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Expression Pattern of Oct-4 in Preimplantation Embryos of Different Species

^a Department of Biotechnology, Institute for Animal Science and Animal Behaviour (FAL), Mariensee, Neustadt, Germany ^b Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany

ABSTRACT

POU transcription factors are involved in transcriptional regulation during early embryonic development and cell differentiation. Oct-4, a member of this family, has been shown to be under strict regulation during murine development. The expression of Oct-4 correlates with the undifferentiated cell phenotype of the mouse preimplantation embryo. In this study, expression of a gene construct consisting of selected parts of the region upstream from the murine Oct-4 gene as promoter/enhancer, enhanced green fluorescent protein (EGFP) as reporter and the five exons of the murine Oct-4 gene (GOF18-PE EGFP) was evaluated in murine, porcine, and bovine preimplantation embryos. For comparison, expression of the endogenous Oct-4 gene was also analyzed in all three species by immunocytochemistry. The transgene construct was microinjected into zygotes cultured in vitro to various developmental stages. The EGFP fluorescence was visualized in developing embryos by excitation with blue light at different days following microinjection and showed similar expression patterns in all three species. Most embryos displayed a mosaic pattern of transgene expression. The EGFP fluorescence was not restricted to the inner cell mass (ICM) but was also seen in trophoblastic cells. An affinity-purified polyclonal antibody specific to Oct-4 was used for immunocytochemical analysis of in vivo- and in vitro-derived bovine and porcine blastocysts and also of in vivo-derived murine blastocysts. In the in vivo-derived murine embryos, Oct-4 protein was detectable in the ICM but not the trophectoderm, whereas in porcine and bovine blastocysts, derived in vivo or in vitro, Oct-4 protein was detected in both the ICM and the trophectoderm. Thus, in the two large animal species, Oct-4 expression from the endogenous gene was clearly not restricted to the pluripotent cells of the early embryo. These results show that Oct-4 regulation differs between these species and that the presence of Oct-4 protein may not be sufficient for selection of undifferentiated cell lines in domestic animals.

conceptus, early development, gene regulation

INTRODUCTION

The POU domain proteins constitute a family of structurally related transcription factors that have been isolated from a variety of organisms [1, 2]. These proteins are characterized by the presence of a conserved DNA-binding domain, the POU domain, originally identified in the mammalian transcription factors Pit-1, Oct-1, and Oct-2 and in the nematode regulating protein unc-86 [1]. Most POU proteins are expressed during embryogenesis, suggesting that they may play a crucial role in development and cell differentiation [3–5]. Oct-4 is a member of Class V of the POU transcription factor family (6–8). Oct-4 cDNAs have been isolated in several laboratories, and the encoded factor has been termed Oct-4 [6] or Oct-3 [9]. The protein sequence, genomic organization, and chromosomal localization of the POU genes are highly conserved [10, 11]. It has been shown that the murine Oct-4 protein shows extensive sequence similarity with the bovine protein, with an overall identity of 81.7%. The percentages of the amino acid identity for exons 1 through 5 between bovine and murine Oct-4 are 71.0%, 95.1%, 95.4%, 84.9%, and 82.8%, respectively [11]. It has also been shown that the amino acid sequence of the human Oct-4 is 87% identical to that of the mouse, and human Oct-4 expression was found in heart, kidney, liver, placenta, spleen, and pancreatic islets using reverse transcription-polymerase chain reaction (RT-PCR) technology [10]. However, it remains to be shown that Oct-4 protein is present and active in these cell types. The high degree of sequence similarity suggests that Oct-4 plays a similar role in all mammalian species.

Oct-4 is the earliest expressed transcription factor that is known to be crucial in murine preimplantation development [9, 12–14], and its effects were recently shown to be dependent on its concentration in the nucleus [15]. Oct-4 mRNA and protein have been found in the unfertilized murine ovum and in the nuclei of subsequent cleavage stages [12, 14, 16, 17]. Oct-4 expression is downregulated during formation of the blastocyst. In the expanded murine blastocyst Oct-4 mRNA and protein are predominantly found in the inner cell mass (ICM) [17, 18]. The ICM differentiates into the epiblast (embryonic ectoderm) and the hypoblast (embryonic endoderm). High levels of Oct-4 protein are found in hypoblast cells as they migrate along the inner surface of the trophectoderm. In the epiblast, Oct-4 expression is high until germ layers start to form. At Day 8.5 of murine gestation Oct-4 becomes restricted to the primordial germ cells [13, 14, 18].

Oct-4 acts as a transcription factor for many genes specifically expressed in pluripotent cells [19]. This expression pattern suggests that Oct-4 is associated with the status of totipotency, both in the preimplantation embryo and in in vitro culture [7, 9, 12, 20, 21]. The high degree of conservation suggests that these genes might have a similar expression pattern that would make Oct-4 a suitable marker for identification of pluripotent and totipotent cells from domestic species.

