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Growth Retardation of Inner Cell Mass Cells in Polyspermic Porcine Embryos Produced In Vitro¹

^a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211 ^b Department of Pathology, School of Medicine, Wonkwang University, Iksan 570–749, Korea

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 ± 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 ± 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 ± 3.4 vs. 39.9 ± 3.9) and TE nuclei (38.5 ± 2.9 vs. 35.7 ± 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pig oocytes matured and fertilized in vitro could develop to the blastocyst stage in vivo [1–3] or in vitro [4–7]. However, the percentage of eggs that develop to the blastocyst stage is still low. One possible factor contributing to this low developmental rate has been thought to be the high incidence of polyspermy [8]. An incidence of polyspermy exceeding 50% was found in pig eggs fertilized in vitro [9–13]. Polyspermy also occurs during human [14–17] and bovine [18–21] in vitro fertilization, with an incidence approaching 10%. Balakier [22] reported that 6% of triploid human zygotes fertilized in vitro developed to blastocyst stage. However, little information is available regarding the ability of polypronuclear pig eggs to develop to the blastocyst stage. In the present study the question whether polypronuclear pig eggs can develop to the blastocyst stage in vitro or in vivo was investigated. In addition, the numbers of inner cell mass (ICM) and trophectoderm (TE) nuclei were compared between two (2PN)- and poly-pronuclear (PPN)-derived blastocysts.

	MATERIALS AND METHODS

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Culture Media

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 [23] supplemented with 10% (v:v) porcine follicular fluid, 10 ng/ml epidermal growth factor, 0.1 mg/ml cysteine, 10 IU/ml eCG (Intervet America Inc., Millsboro, DE), 10 IU/ml hCG (Intervet), 75 µg/ml potassium penicillin G, and 50 µg/ml streptomycin sulfate. The basic medium used for in vitro fertilization (IVF) was essentially the same as described by Abeydeera and Day [24]. This fertilization medium, designated modified Tris-buffered medium (mTBM), consists 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. The culture medium for embryo development was NCSU 23 supplemented with 4 mg/ml BSA (A 8022). Porcine follicular fluid was collected from follicles of 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 at 25–30°C in 0.9% saline containing 75 µg/ml potassium penicillin G and 50 µg/ml streptomycin sulfate. 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 medium supplemented with 0.01% polyvinyl alcohol (PVA) (TL-Hepes-PVA medium) using an Emcon filter (Reproduction Resources, Hebron, IL). The oocytes surrounded by a compact cumulus mass and with evenly granulated cytoplasm were selected and washed twice with 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 4-well multidish (Nunc, Roskilde, Denmark). The medium was covered with warm paraffin oil (light mineral oil; Fisher Scientific) and equilibrated at 39°C, 5% CO₂ in air for at least 2 h prior to use. After 20–22 h of maturation culture, the 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.

IVF

After completion of culture of oocytes for IVM, the oocytes were treated with 0.1% hyaluronidase in NCSU 23 medium to remove cumulus cells and washed three times with mTBM containing 1 mM caffeine and 1 mg/ml BSA (A 7888). After washing, 30–35 denuded oocytes were placed into 50-µl drops of the fertilization medium that had been covered with warm paraffin oil in a 35 x 10-mm² polystyrene culture dish (Becton Dickinson & Co., Lincoln Park, NJ). The dishes were kept in the incubator for about 30 min until spermatozoa were added for fertilization. A frozen semen pellet was thawed and washed three times by centrifugation at 1900 x g for 4 min in Dulbecco's PBS (DPBS; Gibco, Life Technologies, Grand Island, NY) supplemented with 1 mg/ml BSA, 75 µg/ml potassium penicillin G, and 50 µg/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 a 50-µl drop 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. The day of insemination was designated as Day 0.

Selection and Culture of 2NN 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 and washed twice with TL-Hepes medium supplemented with 1 mg/ml BSA (TL-Hepes-BSA medium). Among the fertilized eggs, only 2PN and PPN eggs were selected using a micromanipulator (Leitz, Wetzlar, Germany) under x200 magnification on a warm plate (37°C). Unclear, unfertilized, and fragmented eggs were excluded from experimental groups. After selection, 2PN and PPN eggs were cultured in 0.5 ml of NCSU 23 medium supplemented with 4 mg/ml BSA in a 4-well multidish at 39°C, 5% CO₂ in air for 7 days. Embryo development to the blastocyst stage was evaluated by morphological appearance under a stereomicroscope.

