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Embryo |
Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, Kennett Square, Pennsylvania 19348
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, embryo, gene regulation
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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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Bovine semen was prepared as described previously [13], with minor modifications. Bovine sperm was generously provided by the Hofmann Center for Animal Reproduction (University of Pennsylvania School of Veterinary Medicine, Kennett Square, PA). Subsequent to collection using an artificial vagina, fresh sperm were diluted approximately fivefold in modified Tyrode bicarbonate-buffered medium (SpTALP) and washed three times by centrifugation at 375 x g for 8 min. Capacitation was induced by incubation of sperm at a concentration of 50 x 106 sperm/ml in SpTALP containing 10 µg/ml of heparin for 4 h at 39°C under 5% CO2 in air. In vitro-matured bovine oocytes were obtained from a commercial supplier (BOMED, Inc., Madison, WI). At 19–24 h postmaturation, groups of 8–12 cumulus-oocyte complexes were placed in 100-µl drops of capacitated sperm suspension. After 6–8 h of incubation at 39°C under 5% CO2 in air, cumulus cells were removed from oocytes by pipetting. The presumptive zygotes were cultured in vitro in synthetic oviductal fluid [14] supplemented with fetal bovine serum (HyClone, Logan, UT) at 39°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 as described previously [15, 16].
Preparation of Mouse Embryos
Female C57Bl/6J x C3H/HeN (B6C3) and male ICR mice were purchased from Taconic (Germantown, NY). Female B6C3 mice were superovulated with 7.5 U of eCG, followed 48 h later by 7.5 U of hCG. Cumulus-oocyte complexes were collected 18 h post-hCG, and cumulus cells were removed by hyaluronidase (activity, >5000 IU/mg; 151271; ICN, Aurora, OH) at 50 U/ml in Hepes-buffered CZB medium at 27°C. For recovery of preimplantation-stage embryos, superovulated female B6C3 mice were mated with male ICR mice. Embryos were collected from the oviducts 42 h post-hCG and cultured in microdrops of bicarbonate-buffered CZB medium in 35-mm dishes (430588; Corning, Corning, NY). The CZB medium was supplemented with bovine serum albumin (0.4% w/v; 103700; ICN) and filtered on a cellulose acetate membrane (pore size, 0.22 µm; 09-719A; Fisher, Pittsburgh, PA) before use. Culture drops were overlaid with silicone oil (5 centistokes; DMPSV; Sigma, St. Louis, MO) and incubated under 5% CO2 at 37°C. Embryos were removed and fixed at the following stages: 2-cell (42 h post-hCG), 4-cell (52 h post-hCG), 8-cell (68 h post-hCG), morula (75–97 h post-hCG), and blastocyst (93– 101 h post-hCG). Animals were maintained and used for experimentation according to the guidelines of the Institutional Animal Care and Use Committee of the University of Pennsylvania.
Blastocyst Bisection
Blastocysts were bisected into the ICM-containing half (ICM-half) and the half containing only TE (TE-half) using Ultra Sharp Splitting Blades (AB Technology, Pullman, WA) attached to a micromanipulator.
Whole-Mount In Situ Hybridization of Bovine and Mouse Oocytes and Embryos
Digoxigenin-labeled sense and antisense riboprobes directed against mouse Oct4 mRNA were generated as described previously [17]. For in vitro transcription of a digoxigenin-labeled bovine Oct4 mRNA-specific antisense riboprobe and its sense control, a 517-nucleotide fragment (nucleotides 808–854, 1184–1342, and 1448–1755; GenBank accession no. AF022987) was amplified from bovine blastocysts by reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into pCRII TOPO (Invitrogen, Carlsbad, CA). Bovine and mouse oocytes and embryos were fixed in 4% paraformaldehyde/0.1% glutaraldehyde at room temperature for 30 min. In situ hybridization was performed as described previously [17, 18]. Samples were mounted in microdrops and positioned to localize the ICM.
RT-PCR of Transcripts in Bovine Oocytes and Blastocysts
Semiquantitative RT-PCR was performed on reusable Dynabead Oligo (dT)25 cDNA libraries synthesized from individual embryos as described by Mann et al. [19], with minor modifications. Individual oocytes, embryos, ICM-halves, and pools of four TE-halves were placed in 100 µl of lysis buffer (mRNA Direct Micro Kit; 610.21; Dynal A.S., Oslo, Norway), vortexed, and stored at –80°C. Lysates of individual embryos were incubated with 10 µl of pre-equilibrated Dynabeads and washed with the supplied buffers according to the manufacturer's instructions. The mRNA captured by Dynabeads was reverse transcribed using the Dynabead-coupled Oligo (dT)25 as primer, resulting in a cDNA library covalently bound to the Dynabeads. Beads were resuspended in 10 µl of Reverse Transcriptase buffer (Invitrogen) supplied with 10 mM dithiothreitol (Invitrogen), 0.2 mM each dNTP (Sigma), 20 U of RNase inhibitor (RNasin; Promega), and 50 U Superscript II (Invitrogen) and then incubated for 60 min at 42°C in a rotating hybridization oven. Subsequent to one wash in 50 µl of TNT buffer (50 mM Tris, 1mM EDTA [pH 8], 0.01% IGEPAL, and 0.01% Tween-20), mRNA was removed by incubation at 95°C for 1 min. Dynabeads Oligo (dT)25 cDNA libraries were stored in TNT buffer, and the first-strand cDNA bound to the Dynabeads was used as template for repeated gene-specific, second-strand cDNA synthesis.
