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Embryo |
Centre de recherche en reproduction animale,3 Faculté de médecine vétérinaire, Université de Montréal, CP 5000, St-Hyacinthe, Québec, Canada J2S 7C6
Department of Animal Science,4 McGill University, Macdonald Campus, Ste-Anne-de-Bellevue, Québec, Canada H9X 3V9
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, embryo, implantation, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The proximal stimulus that induces the development of the trophoblast and the ICM of the mink embryo is only partially known. It has been shown that fibroblast growth factor-4 (FGF4) is necessary for proliferation of the mouse trophoblast in vitro [12]. In the mouse, FGF4 is expressed by the zygotic genome, beginning at embryo cleavage; expression persists through preimplantation development [13] and is required for postimplantation proliferation of both ICM and trophoblast [14]. Of the known FGF receptors (FGFRs), FGFR2 expression appears to be the earliest expressed during embryonic development in the mouse [15] and its absence compromises peri- and postimplantation proliferation of both trophoblast and ICM [16].
Mink blastocysts in diapause can survive in coculture with mink uterine cell lines and a low frequency of escape from diapause has been shown to occur under these conditions [17]. Little is known about the cellular events that characterize the reinitiation of embryonic development in mink. In this investigation, the paradigm of prolactin treatment in vivo was employed to terminate diapause. Embryo expansion, protein synthetic capability, and the proliferative capacity of the trophoblast and ICM were then characterized, as was the expression of FGF4 and FGFR2 by embryos and embryo-derived cell lines.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All procedures with live animals were approved by the Comité de déontologie, Faculté de médecine vétérinaire, Université de Montréal. Investigations were carried out over four successive annual breeding seasons using ranch mink of the Dark and Pastel varieties purchased from A. Richard (St. Damase, QC, Canada). Females were bred to two fertile males according to usual commercial farming procedures. Prior to 21 March and 7–9 days after the final mating, embryos in diapause were collected from the first group (0-time) of animals. The remaining animals were injected with 1 mg kg-1 day-1 ovine prolactin (Sigma, Oakville, ON, Canada) to terminate preimplantation delay. Embryos were collected by repeated flushing of the uterine horns with TC-199 medium (Gibco, Burlington, ON, Canada) containing 10% fetal bovine serum (Gibco) from a minimum of 3 and a maximum of 22 animals per collection date, at 1–2-day intervals over 11 days after the initiation of treatment. Groups of embryos were pooled within each day of collection, and the number of embryos per day ranged from 34 to 56, except for Day 1, when the sample comprised 10 embryos collected from three different uteri. Embryo diameters were estimated by ocular micrometer.
DNA and Protein Synthesis
To determine the pattern of acquisition of mitotic capability, embryos were collected in diapause and at Days 3, 5, 9, 11, and 13 after initiation of activation by prolactin (PRL) treatment. They were incubated overnight in the presence of 100 µM bromodeoxyuridine 5'-triphosphate (BrdU, Sigma) to determine DNA synthesis through embryo reactivation, as previously described [18] with minor modification. Following incubation, embryos were liberated from the zona/capsule by treatment with a solution of 0.1% pronase (Sigma), then fixed in 10% formalin for 10 min, permeabilized with 0.5% Triton X-100 (Biopharm, Laval, PQ, Canada) in PBS (Gibco) for 3 h, and washed twice in blocking solution (PBS, 3% BSA, 0.1% Tween 20; Bio-Rad, Richmond, CA) for 20 min at room temperature. Embryos were incubated for 2 h in 10 µl of anti-BrdU monoclonal antibody (Amersham, Oakville, ON, Canada) containing 1 µg/ml DNase, washed in blocking solution, and incubated with fluorescein-labeled goat anti-mouse IgG (Sigma) at 1:100 dilution. Embryos were then washed again in blocking solution and mounted onto slides in Mowiol (Sigma-Aldrich, St. Louis, MO) containing 5 µg/ml Hoechst 33342 (Sigma) to visualize nuclei.
