Pronuclear Location Before the First Cell Division Determines Ploidy of Polyspermic Pig Embryos 1

BIOLOGY OF REPRODUCTION 61, 1340±1346 (1999)

Yong-Mahn Han,3,4 Wei-Hua Wang,3 Lalantha R. Abeydeera,3 Anissa L. Petersen,3 Jae-Hwan Kim,3 Clifton Murphy,3 Billy N. Day,3 and Randall S. Prather2,3 Department of Animal Sciences,3 University of Missouri-Columbia, Columbia, Missouri 65211-5300 Korea Research Institute of Biosciences & Biotechnology,4 Taejon 305-600, Korea

ABSTRACT penetration of the vitellus by several spermatozoa may lead to the formation of chromatin aggregates between adjoining Downloaded from https://academic.oup.com/biolreprod/article/61/5/1340/2734639 by guest on 01 October 2021 Polyspermy occurs frequently in the fertilization of mam- sperm heads that have failed to decondense into accessory malian eggs, but little is known about whether polyspermic eggs pronuclei [2, 22]. Polyploidy in humans mostly results in have developmental ability in vitro or in vivo. We previously spontaneous abortion, but births of tetraploid or triploid reported that poly-pronuclear (PPN; 3 or more pronuclei) pig eggs developed normally to the blastocyst stage despite having children have been reported [23±27]. These live births are fewer inner cell mass cell numbers as compared to blastocysts characterized by severe malformations and multiple anom- derived from two-pronuclear (2PN) eggs. Here it is shown that alies [28±30]. Recently, it has been reported that polysper- most PPN pig eggs have abnormal cleavage patterns (having 3 mic pig eggs develop to the blastocyst stage; however, they or more cells) in the first cell division and retarded development have fewer inner cell mass nuclei as compared to blasto- of pronuclei prior to syngamy as compared to 2PN eggs. Most cysts derived from normal two-pronuclear (2PN) eggs [31]. blastocysts (14 of 18) that developed from PPN eggs showed Little information is available regarding the ability of poly- abnormal ploidy (were haploid, triploid, and tetraploid) whereas spermic eggs to develop to term for two reasons: 1) the 20 of 22 blastocysts derived from 2PN embryos were diploid. lack of experimental material and 2) lack of methods for The size and morphology of most Day 40 fetuses that developed isolating polyspermic eggs. In this study, polyspermic eggs from PPN eggs appeared to be normal. Of 8 Day 40 fetuses were produced after in vitro maturation and fertilization of analyzed, 1 was triploid (XXY) and another was a mosaic with pig oocytes [32]. To isolate the surviving polyspermic eggs, both diploid (XX) and tetraploid cells (frequency of less than the eggs were centrifuged to visualize pronuclei and sub- 10%, XXXX), and the others were diploid. Anomalies of chro- sequently classify them by pronuclear number. Thereafter, mosomal composition were not detected in these fetuses. Five the experiments on development of polyspermic pig eggs live piglets and one dead piglet were born from two recipients could be performed. In addition, the movement of pronuclei of PPN eggs. It is proposed that not all pronuclei of PPN pig in mammalian polyspermic eggs has not been reported; i.e., eggs participate in syngamy, resulting in diploid cells in the con- whether all pronuclei of polyspermic zygotes participate in ceptus. Our data suggest that there are two types of pronuclei syngamy prior to the ®rst cell division. Here we show that location in polyspermic pig eggs and that the resulting ploidy is some poly-pronuclear (PPN) pig eggs can develop quite far determined at the zygote stage before the first cell division according to pronuclear location. into gestation and to term, and that others may correct abnormal ploidy caused by more than 3 pronuclei. INTRODUCTION MATERIALS AND METHODS Polyspermy, or penetration of the egg cytoplasm by more than a single spermatozoon, is one of the principal Culture Media anomalies of fertilization in placental mammals. The inci- Unless otherwise stated, all chemicals used in this study dence of polyspermy in pigs ranges from 5% to 35% [1± were purchased from Sigma Chemical Co. (St. Louis, MO). 