Molecular Human Reproduction, Vol. 8, No. 8, 758-764,
August 2002
© 2002 European Society of Human Reproduction and Embryology
Embryology |
Expression of cystic fibrosis transmembrane conductance regulator during early human embryo development
1 Division of Reproductive Sciences, Department of Obstetrics and Gynaecology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 2 Prenatal Diagnosis Program, the Toronto Hospital General Division, 3 Division of Cell Biology, 4 Department of Biochemistry, The Hospital for Sick Children and 5 Department of Physiology, University of Toronto, M5G 1X8, Ontario, Canada
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Abstract |
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Formation of the blastocyst is one of the first morphological changes in early embryonic development. Ion transport has been shown to be crucial for blastocoele cavity formation and expansion, although the mechanisms that underlie this process are presently unknown. As a transmembrane Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) may participate in ion transport and early blastocoele formation. CFTR mRNA was detected throughout preimplantation embryo development and in the unfertilized oocyte. Immunocytochemistry disclosed the presence of CFTR protein from the 8-cell stage, reaching maximum immunoreactivity at early blastocyst stage embryos. Patch clamp electrophysiology of morulae and blastocysts demonstrated typical CFTR Cl- channel activities in the apical membrane of trophectoderm cells. Thus CFTR is expressed both at mRNA and protein levels in human morulae and blastocysts, and functions as a cAMP-regulated apical membrane Cl- channel. These data suggest that CFTR may contribute to blastocoele formation in the early human embryo.
blastocoele/blastocyst/CFTR/cystic fibrosis/embryo cavitation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Mammalian preimplantation embryonic development is characterized by a series of rapid cell divisions. Maternally derived products deposited in the oocyte during oogenesis control the first cleavage divisions. Further embryonic development, which in mammals requires activation of the embryonic genome, occurs between the 2- to 8-cell stages (Braude et al., 1988
; Telford et al., 1990). Subsequent cleavage stages lead to the first differentiation process, which results in the creation of a blastocyst. The blastocyst contains two morphologically distinct cell types: trophectoderm, which gives rise to the placenta, and the inner cell mass (ICM), which is composed of relatively undifferentiated cells that will develop into the embryo (Cross, 2000).
Knowledge of the physiology of blastocoele fluid formation was initially obtained by extensive studies on the rabbit which has a conveniently large blastocyst, beginning with the independent work of Smith, Gamow and Prescott, and Daniel (Daniel, 1970; Gamow and Prescott, 1970; Smith, 1970). Later studies focused on the mouse, in which the genetic control of the proteins involved in this process could be studied in depth (Biggers et al., 1988; Watson et al., 1992). After the 8-cell stage, the mouse embryo contains tight junctions between the adjacent blastomeres and the cells become tightly sealed with each other. This cellular process, referred to as compaction, results in formation of the morula (Ducibella et al., 1975; Fleming and Johnson, 1988). The next developmental stage requires the first obvious cellular differentiation, accompanied by creation of a liquid filled cavity—the blastocoele. During this process, external cells form the outside layer of trophectoderm, while internal cells establish the ICM. The trophectoderm cells, which represent the first differentiated cell type in development, form a polarized epithelium joined by tight junctions. This cell type represents the first functional epithelium in mammalian embryo development. Vectoral fluid transport by this epithelium into the intercellular space generates the blastocoele cavity (Manejwala et al., 1989) which is crucial for subsequent embryonic development.