In the present study we investigated the potential of individual blastomeres to express from the Oct-4 promoter using the reporter gene construct, GOF18-PE EGFP. This consists of an artificial promoter/enhancer containing elements from the 9-kilobase (kb) region upstream of the murine Oct-4 gene, a reporter gene (enhanced green fluorescent protein), and the five exons of the murine Oct-4 gene. This construct was microinjected into murine, porcine, and bovine zygotes, and after various periods of in vitro culture, expression was monitored by fluorescence microscopy. In addition, we studied expression of the endogenous Oct-4 gene by whole-mount immunocytochemistry using rigorously purified antibodies specific to Oct-4 in blastocysts of these three species. It has previously been demonstrated that the gene expression profiles of in vitro-produced bovine embryos differ from their in vivo counterparts [22, 23]. Differences have also been reported between in vitro- and in vivo-derived porcine embryos [24–26]. For these reasons, we compared the distribution of Oct-4 protein in in vivo- and in vitro-derived porcine and bovine blastocysts.

MATERIALS AND METHODS

Embryo Collection

Murine embryos Female CD2F1 mice were superovulated with a 10 IU eCG injection (Sigma, St. Louis, MO) followed by an injection of 10 IU hCG (Sigma) 46 h later and allowed to mate with males of the same strain. The following morning, mating was confirmed by the presence of a vaginal plug. Zygotes were collected 10 h after fertilization by flushing the excised oviducts with PBS supplemented with 1% NBCS (new born calf serum; Boehringer Mannheim, Indianapolis, IN). The cumulus cells were carefully removed from eggs with hyaluronidase (300 µg/ml in PBS). Following washing, the zygotes were incubated in PBS supplemented with 10% NBCS until microinjection. Blastocysts from superovulated mice were collected by flushing the uterus with PBS/NBCS (1%) at Day 3.5 postfertilization.

Porcine embryos Prepuberal gilts were superovulated and used as donors for porcine zygotes and blastocysts [27, 28]. Superovulation was induced by i.m. injections of 1500 IU eCG followed by 500 IU hCG 72 h later. Twenty-five and 34 h later, the gilts were mated or artificially inseminated. Forty-eight hours after the hCG injection they were killed in the Institute's experimental slaughterhouse. The reproductive tract was immediately removed and transported at 37°C to the laboratory. Each oviduct was flushed twice with 10 ml PBS containing 1% heat-inactivated NBCS to collect zygotes. After washing they were incubated in PBS with 10% NBCS until microinjection. To produce in vitro-derived porcine blastocysts for immunocytochemistry the in vivo-collected zygotes were cultured in NCSU-23 medium [29]. The culture was maintained in a humidified atmosphere composed of 5% CO₂ in air at 38.5°C for up to 7 days until the embryos had reached the blastocyst stage.

In vivo-derived blastocysts were collected by slaughtering donor animals on Days 4 and 5 after mating or artifical insemination and the blastocysts were recovered by flushing each uterine horn with 80 ml PBS/NBCS (1%) [28].

Bovine embryos In cattle, zygotes and blastocysts were obtained after in vitro maturation and in vitro fertilization of cumulus-oocyte complexes (COC) [30, 31]. Briefly, the COC from bovine ovaries were isolated via slicing [32] and subsequently matured in vitro in TCM 199 supplemented with 25 mM Hepes (Sigma), 1 µg/ml estradiol-17ß (Serva, Heidelberg, Germany), 0.5 µg/ml FSH (Folltropin; Vetrepharm, London, ON, Canada), 0.06 IU/ml hCG (Ekluton; Vemie, Kempen, Germany), and 10% estrus cow serum. In vitro fertilization was performed 22–24 h after initiation of maturation. COC were fertilized in Tyrode medium (Fert-Talp) supplemented with 0.56 µg/ml heparin, 1 µM epinephrine, and 10 µM hypotaurin. Swimup separated semen was added to the oil-covered fertilization drops each containing 1 x 10⁶ spermatozoa/ml [33, 34]. Oocytes and sperm were coincubated for 20 h. Cumulus cells were removed by a 6-min incubation in EDTA (0.2%)/trypsin (0.1%) dissolved in PBS/BSA (1%) followed by repeated pipetting.

Bovine zygotes were cultured in 30-µl drops of SOFaaBSA (8 mg/ml fatty acid-free BSA; Sigma), overlaid with silicone oil. Groups of seven to eight embryos were cultured together in a humidified modular incubator chamber at 39°C in 5% CO₂, 7% O₂, 88% N₂ gas mix for 8 days up to the blastocyst stage.

In vivo-derived blastocysts were recovered nonsurgically from superovulated donor cows on Day 8 after estrus by flushing each uterine horn with 400 ml prewarmed PBS supplemented with 1% NBCS using established procedures.