4] and is even higher in pig eggs fertilized in vitro [5±8], The medium used for oocyte maturation (IVM medium) although extensive attempts have been made to reduce was BSA-free North Carolina State University (NCSU) 23 polyspermy [9, 10]. It has been reported that polyspermy medium [33] supplemented with 10% (v:v) porcine follic- of pig eggs in vivo occurs primarily because abnormally ular ¯uid, 0.01 mg/ml epidermal growth factor, 0.1 mg/ml high numbers of competent spermatozoa reach the egg sur- cysteine, 10 IU/ml each of eCG and hCG (Intervet America face more or less simultaneously [11±13]. Polyspermy fre- Inc., Millsboro, DE). The fertilization medium, designated quently occurs in human [14±17] and bovine [18±20] in modi®ed Tris-buffered medium (mTBM), consisted of vitro fertilization, having an incidence of nearly 10% 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2 2H2O, 20 mM among all fertilized oocytes. Polyspermic fertilization in Tris (crystallized free base; Fisher Scienti®c, Fair Lawn, mammals is generally considered pathological, invariably NJ), 11 mM glucose, 5 mM sodium pyruvate, and no an- resulting in developmental failure of the zygote [21]. The tibiotics. Porcine follicular ¯uid was collected from follicles 3±6 mm in diameter, centrifuged at 1900 ϫ g for 30 min Accepted June 25, 1999. at 4ЊC, ®ltered through 1.2-␮m syringe ®lters (Gelman Sci- Received April 26, 1999. ences, Ann Arbor, MI), and stored in aliquots at Ϫ20ЊC 1The material is based upon work supported as a part of the National until use. Cooperative Program on Non-Human In Vitro Fertilization and Preim- plantation Development and was funded by the NICHD through cooperative agreement HD34588, and is a contribution from the Missouri Ag- In Vitro Maturation (IVM) ricultural Experiments Station Journal Series No. 12,831. 2Correspondence: Randall S. Prather, 920 East Campus Drive, Room 162, The ovaries were collected from prepubertal gilts at a Department of Animal Sciences, University of Missouri-Columbia, Columbia, local slaughterhouse and transported to the laboratory in MO 65211-5300. FAX: 573 884 7827; e-mail: [email protected] 0.9% saline containing 75 mg/ml potassium penicillin G 1340 PLOIDY OF POLYSPERMIC EMBRYOS 1341 and 50 mg/ml streptomycin sulfate maintained at 25±30ЊC. same medium and then cultured in 0.5 ml of NCSU me- Immature oocytes were obtained from follicles with a di- dium supplemented with 4 mg/ml BSA (A8022) in each ameter of 3±6 mm using a 20-gauge needle connected to a well of a Nunc 4-well multidish for approximately 12 h 10-ml disposable syringe. Follicular contents were pooled prior to embryo transfer. into a 50-ml conical tube (Fisher Scienti®c) and allowed to sediment. Supernatant was discarded, and the sediment was Embryo Transfer of 2PN and PPN Pig Eggs washed with Tyrode's lactate (TL)-Hepes supplemented with 0.01% polyvinyl alcohol (PVA) medium (TL-Hepes- Experiments were conducted according to Institutional PVA) using an Emcon ®lter (Reproduction Resources, He- Animal Care and Use Committee guidelines. Prepubertal bron, IL). Oocytes surrounded by a compact cumulus mass gilts aged 165±180 days were injected with 1000 IU eCG and having evenly granulated cytoplasm were selected and followed by injection of 500 IU hCG 96 h later. The next washed twice with approximately 3 ml of TL-Hepes-PVA morning they were observed for the onset of standing es- medium. Then 50 oocytes were placed into 0.5 ml of IVM trus, and gilts exhibiting estrus were then used as recipients. medium supplemented with eCG and hCG in each well of The ®rst day of estrus was designated Day 1 of the cycle. Downloaded from https://academic.oup.com/biolreprod/article/61/5/1340/2734639 by guest on 01 October 2021 a Nunc 4-well multidish (Nunc, Roskilde, Denmark). The The synchronized gilts (Day 2) were anesthetized by injec- medium was covered with warm paraf®n oil (light mineral tion of 23 ml of 5% sodium thiopental (Abbott Laborato- ries, North Chicago, IL) followed by 2±5% halothane (Hal- oil; Fisher) and equilibrated at 39ЊC, 5% CO2 in air for at least 2 h prior to use. After 20±22 h of maturation culture, ocarbon Laboratories, River Eagle, NJ) during surgery. The oocytes were washed twice with TL-Hepes-PVA medium gilt was subjected to a midventral laparotomy, and the re- and then further cultured in 0.5 ml of the same IVM me- productive organs including uteri, oviducts, and ovaries dium without hormonal supplements (eCG and hCG) for were exteriorized. Eggs (30 to 35) at the 1-cell stage were 20±22 h. transferred into one oviduct of the recipient to examine the in vivo development of the 2PN and PPN eggs. In Vitro Fertilization (IVF) Examination of Actin Filaments and Microtubules After the end of the IVM period, IVF of pig oocytes was by Confocal Microscopy performed as previously described [32]. Brie¯y, the oocytes were treated with 0.1% hyaluronidase in NCSU 23 medium In some replicates, embryos were ®xed with 3.7% para- to remove cumulus cells and washed 3 times with mTBM formaldehyde in PBS at 4ЊC overnight. The embryos were containing 1 mM caffeine and 1 mg/ml BSA (A 7888). ®rst cultured in a blocking solution of PBS containing 2% After washing, 35±40 denuded oocytes were placed into BSA and 150 mM glycine for 30 min at room temperature. 