Previous reports have shown that Na+ and Cl- play a major role in blastocoele formation (Cross, 1973; Borland et al., 1976; Manejwala et al., 1989). Na/K-ATPase, located in the basolateral membrane of the trophectoderm cells, is necessary for fluid accumulation (Overstrom et al., 1989). However, further details regarding the molecular mechanisms involved in blastocoele function are unclear. Na+ flux into mouse trophectoderm cells is reduced by inhibitors of Na+/H+ exchange and Na+ channels (Manejwala et al., 1989). In rabbit trophectoderm, transepithelial Na+ transport seems to involve amiloride-sensitive Na+ channels and Na+/H+ exchangers (Robinson et al., 1991), as can be observed in other fluid-transporting epithelia. The pathways utilized by Cl- are less well defined. In the mouse embryo, early reports suggested that Cl- transport is passive and paracellular since some Cl- channel inhibitors, including diisothio-cyanatostilbene-disulphonic acid (DIDS) and 2-(3,4-dichlorbenzyl)-5-nitrobenzoic acid, had no effect on Cl- uptake or blastocoele formation (Manejwala et al., 1989). In contrast, blastocoele formation was reduced by furosemide, DIDS and anthracene-9-carboxylic acid in rat (Brison and Leese, 1993) and rabbit embryos (Benos and Biggers, 1983). Interestingly, a previous report showed that both Cl- efflux as well as re-uptake into Cl- depleted blastocoele, occurs via a Cl- channel-like mechanism (Zhao et al., 1997). However, the identity of this Cl- channel remains unknown.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein encoded by the cystic fibrosis (CF) gene (Riordan et al., 1989). CFTR is a cAMP-regulated Cl- channel (Bear et al., 1992) which is expressed in epithelial cells in a number of adult tissues, including the pancreas, intestine, lungs and salivary glands (Kartner et al., 1992). It plays a role in transepithelial ion and fluid transport by providing a pathway for Cl- across the apical membrane. CFTR has been detected in mid-trimester fetal tissue (Harris et al., 1991; McCray et al., 1992) and the CFTR mRNA transcript has been found in human fetuses as early as 10 weeks gestation (Tizzano et al., 1993). However, CFTR expression and its possible involvement in human preimplantation embryo development have not been established. We report here that CFTR is expressed during early human embryo development, and that it functions as a cAMP-regulated Cl- channel in apical membranes of the human blastocyst. Moreover, maternally deposited mRNA for CFTR protein may explain the mechanism of how CFTR mutant blastocysts cavitate and are able to develop beyond the implantation stage.
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Materials and methods |
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Human samples
Human cumulus cells, unfertilized oocytes and spare preimplantation embryos were obtained from the IVF programme at The Toronto Hospital, General Division. Patients who chose not to freeze their spare embryos for future transfers were asked to donate these embryos for research and their informed consent was obtained. This research was approved by the Committee for Research on Human Subjects of the Toronto Hospital. Donated spare embryos were incubated in HAM's-F10 media (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated human serum. Morphological assessment of the developing embryos was recorded daily until they reached the blastocyst stage,
5–6 days after oocyte insemination.
Nucleic acid preparation from a small number of cells and RT–PCR
We used RT–PCR for a small number of cells as previously described (Brady and Iscove, 1993). To ensure adequate nucleic acid extraction, we used one blastocyst or morula, 3–5 embryos in early cleavage stages or 100 cumulus cells, which were loaded into guanidinium thiocyanate solution. Total nucleic acid was recovered from each sample by ethanol precipitation using glycogen as a carrier. The pellet of nucleic acid obtained from the embryos and cumulus cells was reverse transcribed as previously described (Jurisicova et al., 1996). RNA from first trimester placenta or intestinal epithelium was used as a positive control (up to 1 µg total). The RT product was divided into two portions for PCR amplification of CFTR and ß2-microglobulin (ß2m) transcripts. Thirty-five cycles of PCR, which included denaturation at 95°C for 1 min, annealing at 61°C for 1.5 min and extension at 72°C for 2 min, were performed in a PCR Thermal Cycler (Perkin-Elmer). Following PCR, the amplified products were separated on a 2% agarose gel along with a 1 kb DNA ladder. To differentiate between the possible genomic contamination, primers were designed to span the two last exons (23 and 24) of the CFTR gene. The desired amplified product of the CFTR cDNA is only 278 bp. The sequence of the primers was as follows: CF1 5'-CACAGGATAGAAGCAATGCTG-3' and CF2 5'-CATGAGCAAATGTCCCATGAC-3'. The specificity of the CFTR cDNA amplified fragment was confirmed by restriction digestion with the Alu I enzyme, which was expected to cut the RT–PCR product into two 140 bp products. Primers for ß2m were used as previously described (Jurisicova et al., 1996).