To estimate protein synthesis, newly flushed embryos were incubated in TC-199 medium in the presence of 35S-L-methionine (New England Nuclear, Guelph, ON, Canada) at a concentration of 10 mCi/ml at 37°C for 2 h. They were then washed twice for 10 min in Tris-buffered saline, pH 7.6 (20 mM Tris, 137 mM NaCl, and 1 M HCl). Embryos were lysed in SDS cold dissociation buffer and 35S-methionine incorporation was determine as described by Bell et al. [19].
Culture of Trophoblast- and ICM-Derived Cells
To determine the growth potential of the trophoblast and ICM, embryos from the pool collected during diapause (n = 3), at Day 5 (n = 5), and Day 9 (n = 5) after initiation of activation were employed. ICM and trophoblast tissues were visualized by microscopy and manually dissected. Cell populations were plated onto a mouse fetal fibroblast feeder monolayer. The entire ICM was cultured in mouse embryonic stem (ES)-cell medium [20], while segments of trophoblast were incubated in trophoblast stem (TS)-cell medium containing 25 µg/ml FGF4 (Sigma) as described by Tanaka et al. [12]. The medium was changed at 2- to 3-day intervals, and the capacity of ICM and trophoblast explants to proliferate was determined by daily inspection of the colonies over 7 days. In cultures that proliferated, cells were passaged at confluence by scalpel or by trypsinization (0.25% trypsin EDTA supplemented with 10% chicken serum; Gibco). The trophoblast monolayers developed vesicular outgrowths (Fig. 1); these were removed by pipetting and were plated into gelatin-coated wells, with fibroblast-conditioned TS-cell medium to replace the fibroblast monolayer.
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To estimate the rate of growth of the trophoblast cell populations and their dependence on FGF4 for proliferation [12], cells were incubated in medium with or without FGF4 supplementation. Growth was assessed first by determining the maximum diameter of the consequent colonies, measured by ocular micrometer at 6, 24, or 48 h after plating. The growth curves were determined for trophoblast cells over 7 day in culture in fibroblast-conditioned medium in the presence or absence of FGF4. Cultures were terminated and cells were fixed with cold methanol and stored at -20°C until staining and counting. Cells were then washed for 5 min with ice-cold PBS, incubated in the dark for 5 min with 100 ng/ml 4'-6-diaminino-2-phenylindol (DAPI; Sigma), and again washed for 5 min with PBS. Ten preassigned fields of each culture well were photographed under epifluorescence at 100x and all nuclei were enumerated.
Cells from both ICM and trophoblast cultures were frozen on liquid N2 and preserved for periods of several weeks before thawing and reconstituting of cultures.
RNA Isolation
Blastocysts recovered by uterine flushing were washed in PBS and frozen in separate tubes in liquid nitrogen. Total RNA was extracted with RNeasy Protect Mini kit (Qiagen, Mississauga, ON, Canada), with modifications to the standard protocol. Briefly, embryonic cells were disrupted with 350 µl of RLT buffer + ß-mercaptoethanol, followed by a 20-sec vortex and the addition of 350 µl 70% ethanol. The column was washed once with 400 µl RW1 buffer, and a second time with 400 µl RPE buffer. RNA was eluted with 22 µl dimethyl pyrocarbonate-treated water for 10 min. From this volume, 8.8 µl was used for reverse transcription of mRNA.
RNA isolation from the trophoblast, ICM cells, and the cells of the fibroblast feeder layer was achieved according to the standard procedures with Qiaquick RNA isolation kit (Qiagen). An aliquot of 1 µg of the total RNA was used for reverse transcription.