50-␮l drops of fertilization medium that had been covered After washing for 1 h, the embryos were stained with 10 with warm paraf®n oil in a 35 ϫ 10-mm2 polystyrene cul- IU/ml rhodamine-phalloidin (for actin ®laments) or 1 ␮g/ ture dish (Becton Dickinson, Lincoln Park, NJ). The oo- ml ¯uorescein isothiocyanate (FITC)-conjugated anti-␣-tu- cytes were incubated for about 30 min before spermatozoa bulin (for microtubules) for 1 h at 39ЊC in PBS-Tween 20 were added. A frozen semen pellet was thawed and washed (0.1%, v:v). After 2 washes in PBS-Tween solution for 2 h 3 times by centrifugation at 1900 ϫ g for 4 min in Dul- at room temperature, the embryos were stained with 100 becco's PBS (DPBS; Gibco Life Technologies Inc., Grand nM YO-Pro-1 iodide or 10 ␮g/ml propidium iodide for 5± Island, NY) supplemented with 1 mg/ml BSA, 75 mg/ml 10 min for detection of DNA. The embryos were mounted potassium penicillin G, and 50 mg/ml streptomycin sulfate on glass slides and examined by confocal microscopy. Con- (pH 7.2). At the end of the washing procedure, the sperm focal microscopy was performed by using a Bio-Rad (Rich- pellet was resuspended in the fertilization medium of mond, CA) MRC-600 equipped with a krypton argon ion. mTBM containing 1 mM caffeine and 1 mg/ml BSA. After The laser scanning for an image was repeated 5 times for appropriate dilution, 50 ␮l of the sperm suspension was 5 sec to improve the signal-to-noise ratio. added to 50 ␮l of the fertilization medium containing oocytes to give a ®nal sperm concentration of 1.5 ϫ 105 cells/ Ploidy of Embryos and PPN-Derived Fetuses ml. Oocytes were coincubated with spermatozoa for 5±6 h at 39ЊC in an atmosphere of 5% CO in air. Porcine embryonic ®broblasts were isolated from the 2 whole body of each Day 40 fetus from which the head and internal organs had been cut off. The primary culture was Classification of 2PN and PPN Eggs in a 100-mm dish in DMEM (Dulbecco's modi®ed Eagle's Approximately 10 h after insemination, the eggs were medium) supplemented with 10% fetal bovine serum at centrifuged at 12 000 ϫ g in a microcentrifuge (Fisher Sci- 39ЊC, 5% CO2 in air. The subcultures were seeded with 1± enti®c) for 10 min to stratify the cytoplasm and permit vi- 2 million cells and cultured for 2±3 days (at least a week sualization of the pronuclei [34] and then washed twice for the triploid cells) until con¯uent. Fibroblasts of the 4th with TL-Hepes medium supplemented with 1 mg/ml BSA passage were cultured in DMEM containing colcemid (0.05 (TL-Hepes-BSA medium). According to pronuclear num- ␮g/ml) overnight, harvested, and treated in hypotonic so- bers, the eggs were individually classi®ed into 2PN and lution (0.6% sodium citrate) for 30 min at room tempera- PPN eggs by use of a micromanipulator (Leitz, Wetzlar, ture. The cells were ®xed with methanol:glacial acetic acid Germany) under ϫ200 magni®cation on a warm plate (3:1) twice and then dropped on glass slides. After air dry- (37ЊC). Unclear, unfertilized, and fragmented eggs were ex- ing, the slides were stained with 5% (v:v) Giemsa for 5 min cluded from experimental groups. The validity of assign- and washed with distilled water. Metaphase spreads were ments of these eggs after classi®cation was ®rst evaluated observed on a microscope under ϫ600 magni®cation. In by using an aceto orcein staining method. The accuracy rate the case of embryo samples, blastocysts were cultured in for classi®cation into 2PN and PPN eggs was 94.6% (105 NCSU medium containing colcemid (0.05 ␮g/ml) over- of 111) and 96.8% (61 of 63), respectively. After classi®- night. Thereafter, a single blastocyst was treated with 0.56% cation, 2PN and PPN eggs were washed twice with the KCl for 5 min, directly mounted on a glass slide, and then 1342 HAN ET AL.

