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a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211-5300
b Korea Research Institute of Biosciences & Biotechnology, Taejon 305-600, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The medium used for oocyte maturation (IVM medium) was BSA-free North Carolina State University (NCSU) 23 medium [33] supplemented with 10% (v:v) porcine follicular fluid, 0.01 mg/ml epidermal growth factor, 0.1 mg/ml cysteine, 10 IU/ml each of eCG and hCG (Intervet America Inc., Millsboro, DE). The fertilization medium, designated modified Tris-buffered medium (mTBM), consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2 2H2O, 20 mM Tris (crystallized free base; Fisher Scientific, Fair Lawn, NJ), 11 mM glucose, 5 mM sodium pyruvate, and no antibiotics. Porcine follicular fluid was collected from follicles 3–6 mm in diameter, centrifuged at 1900 x g for 30 min at 4°C, filtered through 1.2-µm syringe filters (Gelman Sciences, Ann Arbor, MI), and stored in aliquots at -20°C until use.
In Vitro Maturation (IVM)
The ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory in 0.9% saline containing 75 mg/ml potassium penicillin G and 50 mg/ml streptomycin sulfate maintained at 25–30°C. Immature oocytes were obtained from follicles with a diameter of 3–6 mm using a 20-gauge needle connected to a 10-ml disposable syringe. Follicular contents were pooled into a 50-ml conical tube (Fisher Scientific) and allowed to sediment. Supernatant was discarded, and the sediment was washed with Tyrode's lactate (TL)-Hepes supplemented with 0.01% polyvinyl alcohol (PVA) medium (TL-Hepes-PVA) using an Emcon filter (Reproduction Resources, Hebron, IL). Oocytes surrounded by a compact cumulus mass and having evenly granulated cytoplasm were selected and washed twice with approximately 3 ml of TL-Hepes-PVA medium. Then 50 oocytes were placed into 0.5 ml of IVM medium supplemented with eCG and hCG in each well of a Nunc 4-well multidish (Nunc, Roskilde, Denmark). The medium was covered with warm paraffin oil (light mineral oil; Fisher) and equilibrated at 39°C, 5% CO2 in air for at least 2 h prior to use. After 20–22 h of maturation culture, oocytes were washed twice with TL-Hepes-PVA medium and then further cultured in 0.5 ml of the same IVM medium without hormonal supplements (eCG and hCG) for 20–22 h.
In Vitro Fertilization (IVF)
After the end of the IVM period, IVF of pig oocytes was performed as previously described [32]. Briefly, the oocytes were treated with 0.1% hyaluronidase in NCSU 23 medium to remove cumulus cells and washed 3 times with mTBM containing 1 mM caffeine and 1 mg/ml BSA (A 7888). After washing, 35–40 denuded oocytes were placed into 50-µl drops of fertilization medium that had been covered with warm paraffin oil in a 35 x 10-mm2 polystyrene culture dish (Becton Dickinson, Lincoln Park, NJ). The oocytes were incubated for about 30 min before spermatozoa were added. A frozen semen pellet was thawed and washed 3 times by centrifugation at 1900 x g for 4 min in Dulbecco's PBS (DPBS; Gibco Life Technologies Inc., Grand Island, NY) supplemented with 1 mg/ml BSA, 75 mg/ml potassium penicillin G, and 50 mg/ml streptomycin sulfate (pH 7.2). At the end of the washing procedure, the sperm pellet was resuspended in the fertilization medium of mTBM containing 1 mM caffeine and 1 mg/ml BSA. After appropriate dilution, 50 µl of the sperm suspension was added to 50 µl of the fertilization medium containing oocytes to give a final sperm concentration of 1.5 x 105 cells/ml. Oocytes were coincubated with spermatozoa for 5–6 h at 39°C in an atmosphere of 5% CO2 in air.