Reverse Transcription-Polymerase Chain Reaction
Isolated RNA was treated with DNase 1 RNase free (Ambion, Austin, TX), and reverse transcribed into cDNA with Superscript RNase H- enzyme (Invitrogen, Carlsbad, CA) according to manufacturer instructions. Polymerase chain reactions (PCRs) were performed on a thermal cycler (MJ Research, Scarborough, ON, Canada) with 1 U of Taq DNA polymerase (Amersham) per µl of reaction in a final volume of 20 µl. One microliter of the reverse transcription product was used for each PCR for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and FGFR2; 2.0 µl reverse transcription product + 10% Q solution (Qiagen) were required to detect FGF4 in each PCR. The FGFR2 primer sequences were designed with the Oligo 4.0 program (National Biosciences Inc., Plymouth, MN) and based on an homology among Canis familiaris (GeneBank accession number AF211257), Homo sapiens (Z71929), Mus musculus (NM_010207) and Oryctolagus cuniculus (AF184968), FGFR2 sense: 5'-GGA CAG TGC TTA TTG GGG AGT AC-3', reverse: 3'-GCG ATG CTC CTG CTT AAA CTC CT-5'. FGF4 primers were bovine specific [21]. GAPDH primers were designed from the mink GAPDH mRNA partial sequence (GeneBank accession number AF076283), GAPDH sense: 5'-CCT GCT TCA CCA CCT TCT TG-3', reverse: 3'-GTC CAT GCC ATC ACT GCC AC-5'.
Data Analyses
Data were analyzed by means of least-square analysis of variance in the General Linear model procedures of SAS (SAS Institute, Cary, NC). Following confirmation of a significant F value, comparisons among means were made by the Tukey honestly significant difference test. Regression analysis was performed for in vitro proliferation experiments. Significance was established at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Embryos in diapause displayed a consistent diameter approximating 0.23 mm (Fig. 2). This parameter was constant through Day 3 after treatment with prolactin followed by a progression of embryo expansion to maxima on Day 13 following initiation treatment. The largest embryos recovered were 2.0 mm in diameter. In approximately half of the animals examined at Day 13, there were implantation chambers in the uterus and evidence of implantation in the form of proliferation of trophoblastic knobs and trophoblast invasion into the endometrial epithelium (Fig. 3).
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Embryo DNA and Protein Synthesis
BrdU incorporation was measured to determine DNA synthesis throughout embryo reactivation. No uptake of BrdU could be detected in embryos in diapause, suggesting that DNA synthesis was not underway. By Day 3 after initiation of prolactin treatment, there was substantial BrdU incorporation into embryonic cell nuclei in both the trophoblast and ICM compartments (Fig. 4). By Day 5, virtually all of the cells in the embryo displayed BrdU incorporation, persisting through Days 9, 11, and 13 (Fig. 4). As noted above, there was substantial trophoblastic outgrowth in some embryos recovered at Day 13 after prolactin-mediated reactivation, and intense BrdU staining defined the ICM of the embryo (Fig. 4).
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Protein synthesis in the embryo was assayed during the escape from diapause by determination of the incorporation of 35S-methionine. Embryos in diapause displayed the lowest levels of methionine incorporation (Fig. 5). The first significant increase could be detected at 72 h after initiation of PRL treatment (P < 0.05), and a logarithmic increase was observed up to Day 11, 2 days before implantation (Fig. 5, P < 0.01).
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The Growth Potential of ICM and Trophoblast
To further characterize termination of embryonic diapause, ICM and trophoblast cells were dissected from embryos in diapause and embryos from Day 5 or Day 9 after PRL reactivation and their potential for mitosis was examined in vitro. FGF4 was added to the trophoblast stem cell medium to maintain replication, presumably in the absence of differentiation, as shown in the mouse [12]. With a single exception (Table 1), neither trophoblast nor ICM cells from embryos collected during diapause showed potential for growth in vitro. Trophoblasts from Day 5 embryos yielded cultures of trophoblast cells that displayed the capacity to replicate in vitro. This potential for mitosis of the trophoblast was greatly reduced in embryos taken at Day 9 after activation (Table 1). ICM cultures displayed a different pattern of replication in that the majority of the cultures from embryos at Day 5 after activation did not appear to replicate in vitro, while all cultures from Day 9 demonstrated vigorous replication (Table 1 and Fig. 6). ICM and trophoblast cells exhibited different morphology (Fig. 6). Cells from the ICM, which have the potential to form the embryo proper, were cuboidal or epithelial-like throughout the culture (Fig. 6A). In contrast, the trophoblastic cells in the center of explants were smaller, rounded, and contained larger nuclei, replete with perinuclear lipoid inclusions (Fig. 6B). Those at the periphery acquired a pavement block phenotype, while retaining a higher nucleus:cytoplasm ratio than the ICM explants (Fig. 6B). As cultures progressed, trophoblastic vesicles formed and detached from the monolayer (Fig. 1).