TABLE 2. Proﬁles of Day 40 fetuses that developed from PPN pig eggs. Implanted/ Pig transferred Fetus size/ no. (%) fetus number Ploidy 184-5 7/35 (20.0) 5.0–5.5 cm/6 Not analyzed 2.5 cm/1 43-3 2/30 (6.7) 5.0–5.5 cm/2 2 Fetuses: diploid 51-7 8/35 (23.7) 5.6–6.0 cm/4 4 Fetuses: diploid 5.0–5.5 cm/2 1 Fetus: di- & tetraploid 2.5 cm/1 1 Fetus: triploid absorption/1 80-4a 2/16 (12.5) 5.5 cm/2 2 Fetuses: diploid a One recipient received 2PN eggs as a control. Downloaded from https://academic.oup.com/biolreprod/article/61/5/1340/2734639 by guest on 01 October 2021

RESULTS Development of PPN Pig Eggs 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). More- over, 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 FIG. 1. Evidence of syngamy of pronuclei in a PPN egg just before the ﬁrst cell division. All the pronuclei of a PPN egg were centrally located were carried out on the stillborn piglet, there were no his- 24 h after insemination (A). Note that in this example each pronucleus tological lesions. It was determined that the stillborn piglet has a single prominent nucleolus. Pronuclei development was observed suffocated before parturition as the lungs failed to ¯oat in at intervals of 1 h under an inverted microscope. When the membranes formalin, and umbilical hematomas were not present. The of the pronuclei disappeared, the egg was ﬁxed and then stained with results demonstrate that PPN pig eggs have in vivo devel- FITC-conjugated anti-␣-tubulin antibody and propidium iodide. Only one opmental ability to late gestation and term. set of metaphase chromosomes was detected by confocal microscopy (B). Ploidy of PPN-Derived Embryos ®xed. The subsequent methods were the same as described Ploidy of PPN embryos was ®rst investigated at the blas- above for ®broblasts. For blood samples, metaphase spreads tocyst stage. In contrast to 2PN embryos (2 of 22), most were prepared as described by Verma and Babu [35]. Pig blastocysts (14 of 18) that developed from PPN eggs chromosomes were karyotyped according to standard kar- showed abnormal ploidy, i.e., they were haploid, triploid, yotype of the domestic pig [36]. and tetraploid (Table 3). Karyosyngamy of all pronuclei in the zygote stage was con®rmed by confocal microscopy (Fig. 1). When a PPN egg was stained with anti-␣-tubulin TABLE 1. Results after transfer of PPN pig eggs fertilized in vitro. antibody and propidium iodide just after the disappearance of pronuclei membranes, only one set of metaphase chro- Embryo No. of eggs mosomes was observed. This embryo should be triploid. Pig no. status transferred Remarks To examine the ploidy of fetuses derived from PPN eggs, 184-5 PPN 35 7 Fetuses (Day 40) embryonic ®broblasts were isolated from each fetus and 43-3 PPN 30 2 Fetuses (Day 40) their ploidy was determined by cytological analysis. To val- 51-7 PPN 35 7 Fetuses (Day 40) 95-1 PPN 35 Heat (Day 60) idate our classi®cation of eggs, more than 20 metaphase 99-1 PPN 30 Heat (Day 97) spreads for each fetus were observed under ϫ600 magni- 203-7 PPN 35 2 Live piglets ®cation: one had triploid cells (XXY) and another was mix- 242-7 PPN 35 3 Live piglets oploid with both diploid (XX) and tetraploid cells (fre- 1 Stillborn quency of less than 10%, XXXX), while the others had 80-4 2PN 16 2 Fetuses (Day 40) diploid cells (Table 2). Chromosome numbers on metaphase 236-2 2PN 20 2 Live piglets spreads of triploid and tetraploid cells were correct, repre- PLOIDY OF POLYSPERMIC EMBRYOS 1343

TABLE 3. Ploidy of 2PN and PPN embryos that developed to blastocysts. No. of embryos showing No. of Metaphase Group embryosa spreadsb Haploid Diploid Triploid Tetraploid 2PN 26/46 71/154 20 1 1(Mc) (90%) (5%) (5%) PPN 21/39 37/64 5 4 7 2(Md) (28%) (22%) (39%) (11%) a No. of embryos with metaphase spreads/no. of embryos examined. b Countable metaphase spreads/total metaphase spreads. c M means a mosaic embryo of di- and tetraploid. d M means one of these was a mosaic of di- and tetraploid. Downloaded from https://academic.oup.com/biolreprod/article/61/5/1340/2734639 by guest on 01 October 2021 senting 57 and 76, respectively. Thus, there appeared to be of distribution of pronuclei in the PPN zygotes. In the ®rst, no other karyotypic anomalies in these fetuses (Fig. 2). It all the pronuclei progressed to the center of the oocyte. In was hypothesized that if all the pronuclei of PPN zygotes the second, 1 female and 1 male pronucleus were centrally were to participate in syngamy, a mixed sex (XX and XY) located and the other male pronucleus was located eccen- might be observed on the metaphase spreads from a single trically. These different types of pronuclear location in PPN fetus because two sperm with different sex chromosomes eggs were detected again by confocal microscopy after might penetrate into the cytoplasm of a single oocyte. How- treatment with anti-␣-tubulin antibody and propidium io- ever, metaphase spreads of different sex were not detected in the diploid fetuses. The growth of ®broblast cells derived from the triploid fetus was slow in culture in vitro until con¯uent 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 ®rst cell division.