Quantitative analysis of CFTR mRNA expression
In order to quantitate expression of the CFTR transcript during preimplantation embryo development, we used a RT–PCR dot blot-based assay (Rambhatla et al., 1995). Three samples of cDNA at each developmental stage, derived from a single oocyte or embryo, were amplified as previously described (Brady and Iscove, 1993). The amplified material was then analysed by hybridization of dot blots with random prime radiolabelled cDNA probes, followed by quantitation of signals on a phosphorimager as described (Rambhatla et al., 1995). The cDNA probes used for analysis recognize the 3' untranslated regions (3' UTR) of ß-actin and CFTR, and were either cloned from an oligo dT-primed human full term placenta cDNA library (ATCC, Rockville, USA) or amplified by RT–PCR using primers located within 3' UTR (5610–6050 bp) of the CFTR transcript (accession no. M28668). The identity of both clones was confirmed by sequencing using a dsDNA Cycle Sequencing Kit (Gibco).
Immunocytochemistry
A mouse monoclonal CFTR antibody of isotype IgG1-kappa (M3A7) was used as the primary antibody. This antibody recognizes an intracytoplasmic domain of the human CFTR protein between amino acids 1195–1480 (Kartner et al., 1992). As a secondary antibody, we used biotinylated swine immunoglobulin (Multilink; Dako, Denmark). The complex was identified by streptavidin conjugated to Texas Red (Calbiochem, La-Jolla, CA, USA).
Morulae and blastocysts were washed twice with phosphate-buffered saline (PBS) and either fixed on slides with cold acetone or processed as whole mounts. Fixed samples were washed in PBS for 10 min at room temperature, preincubated in PBS + 0.1% Triton-X supplemented with 10% normal swine serum (PTS) for 10 min, and then incubated with M3A7 antibody (20 µg/ml; 4°C, overnight). Following two washes in PBS, the embryos were re-incubated in 1:150 dilution of Multilink for 30 min. A final incubation was performed at 4°C in Streptavidin–Texas Red in PBS for 45 min (1:200 dilution), followed by three washes in cold PBS (10 min each) and counterstaining with nuclear fluorochrome, DAPI. Control embryos were exposed to the same conditions omitting M3A7 antibody in the primary incubation or were exposed to non-specific IgG antibody as previously described (Kartner et al., 1992). All samples were examined using a Zeiss Axioplan Universal microscope and photographs were taken with a Zeiss microscope Camera MC 100.
Electrophysiology
Oocytes or embryos were transferred onto a glass coverslip and allowed to stick on the stage of an inverted microscope mounted in a perfusion chamber, and visualized with video enhancement. Prior to transferring the samples to the chamber, the zona pellucida was removed using acid Tyrode's solution and the embryos were extensively washed in fresh culture medium.
The patch clamp technique was used to record single channel currents in cell-attached and excised (inside-out) patches. Recordings were usually carried out with identical solutions in the pipette and bath, using either (i) an NMDG-Cl solution (in mmol/l): 120 N-methyl-D-glutamate (NMDG), 2 MgCl2, 0.1 CaCl2, 1.1 EGTA, 10 HEPES, 5 glucose, pH 7.4 with HCl; or (ii) a CsCl solution (in mmol/l): 120 CsCl, 2 MgCl2, 0.1 CaCl2, 1.1 EGTA, 10 HEPES, 5 glucose, pH 7.4 with CsOH. Data are shown for experiments performed with the NMDG solutions. Similar results were obtained with the Cs+ solution (data not shown). In asymmetrical conditions, NMDG-Cl was reduced to 20 mmol/l in the bath and 200 mmol/l glucose was added to balance the osmotic pressure. The patch pipettes used (<0.5 µm diameter) had a resistance of 15–30 megaohms and were heat polished. In cell-attached recordings, intracellular levels of cAMP were elevated by addition to the bath of a cocktail (FIC) containing forskolin (1–10 µmol/l), 3-isobutyl-1-methylxanthine (IBMX; 10–100 µmol/l) and chlorophenylthio-cAMP (CPT-cAMP; 10–100 µmol/l). In excised patches, the catalytic subunit of protein kinase A (PKA; 90 nmol/l) and ATP (1 mmol/l) were applied to the bath solution. Treatments were added directly as a stock solution to achieve the desired concentration, or by perfusion (3–4 ml/min). All experiments were performed at room temperature. Currents were amplified (Axopatch-1D; Axon Instruments, Inc.), digitized (Macintosh computer via ITC-16 interface; Instrutech Corp.) and saved directly onto the hard disk (Pulse+PulseFit software; HEKA Electronik). Data were analysed using Pulse+PulseFit and MacTac (Skalar Inst.). Open probability (Po) of single channels was determined for 20 s periods of recording. Data were expressed as original values or mean ± SEM, and curves were fitted using simple regression analysis. X/Y indicates that X out of total Y patches responded to a drug.