Oct-4 Reporter Construct

The gene construct (GOF18-PE EGFP) was produced by inserting the EGFP reporter gene between a modified 9-kb promoter fragment of the murine Oct-4 and its structural gene. The modified promoter was produced by removing the proximal enhancer element (PE) from the 18-kb genomic Oct-4 fragment (GOF). The remaining elements of the 9-kb Oct-4 promoter fragment included the GC-box, the hormone response elements (HREs), the binding site for the transcription factor ELP and the distal element (DE) [7]. The EGFP-1 gene (Clontech, Palo Alto, CA) was inserted in frame into an MluI site that had been introduced in front of the initiaton codon ATG of Oct-4 [17]. The construct GOF18-PE EGFP (Fig. 1) was excised from the plasmid pBluescript II by digestion with NotI, and the resulting 21.2-kb fragment was separated by electrophoresis on a 0.6% agarose gel. The fragment was excised from the gel and purified using the Qiagen gel purification kit. Purified DNA was quantified by optical density and diluted to 7 ng/µl in TE buffer, pH 7.4 (1 mM Tris-Cl, 0.1 mM EDTA). Aliquots of the DNA solution were stored at -20°C prior to microinjection.

View larger version (5K): [in this window] [in a new window]   FIG. 1. Schematic presentation of the construct GOF18-PE EGFP. Black boxes represent the exons of Oct-4. The position of EGFP insertion is indicated

Microinjection and Culture of Zygotes

Murine zygotes were placed on a glass microscope slide in a 500-µl drop of PBS/NBCS (10%) under the micromanipulator, and the gene construct was injected into pronuclei at 320x magnification. Prior to microinjection, bovine and porcine zygotes were transferred into a microcentrifuge tube and centrifuged for 3 min or 6 min at 15 000 x g, respectively, to displace lipids and visualize the pronuclei.

Swelling of the pronuclei was taken as the criterion indicative of successful microinjection. After injection, intact zygotes were returned to culture. The day of microinjection and the beginning of in vitro culture was defined as Day 1 of embryonic development. Murine embryos were cultured in 100-µl drops of M16 medium (Gibco-BRL, Eggenstein, Germany) in a humidified atmosphere of 5% CO₂ and 95% air at 38.5°C for 5 days by which time they had developed into blastocysts. Bovine zygotes were cultured for 9 days in SOFaaBSA under incubation conditions described above to produce blastocysts. In vitro development of porcine embryos was observed in four-well tissue culture plates (Nunc, Roskilde, Denmark) filled with 500 µl of culture medium NCSU-23 for 8 days under the same conditions described for mice. Control embryos without any manipulation were placed directly into culture media after flushing or fertilization.

Determination of EGFP Fluorescence in the Embryos

The EGFP fluorescence was evaluated on Days 3 and 5, respectively, after fertilization for mice, and on Days 5 and 8, respectively, after fertilization for cow and pig embryos using a fluorescent microscope with an excitation-filter at 488 nm and a bandpass (500 to 530 nm) emission-filter. At the same time, the cleavage rate (more than two blastomeres) and percentage of embryos that developed to morulae and blastocysts were recorded. The proportion of positive embryos (i.e., those with at least one fluorescing blastomere) and the distribution of fluorescing blastomeres within each embryo was assessed. In addition, the proportion of EGFP-positive blastomeres was determined in relation to all blastomeres within the embryo.

Polyclonal Oct-4 Antiserum

The antiserum used was a polyclonal antibody raised in rabbits against Oct-4. Antibodies were enriched from the serum by a Prot A Sepharose column. A rigorous purification procedure using a double affinity column was necessary to remove cross-reactivity with other POU proteins [14]. The specificity of the Oct-4 antibody was confirmed immunohistochemically on Western blots that included Oct-4 protein, the POU region of Oct-1, and the Oct-6 protein.

Immunocytochemistry

Whole-mount immunocytochemistry was performed on blastocysts from mouse, cow, and pig. Following double washing in PBS supplemented with BSA (4 mg/ml), the embryos were fixed for 30 min in freshly prepared 3% paraformaldehyde in PBS and permeabilized by incubation in PBS containing 0.2% Triton X-100 (Koch-Light Laboratories Ltd., Haverhill, Suffolk, UK). Nonspecific immunoreactions were blocked with 3% FCS (fetal calf serum) and 0.1% Tween-20 (Sigma) in PBS. Quenching of nonspecific binding on free aldehyde groups was accomplished with 50 mM glycine (Sigma) in Tween/FCS blocking buffer for 15 min. Following this pretreatment, the affinity-purified primary polyclonal antibody raised against Oct-4 and diluted 1:400 in PBS was applied for 30 min at room temperature. To remove all free Oct-4 antibodies, the embryos were washed extensively for 5 to 10 min in blocking buffer. After washing, the samples were incubated with secondary Alexa 488-conjugated goat antirabbit IgG (Molecular Probes, Eugene, OR) diluted to 1:2000 for 20 min at room temperature. After additional washing the embryos were incubated in PBS containing propidium iodide (30 µg/10 ml PBS) for 2 h in the dark. They were then mounted on slides in 10% DABCO solution in glycerol (1,4-diazabicyclo [2,2,2]-octane; Sigma) after going through an increasing glycerol series (5%, 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90%). Controls were produced using only the secondary antibody or by omission of both antibodies.