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 com- petence in early embryos because actin ®laments 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 Ͻ 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 ®rst cell division. 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 ®rst cell division. To determine this, the location (development) of FIG. 2. Triploid (A) or tetraploid chromosomes (B) of PPN-derived fe- pronuclei in PPN eggs was examined at 24 h after insem- tuses. Fibroblast cells were established from each fetus, and their ploidy ination. Approximately half of 2PN zygotes (46%, 24 of was then examined by a cytological method. Of 8 fetuses analyzed, 1 had triploid cells (XXY) and another was a mosaic with both diploid (XX) 52) had already developed to metaphase and cleaved eggs, and tetraploid cells (frequency of less than 10%, XXXX), while the others while most PPN zygotes (88%, 43 of 49) still had pronuclei had diploid cells. Number of metaphase chromosomes was correct in (Fig. 3). It was found that there were two different types triploid (n ϭ 57) or tetraploid cells (n ϭ 76). 1344 HAN ET AL.

FIG. 3. Pronuclear development of 2PN and PPN eggs at 24 h after insemination. The experiments were repeated 3 times. Downloaded from https://academic.oup.com/biolreprod/article/61/5/1340/2734639 by guest on 01 October 2021 dide (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 partici- pated in syngamy, and the other pronucleus was located eccentrically. One fertilized egg had one telophase chromosome set just before the ®rst 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. FIG. 4. Development of pronuclei in 2PN and PPN pig zygotes. The Thus, the abnormal nuclear divisions could explain how eggs were ﬁxed with 3.7% paraformaldehyde 24 h after insemination and treated with FITC-conjugated anti-␣-tubulin antibody and propidium io- some PPN pig eggs divided into more than 3 cells at the dide. The microtubule distribution and DNA were detected by confocal ®rst cleavage (Table 4). These results were also consistent microscopy. Panels A–D and E–H eggs show 2PN and PPN zygotes, re- with karyotypes of PPN-derived blastocysts or fetuses that spectively. Microtubules were distributed in the cytoplasm around pro- showed a variety of karyotypes, i.e., haploid, diploid, trip- nuclei (A, E, and F) at pronuclear stage and in the spindles at metaphase loid, and tetraploid. (B) or telophase (C, D, G, and H) just before the ﬁrst cell division.

DISCUSSION (2PN) eggs [31]. When the subsequent developmental abil- In Vivo Development of PPN Pig Eggs ity of PPN embryos was investigated, most Day 40 fetuses appeared to be normal in size and morphology compared To our knowledge, the present study is the ®rst detailed to 2PN-derived fetuses, although 2 fetuses appeared dead report on in vivo development of early mammalian poly- and were small (Table 2). Moreover, live piglets were born spermic embryos. Pig oocytes matured and fertilized in vi- from recipients of PPN eggs (Table 1). Thus, the present tro could develop to blastocysts after transfer of fertilized study demonstrates that some PPN pig eggs develop nor- oocytes to the oviduct [38±41], and under in vitro culture mally to term. 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 Ploidy of PPN-Derived Embryos high incidence of polyspermy [44, 45]. However, we pre- In humans, triploid fetuses are usually retarded in growth viously reported that PPN eggs developed to the blastocyst and have severe anatomic defects of the head, heart, and stage at a developmental rate similar to that of normal extremities [46]. The development of rat triploid embryos

TABLE 4. Nuclei numbers of the cleaved eggs at 36 hours after insemination.a Number of nuclei Cell No. of eggs Group stageb examined 123456 2-cell (I) 35 32 2 1 2-cell (II) 15 13 3-cell 10 9 1 2PN Ͼ4-cell 21 2 10 6 32 2-cell (I) 19 15 1 2-cell (II) 7 3 7 3-cell 16 4 4 5 2 PPN Ͼ4-cell 40 1 7 6 21 51 a This is a summation of 6 replications. b 2-cell (I) means a normal 2-cell egg with 2 blastomeres, while (II) denotes an abnormal 2-cell egg with 2 blastomeres and 1 smaller blastomere. PLOIDY OF POLYSPERMIC EMBRYOS 1345

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 ®nd any ex- plicit data supporting this mechanism, although most PPN eggs divided into eggs of 3 or more cells at the ®rst 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 ®rst 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 ®rst mitotic division