Classification of 2PN and PPN Eggs
Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g in a microcentrifuge (Fisher Scientific) for 10 min to stratify the cytoplasm and permit visualization of the pronuclei [34] and then washed twice with TL-Hepes medium supplemented with 1 mg/ml BSA (TL-Hepes-BSA medium). According to pronuclear numbers, the eggs were individually classified into 2PN and PPN eggs by use of a micromanipulator (Leitz, Wetzlar, Germany) under x200 magnification on a warm plate (37°C). Unclear, unfertilized, and fragmented eggs were excluded from experimental groups. The validity of assignments of these eggs after classification was first evaluated by using an aceto orcein staining method. The accuracy rate for classification into 2PN and PPN eggs was 94.6% (105 of 111) and 96.8% (61 of 63), respectively. After classification, 2PN and PPN eggs were washed twice with the same medium and then cultured in 0.5 ml of NCSU medium supplemented with 4 mg/ml BSA (A8022) in each well of a Nunc 4-well multidish for approximately 12 h prior to embryo transfer.
Embryo Transfer of 2PN and PPN Pig Eggs
Experiments were conducted according to Institutional Animal Care and Use Committee guidelines. Prepubertal gilts aged 165–180 days were injected with 1000 IU eCG followed by injection of 500 IU hCG 96 h later. The next morning they were observed for the onset of standing estrus, and gilts exhibiting estrus were then used as recipients. The first day of estrus was designated Day 1 of the cycle. The synchronized gilts (Day 2) were anesthetized by injection of 23 ml of 5% sodium thiopental (Abbott Laboratories, North Chicago, IL) followed by 2–5% halothane (Halocarbon Laboratories, River Eagle, NJ) during surgery. The gilt was subjected to a midventral laparotomy, and the reproductive organs including uteri, oviducts, and ovaries were exteriorized. Eggs (30 to 35) at the 1-cell stage were transferred into one oviduct of the recipient to examine the in vivo development of the 2PN and PPN eggs.
Examination of Actin Filaments and Microtubules by Confocal Microscopy
In some replicates, embryos were fixed with 3.7% paraformaldehyde in PBS at 4°C overnight. The embryos were first cultured in a blocking solution of PBS containing 2% BSA and 150 mM glycine for 30 min at room temperature. After washing for 1 h, the embryos were stained with 10 IU/ml rhodamine-phalloidin (for actin filaments) or 1 µg/ml fluorescein isothiocyanate (FITC)-conjugated anti-
-tubulin (for microtubules) for 1 h at 39°C in PBS-Tween 20 (0.1%, v:v). After 2 washes in PBS-Tween solution for 2 h at room temperature, the embryos were stained with 100 nM YO-Pro-1 iodide or 10 µg/ml propidium iodide for 5–10 min for detection of DNA. The embryos were mounted on glass slides and examined by confocal microscopy. Confocal microscopy was performed by using a Bio-Rad (Richmond, CA) MRC-600 equipped with a krypton argon ion. The laser scanning for an image was repeated 5 times for 5 sec to improve the signal-to-noise ratio.
Ploidy of Embryos and PPN-Derived Fetuses
Porcine embryonic fibroblasts were isolated from the whole body of each Day 40 fetus from which the head and internal organs had been cut off. The primary culture was in a 100-mm dish in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum at 39°C, 5% CO2 in air. The subcultures were seeded with 1–2 million cells and cultured for 2–3 days (at least a week for the triploid cells) until confluent. Fibroblasts of the 4th passage were cultured in DMEM containing colcemid (0.05 µg/ml) overnight, harvested, and treated in hypotonic solution (0.6% sodium citrate) for 30 min at room temperature. The cells were fixed with methanol:glacial acetic acid (3:1) twice and then dropped on glass slides. After air drying, the slides were stained with 5% (v:v) Giemsa for 5 min and washed with distilled water. Metaphase spreads were observed on a microscope under x600 magnification. In the case of embryo samples, blastocysts were cultured in NCSU medium containing colcemid (0.05 µg/ml) overnight. Thereafter, a single blastocyst was treated with 0.56% KCl for 5 min, directly mounted on a glass slide, and then fixed. The subsequent methods were the same as described above for fibroblasts. For blood samples, metaphase spreads were prepared as described by Verma and Babu [35]. Pig chromosomes were karyotyped according to standard karyotype of the domestic pig [36].