Confocal Laser Scanning Microscopy

The embryos were analyzed on the EMBL confocal scanning laser microscope. An excitation wavelength of 488 nm was selected from an argon-ion laser to excite the Alexa-conjugated secondary antibody and 529 nm wavelength to excite propidium iodide. Images of serial optical sections were recorded every 1.5- to 2-µm vertical step along the Z-axis of each embryo. Three-dimensional images were constructed using appropriate software written for the EMBL confocal microscope.

Statistical Analysis

Percentages of zygotes that had cleaved and formed blastocysts and showed EGFP expression were calculated as a proportion of total injected or noninjected zygotes that continued to develop. To determine differences in cleavage rate and blastocyst formation, the chi-square test was performed using 2 by 2 contingency table classifications. Immunocytochemical results were confirmed in at least two replicate experiments.

RESULTS

Expression of GOF18-PE EGFP During Early Development

A total of 241, 161, and 208 zygotes from mouse, pig, and cow, respectively, were injected with the experimental gene construct GOF18-PE EGFP. Cleavage rates and the proportion of cleaved embryos that developed further to the blastocyst stage are shown in Table 1.

View this table: [in this window] [in a new window]   TABLE 1. Survival of murine, porcine, and bovine embryos following microinjection of the construct GOF18–{}PE EGFP

Examination at Day 3 (mice) and Day 5 (pigs and cattle) revealed two- and four-cell embryos but also zygotes that had failed to divide. The numbers of embryos showing fluorescence both before and after major activation of the embryonic genome (MET) are shown in Table 2. Murine MET occurs at the 2-cell stage, porcine MET takes place at the 4-cell stage, and bovine MET occurs at the 8- to 16-cell stage [35]. The overall cleavage rate of murine and bovine embryos was significantly higher in the noninjected controls than in the injected groups, but no significant differences were observed between the cleavage rate of the noninjected and injected groups of porcine embryos. The proportion of cleaved porcine and bovine embryos that developed to blastocysts was not affected by injection. However, the development of murine blastocysts was higher in noninjected than in microinjected zygotes.

The ability to express the experimental gene construct (GOF18-PE EGFP), as indicated by the presence of green fluorescence, was observed in all developmental stages from the zygote up to the blastocyst stage and in all cell types in all three species. Varying degrees of green fluorescence intensity were observed in 16.2%, 34.2%, and 9.6% of murine, porcine, and bovine microinjected embryos, respectively (Table 3). As shown in Table 2, most of the fluorescing embryos displayed a mosaic pattern of expression (Fig. 2). While the proportion of positive cells varied considerably, most embryos showed fluorescence in less than half of their blastomeres. Only a few two-cell stages and a single murine blastocyst exhibited fluorescence in all blastomeres. In blastocysts, fluorescence was frequently restricted to a few or even single blastomeres. The intensity of fluorescence also varied considerably and embryos typically contained a range of strongly fluorescing, weakly fluorescing, and nonfluorescing blastomeres. The distribution of positive blastomeres was random. There was no relationship between EGFP/Oct-4 expression and developmental stage. Contrary to expectations, fluorescence was not restricted to the cells of the ICM but was also seen in trophectodermal cells (Fig. 3).

View larger version (127K): [in this window] [in a new window]   FIG. 2. Porcine two- and four-cell stages showing a mosaic expression pattern of the microinjected construct GOF18-PE EGFP. In two embryos only one blastomere each displays EGFP fluorescence

View larger version (121K): [in this window] [in a new window]   FIG. 3. The EGFP fluorescence of a few ICM and trophectoderm cells in a porcine blastocyst derived from microinjection of GOF18-PE EGFP into the pronucleus of a zygote

Oct-4 Protein Distribution in Blastocysts

A total of 42 in vivo-derived murine blastocysts were assessed by immunocytochemistry. Among these, the pattern of Oct-4 immunostaining depended on the developmental stage of the embryo. In early blastocysts, Oct-4 protein was detected in the ICM as well as in trophectoderm. The Oct-4 protein was in all cases localized to the nucleus. During formation and maturation of ICM and trophectoderm, the Oct-4 signal disappeared from the trophectoderm, and in expanded blastocysts the Oct-4 signal was restricted to the ICM (Fig. 4).

View larger version (90K): [in this window] [in a new window]   FIG. 4. Murine in vivo-derived expanded blastocyst with the Oct-4 signal (yellow fluorescence) concentrated to the ICM cells. Slight staining of trophectodermal cells can also be seen

Examination of bovine blastocysts showed no such relationship. In contrast to murine blastocysts, immunostaining of Oct-4 in 108 fully expanded, in vitro-derived bovine blastocysts revealed Oct-4 in the ICM as well as in trophectodermal cells (Fig. 5). The Oct-4 signal was observed in the cytoplasm as well as in nuclei of these cells. A similar picture was seen for 43 in vivo-derived bovine blastocysts.