after fertilization is controlled by the sperm centriole, which Downloaded from https://academic.oup.com/biolreprod/article/61/5/1340/2734639 by guest on 01 October 2021 must be present for proper embryo cleavage [54±56]. In the sea urchin, fertilization by two sperm results in a trip- FIG. 5. A diagram representing the developmental mechanisms of PPN loid nucleus. Since the sperm centriole divides to form the pig eggs. There were two types of pronuclei location in the PPN eggs. In two poles of a miotic apparatus, instead of a bipolar mitotic one type (I), all the pronuclei were centrally located. In the other (II), 1 spindle separating the chromosomes into 2 cells, the triploid female and 1 male pronucleus were close, but the other was located chromosomes would divide into as many as 4 cells. Third, eccentrically. In type I fertilizations, either the embryo developed as a both a female and a male pronucleus of a PPN zygote will triploid (A) or correct ploidy was established in some cells (B) and development continued. Alternatively, in type II fertilizations, a diploid/an- participate in cleavage and the other male pronucleus may euploid mosaic was established with division to the 3- or 4-cell stage divide separately, giving rise to a mosaic of 1 haploid and directly from the 1-cell stage. The present study suggests that the two types 2 diploid cells (Fig. 5, C) or 2 haploid and 2 diploid cells of pronuclear location result in embryos that have different ploidy. (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 is reported to be identical to that of diploid embryos up to mosaic embryos were rarely shown in sheep and were as- about 10 days of gestation [47]. Rabbit triploid embryos cribed to polyspermic fertilization [57, 58]. However, it is are described as being morphologically normal at 15 days unknown whether the haploid cell(s) degenerates later dur- of gestation and retarded by only about 1 day compared to ing embryogenesis, or even whether it fuses with one of normal diploid embryos [48]. Diandric triploid mouse em- the blastomeres. The fate of these haploid cells in poly- bryos produced by nuclear manipulation are capable of sur- spermic embryos may be elucidated if the molecular mark- viving up to the forelimb bud stage and possess about 25 ers speci®c for pig sperm are utilized. In the yolky eggs of pairs of somites. They appear to be morphologically normal certain birds, reptiles, and salamanders, several sperm enter but are considerably smaller than fertilized embryos ana- the egg cytoplasm at fertilization. Through an unknown lyzed at similar stages of development [49, 50]. The reduc- mechanism, all but one of these sperm are induced to dis- tion in size of postimplantation triploid embryos compared integrate in the cytoplasm after fusion of the egg pronucleus with developmentally matched controls may result from with 1 of the sperm pronuclei [59]. Whatever the mecha- their reduced cell number, possibly due to their slowed nism, only 1 haploid sperm nucleus is allowed to fuse with cleavage rate during the preimplantation period [51]. In the the haploid nucleus of the egg. Here, we propose that the present study, a triploid pig fetus at Day 40 of gestation developmental mechanism of PPN pig eggs may be anal- appeared to be morphologically normal in size, although ogous to that of yolky eggs. The results for live piglets with the growth of ®broblast cells derived from the fetus was diploid cells support the third model. The present study slow in culture in vitro until con¯uence as compared to that suggests that the two types of pronuclear location in poly- of diploid cells. Another fetus was a mosaic of diploid and spermic pig eggs result in embryos that have differing ploi- tetraploid nuclei (Table 2). Diploid and tetraploid mosai- dy. This correction of ploidy can likely occur at the 1-cell cism has been observed in the trophoblasts of pigs [52] and stage before the ®rst cell division. sheep [53] after Day 10 in vivo. Mosaic embryos may develop to fetuses if polyploid cells enter the trophoblast lin- eage and participate in the formation of extraembryonic REFERENCES structures. Live piglets with diploid nuclei developed from PPN eggs, although some blastocysts and fetuses derived 1. Hancock JL. Polyspermy of pig ova. Anim Prod 1959; 1:103±106. from PPN embryos were multiploid (Tables 2 and 3). Thus, 2. Hunter RHF. The effects of delayed insemination on fertilization and early cleavage in the pig. J Reprod Fertil 1967; 13:133±147. the ploidy of PPN embryos may change during early em- 3. Hunter RHF. Local action of progesterone leading to polyspermic fer- bryonic development. tilization in pigs. J Reprod Fertil 1972; 31:433±444. 4. Hunter RHF. Polyspermic fertilisation in pigs after tubal deposition of Models of Embryonic Development of PPN Pig Eggs excessive numbers of spermatozoa. J Exp Zool 1973; 183:57±64. 5. Nagai T, Niwa K, Iritani A. Effect of sperm concentration during Although more experiments are needed to test these hy- preincubation in a de®ned medium on fertilization in vitro of pig fol- potheses, some possible mechanisms in relation to embry- licular oocytes. J Reprod Fertil 1984; 70:271±275. onic development of polyspermic pig eggs are proposed. 6. Wang WH, Okuda K, Niwa K. In vitro penetration of pig oocytes First, all the pronuclei of a PPN egg participate at the ®rst matured in culture by frozen-thawed ejaculated spermatozoa. J Reprod Fertil 1991; 93:491±499. cleavage, forming two identical daughter cells that then de- 7. Wang WH, Abeydeera LR, Okuda K, Niwa K. Penetration of porcine velop to blastocysts and fetuses (Fig. 5, A). This hypothesis oocytes during maturation in vitro by cryopreserved, ejaculated sper- was illustrated by the appearance of triploid or tetraploid matozoa. Biol Reprod 1994; 50:510±515. cells in blastocysts or ®broblasts derived from PPN pig 8. Wang WH, Abeydeera LR, Cantley TC, Day BN. Effects of oocyte 1346 HAN ET AL.