Bovine Oct4 transcript and, as endogenous control, poly(A) polymerase (PolyA) transcript were amplified from each individual Dynabead cDNA library. Second-strand cDNA was synthesized from each library by performing one PCR cycle in 20 µl of PCR mix (1.5 mM MgCl2, 0.2 mM each dNTP, and 5 U/µl of Taq polymerase; Promega; in 1x buffer supplied by the manufacturer) containing 2 µM gene-specific forward primer. After denaturation at 95°C for 1 min, supernatant containing second-strand cDNA was mixed with 20 µl of PCR mix supplied with 2 µM gene-specific reverse primer, and 30 additional PCR cycles were performed as follows: 94°C for 30 sec, 54°C for 30 sec, and 72°C for 30 sec. Forward and reverse primers for bovine Oct4 were 5'-GGTTCTCTTTGGAAAGGTGTTC-3' and 5'-ACACTCGGACCACGTCTTTC-3', respectively (GenBank accession no. AF022987 [7, 20]). Sequences of forward and reverse primers for PolyA were 5'-GTTGCAGGGTAACCGATGAA-3' and 5'-TGTTGTGGGTATGCTGGTGT-3', respectively (GenBank accession no. X63436 [20]). Amplification conditions for PolyA were as described above, but with an annealing temperature of 57°C.
The PCR products for Oct4 (314 base pairs [bp]) and PolyA (361 bp) were electrophoresed in ethidium bromide-stained 3% agarose gels. The density of signal was measured using digital photography and NIH Image software (National Institutes of Health, Bethesda, MD), and the signal for Oct4 in each sample was standardized to that of PolyA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence and spatial distribution of Oct4 transcript in bovine oocytes and IVF embryos at the 2- to 4-cell (Day 1), 8-cell (Day 2), 16-cell (Days 3–4), compacted morula (Days 4–5), early blastocyst (Day 5), and expanded blastocyst (Days 6–7) stages were analyzed by in situ hybridization with a bovine Oct4 transcript-specific antisense riboprobe (see Materials and Methods). More than five oocytes or embryos of each developmental stage were analyzed from two and seven replicates for the oocyte/ cleavage/morula and blastocyst stages, respectively. For comparison with a species in which Oct4 expression has been previously characterized, mouse embryos were assessed in parallel at preimplantation stages using a mouse transcript-specific antisense riboprobe [17]. Both mouse and bovine oocytes contained maternal Oct4 transcript (Figs. 1a and 2a), but at a relatively low level compared to postcompaction stages (Figs. 1, e–h, and 2, f–h). In mouse embryos, Oct4 transcript was not detected at the 2- and 4-cell stages and was detected at a very low level at the precompaction 8-cell stage (Fig. 1, b–d). Similarly, bovine Oct4 transcript signal was not visible in 2-, 4-, and 8-cell bovine embryos, whereas 16-cell embryos exhibited a very weak signal compared to the respective negative controls (Fig. 2, b–e and i–k). In embryos of both species, Oct4 mRNA levels increased dramatically with compaction (Figs. 1, e and f, and 2f). In both mouse and bovine blastocysts (Figs. 1, g and h, and 2, g and h), Oct4 mRNA was clearly downregulated in the TE and restricted to the ICM. Embryos processed without riboprobe (Fig. 2, i–l) are shown for comparison. A strong ICM-specific signal was detected in all of 39 expanding and hatched bovine blastocysts analyzed (Fig. 2h).