Aceto Orcein Staining

To validate the reliability of the selection method used in this experiment, the number of pronuclei in eggs classified as 2PN and PPN was confirmed in the zygote stage through aceto orcein staining. Briefly, 2PN and PPN eggs were mounted on glass slides, fixed in 25% (v:v) acetic acid in ethanol at room temperature for 48–72 h, and stained with 1% (w:v) orcein in 45% (v:v) acetic acid. Number of pronuclei was counted by using a microscope (Leitz) with phase-contrast optics under x200 or x400 magnification.

Embryo Transfer

Experiments were conducted according to institutional Animal Care and Use Committee guidelines. Classified eggs (Day 1) were transferred into each oviduct of a synchronized recipient and then recovered at 6 days after transfer by retrograde flushing. Briefly, gilts with natural estrus were used as recipients. The day of estrus was designated as Day 1 of estrus. The synchronized gilts (Day 2) were anaesthetized by injection of 23 ml of 5% sodium thiopental (Abbott Laboratories, North Chicago, IL) followed by 2–5% halothane (Halocarbon Laboratories, River Edge, NJ) during surgery. The gilt was subjected to midventral laparotomy, and the reproductive organs including uteri, oviducts, and ovaries were exteriorized. To prevent the transport of embryos from one uterine horn to the other, both uteri near the cervix were tightly tied with sterilized umbilical tape. Ten to twenty embryos were transferred into each oviduct of a single recipient using a tomcat catheter (Sherwood Medical, St. Louis, MO). At 6 days after transfer, the embryos were recovered from each uterus by retrograde flushing.

Number of ICM and TE Nuclei

Numbers of ICM and TE nuclei of pig blastocysts were investigated by a modified immunosurgery method [7]. Briefly, blastocysts were treated with 0.5% protease to remove zona pellucida. Zona-free blastocysts were incubated in 50 µl of TL-Hepes-PVA medium supplemented with 10% (v:v) rabbit anti-pig whole serum at 39°C for 40 min. The embryos were incubated in 50 µl of TL-Hepes-PVA medium supplemented with 10% (v:v) guinea pig serum as complement, 4 µg/ml bisbenzimide (Hoechst 33342), and 100 µg/ml propidium iodide. After 1 h, the embryos were washed with TL-Hepes-PVA medium and individually mounted on a glass slide. The number of ICM and TE nuclei in a single embryo was counted by using a fluorescence microscope (Nikon Corp., Tokyo, Japan).

Statistical Analysis

Developmental rates to blastocysts of 2PN and PPN eggs cultured in vitro or in vivo were evaluated by chi-square analysis. Differences for mean values of ICM and TE nuclei numbers between 2PN- and PPN-derived blastocysts were determined by calculating least significant differences using the Multivariate General Linear Hypothesis of Systat [25].

	RESULTS

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In preliminary experiments, the proportion of 2PN, PPN, and other (fragmented and unfertilized) eggs after IVF was 41.6% (185 of 444), 26.4% (117 of 444), and 32.0% (142 of 444), respectively. The developmental rate (21.8%, 213 of 977) to blastocyst stage of the centrifuged eggs was similar to that (26.7%, 59 of 221) of control (noncentrifuged) eggs. This result indicated that there was no detrimental effect of centrifugation on the in vitro development of IVF-derived pig eggs.