maturation media on development of pig embryos produced by in 34. Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, vitro fertilization. J Reprod Fertil 1997; 111:101±108. Palmiter RD, Brinster RL. Production of transgenic rabbits, sheep and 9. Hunter RHF, Nichol R. Capacitation potential of the fallopian tube: a pigs by microinjection. Nature 1985; 315:680±683. study involving surgical insemination and the subsequent incidence of 35. Verma RS, Babu A. Human Chromosomes: Manual of Basic Tech- polyspermy. Gamete Res 1988; 21:255±266. niques. New York: Pergamon Press; 1989: 6±9. 10. Hunter RHF. Ovarian control of very low sperm/egg ratios at the com- 36. Gustavsson I. Standard karyotype of the domestic pig. Hereditas 1988; mencement of mammalian fertilisation to avoid polyspermy. Mol Re- 109:151±157. prod Dev 1996; 44:417±422. 37. Barnett DK, Clayton MK, Kimura J, Bavister BD. Glucose and phos- 11. Day BN, Polge C. Effects of progesterone on fertilization and egg phate toxicity in hamster preimplantation embryos involves disruption transport in the pig. J Reprod Fertil 1968; 17:227±230. of cellular organization, including distribution of active mitochondria. 12. Hunter RHF, Leglise PC. Polyspermic fertilization following tubal sur- Mol Reprod Dev 1997; 48:227±237. gery in pigs, with particular reference to the role of the isthmus. J 38. Mattioli M, Bacci ML, Galeati G, Seren E. Developmental compe- Reprod Fertil 1971; 24:233±246. tence of pig oocytes matured and fertilized in vitro. Theriogenology 13. Polge C. Fertilization in the pig and horse. J Reprod Fertil 1978; 54: 1989; 46:1201±1207. 461±470. 39. Yoshida M, Ishigaki K, Kawagishi H. Blastocyst formation by pig

14. Rudak E, Dor J, Mashiach S, Nebel L, Goldman B. Chromosome embryos resulting from in vitro fertilization of oocytes matured in Downloaded from https://academic.oup.com/biolreprod/article/61/5/1340/2734639 by guest on 01 October 2021 analysis of multipronuclear human oocytes fertilized in vitro. Fertil vitro. J Reprod Fertil 1990; 88:1±8. Steril 1984; 41:538±545. 40. Funahashi H, Stumpf TT, Terlouw SL, Cantley T, Rieke A, Day BN. 15. Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oo- Developmental ability of pig oocytes matured and fertilized in vitro. cytes: altered cleavage patterns and subsequent karyotypic analysis of Theriogenology 1994; 41:1425±1433. embryos. Biol Reprod 1987; 37:395±401. 41. Machaty Z, Day BN, Prather RS. Development of early porcine em- 16. Plachot M, Crozet N. Fertilization abnormalities in human in vitro bryos in vitro and in vivo. Biol Reprod 1998; 59:451±455. fertilization. Hum Reprod Suppl 1992; 7:89±94. 42. Funahashi H, Cantley TC, Stumpf TT, Terlouw S, Day BN. In vitro 17. Yie SM, Collins JA, Daya S, Hughes E, Sagle M, Younglai EV. Poly- development of in vitro-matured porcine oocytes following chemical ploidy and failed fertilization in in-vitro fertilization are related to activation or in vitro fertilization. Biol Reprod 1994; 50:1072±1077. patient's age and gamete quality. Hum Reprod 1996; 11:614±617. 43. Rath D, Niemann H, Torres CRL. In vitro development to blastocysts 18. Iwasaki S, Shioya Y, Masuda H, Hanada A, Nakahara T. Incidence of of early pig embryos produced in vivo and in vitro. Theriogenology chromosomal anomalies in early bovine embryos derived from in vitro 1996; 43:913±921. fertilization. Gamete Res 1989; 22:83±91. 44. Nagai T, Takahashi T, Masuda H, Shioya Y, Kuwayama M, Fukushima 19. Iwasaki S, Hamano S, Kuwayama M, Yamashita M, Ushijima H, Na- M, Iwasaki S, Hanada A. In-vitro fertilization of pig oocytes by frozen gaoka S, Nakahara T. Developmental changes in the incidence of chro- boar spermatozoa. J Reprod Fertil 1988; 84:585±591. mosome anomalies of bovine embryos fertilized in vitro. J Exp Zool 45. Niwa K. Effectiveness of in vitro maturation and in vitro fertilization 1992; 261:70±85. techniques in pigs. J Reprod Fertil Suppl 1993; 48:49±59. 20. Kawarsky SJ, Basrur PK, Stubbings RB, Hansen PJ, King WA. Chro- 46. Szulman AE, Philippe E, Boue JG, Boue A. Human triploidy: asso- mosomal abnormalities in bovine embryos and their in¯uence on de- ciation with partial hydatidiform moles and nonmolar conceptuses. velopment. Biol Reprod 1996; 54:53±59. Hum Pathol 1981; 12:1016±1021. 