact oocyte. However, the degree of swelling after nuclear transfer was not different between the different types of nuclear transfers, i.e., blastomere vs. karyoplast and onehalf oocyte vs. intact oocyte. Interestingly, the retrospective analysis suggested that nuclear diameter decreases during the early cleavage stages of the pig, but that the degree of swelling after transfer was similar for the 4-, 8- and 16-cell stage nuclei. This again suggests that the components responsible for the observed swelling are not limiting as is the case when multiple fertilization occurs in mouse oocytes (Czolowska et al., '84). Thus these results suggest that the morphological observation of nuclear swelling is not limited by the amount of cytoplasm represented by the two different sources used in this study. It should, however, be noted that the degree of nuclear swelling may not be an indicator of subsequent development. Similar experiments evaluating the development potential of these types of nuclear transfers need t o be completed.

ACKNOWLEDGMENTS This manuscript was prepared while supported by the Cooperative State Research Service, U.S. Department of Agriculture under agreement No. 88-37240-3755, the University of WisconsinMadison College of Agriculture and Life Sciences, and is a contribution from the Missouri Agricultural Experiment Station Journal Series Number 10,964. LITERATURE CITED Bavister, B.D., M.L. Leibfried, and G. Lieberman (1983) Development of preimplantation embryos of the golden ham-

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