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Provenance of FGF4 and Its Effects on Mink Trophoblast Cell Cultures
We then examined the occurrence of expression of FGF4 and FGFR2, the isoform of its receptor associated with early embryo development in the mouse [16]. Figure 7 depicts the results of PCR amplification of FGF4, FGFR2, and the control housekeeping gene, GAPDH, in reverse-transcribed RNA representative of groups of 3–5 whole embryos collected during diapause and at various times after reactivation. FGF4 transcript abundance was low during diapause, followed by an increase to a relatively constant level from Day 3 to Day 11 after activation. FGF4 transcripts were not detected in cultured trophoblast cells nor were they present in the fibroblast feeder layer. They were abundant in the ICM in culture. FGFR2 mRNA was not detected in the whole embryo until Day 5 after activation, after which time it was present. At Day 9, there was an apparent reduction in some samples. Strong expression of FGFR2 was observed in both trophoblast cultures and in the feeder layer of fibroblasts. Transcript abundance was low but detectable in ICM cultures that had been isolated from the feeder layer (Fig. 7). Together, the evidence indicates that the ICM is the source of FGF4 and its cognate embryonic receptor, FGFR2, is present on trophoblast and ICM cells, beginning soon after activation of the embryo.
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To further define the role of FGF4 in trophoblast proliferation, we plated trophoblastic vesicles in gelatin-coated wells in the presence and absence of this growth factor. The consequent colonies were approximately round, and we measured proliferation by determination of the maximum diameter of each. As can be seen in Figure 8a, trophoblast cultures did not change over 48 h of culture in the absence of FGF4. In contrast, significant growth and expansion of cultures was observed as early as 6 h after addition of FGF4 in fibroblast-conditioned medium, and the cultures containing this growth factor had persistent growth through 48 h to approximately threefold contemporary control values (Fig. 8a). Growth over 7 days, estimated by counting nuclei (Fig. 8b), indicated that, after an initial period of relatively slow proliferation, there was rapid cell replication in cultures containing FGF4, relative to control cultures subjected to fibroblast-conditioned medium without FGF4. Regression analysis indicated highly significant (P < 0.01) linear growth of both cultures for the period from 3 to 7 days after initiation of culture. The doubling time, calculated from the slope of the regression line, was 1.4 days for cultures containing FGF4, compared with 4.0 days in the absence of the growth factor. Cultures containing FGF4 reached confluence at 8–9 days after initiation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Embryo Expansion in the Reactivated Blastocyst
By sampling of a large number of animals between diapause and around the time of expected implantation, Stoufflet et al. [25] demonstrated that the diameter of mink blastocysts recovered from the uterus varies from 0.2 to 2.0 mm, presumably indicating a progression of expansion from diapause to implantation. We provide the first data on the sequence of expansion by showing that embryo volume increases gradually in the first few days after reactivation, followed by a more rapid increase in volume during Days 9–13. A similar spectrum of embryo expansion has been described for the spotted skunk [2]. The mink model is consistent in some ways with the roe deer, where the embryo first expands slowly at the termination of diapause [26]. In the roe deer, this is followed by rapid elongation of the embryo in the ruminant pattern until the time of implantation [26].