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously reported that PPN pig eggs could develop to the blastocyst stage in vitro; however, they have a smaller number of inner cell mass nuclei than do blastocysts derived from 2PN eggs. These results led us to examine whether the PPN eggs continue to develop after implantation. Sixteen fetuses were obtained at 40 days after transfer from three animals that had received PPN eggs (Table 1). Implantation rate (17.0%, 17 of 100) of PPN eggs was similar to that (12.5%, 2 of 16) of 2PN eggs (Table 2). Moreover, the size and morphology of most PPN fetuses appeared to be normal in comparison to 2PN fetuses, although 2 fetuses appeared dead and were small (about 2.5 cm). In order to test whether PPN embryos can develop to term, pregnant recipients were allowed to continue beyond Day 40. While two recipients showed estrus on Day 60 (#95–1) and Day 97 (#99–1), respectively, two others (#203–7 and #242–7) delivered 5 live piglets and 1 stillborn piglet. The live piglets appeared to be normal at birth and, except for one that was crushed, are growing well at present. When histopathological, bacteriological, and serological tests were carried out on the stillborn piglet, there were no histological lesions. It was determined that the stillborn piglet suffocated before parturition as the lungs failed to float in formalin, and umbilical hematomas were not present. The results demonstrate that PPN pig eggs have in vivo developmental ability to late gestation and term.
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Ploidy of PPN-Derived Embryos
Ploidy of PPN embryos was first investigated at the blastocyst stage. In contrast to 2PN embryos (2 of 22), most blastocysts (14 of 18) that developed from PPN eggs showed abnormal ploidy, i.e., they were haploid, triploid, and tetraploid (Table 3). Karyosyngamy of all pronuclei in the zygote stage was confirmed by confocal microscopy (Fig. 1). When a PPN egg was stained with anti--tubulin antibody and propidium iodide just after the disappearance of pronuclei membranes, only one set of metaphase chromosomes was observed. This embryo should be triploid.
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To examine the ploidy of fetuses derived from PPN eggs, embryonic fibroblasts were isolated from each fetus and their ploidy was determined by cytological analysis. To validate our classification of eggs, more than 20 metaphase spreads for each fetus were observed under x600 magnification: one had triploid cells (XXY) and another was mixoploid with both diploid (XX) and tetraploid cells (frequency of less than 10%, XXXX), while the others had diploid cells (Table 2). Chromosome numbers on metaphase spreads of triploid and tetraploid cells were correct, representing 57 and 76, respectively. Thus, there appeared to be no other karyotypic anomalies in these fetuses (Fig. 2). It was hypothesized that if all the pronuclei of PPN zygotes were to participate in syngamy, a mixed sex (XX and XY) might be observed on the metaphase spreads from a single fetus because two sperm with different sex chromosomes might penetrate into the cytoplasm of a single oocyte. However, metaphase spreads of different sex were not detected in the diploid fetuses. The growth of fibroblast cells derived from the triploid fetus was slow in culture in vitro until confluent as compared to that of diploid cells (data not shown). Blood samples of 5 live piglets were examined, and all were found to be diploid. These results indicate that some polyploid eggs can develop to blastocysts or fetuses, suggesting indirectly that chromosomes from multiple pronuclei can participate in karyosyngamy in the zygote stage just before the first cell division.
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How Can PPN Pig Eggs Develop to Blastocysts or Fetuses?
A variety of ploidy was observed in PPN-derived embryos or fetuses (Tables 2 and 3). On the basis of these results, we hypothesized that PPN eggs might have a different cytoskeletal distribution due to additional sperm as compared to 2PN eggs. As an example, it is known that F-actin may be a marker for evaluating developmental competence in early embryos because actin filaments are related to the dynamic movement of organelles during early mammalian development [37]. While no differences in F-actin distribution between the 2 groups were detected (data not shown), the cleavage pattern of PPN eggs was different from that of 2PN eggs at 36 h after insemination (Table 4). The majority of 2PN eggs that had cleaved at that time were at the 2-cell stage (61.7%, 50 of 81) and almost all of the 2-cell eggs (90.0%, 45 of 50) had 2 nuclei, whereas most PPN eggs had divided into 3 or more cells. That is, the proportion of embryos with more than 3 cells in the PPN group (68.3%, 56 of 82) was higher (P < 0.001) than that for 2PN eggs (38.3%, 31 of 81). Moreover, PPN eggs that divided into 3 or more cells in the main had 3 or more nuclei. Thus, the results indicated that most PPN pig eggs showed abnormal cleavage at the first cell division.
