MOLECULAR REPRODUCTION AND DEVELOPMENT 29:124-128 (1991)

3H-UridineIncorporation in Early Porcine Embryos M. FREITAG, H.H. DOPKE, H. NIEMANN, AND F. ELSAESSER Institut fur Tierzucht und Tierverhalten (FAL), Mariensee, Neustadt, Germany

ABSTRACT The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two-cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 KM 3Huridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. N o RNA synthesis was detected at the two-cell stage. From the four-cell to the morula stage, 3H-uridine incorporation per embryo increased about ninefold ( P < 0,001); in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. O n a per cell basis, 3H-uridine incorporation was not different from the four-cell stage up to the zona pelIucida-intact blastocyst and amounted to 0.290.37 fmol 3H-uridine incorporation/cell/3 hr. In hatched blastocysts, 3H-uridine incorporation per blastomere was increased ( P < 0.01 compared with younger stages) and amounted to 0.86 fmol 3H-uridine incorporation/cell/3 hr. It is concluded that 1 ) the rate of uridine incorporation depends on the cell stage in zona pellucida-intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages. Key Words: Pig embryo, RNA synthesis, Blastocysts

INTRODUCTION The early embryonic development in mammals immediately after fertilization is controlled by a n oocyte derived genomic program. The onset of the embryonic genomic activity varies among species and ranges from the two-cell stage in mice to the eight- to 16-cell stage in human, rabbit, sheep, and cow (Flach et al., 1982; Manes, 1973; Tesarik et al., 1986; Crosby et al., 1988; Camous et al., 1987; Barnes and Eyestone, 1990). In the pig, the embryonic genome is activated at the fourcell stage, a s indicated by uridine incorporation into the nucleoplasm and ultrastructural features such as a

0 1991 WILEY-LISS, INC.

functional restructuring of the nucleoli (Tomanek et al., 1989). Until now, the ontogenetic pattern of the RNA synthesis as a measure for porcine embryonic genomic activity up to the stage of the newly hatched blastocyst has not been studied systematically. It was the purpose of the present study to characterize the ontogeny of 3H-uridine incorporation a s a measure of RNA synthesis in porcine embryos up to the stage of the newly hatched blastocyst.

MATERIALS AND METHODS A total of 568 morphologically intact embryos from 26 superovulated prepubertal German Landrace gilts (194 ? 18 days of age, 97.1 k 10.3 kg body weight; mean k s.d.) were used for these experiments. Superovulation was induced by intramuscular injection of 1,500 IU pregnant mare’s serum gonadotropin (PMSG) (Seragon; Ferring, Kiel, Germany) followed 72 h r later by 500 IU human chorionic gonadotropin (hCG) (Ekluton; Vemie, Kempen, Germany). Gilts were mated or artificially inseminated 24 and 48 h r after hCG injection. Embryos were collected a t slaughter according to the following time schedule: two-cell embryos 36 hr, four-cell embryos 48 hr, eight-cell embryos 84 hr, morulae 96 hr, blastocysts and hatched blastocysts 108 h r after the second mating ( = day 0). At slaughter in the Institute’s slaughterhouse, genital tracts were removed immediately after bleeding and were carried in prewarmed containers to the laboratory. Within 10-15 min after slaughter, uterine horns were flushed twice with 40 ml phosphate-buffered saline (PBS; Serva, Heidelberg, Germany) supplemented with 1%heat-inactivated (30 min a t 56°C) newborn calf serum (NBCS; Boehringer, Mannheim, No. 295957). After recovery from flushing medium, embryos were evaluated a t x 94 magnification for fertilization, stage of development, and morphological criteria (Niemann et al., 1983).

Received October 30, 1990; accepted December 19, 1990. Address reprint requests to Dr. H. Niemann, Institut fur Tierzucht und Tierverhalten, (FAL), Mariensee, 3057 Neustadt 1, Germany.

URIDINE INCORPORATION IN PORCINE EMBRYOS

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TABLE 1. 3H-UridineIncorporation per Embryo After 3 hr of Incubation in KRB Medium Containing 9 p M 3H-Uridine Stage of development Two-cell Four-cell Eight-cell Morula Blastoc. Exp. bl. Hatched bl.

d.p.m. Incorp. (mean f s.e.m.1 -

66 t 5.8 189 t 10.6* 617 t 62.4* 957 t 113 2168 +- 767 9012 2 1094*

fmol 3H-Uridine incorp. 1.1 3.1

9.9 15.4 34.7 144.4

Percent increase over previous stage -

186 226 55 127 316

Number of Replicates Embryos 8 57 21 150 16 119 13 107 12 74 3 20 7 41

*P < 0.001 compared with the previous stage of development.