Increases in the volume of the mink blastocyst have been attributed to fluid uptake, based on the apparent low levels of protein synthesis recorded during embryo expansion [8]. This conclusion is not in agreement with the observations in the present investigation, as the first definable increase in volume follows the first definable increase in protein synthesis (35S-methionine uptake) by 2 days. Further, new protein synthesis, rather than solely fluid uptake [27], appears to be the mechanism for growth and expansion from Day 5 after activation through implantation. Our 35S-methionine uptake data demonstrating lowest incorporation during diapause are in agreement with reports indicating that only a basal level of amino acid uptake is present in spotted skunk embryos in diapause [28, 29]. The current results, along with previous studies in the skunk [1, 29], suggest the rate of protein synthesis increases somewhat slowly after escape from obligate diapause. The early stages of activation of the roe deer embryo are similarly characterized by modest uptake of 35S-methionine and, as in the mink, the logarithmic phase is observed later in the activation process [30]. This is in contrast with observations of a precipitous increase in amino acid uptake following reinitiation of development in the mouse blastocyst at the termination of facultative diapause [31]. The more rapid response in the mouse may reflect the fact that the rodent embryo implants within 48 h after activation, while the current study shows that the mustelid embryos implant some 10 days after the first measurable indication of protein synthesis.
An earlier study by Polejaeva et al. [32] indicated that there were direct effects of prolactin on increasing development of mink embryos in culture. Doses of 5–10 µg/ml were required, and embryos collected 13–16 days after mating responded to prolactin, while those collected on the seventh day after copulation did not. The mean diameter of the older blastocysts was 0.4 mm, similar to activated embryos in the present investigation, indicating that embryos that responded to prolactin in vitro had already escaped from diapause. It is well known that elevations in prolactin precede implantation [33] and that prolactin alone induces embryo implantation in the hypophysectomized mink [6]. It is possible that the exogenous prolactin we employed to terminate diapause also had direct effects on embryo development.
DNA Synthesis and Cell Proliferation Following Activation
In the present study, BrdU incorporation was not observed in mink embryos in diapause, in keeping with previous observations of an absence of mitotic activity [7] and 3H-thymidine uptake [28] in developmentally arrested mink embryos. With a single exception, none of the ICM or the trophoblast explants from embryos in diapause in the present study proved capable of proliferation, further supporting the view that mitotic activity is arrested during the delay that precedes implantation in the mink. At Day 3 after activation, there was clear evidence of BrdU incorporation throughout the mink trophoblast and in the ICM, indicating that the S-phase of the cell cycle had been reinitiated and was occurring during the 16 h of BrdU incubation. At Day 5, all trophoblast explants displayed the capacity for replication in vitro, as judged by the growth of colonies over 7 days, while the ICM proved less capable of in vitro proliferation. Concurrent observations of BrdU incorporation and in vitro proliferation at Day 9 after activation indicated that both trophoblast and ICM cells were in the S-phase of the cell cycle but that proliferation of the trophoblast in culture was restricted. It has been reported that the mink trophoblast displays chromosomal polyteny resulting from endoreduplication before implantation [34]. This bears some similarity to the rodent trophectoderm, which has been shown to undergo endoreduplication to form the trophoblast giant cells during the process of terminal differentiation of this tissue [35]. The current observation that the mink trophoblast synthesizes DNA at Day 9 after activation, coupled with the view that the trophoblast appears not to readily proliferate in vitro, suggests that endoreduplication may be occurring.