21. Austin CR. Fertilisation and transport of the ovum: In: Hartman CG 47. Piko L, Bomsel-Helmreich O. Triploid rat embryos and other chro- (ed.), Mechanism Concerned with Conception. Oxford: Pergamon mosomal deviants after colchicine treatment and polyspermy. Nature Press; 1963: 285±320. 1960; 186:737±739. 22. Hunter RHF. Sperm-egg interactions in the pigs: monospermy, exten- 48. Bomsel-Helmreich O. Fate of heteroploid embryos. Adv Biosci 1971; sive polyspermy, and the formation of chromatin aggregates. J Anat 6:381±403. 1976; 122:43±59. 49. Kaufman MH, Lee KKH, Speirs S. In¯uence of diandric and digynic 23. Uchida IA, Freeman VC. Triploidy and chromosomes. Am J Obstet triploid genotypes on early mouse embryogenesis. Development 1989; Gynecol 1985; 151:65±69. 105:137±145. 24. Sherard J, Bean C, Bove B, Delduca V, Esterly KL, Kavish HJ, Mun- 50. Kaufman MH. New insights into triploidy and tetraploidy, from an shi G, Reamer JF, Suazo G, Wilmoth D, Dahlke MB, Weiss C, Bor- analysis of model systems for these conditions. Hum Reprod 1991; 6: gaonkar DS. Long survival in a 69XXY triploid male. Am J Med 8±16. Genet 1986; 25:307±312. 51. Henery CC, Kaufman MH. Cleavage rate of diandric triploid mouse 25. Shiono H, Azumi J, Fujiwara M, Yamajaki H, Kikuchi K. Tetraploidy embryos during the preimplantation period. Mol Reprod Dev 1992; 32:251±258. in a 15-month old girl. Am J Med Genet 1988; 29:543±547. 52. Long SE, Williams CV. A comparison of the chromosome comple- 26. Roberts HE, Saxe DF, Muralidharan K, Coleman KB, Zacharias JF, ment of inner cell mass and trophoblast cells in Day-10 pig embryos. Fernhoff PM. Unique mosaicism of tetraploidy and trisomy 8: clinical, J Reprod Fertil 1982; 66:645±648. cytogenetic, and molecular ®ndings in a live-born infant. Am J Med 53. Murray JD, Moran C, Boland MP, Nancarrow CD, Sutton R, Hoskin- Genet 1996; 62:243±246. son RM, Scaramuzzi RJ. Polyploid cells in blastocysts and early fe- 27. Dean J, Cohen G, Kemp J, Robson L, Tembe V, Hasselaar J, Webster tuses from Australian Merino sheep. J Reprod Fertil 1986; 78:439± B, Lammi A, Smith A. Karyotype 69, XXX/47, XX, ϩ15 in a 2±1/2 446. year old child. J Med Genet 1997; 34:246±249. 54. Palermo G, Munne S, Cohen J. The human zygote inherits its mitotic 28. Pitt D, Leversha M, Sin®eld C, Campbell P, Anderson R, Bryan D, potential from the male gamete. Hum Reprod 1994; 9:1220±1225. Rogers J. Tetraploidy in a liveborn infant with spina bi®da and other 55. Asch R, Simerly C, Ord T, Ord VA, Shatten G. The stages at which anomalies. J Med Genet 1981; 18:309±311. human fertilization arrests: microtubule and chromosome con®gura- 29. Doshi N, Surti U, Szulman AE. Morphologic anomalies in triploid tions in inseminated oocytes which failed to complete fertilization and liveborn fetuses. Hum Pathol 1983; 14:716±723. development in humans. Hum Reprod 1995; 10:1897±1906. 30. Kjaer I, Keeling JW, Smith NM, Hansen BF. Pattern of malformations 56. Kim NH, Lee JW, Jun SH, Lee HT, Chung KS. Fertilization of porcine in the axial skeleton in human triploid fetuses. Am J Med Genet 1997; oocytes following intracytoplasmic spermatozoon or isolated sperm 72:216±221. head injection. Mol Reprod Dev 1998; 51:436±444. 31. Han YM, Abeydeera LR, Kim JH, Day BN, Moon HB, Prather RS. 57. Long SE, Williams CV. Frequency of chromosomal abnormalities in Growth retardation of inner cell mass cells in polyspermic pig embry- early of the domestic sheep (Ovis aries). J Reprod Fertil 1980; 58: os produced in vitro. Biol Reprod 1999; 60:1110±1113. 197±201. 32. Abeydeera LR, Day BN. Fertilization and subsequent development in 58. Murray JD, Boland MP, Moran C, Sutton R, Nancarrow CD, Scara- vitro of pig oocytes inseminated in a modi®ed Tris-buffered medium muzzi RJ, Hoskinson RM. Occurrence of haploid and haploid/diploid with frozen-thawed ejaculated spermatozoa. Biol Reprod 1997; 57: mosaic embryos in untreated and androstenedione-immune Australian 729±734. Merino sheep. J Reprod Fertil 1985; 74:551±555. 33. Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil Suppl 59. Elinson RP. Fertilization in amphibians: the ancestry of the block to 1993; 48:61±73. polyspermy. Int Rev Cytol 1986; 101:59±100.