After collection, embryos were randomized and washed four times in sterile flushing medium. They were then cultured in small cups (Greiner, Nurtingen, Germany, No. 618380) containing 50 p1 medium at 37°C with 5% C 0 2 in air and in a humidified atmosphere. The culture medium was composed of modified Krebs-Ringer bicarbonate medium (KRB) (Davis and Day, 1978) without bovine serum albumin (BSA) but supplemented with 10% heat-inactivated lamb serum (Sebio, Walchsing, Germany; No. 10096) (Niemann e t al., 1983). A total of 568 embryos from the two-cell to the hatched blastocyst stage were collected and grouped according to developmental stage. On the average, seven embryos per replicate were incubated in KRB medium with 9 pM 3H-uridine (Amersham Buchler, Braunschweig, Germany; specific activity 1.04 TBq/ mmol) for 3 hr. The uridine stock solution (37 MBq/ml) was dissolved in water and added to the culture medium. For the calculation of RNA synthesis on a per cell basis, embryos were considered to contain the following number of blastomeres; morulae 32, blastocysts 59, expanded blastocysts 95, and recently hatched blastocysts 240 (Niemann et al., 1983; Papaioannou and Ebert, 1988). RNA synthesis was determined according to the method of Warner and Tollefson (1977). At the end of the incubation period, RNA synthesis was stopped by adding 100 pg carrier RNA (yeast RNA, type 111; Sigma No. R-7125; stock solution 4 pg/ml bidistilled water) to each cup and freezing the cup contents in liquid nitrogen. The freeze-thaw process was repeated five times to disrupt cell membranes and to release nucleic acids into the medium. To isolate RNA, the cup contents were spotted on DEAE cellulose filter discs (Whatman DE 81, diameter 23 mm; Schutt, Gottingen, Germany). Each cup was washed with 75 pl 0.01 M Tris buffer (pH 7.5) with 0.5% BSA, and the washing was spotted on a separate filter. Blanks (at least three per experimental group) consisted of the incubation medium and washing of cups without embryos. To remove unincorporated 3H-uridine, filters were washed for 12-14 hr in 0.5 M sodium phosphate buffer at pH

9.2. The washing buffer was changed three times at 10 min intervals, then after 20 min, 30 min, and 3 hr. Filters remained in the last washing buffer for 8-10 hr overnight. Finally, they were rinsed twice in distilled water and once in 95% ethanol and ether. After air drying, radioactivity on the filters was counted in a liquid scintillation counter (Packard 2000 CA with luminescence and quench correction) using 10 ml of scintillation fluid (Rialuma; Baker Chemicals, Deventer, The Netherlands). Radioactivity was expressed as d.p.m. per embryo after subtraction of background activity, which amounted to 1,187 * 88 d.p.m. The specificity of 3H-uridine incorporation was verified by RNase treatment of four-cell, eight-cell, and morula/blastocyst stages. Eight embryos per group were incubated and were a t the end of the 3 h r incubation period transferred to 50 p1 buffer (1M NaC1, 2 mM EDTA, 40 mM Tris, pH 7.5) supplemented with 9 pM 3H-uridine. After the freeze-thaw procedure, 100 pg RNase (Sigma No. R-5503) dissolved in 10 g/liter Tris NaCl buffer (10 mM Tris, 15 mM NaC1, pH 7.5) was added. After a 2 h r incubation period a t 37"C, the assay for RNA synthesis was performed as described above. Differences among groups were calculated by oneway analysis of variance for completely randomized samples followed by a Scheffe test. Before calculation, data for developmental rates were transformed with arcsin (Snedecor, 1959), and calculation of RNA synthesis per cell was transformed by log 10 to create homogeneity of variance (Bartlett test). Differences were considered statistically significant at P < 0.05. Data are expressed as mean ? s.e.m.

RESULTS After 3 h r of incubation, a significant incorporation of 3H-uridine was measured in all embryonic stages but the two-cell stage (Table 1). In two-cell embryos, radioactivity above background levels could be determined in only one of eight replicates even though preliminary experiments demonstrated embryonic 3 H - ~ r i dine uptake. RNase treatment reduced radioactivity to

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M. FREITAG ET AL. d.p.mJ ’ cell

H -widin e per cell - 0.9

60 -

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-0.75

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-045

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-0.15

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(21)

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fertilization

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bl. exp. (11)

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(3)

hatched M. (6)

Fig. 1. 3H-uridine incorporation per blastomere after 3 hr in vitro incubation (mean -+ s.e.m.; N = replicates of about seven embryos). Differences between hatched blastocysts and younger stages were significant (P< 0.01).