FGF4 and FGFR2 Expression in Mink Embryos and Embryo-Derived Tissues
In rodents, FGF4 is not required for preimplantation development, as null mutation of the FGF4 gene does not impair the advance of the mouse embryo to the blastocyst stage [14]. Of the known FGF receptors, FGFR2 expression appears to be the earliest in embryonic development in the mouse [15]. Targeted disruptions of the FGF4 [14] and FGFR2 [16] genes are embryo lethal and result in the same phenotype, i.e., interference with peri- and postimplantation proliferation of both trophoblast and ICM. The current results demonstrate that FGF4 is expressed in the mink embryo during diapause and following activation, while FGFR2 was first seen at Day 5 after activation. The substantial BrdU incorporation into embryos collected at Day 3 after activation, a time when the FGFR2 expression was absent, suggests that early proliferative events may not be mediated by this FGF receptor isoform. The abundance of signal for FGF4 and FGFR2 at Day 5 and thereafter suggests that FGF4, acting through the FGFR2, may participate in the events associated with postactivation development in the carnivore embryo.
ICM and Trophoblast Cell Lines Derived from the Mink Embryo
We demonstrate that the separated components of the mink embryo, the ICM and the trophoblast, can be cultured, passaged, frozen, thawed, and cultures reconstituted. Mixed cultures of mink ICM and trophoblast have previously been reported [23, 32], and there is a report of isolation of mink ICM by microsurgery and subsequent culture [23]. Sukoyan et al. [23] demonstrated that the ICM lines from the mink embryo had the characteristics of embryonic stem cells [36]. The current mink ICM lines, as well as others that we have derived from preimplantation embryos, did not differ in any obvious way from those previously reported [23].
This appears to be the first demonstration of isolation of trophoblast cells and the establishment of trophoblast cell lines from the carnivore embryo. FGF4 clearly enhances mink trophoblast cell attachment and replication in vitro. This concurs with the rodent model, where FGF4 promotes proliferation of trophoblast cells [12]. We report herein that FGF4 transcripts are present in whole embryos and in ICM cultures but are absent in cultured trophoblast cells. In the mouse, it has been shown that FGF4 is expressed by the ICM [37] in cells derived from the mink trophoblast. We suggest that the ICM of the mink embryo is the source of FGF4 for trophoblast proliferation and that its expression increases rapidly with embryo activation.
It is not currently known whether mink trophoblast cell lines we have isolated have stem cell potential, i.e., the capacity for continued proliferation and totipotency [36]. In the mouse model, trophoblast cells derived from embryos at either 3.5 or 6.5 days after fertilization display characteristics of differentiation in the form of expression of lineage-specific genes of the incipient placenta [38]. Nonetheless, in the presence of FGF4, mouse trophoblast cells retain their stem cell totipotency if transplanted back into the embryo, while removal of the growth factors drives them toward terminal differentiation into giant cells in vitro [36]. In this study, we show that the proliferative capability of mink trophoblast cells derived from Day 9 after activation of the embryo is consistently less than cells derived from Day 5, even in the presence of FGF4. Thus, it may be that differentiation takes place in the mink trophoblast before implantation and earlier in the developmental sequence than in the mouse model [38].
In summary, we have investigated the process of escape from diapause by the mink embryo and have profiled changes that take place in diameter and protein and nucleic acid synthesis. The first changes observed are in embryo size, apparently due to increased fluid uptake, and protein and DNA synthesis show marked increases within 72 h after the reinitiation of development. The ICM of the mink blastocyst expresses FGF4, and both trophoblast and ICM express receptors for FGF4, and these appear to play a role in preimplantation proliferation of embryonic components. We have examined the ability of embryo components taken at different times after initiation of embryo activation to proliferate in vitro and have demonstrated that the trophoblast from embryos at Day 5 after activation proliferates more readily than that from diapause or from Day 9 after activation, while cells from the inner cell mass replicate at a greater rate at Day 9. These findings raise a number of questions, including whether these cell lines have the characteristics of trophoblast stem cells and whether the endoreduplication that is believed present in vivo can be recapitulated in trophoblast lines in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: B.D. Murphy, CRRA, 3200 rue Sicotte, St-Hyacinthe, QC, Canada J2S 7C6. FAX: 450 778 8103; murphyb{at}medvet.umontreal.ca
Received: 7 April 2003.
First decision: 27 April 2003.
Accepted: 16 October 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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