View larger version (46K): [in this window] [in a new window]   FIG. 5. Bovine in vitro-produced blastocyst showing Oct-4 protein (yellow fluorescence) in the ICM and the trophectoderm

The distribution pattern of Oct-4 protein in 23 porcine in vitro-derived embryos and in 86 in vivo-derived embryos was similar to that observed in bovine blastocysts in that Oct-4 protein was present both in the ICM and in the trophectoderm (Fig. 6). A diffuse distribution of the Oct-4 signal was present in both cytoplasm and nuclei. No staining was observed in control embryos in which the primary antibody had been omitted (data not shown).

View larger version (55K): [in this window] [in a new window]   FIG. 6. Porcine in vivo-produced blastocyst showing Oct-4 protein (yellow fluorescence) in the ICM and trophectoderm. Accessory sperm in the zona pellucida are visible by the red propidium iodide fluorescence

DISCUSSION

This is the first study in which it was shown that an artificial promoter sequence based on modification to the 9-kb region upstream of the murine Oct-4 gene functions in three different mammalian species: mice, cattle, and pigs. This indicates that these species have similar control mechanisms for this promoter sequence and importantly opens the possibility of using a cell sorter to selecting cells that are expressing Oct-4 at a specific level. The results also demonstrated that this promoter was active in all cell types of all preimplantation stages in all three species, although the proportion of EGFP-positive embryos differed somewhat between the three species. This is consistent with previous observations [36, 37] that indicate that the expression of an injected transgene is not noticeably affected by factors controlling major activation of embryonic genome (MET).

A direct comparison of microinjection with in vitro- and in vivo-derived porcine zygotes showed that in vivo embryos had a higher cleavage rate [38]. The present study supports this generalization to the extent that the cleavage rate following microinjection of the in vitro-produced bovine zygotes was lower than that of the in vivo-derived murine and porcine zygotes. The fact that there was also a lower yield of fluorescing embryos from the microinjection of bovine zygotes supports the hypothesis that there are other factors unique to bovine embryos that make them more resistant to this stress than other species [39–41].

Mosaicism in the expression patterns and in the expression intensity of injected transgenes has been observed in several laboratories [36, 37, 42–46], and while some laboratories have reported a lower proportion of mosaicism under similar conditions [37, 46], it would appear that mosaicism is a characteristic of the injection procedure itself and not of a particular gene construct [42–45]. The occurrence of mosaics can be explained by the timing of integration, unequal distribution of the nonintegrated DNA during cell division, extrachromosomal replication of the injected DNA [36, 46], or loss of an integrated transgene during early development [47]. Our findings are consistent with a recent report showing that embryos expressing a microinjected gene uniformly were transgenic, while embryos with mosaic expression were nontransgenic [48].

To gain insight into the expression pattern of the endogenous Oct-4 gene, the spatial localization of Oct-4 protein was determined by immunocytochemistry using a well-characterized polyclonal antibody with no cross-reactivity to other POU domain proteins as determined by Western blot analysis. We focused on the blastocyst stage when the first differentiated tissue of the preimplantation embryo appears. In murine blastocysts, Oct-4 protein was not detectable in cells of the trophectoderm as reported in earlier studies [13, 14, 17]; however, even in fully expanded bovine and porcine blastocysts, both cell types were positive. Previously, Oct-4 mRNA and protein were observed in in vitro-produced preattachment stage bovine embryos using antibodies whose specificity was determined empirically by testing on differentiated and undifferentiated P19 cells [11]. Furthermore Oct-4 mRNA was detected by reverse transcription-T7 RNA-dependent amplification in both the trophectoderm and the primitive ectoderm at Days 10 and 11 of porcine pregnancy [49]. The single major difference between murine, porcine, and bovine blastocysts was that Oct-4 protein was not detected in the murine trophectoderm.

The early preimplantation development of mouse, pig, and cow is similar in that the embryos of these mammalian species all progress through three major morphogenetic transitions, compaction, cavitation, and expansion, finally leading to hatching and implantation. However, some differences are evident. One difference between murine embryos and those of larger mammals is in the timing of genetic and morphological transitions [35, 50]. Another difference is that cell fates in murine preimplantation development are more stringently regulated. For example, according to the inside/outside hypothesis, the two cell populations producing ICM and trophectoderm in the mouse are derived from the inner and outer cell layers of the morula [51]. In porcine and bovine embryos, compaction and ICM allocation seem to be independent processes and allocation may even be random [52]. The delayed downregulation of Oct-4 in large mammals may be the consequence of the lengthened period of preimplantation development. It may be necessary to study Oct-4 expression in porcine and bovine blastocysts between Days 8–15 and 8–23, respectively, to observe downregulation of Oct-4 expression in the trophectoderm in these species.