To examine whether polyspermic eggs can develop to the blastocyst stage, 2PN and PPN eggs were selected by pronuclei number and cultured in vitro. The validation of assignments of these eggs after classification was first evaluated on a sample of embryos by using an aceto orcein-staining method. The accuracy of the classification in 2PN and PPN groups was 94.6% (105 of 111) and 96.8% (61 of 63), respectively. After 7 days of culture (Table 1), the developmental rate of PPN eggs to blastocysts (40.7%, 61 of 150) was not different (p > 0.1) from that of 2PN eggs (53.6%, 67 of 125). The aim of the next experiment was to determine whether there were differences in the relative numbers of ICM and TE between 2PN- and PPN-derived blastocysts. As shown in Figure 1A, ICM nuclei numbers (4.2 ± 0.8) of PPN-derived blastocysts (n = 37) were significantly lower (p < 0.001) than those for (8.2 ± 0.7) of 2PN-derived blastocysts (n = 48). However, no differences (p > 0.05) in the number of total (39.9 ± 3.9 vs. 46.7 ± 3.4) or TE nuclei (35.7 ± 3.3 vs. 38.5 ± 2.9) were detected. From the experiments described above, it was concluded that PPN eggs could develop to the blastocyst stage at a frequency not different from that for 2PN, although having a smaller number of ICM nuclei than 2PN embryos.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Cell numbers of 2PN- and PPN-derived blastocysts that developed in vitro (A) or in vivo (B). Pig eggs derived from in vitro maturation and fertilization were classified into 2PN and PPN groups by pronuclear number and then cultured in NCSU medium at 39°C, 5% CO2 in air for 7 days in vitro (A) or cultured for 6 days in vivo (B). Number of ICM nuclei of PPN-derived blastocysts cultured in vitro or in vivo were significantly lower than those of 2PN-derived blastocysts (p < 0.01), but no differences were detected in the number of TE or total nuclei (p > 0.05). The results suggest that the growth of ICM cells in PPN eggs slowed in early development.

Embryo culture systems designed for mammalian eggs in vitro, especially pig eggs, are not comparable to in vivo development [6, 7]. To confirm the results of in vitro development of PPN eggs, the classified eggs were transferred into each oviduct of a synchronized recipient and recovered at 6 days after transfer. The developmental rate of 2PN and PPN eggs to the blastocyst stage was not different (p > 0.1), 33.7% (22 of 66) and 27.4% (23 of 84), respectively. ICM nuclei number (10.5 ± 4.4) of PPN-derived blastocysts (n = 15) was lower (p < 0.01) than that (24.2 ± 3.9) of 2PN-derived blastocysts (n = 19). However, there were no differences (p > 0.1) in the number of total (64.5 ± 10.5 vs. 76.1 ± 9.3) or TE (54.0 ± 9.5 vs. 51.9 ± 6.7) nuclei. Thus, the number of ICM and TE nuclei of embryos cultured in vivo showed a pattern similar to that for in vitro-produced blastocysts, except that embryos cultured in vivo for 6 days had a higher cell number than in vitro-produced blastocysts (Fig. 1B). When the transferred embryos were recovered at 7 days after transfer, in both groups all of the embryos recovered (10 of 10 and 4 of 4, 2PN and PPN respectively) had developed to hatched blastocyst stage. They had a higher number of ICM and TE nuclei than Day 6 or in vitro-produced Day 7 blastocysts. However, differences in ICM cell number (80.9 ± 10.3 vs. 33.3 ± 16.2) were also detected between 2PN (n = 10) and PPN embryos (n = 4) (p < 0.05), although there was no difference (p > 0.1) in numbers of total (216.3 ± 27.3 vs. 141.5 ± 43.1) and TE nuclei (135.4 ± 19.4 vs. 108.3 ± 30.6).

These results led us to examine whether the PPN eggs continue to develop after implantation. The 2PN and PPN eggs were transferred into separate oviducts of a single recipient. Six and three had developed from 2PN and PPN eggs, respectively, on 21 days after transfer. Thus, some PPN eggs developed to fetuses, although they showed a lower developmental percentage (15.8%) as compared to 2PN eggs (46.2%) (p > 0.1). The size of 6 2PN-derived fetuses from crown to rump was 3.1, 3.0, 2.8, 2.8, 2.7, and 2.5 cm (mean; 2.85 ± 0.098 cm), and that of 3 PPN-derived fetuses was 2.7, 2.2, and 2.2 cm (mean; 2.37 ± 0.14). Thus, the size of fetuses that developed from PPN eggs was smaller (p < 0.05) than that of 2PN-derived fetuses. However, histological analyses showed no major developmental anomalies in PPN-derived fetuses (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To our knowledge, the present study is the first report on the in vitro development of early polyspermic pig embryos. Information on development of polyspermic embryos is lacking for two reasons: the lack of 1) experimental materials and 2) methods for isolating polyspermic eggs. In previous studies, pig eggs fertilized in vitro showed a high incidence of polyspermy [9, 13]. Here, zygotes were centrifuged for visualization of pronuclei [26] and then classified into 2PN and PPN eggs. Thereafter, the developmental ability of these polyspermic pig eggs could be evaluated.