background levels, verifying the specificity of 3 H - ~ r i dine incorporation into RNA. At the four-cell stage, radioactivity was above background levels in all 21 replications and amounted to 1.1 fmol(66 +- 5.8d.p.m.1 3H-uridine incorporation per embryo. 3H-uridine incorporation increased with time after fertilization and developmental stage of the embryo (Table 1). The increase in 3H-uridine incorporation per embryo differed, however, among developmental stages and amounted to about 200% between the fourcell and eight-cell stages and the eight-cell and morula stages (P < 0.001). It was not significantly different between the morula and the blastocyst and the blastocyst and expanded blastocyst stages and amounted to 55% and 130%, respectively. In hatched blastocysts, 3H-uridine incorporation increased by 300% (P < 0.001) compared with expanded blastocysts. 3H-uridine incorporation per blastomere (Fig. 1)was similar among zona pellucida-intact embryos, i.e., fourcell embryos to expanded blastocysts, and amounted to 0.29 to 0.37 fmol (16.9 6.6 to 23.1 1.4 d.p.m.1 3Huridine incorporation. In hatched blastocysts, genomic activity was increased to 0.86 fmol (51.4 4.6 d.p.m.) 3H-uridine incorporation (P < 0.01 compared with younger stages).

*

*

DISCUSSION In this study, rates of RNA synthesis were determined indirectly via incorporation of 3H-uridine, which is taken up by embryos and integrated into the endogenous UTP pool. This incorporation was observed for the first time at the four-cell stage. Uridine incorporation indicates RNA synthesis, since uridine is neither

incorporated into DNA nor metabolized to cytidine by early embryos (Woodland and Graham, 1969). The 3Huridine concentration of 9 pM was based on preliminary experiments with mouse embryos demonstrating highest repeatability of the assay system. The specificity of the RNA assay was further confirmed by RNase treatment that reduced radioactivity to background levels. RNA isolation by binding the nucleic acid to DEAE cellulose filters was preferred over precipitation on glass fiber filters because background activity is less and recovery rates are higher when working with small volumes (Litman, 1968). Background radioactivity was relatively high in our experiments. However, in all 21 replicates at the four-cell stage, 3H-uridine incorporation was well above background levels, which should allow the conclusion of first RNA synthesis at this stage. It must be taken into account, however, that the endogenous UTP and ATP pool sizes were not determined, so the amounts of RNA synthesis could not be measured and results were expressed as rate of 3Huridine incorporation. Although the ATP pool was relatively constant during preimplantation development in the mouse, the UTP pool increased from the one-cell to the blastocyst stage at different rates (Clegg and Piko, 1977). If indeed the UTP pool size changes during early porcine embryonic development, this may affect the rates of RNA synthesis. Embryos were incubated for 3 h r to allow recovery after the collection procedure and adaptation to the altered culture medium and to ensure equilibration of cytoplasmic and nuclear UTP pools. On the other hand, the incubation period is short enough to prevent embryo development to subsequent morphological stages.

URIDINE INCORPORATION IN PORCINE EMBRYOS Results were not expressed on a n hourly basis, since due to the “start effect” the first hour will likely not reflect the physiological amount of RNA synthesis. Moreover, we could not determine RNA synthesized within 1 hr, since RNA half-lives are not known. Our results demonstrate a lack of genomic activity in porcine embryos prior to the four-cell stage. This is in agreement with studies of Tomdnek et al. (1986) that also suggested first RNA synthesis at the four-cell stage. The ontogenetic pattern of RNA synthesis is only poorly understood but speculatively seems to differ among species. The amount of 3H-uridine incorporation per blastomere was not significantly different between the four-cell stage and the blastocyst stage in pigs in our study and in hamster embryos (Hutz et al., 1984). In mouse embryos, however, RNA synthesis remained low until the four-cell stage and increased 3.4fold between the four-cell and the morula stages and then declined in blastocyst stages (Clegg and Piko, 1977). This sharp rise in genomic activity coincides with the differentiation into inner cell mass and trophectoderm beginning at the eight-cell stage (Pakrasi and Dey, 1984) and is accompanied by changes in protein synthesis and energy metabolism (Ellem and Gwatkin, 1968; Clegg and Piko, 1977; Wales, 1986). At the same time, the number of ribosomes increases (Piko and Clegg, 1982) and cell content of maternal mRNA templates decreases (Adamson and Gardner, 1979). Furthermore, in mouse embryos, it was observed that the number of small ribonucleic proteins increased significantly from the two-cell stage to the blastocyst stage, which correlated with the capacity for RNA synthesis (Prather et al., 1990). Additionally, the amount of uridine incorporation in mouse embryos was augmented in direct proportion to the amount of granularity of the nucleoli (Hillman and Tasca, 1969).As a potential explanation for these species differences in genomic activity, we suggest that in hamster and pig embryos the process of differentiation might be stretched over a longer period of embryo development and thus no pronounced increase a t a certain stage would be observed. At the same time, maternal mRNA templates might be more stable and embryo development less dependent on its own genomic activity. Similar to our results, Tomanek et al. (1989) found a significantly higher RNA synthesis in eight-cell stages compared with younger stages, and no further increase was reported for morulae. In our experiments, 3 H - ~ r i dine incorporation increased ninefold from the four-cell stage to the morula stage. Our study was conducted with in vivo-grown embryos since it had recently been shown that in vivo- and in vitro-grown porcine embryos can display different ultrastructural features (Hyttel and Niemann, 1990). Thus the findings of this study reflect the in vivo situation. In summary, this study defined for the first time the ontogenetic pattern of 3H-uridine incorporation as a measure for RNA synthesis in the porcine embryo up to