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Relative Levels of Oct4 Transcript in Bovine Oocytes and Embryos
To confirm that the differences in signal intensity as visualized by in situ hybridization between oocytes and various preimplantation stages correlated with the true level of expression, we performed semiquantitative RT-PCR on individual oocytes and blastocysts. Figure 3A shows amplification of Oct4 transcript and, as an endogenous control, PolyA transcript from three individual bovine oocytes (lanes 2–4) and blastocysts (lanes 5–7). The PCR amplification was limited to 30 cycles for comparison during the exponential stage of amplification. The Oct4/PolyA values of the oocytes (0.017–0.784) were much lower than those of the blastocysts (2.647–3.308), which is consistent with the results obtained by situ hybridization (Fig. 2). In three repetitions, the level of Oct4 transcript in blastocysts was consistently more than threefold that of oocytes (Fig. 3, A and B; compare lane 3 [oocyte] and lane 10 [blastocyst]). Oct4 transcript levels in 2-, 4-, and 8-cell embryos were very low (<0.01 the level of the oocyte) (Fig. 3A, lanes 4–7) but were detectable at the 16-cell stage (Fig. 3B, lane 8). Comparison of Oct4 transcript levels detected in whole blastocysts (Fig. 3C, lane 1), the ICM-half of a blastocyst (Fig. 3C, lane 2), and a pool of four TE-halves of blastocysts (Fig. 3C, lane 3) confirms restriction of Oct4 transcript to the ICM of the bovine blastocyst as visualized by in situ hybridization (Fig. 2, g and h).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During preimplantation development, the distribution of Oct4 transcript in bovine embryos is very similar to that in the mouse (Fig. 4). In both species, the onset of embryonic expression of Oct4 mRNA occurs at one or two cell cycles after genomic activation (e.g., at the 8-cell stage in the mouse and the 16-cell stage in the bovine). Furthermore, transcript levels significantly increase subsequent to compaction. Both mouse and bovine blastocysts express Oct4 mRNA in the ICM, whereas transcription of Oct4 is downregulated in the TE. Therefore, regulation of transcription is conserved both spatially and developmentally, which is consistent with the high homology of the regulatory regions between the mouse and bovine Oct4 genes [8].
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Contradictory to previous analyses by RT-PCR [7, 20], Oct4 transcript is not present in bovine cleavage-stage embryos until the precompaction morula stage, as determined by in situ hybridization. Previous analyses of Oct4 transcription by RT-PCR reported the presence of bovine Oct4 transcript at all stages of preimplantation development [7, 20], with high levels in the oocyte and blastocyst and low levels in the early cleavage stages [20]. Our results from situ hybridization and RT-PCR indicate that maternal transcript in the oocyte is much less abundant than embryonic transcript in the compacted morula and in the blastocyst. Recent findings indicate that Oct4 transcript levels in bovine oocytes are higher than in early cleavage stages but are 20-fold lower than in blastocysts [21]; in this report, levels of transcript from pools of 40 oocytes/embryos at each stage were analyzed by real-time PCR, and our results from individual oocytes/embryos are consistent with these published results.
Despite high similarity in transcript distribution, Oct4 protein distribution is clearly heterogeneous between mammalian species. Whereas Oct4 protein is absent from the TE of the expanded mouse and monkey blastocyst, it is found in both ICM and TE cells of the bovine blastocyst at the early and the expanded stages [7, 12]. Because our observations show that Oct4 transcript is already downregulated in the TE of the bovine blastocyst at Day 7, Oct4 protein detected in the TE at later stages must have been produced at or before the blastocyst stage and, therefore, must be stable for at least several days. Thus, although commitment to a trophectodermal fate has been made on a transcriptional level, protein clearance appears to be delayed. This is, to a lesser degree, also observed in the mouse, in which subsequent to transcriptional downregulation of Oct4 in the TE of the blastocyst, substantial amounts of protein remain in the TE and clearance is not observed until the expanded blastocyst stage [5, 12]. The disparity between cessation in transcription and presence of protein indicates that Oct4 protein is either more stable or is not actively eliminated in the TE of bovine embryos subsequent to the blastocyst stage. The persistence of Oct4 protein in the bovine TE compared to the mouse could be attributed to differences in trophectodermal function. Implantation of the bovine embryo is delayed compared to the mouse, and blastocysts expand and elongate before implantation, with extensive proliferation of the TE. Bovine Oct4 acts as a repressor of TE-specific genes, such as interferon-
, and is implicated as a regulator of trophectodermal differentiation [22]. Oct4 protein maintained in the TE may suppress the expression of extraembryonic lineage-specific genes during preimplantation development and allow extensive proliferation of the TE before implantation.
Establishing Oct4 as a marker for pluripotent cell populations has application for animal cloning and establishment of ES cells in domestic species. Aberrant spatial distribution and levels of Oct4 transcript are observed in a majority of mouse clone blastocysts and blastocyst outgrowths, and ES cell lines can be established from cloned blastocysts with high levels of Oct4 expression [17]. Therefore, examining the expression pattern of Oct4 transcript in bovine clones may be an effective means for evaluating nuclear reprogramming in nuclear transfer. Because placental abnormalities are frequently observed in cloned animals, analysis of Oct4 expression in respect to its interacting protein partners, particularly those involved in extraembryonic lineage differentiation, may provide important information concerning the abnormalities in somatic cell clones.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: K. John McLaughlin, Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, Kennett Square, PA 19348. FAX: 610 925 8121; kjmclaug{at}vet.upenn.edu
Received: 5 March 2004.
First decision: 2 April 2004.
Accepted: 25 June 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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