Following blastulation, greater differences in development are observed between the three species examined in this study. Murine embryos form an egg cylinder stage, whereas in livestock germinal disc-stage embryos are observed [53]. In the mouse, hatching and implantation occur more or less simultaneously, whereas in domestic species development is characterized by delayed implantation [50, 54]. In particular, the trophectoderm has characteristic differences among these three species. In mice, expansion of the blastocyst is relatively modest and the growth of the trophoblast is accomplished by mitosis only among the trophectoderm cells overlying the ICM [55]. Murine trophectoderm cells are end cells and are dependent upon contact with the ICM to retain their ability to proliferate. In fact, it has been proposed that murine ICM cells may become externalized and as such contribute to the trophectoderm lineage [56]. In contrast, the porcine trophoblast continues dividing and expands several orders of magnitude prior to implantation [57]. The bovine trophoblast also expands more than the murine trophoblast but not quite so extensively as the porcine trophoblast [50]. In mice, the decrease of Oct-4 levels in the trophoblast and the increase of Oct-4 protein in the primitive endoderm (hypoblast) suggest that as a transcription factor Oct-4 regulates both genes involved in determining cell commitment and genes involved in the regulation of proliferation in specific cell lineages [17]. Our findings support this hypothesis in that Oct-4 levels decreased in the trophectoderm of murine blastocysts at the time when proliferation is terminated but remained higher in the trophectoderm of the two domestic animal species that show high proliferative activity at this stage of development.

In addition to the comparison of endogenous Oct-4 protein distribution between murine, porcine, and bovine blastocysts, the distribution within in vitro-produced bovine and porcine blastocysts was compared to that of their in vivo counterparts. Several studies have demonstrated that in vitro-produced bovine and porcine embryos display a number of marked differences compared with their in vivo-derived counterparts, including morphology, color, cell number and cell size, timing of development [24, 25, 58], cellular damage (broken DNA) [26], and resistance to freezing [59, 60]. In bovine and murine embryos it has been demonstrated, that a variety of genes are differently expressed in in vivo-derived embryos compared with embryos produced in vitro [23]. In contrast, no differences were detected between in vivo- and in vitro-produced bovine or porcine blastocysts with regard to the Oct-4 protein distribution in the present study. It remains to be determined whether differences exist in the endogenous Oct-4 mRNA levels. Although there were no differences in the Oct-4 protein distribution between in vivo- and in vitro-produced embryos, the analysis of other such markers of embryo development could be useful for the evaluation of in vitro-produced embryos.

Oct-4 is presumed to be a critical factor controlling murine, bovine, and porcine preimplantation embryonic development. The striking finding in the present study is that Oct-4 protein in pig and cow was detected in the ICM and in the trophectoderm. In the mouse, Oct-4 expression correlates with the undifferentiated cell type, suggesting that Oct-4 is a marker for pluripotency. Its expression is crucial for maintenance of the pluripotent phenotype in nascent murine ICM cells [61], although residual Oct-4 protein is occasionally detected in cells of the murine trophectoderm in early blastocysts [17]. The detection of Oct-4 protein in the trophectoderm of porcine and bovine expanded blastocysts indicates that it may be the biological activity of Oct-4, and not simply its presence, that correlates with the embryonal stem cell type. Cytokines, such as leukemia inhibitory factor, are similarly important for maintenance of the undifferentiated/pluripotent state in embryonic stem cell cultures [62]. Recently, it was demonstrated that the level of Oct-4 expression is responsible for the maintenance or loss of pluripotency in preimplantation embryos [15].

In conclusion, data from our experiments reveal that there are marked differences in Oct-4 regulation between mice and larger mammals—especially the pig, which had not been previously studied. This study has established that a single construct, based on the 9-kb promoter sequence of the murine Oct-4 gene (GOF18-PE EGFP) and green fluorescent protein, is expressed in three different mammalian species: mice, cattle, and pigs. It is now recognized that the mere presence of Oct-4 protein or Oct-4 transcription do not define pluripotency because it is the level of expression that is the key to regulation [15], and it is necessary to measure Oct-4 activity quantitatively. Further experiments are needed to confirm that the level of bovine and porcine Oct-4 expression, as indicated by GOF18-PE EGFP, regulates pluripotency in the same manner as in the murine system. If this is the case, this construct will serve as a tool for selection of pluripotent cells.

ACKNOWLEDGMENTS

The authors thank Klaus-Gerd Hadeler for collection of in vivo bovine embryos and Doris Herrmann for the technical assistance during collection of porcine zygotes and in vivo-derived porcine blastocysts. Thanks also to the staff of the EMBL for their support and assistance in confocal microscopy.

FOOTNOTES

First decision: 26 April 2000.

¹ Correspondence: Heiner Niemann, Department of Biotechnology, Institut für Tierzucht und Tierverhalten, (FAL) Mariensee, 31535 Neustadt, Germany. FAX: 49 5034 871 101; niemann{at}tzv.fal.de

² Current address: University of Pennsylvania, New Bolton Center, Center for Animal Transgenesis and Germ Cell Research, Philadelphia, PA 19348.

Accepted: July 25, 2000.

Received: March 27, 2000.