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RT–PCR detection of CFTR and ß2m in human embryos
RT–PCR analysis of RNA isolated from intestine with primers CF1 and CF2 resulted in the expected 278 bp CFTR product. Cumulus cell samples obtained from five different patients, as well as first trimester placenta, were also positive for CFTR expression. A ß2m specific product (123 bp) was amplified from all samples analysed. CFTR transcript was detected in all human embryo samples analysed from the 8- to 12-cell stage onwards (n = 3 for each stage; data not shown). Expression persisted throughout the morula and blastocyst stages. All these developmental stages were also positive for ß2m expression, which served as the internal control. The specificity of the CFTR cDNA amplified fragment from human blastocysts was also determined by restriction digestion analysis with the Alu I enzyme, which yielded the expected 140 and 138 bp fragments.
In order to estimate the abundance of CFTR transcript throughout human preimplantation embryo development, we used quantitative RT–PCR dot blot analysis. Results are shown in Figure 1A and revealed that CFTR mRNA is abundantly stored in unfertilized oocytes and 2-cell stage embryos and later appears to be re-expressed after activation of the embryonic genome from the 8-cell stage onwards, with the highest levels observed in blastocysts. Figure 1B depicts the raw data obtained for the level of CFTR transcripts for three independent samples shown in Figure 1A. These results suggest that CFTR expression levels are highest in blastocysts.
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Detection of CFTR protein during human preimplantation development
No specific CFTR fluorescence was observed in the cytoplasm of unfertilized oocytes, polypronuclear zygotes or early cleavage stage embryos (2- to 4-cell stage) using indirect immunocytochemistry with a CFTR-specific antibody (Figure 2A–D
). In addition, no specific CFTR staining was found on the surface of either oocytes or embryos when non-specific IgG antibody was used or when the primary antibody was omitted (Figure 2G,H). The first developmental stage exhibiting cytoplasmic but no membrane staining of CFTR was at the 8-cell stage (Figure 2E). Strong staining persisted in an 8- to 16-cell embryo (Figure 2F).
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At the early blastocyst stage, strong antibody-associated fluorescence was observed in both the trophectoderm and the ICM (Figure 3C
). At the expanded blastocyst stage staining appeared to be predominately localized to membrane compartments (Figure 3D). In these cases, where the blastocoele was extensively filled with fluid, acetone fixation of the embryos affected the morphology and it was sometimes difficult to localize the ICM within the blastocoele cavity. However, in a few hatching blastocysts (n = 3) staining was observed in the ICM area and the overlaying polar trophectoderm with minimal to no staining in the mural trophectoderm (Figure 3F).