The data show that polyspermic eggs can develop to the blastocyst stage at the same percentage as normal eggs. The subsequent development of the transferred embryos depends on the viability of ICM, which gives rise to the embryo proper [27]. Therefore, the ratio of ICM and TE cells during preimplantation development may be one marker by which the developmental competence of mammalian embryos can be evaluated. In the present study, interestingly, PPN-derived blastocysts had significantly fewer ICM nuclei than 2PN-derived blastocysts, but there was no difference in numbers of TE and total nuclei between the two groups (Fig. 1). These results suggest that the growth of ICM cells in PPN embryos was slowed during early development while the growth of TE was normal as compared to that of 2PN embryos. The total cell number of these blastocysts is comparable to results in previous reports (34–56) [28].

Additional data showed that approximately half of 2PN zygotes (46.2%, 24 of 52) developed to metaphase or cleaved eggs at 24 h after insemination, while most PPN zygotes (87.8%, 43 of 49) still had pronuclei (unpublished results). This observation may partially explain why PPN-derived blastocysts have a smaller number of ICM nuclei than 2PN-derived blastocysts. It suggests that the early development of polyspermic pig eggs is retarded from the pronuclear stage. Kawarsky et al. [21] reported that polyploid bovine embryos developed to blastocysts at a slower rate than diploid embryos. Similarly, triploids and tetraploids among in vivo-derived bovine embryos possessed lower cell numbers relative to their diploid counterparts [29]. The possibility exists that the duration of the cell cycle is increased in polyploids because of the time needed for the replication of additional DNA, and that the prolonged cell cycle time may be responsible for the decrease in development. Studies on murine embryos led to conflicting results: in some studies, triploid embryos cleaved more slowly than diploid [30], whereas other reports indicate no difference in cleavage rates between these groups [31–33]. The difference in these results may be due to the different techniques used to induce polyploidy.

Here it is suggested that the size of Day 21 fetuses that developed from PPN eggs was small compared to that of 2PN-derived fetuses. However, no developmental anomalies were histologically detected in PPN-derived fetuses. These results suggest that polyspermic pig embryos may be developmentally retarded, which may be a result of a smaller number of ICM nuclei at the blastocyst stage. In rodents and rabbits, triploid embryos survive into the early postimplantation period, although no development to term has so far been reported. The development of rat triploid embryos is reported to be identical to that of diploid embryos up to about 10 days of gestation [34]. 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 [35]. Diandric triploid mouse embryos produced by nuclear manipulation developed to the 15- to 25-somite stage, appearing to be morphologically normal, but were considerably smaller than fertilized embryos analyzed at similar stages of development [36]. In humans, triploid fetuses are usually growth retarded and have severe anatomic defects of the head, heart, and extremities [37]. The reduction in size of postimplantation mouse triploid embryos compared to developmentally matched controls may result from their reduced cell number, possibly due to their slowed cleavage rate during the preimplantation period [32]. The data suggest that growth retardation of PPN embryos may result from the reduced number of ICM, but not TE or total cells.

	FOOTNOTES

 
¹ This material is based upon work supported as a part of the National Cooperation on Non-Human In Vitro Fertilization and Preimplantation Development and was funded by the NICHD through cooperate agreement HD34588, and is a contribution from the Missouri Agricultural Experiments Station Journal Series No. 12,839.

² Correspondence. FAX: 573 882 6827; pratherr{at}missouri.edu

³ Current address: Korean Research Institute of Biosciences & Biotechnology, Taejon 305–600, Korea.

Accepted: December 8, 1998.

Received: September 4, 1998.

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