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The next question was how the PPN zygotes make diploid or mutiploid cells. If a PPN zygote were to develop to a triploid fetus as shown in Table 2, 3 pronuclei of the zygote should participate in syngamy prior to the first cell division. To determine this, the location (development) of pronuclei in PPN eggs was examined at 24 h after insemination. Approximately half of 2PN zygotes (46%, 24 of 52) had already developed to metaphase and cleaved eggs, while most PPN zygotes (88%, 43 of 49) still had pronuclei (Fig. 3). It was found that there were two different types of distribution of pronuclei in the PPN zygotes. In the first, all the pronuclei progressed to the center of the oocyte. In the second, 1 female and 1 male pronucleus were centrally located and the other male pronucleus was located eccentrically. These different types of pronuclear location in PPN eggs were detected again by confocal microscopy after treatment with anti--tubulin antibody and propidium iodide (Fig. 4). Microtubules were distributed around pronuclei and spindles. As shown in Figure 4E, 3 pronuclei of the egg were located in the center. Another type is shown in Figure 4F; i.e., 1 male and 1 female pronucleus participated in syngamy, and the other pronucleus was located eccentrically. One fertilized egg had one telophase chromosome set just before the first cell division, while 1 male pronucleus remained in the egg (Fig. 4G). This embryo might have been dividing into 3 cells. Another zygote had one set of chromatin in anaphase and one in telophase (Fig. 4H), suggesting that the telophase (left) was derived from syngamy and the anaphase (right) from the other male pronucleus. This embryo probably would divide into 4 cells. Thus, the abnormal nuclear divisions could explain how some PPN pig eggs divided into more than 3 cells at the first cleavage (Table 4). These results were also consistent with karyotypes of PPN-derived blastocysts or fetuses that showed a variety of karyotypes, i.e., haploid, diploid, triploid, and tetraploid.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To our knowledge, the present study is the first detailed report on in vivo development of early mammalian polyspermic embryos. Pig oocytes matured and fertilized in vitro could develop to blastocysts after transfer of fertilized oocytes to the oviduct [38–41], and under in vitro culture conditions [32, 42, 43], although the percentage that developed to the blastocyst stage was still low. One reason for this low developmental frequency was thought to be the high incidence of polyspermy [44, 45]. However, we previously reported that PPN eggs developed to the blastocyst stage at a developmental rate similar to that of normal (2PN) eggs [31]. When the subsequent developmental ability of PPN embryos was investigated, most Day 40 fetuses appeared to be normal in size and morphology compared to 2PN-derived fetuses, although 2 fetuses appeared dead and were small (Table 2). Moreover, live piglets were born from recipients of PPN eggs (Table 1). Thus, the present study demonstrates that some PPN pig eggs develop normally to term.
Ploidy of PPN-Derived Embryos
In humans, triploid fetuses are usually retarded in growth and have severe anatomic defects of the head, heart, and extremities [46]. The development of rat triploid embryos is reported to be identical to that of diploid embryos up to about 10 days of gestation [47]. Rabbit triploid embryos are described as being morphologically normal at 15 days of gestation and retarded by only about 1 day compared to normal diploid embryos [48]. Diandric triploid mouse embryos produced by nuclear manipulation are capable of surviving up to the forelimb bud stage and possess about 25 pairs of somites. They appear to be morphologically normal but are considerably smaller than fertilized embryos analyzed at similar stages of development [49, 50]. The reduction in size of postimplantation triploid embryos compared with developmentally matched controls may result from their reduced cell number, possibly due to their slowed cleavage rate during the preimplantation period [51]. In the present study, a triploid pig fetus at Day 40 of gestation appeared to be morphologically normal in size, although the growth of fibroblast cells derived from the fetus was slow in culture in vitro until confluence as compared to that of diploid cells. Another fetus was a mosaic of diploid and tetraploid nuclei (Table 2). Diploid and tetraploid mosaicism has been observed in the trophoblasts of pigs [52] and sheep [53] after Day 10 in vivo. Mosaic embryos may develop to fetuses if polyploid cells enter the trophoblast lineage and participate in the formation of extraembryonic structures. Live piglets with diploid nuclei developed from PPN eggs, although some blastocysts and fetuses derived from PPN embryos were multiploid (Tables 2 and 3). Thus, the ploidy of PPN embryos may change during early embryonic development.