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the newly hatched blastocyst and demonstrated that the blastomere gains RNA-synthesizing capacity at the four-cell stage, and this capacity remains relatively constant until hatching, when a significant increase occurs. Our findings should facilitate the planning of more detailed molecular studies into the RNA-synthetic activity of early porcine embryos, which should include measurements of RNA precursor pools and characterization of the main groups of synthesized RNA.

ACKNOWLEDGMENTS This work was supported by Deutsche Forschungsgemeinschaft grants EL 4619-1 and EL 4619-2. REFERENCES Adamson ED, Gardner RL (1979): Control of early development. Br. Med. Bull. 35113-119. Barnes FL, Eyestone WH (1990): Early cleavage and the maternal zygotic transition in bovine embryo. Theriogenology 33:141-152. Camous S, Kopecny V, Flechon J E (1987):Autoradiographic detection of the earliest stage of (3H)-uridineincorporation into the cow embryo. Biol Cell 58:195-200. Clegg KB, Pik6 L (1977):Size and specific activity of the UTP pool and overall rates of RNA synthesis in early mouse embryos. Dev Biol 58:76-95. Clegg KB, Pik6 L (1982):RNA synthesis and cytoplasmic polyadenylation in the one-cell mouse embryo. Nature 295342-345. Crosby JM, Gandolfi F, Moor RM (1988):Control of protein synthesis during cleavage of sheep embryos. J Reprod Fertil 82:769-775. Davis DL, Day BN (1978): Cleavage and blastocyst formation by pig eggs in vitro. J Anim Sci 46:1043-1053. Ellem KAO, Gwatkin RBL (1968): Pattern of nucleic acid synthesis in the early mouse embryo. Dev Biol 18:311-330. Flach G, Johnson MH, Braude PR, Taylor RAS, Bolton VN (1982): The transition from maternal to embryonic control in the 2-cell mouse embryo. EMBO J 1:681-686. Hillman N, Tasca R J (1969): Ultrastructural and autoradiographic studies of mouse cleavage stages. Am J Anat 126:151-173. Hutz RJ, Ghosh M, Dukelow WR (1984): Steroid uptake and 3 H - ~ r i dine incorporation by early hamster and squirrel monkey (in vitro fertilized) embryos: effects of ovulatory regimen. Zoo1 Sci 1:771776. Hyttel P, Niemann H (1990): Ultrastructure of porcine embryos following development in vitro versus in vivo. Mol Reprod Dev 27: 136-144. Litman RM (1968): A desoxyribonucleic acid polymerase from Micrococcus luteus (Micrococcus lysodeikticus) isolated on deoxyribonucleic acid cellulose. J Biol Chem 243:6222-6233. Manes C (1973): The participation of the embryonic genome during early cleavage in the rabbit. Dev Biol 32:453-459. Niemann H, Illera MJ, Dziuk PJ (1983):Developmental capacity, size and number of nuclei in pig embryos cultured in vitro. Anim Reprod Sci 5311-321. Pakrasi PL, Dey SK (1984): Role of calmodulin in blastocyst formation in the mouse. J Reprod Fertil71:513-517. Papaioannou VE, Ebert KM (1988):The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development 102:793-803. Piko L, Clegg KB (1982): Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos. Dev Biol 98:362378. Prather R, Simerly C, Schatten G , Pilch DR, Lob0 SM, Marzluff WF, Dean WL, Schultz GA (1990): U3 sn RNPs and nucleolar development during oocyte maturation, fertilization and early embryogenesis in the mouse: U3 snRNA and snRNPs are not regulated coordinate with other snRNAs and snRNPs. Dev Biol 138:247-255.

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3H-uridine incorporation in early porcine embryos.

The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos fro...
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