REFERENCES

1. Herr W, Sturm RA, Clerc RG, Corcoran LM, Baltimore D, Sharp PA, Ingraham HA, Rosenfeld MG, Finney M, Ruvkun G. The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene product. Genes Dev 1988; 2:1513–1516.
2. Schöler HR. Octamania: the POU factors in murine development. Trends Genet 1991; 7:323–329.
3. Ruvkun G, Finney M. Regulation of transcription and cell identity by POU domain proteins. Cell 1991; 64:475–478.
4. Verrijzer CP, van der Vliet PC. POU domain transcription factors. Biochim Biophys Acta 1993; 1173:1–21.
5. Ryan AK, Rosenfeld MG. POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev 1997; 11:1207–1225.
6. Schöler HR, Ruppert S, Suzuki N, Chowdhury K, Gruss P. New type of POU domain in germ line-specific protein Oct-4. Nature 1990; 344:435–439.
7. Ovitt CE, Schöler HR. The molecular biology of Oct-4 in the early mouse embryo. Mol Hum Reprod 1998; 4:1021–1031.
8. Pesce M, Anastassiadis K, Schöler HR. Oct-4: lessons of totipotency from embryonic stem cells. Cell Tissue Organs 1999; 165:144–152.
9. Okamoto K, Okazawa H, Okuda A, Sakai M, Muramatsu M, Hamada H. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 1990; 60:461–472.
10. Takeda J, Seino S, Bell GI. Human Oct3 gene family: cDNA sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues. Nucleic Acids Res 1992; 20:4613–4620.
11. van Eijk MJ, van Rooijen MA, Modina S, Scesi L, Folkers G, van Tol HT, Bevers MM, Fisher SR, Lewin HA, Rakacolli D, Galli C, de Vaureix C, Trounson AO, Mummery CL, Gandolfi F. Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1. Biol Reprod 1999; 60:1093–1103.
12. Schöler HR, Balling R, Hatzopoulos AK, Suzuki N, Gruss P. Octamer binding proteins confer transcriptional activity in early mouse embryogenesis. EMBO J 1989; 8:2551–2557.
13. Schöler HR, Dressler GH, Balling R, Rohdewohld H, Gruss P. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J 1990; 9:2185–2195.
14. Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby PW, Staudt LM. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 1990; 345:686–692.
15. Niwa H, Miyazaki J-I, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000; 24:372–376.
16. Yeom YI, Ha HS, Balling R, Schöler HR, Artzt K. Structure, expression and chromosomal location of the Oct-4 gene. Mech Dev 1991; 35:171–179.
17. Palmieri SL, Peter W, Hess H, Schöler HR. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 1994; 166:259–267.
18. Pesce M, Wang X, Wolgemuth DJ, Schöler HR. Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech Dev 1998; 71:89–98.
19. Saijoh Y, Fujii H, Men C, Sato M, Hirota Y, Nagamatsu S, Ikeda M, Hamada H. Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor. Genes Cells 1996; 1:239–252.
20. Yeom YI, Fuhrmann G, Ovitt CE, Brehm A, Ohbo K, Gross M, Hübner K, Schöler HR. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 1996; 122:881–894.
21. Schöler HR, Hatzopoulus AK, Balling R, Suzuki N, Gruss P. A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J 1989; 8:2543–2550.
22. Abe H, Otoi T, Tachikawa S, Yamashita S, Satoh T, Hoshi H. Fine structure of bovine morulae and blastocysts in vivo and in vitro. Anat Embryol 1999; 199:519–527.
23. Niemann H, Wrenzycki C. Alterations of expression of developmentally important genes in preimplantation bovine embryos by in in vitro culture conditions: implications for subsequent development. Theriogenology 2000; 53:21–34.
24. Hyttel P, Niemann H. Ultrastructure of porcine embryos following development in vitro versus in vivo. Mol Reprod Dev 1990; 27:136–144.
25. Machaty Z, Day BN, Prather RS. Development of early porcine embryos in vitro and in vivo. Biol Reprod 1998; 59:451–455.
26. Long CR, Dobrinsky JR, Garrett WM, Johnson LA. Dual labeling of the cytoskeleton and DNA strand breaks in porcine embryos produced in vivo and in vitro. Mol Reprod Dev 1998; 51:59–65.
27. Eckert J, Tao T, Niemann H. Ratio of inner cell mass and trophoblastic cells in blastocysts derived from porcine 4- and 8-cell embryos and isolated blastomeres cultured in vitro in the presence or absence of protein and human leukemia inhibitory factor. Biol Reprod 1997; 57:552–560.
28. Reichelt B, Niemann H. Generation of identical twin piglets following bisection of embryos at the morula and blastocyst stage. J Reprod Fertil 1994; 100:163–172.
29. Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil 1993; 48(suppl):61–73.
30. Wrenzycki C, Hermann D, Carnwath JW, Niemann H. Expression of the gap junction gene connexin 43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo. J Reprod Fertil 1996; 108:17–24.
31. Wrenzycki C, Hermann D, Carnwath JW, Niemann H. Expression of RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with bovine serum albumin (BSA). J Reprod Fertil 1998; 112:387–398.
32. Eckert J, Niemann H. In vitro maturation, fertilization and culture to blastocysts of bovine oocytes in protein-free media. Theriogenology 1995; 43:1211–1225.
33. Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986; 25:591–600.
34. Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38:1171–1180.
35. Telford NA, Watson AJ, Schultz GA. Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 1990; 26:90–100.
36. Chauhan MS, Nadir S, Bailey TL, Pryor AW, Butler SP, Notter DR, Velander WH, Gwazdauskas FC. Bovine follicular dynamics, oocyte recovery, and development of oocytes microinjected with a green fluorescent protein construct. J Dairy Sci 1999; 82:918–926.
37. Kubisch HM, Hernandez-Ledezma JJ, Larson MA, Sikes JD, Roberts RM. Expression of two transgenes in in vitro matured and fertilized bovine zygotes after DNA microinjection. J Reprod Fertil 1995; 104:133–139.
38. Koo DB, Kim NH, Lim JG, Lee SM, Lee HT, Chung KS. Comparison of in vitro development and gene expression of in vivo and IVM/IVF derived porcine embryos after microinjection of foreign DNA. Theriogenology 1997; 48:329–340.
39. Eyestone WH. Challenges and progress in the production of transgenic cattle. Reprod Fertil Dev 1994; 6:647–652.
40. Wall RJ. Transgenic livestock: progress and prospects for the future. Theriogenology 1996; 45:57–68.
41. Wall RJ, Kerr DE, Bondioli KR. Transgenic dairy cattle: genetic engineering on a large scale. J Dairy Sci 1997; 80:2213–2254.
42. Takeda S, Toyoda Y. Expression of SV40-LacZ gene in mouse preimplantation embryos after pronuclear microinjection. Mol Reprod Dev 1991; 30:90–94.
43. Lemme E, Eckert J, Carnwath JW, Niemann H. Expression of 6WTK-LacZ gene construct in vitro produced bovine embryos following microinjection into pronuclei or cytoplasm [abstract]. Theriogenology 1994; 41:236.
44. Kubisch HM, Larson MA, Funahashi H, Day BN, Roberts RM. Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos. Theriogenology 1995; 44:391–401.
45. Chan AWS, Kukolj G, Skalka AM, Bremel RD. Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection. Mol Reprod Dev 1999; 52:406–413.
46. Burdon TG, Wall RJ. Fate of microinjected genes in preimplantation mouse embryos. Mol Reprod Dev 1992; 33:436–442.
47. Ellison AR, Wallace H, Al-Shawi R, Bishop JO. Different transmission rates of Herpesvirus thymidine kinase reporter transgenes from founder male parents and male parents of subsequent generations. Mol Reprod Dev 1995; 41:425–434.
48. Kato M, Yamanouchi K, Ikawa M, Okabe M, Naito K, Tojo H. Efficient selection of transgenic mouse embryos using EGFP as a marker gene. Mol Reprod Dev 1999; 54:43–48.
49. Piedrahita JA, Paquin Platts DD, Bazer FW, Finnell RH. Differential gene expression between embryonic ectoderm and trophectoderm in the preimplantation porcine embryo [abstract]. Theriogenology 1994; 41:274.
50. Betteridge KJ, Flechon JE. The anatomy and physiology of pre-attachment bovine embryos. Theriogenology 1988; 29:155–186.
51. Johnson MH, Maro B. Time and space in the mouse early embryo: a cell biological approach to cell diversification. In: Rossant J, Pedersen RA (eds.), Experimental Approaches to Mammalian Embryonic Development. Cambridge: Cambridge University Press; 1986: 35–65.
52. Boerjan M, te Kronnie G. The segregation of inner and outer cells in porcine embryos follows a different pattern compared to the segregation in mouse embryos. Roux's Arch Dev Biol 1993; 203:113–116.
53. Evans MJ, Notarianni E, Laurie S, Moor RM. Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocysts. Theriogenology 1990; 33:125–128.
54. Stewart CL. Prospects for the establishment of embryonic stem cells and genetic manipulation of domestic animals. In: Pedersen RA, McLaren A, First NL (eds.), Animal Applications of Research in Mammalian Development. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1991: 267–284.
55. Gardner RL. Cell lineage and differentiation during growth of the early mammalian embryo. Proc Nutr Soc 1990; 49:269–279.
56. Cruz YP, Pedersen RA. Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers. Dev Biol 1985; 112:73–83.
57. Papaioannou VE, Ebert KM. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development 1988; 102:93–803.
58. Greve T, Callesen H, Hyttel P, Avery B. From oocyte to calf: in vivo and in vitro. In: Greppi GF, Enne G (eds.), Animal Production and Biotechnology. Paris: Elsevier, Biofutur; 1995: 71–97.
59. Leibo SP, Loskutoff NM. Cryobiology of in vitro-derived bovine embryos. Theriogenology 1993; 43:81–94.
60. Niemann H. Advances in cryopreservation of bovine oocytes and embryos derived in vitro and in vivo. In: Enne G, Greppi GF, Lauria A (eds.), Reproduction and Animal Breeding: Advances and Strategies. Paris: Elsevier Biofutur; 1995: 117–128.
61. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Schöler H, Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct-4. Cell 1998; 95:379–391.
62. Stewart CL. Leukemia inhibitory factor and the regulation of preimplantation development of the mammalian embryo. Mol Reprod Dev 1994; 39:233–238.

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