Models of Embryonic Development of PPN Pig Eggs
Although more experiments are needed to test these hypotheses, some possible mechanisms in relation to embryonic development of polyspermic pig eggs are proposed. First, all the pronuclei of a PPN egg participate at the first cleavage, forming two identical daughter cells that then develop to blastocysts and fetuses (Fig. 5, A). This hypothesis was illustrated by the appearance of triploid or tetraploid cells in blastocysts or fibroblasts derived from PPN pig eggs (Tables 2 and 3, and Fig. 2). Second, all the pronuclei participate in syngamy, but the egg divides into 3 or 4 cells like polyspermic sea urchin eggs. We did not find any explicit data supporting this mechanism, although most PPN eggs divided into eggs of 3 or more cells at the first cleavage (Table 4). Kola et al. [15] reported that 62% (18 of 29) of tripronuclear human zygotes cleaved directly to 3-cell embryos at the first cleavage division and that all of these embryos examined by chromosomal analysis had a highly abnormal chromosome composition. In the present study, however, anomalies of chromosome composition, other than ploidy, were not detected in fetuses that developed from PPN pig eggs. Interestingly, the first mitotic division after fertilization is controlled by the sperm centriole, which must be present for proper embryo cleavage [54–56]. In the sea urchin, fertilization by two sperm results in a triploid nucleus. Since the sperm centriole divides to form the two poles of a miotic apparatus, instead of a bipolar mitotic spindle separating the chromosomes into 2 cells, the triploid chromosomes would divide into as many as 4 cells. Third, both a female and a male pronucleus of a PPN zygote will participate in cleavage and the other male pronucleus may divide separately, giving rise to a mosaic of 1 haploid and 2 diploid cells (Fig. 5, C) or 2 haploid and 2 diploid cells (Fig. 5, D). Thereafter, if 1 or more haploid cells die later during embryo development, this embryo will become diploid and then subsequently develop to term. Haploid/diploid mosaic embryos were rarely shown in sheep and were ascribed to polyspermic fertilization [57, 58]. However, it is unknown whether the haploid cell(s) degenerates later during embryogenesis, or even whether it fuses with one of the blastomeres. The fate of these haploid cells in polyspermic embryos may be elucidated if the molecular markers specific for pig sperm are utilized. In the yolky eggs of certain birds, reptiles, and salamanders, several sperm enter the egg cytoplasm at fertilization. Through an unknown mechanism, all but one of these sperm are induced to disintegrate in the cytoplasm after fusion of the egg pronucleus with 1 of the sperm pronuclei [59]. Whatever the mechanism, only 1 haploid sperm nucleus is allowed to fuse with the haploid nucleus of the egg. Here, we propose that the developmental mechanism of PPN pig eggs may be analogous to that of yolky eggs. The results for live piglets with diploid cells support the third model. The present study suggests that the two types of pronuclear location in polyspermic pig eggs result in embryos that have differing ploidy. This correction of ploidy can likely occur at the 1-cell stage before the first cell division.
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FOOTNOTES |
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2 Correspondence: Randall S. Prather, 920 East Campus Drive, Room 162, Department of Animal Sciences, University of Missouri-Columbia, Columbia, MO 65211-5300. FAX: 573 884 7827; pratherr{at}missouri.edu
Accepted: June 25, 1999.
Received: April 26, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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