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

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Pronuclear Location Before the First Cell Division Determines Ploidy of Polyspermic Pig Embryos 1 BIOLOGY OF REPRODUCTION 61, 1340±1346 (1999) Pronuclear Location Before the First Cell Division Determines Ploidy of Polyspermic Pig Embryos 1 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 ®rst 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 ®rst cell division ac- cording to pronuclear location. into gestation and to term, and that others may correct ab- normal 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 3 g for 30 min Accepted June 25, 1999. at 48C, ®ltered through 1.2-mm syringe ®lters (Gelman Sci- Received April 26, 1999. ences, Ann Arbor, MI), and stored in aliquots at 2208C 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 coop- erative 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±308C. 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 398C, 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 48C 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-ml 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 3 10-mm2 polystyrene cul- IU/ml rhodamine-phalloidin (for actin ®laments) or 1 mg/ ture dish (Becton Dickinson, Lincoln Park, NJ).
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