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Activity of CFTR at the blastocyst stage
The patch clamp technique was used to determine whether CFTR functions as a regulated Cl- channel in the apical membrane of trophectodermal cells. Single channel activities in the apical plasma membranes of human blastocysts were recorded in both cell-attached and inside-out patches. In unstimulated blastocysts, no channels were observed in the cell-attached configuration. Addition of a cocktail containing forskolin (1–10 µmol/l), IBMX and CPT-cAMP (each 10–100 µmol/l) activated low-conductance channels in 5/7 patches (Figure 4A
). Upon excision of the patch the channel activities disappeared, but subsequent addition of the catalytic subunit of PKA (90 nmol/l) and ATP (1 mmol/l) to the bath activated similar channels in 5/5 inside-out patches (Figure 4A). The activities sometimes appeared as single channels, but more often multiple channels were observed (Figure 4C). Single channels had a conductance of 6–10 pS and a linear I-V relationship (Figure 4B). The channels were Cl- selective, since imposing a Cl- gradient across the patch (120 mmol/l Cl-ext/20 mmol/l Cl-cyt) shifted the I-V curve to negative voltages, as predicted for Cl- selective channels (Figure 4B). The channel conductance was inhibited by the presence of 60 mmol/l iodine on the extracellular side of the channel (Figure 4C,D), whereas the channels were insensitive to the anion transporter inhibitor DIDS (Figure 4E). The open probability Po in single channel recordings was 0.58. Open and closed time histograms of single channel currents showed one open and one closed state (23 and 17 ms respectively) when measured at +60 mV. The characteristics of these channels resemble CFTR Cl- channels observed in the plasma membranes of many cell types (Welsh et al., 1992).
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Discussion |
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Compaction of embryonic blastomeres at the 8-cell stage in mice and the 16-cell stage in humans results in the formation of external and internal blastomeres. The internal cells develop into the ICM which gives rise to the embryo proper. In contrast, the external cells become polarized, with apical membranes containing amino acid transport systems (DiZio and Tasca, 1977
) and Na+ channels (Manejwala et al., 1989; Robinson et al., 1991). In addition, the apical membranes have `apposed' basolateral regions which are rich in gap and tight junctions and have high levels of Na+-K+-ATPase and the cell adhesion molecule uvomorulin (Vestweber et al., 1987). The differentiation of trophectoderm cells into polarized epithelial cells is hypothesized to result from asymmetric cell–cell contacts which lead to an ionic gradient across the blastomeres (Wiley et al., 1990). The development of trophectoderm polarity is in turn proposed to promote vectoral transport of solutes and water, with the consequent production of blastocoele fluid. Expansion of the embryo, and blastocoele formation, are essential for further differentiation of the ICM and for hatching of the embryo from the zona pellucida.
Recently it was proposed that water transport via aquaporins, major intrinsic membrane proteins that are present in the trophoblast cells of the preimplantation embryo, are involved in blastocyst formation (Offenberg et al., 2000). These proteins, 10 of which have been identified to date in mammals, function as molecular water channels in somatic epithelia that allow for the rapid flow of water across plasma membranes. Preliminary results of aquaporin inhibitors strongly suggest a role for these water channels in blastocyst formation (Watson and Barcroft, 2001). Thus, if CFTR facilitates water and chloride transport, clear evidence exists regarding the redundancy of molecular pathways in this system.
Previous studies in rabbits have shown that fluid accumulation in the blastocoele is secondary to active transport of Na+ and Cl- (Cross, 1973; Borland et al., 1976), although the exact mechanisms responsible for blastocoele formation remain unclear. This ionic transport necessitates entry of the ions through the apical side of the cells and extrusion from the basolateral side mediated by Na+-K+-ATPase (DiZio and Tasca, 1977; Benos, 1981; Wiley, 1984). Manejwala et al. confirmed the crucial role of Na+ and Cl-, but not K+ in mouse blastocoele formation by demonstrating that elimination of Na+ or Cl- from the medium resulted in a 2-fold reduction in the rate of blastocoele expansion, while elimination of K+ had no effect (Manejwala et al., 1989). Furthermore, the use of Na+ transport inhibitors indicated that Na+ transport is mediated by several routes, including Na+ channels, a Na+/H+ exchanger and the ouabain-sensitive Na+-K+-ATPase. Also present throughout preimplantation mouse embryo development are bicarbonate/chloride exchangers which regulate intracellular pH and which are necessary for embryo viability (Zhao et al., 1995; Zhao and Baltz, 1996). Swell-activated anion channel activity has also been characterized in early preimplantation mouse embryos (Kolajova et al., 2001).
Cl- depleted medium strongly inhibits mouse blastocyst formation (Manejwala et al., 1989; Brison and Leese, 1993). The use of Cl- transport inhibitors has yielded conflicting results, and it is still not clear whether Cl- transport is transcellular or paracellular. The results of our study suggest that CFTR may play a role in fluid transport in the human blastocoele by providing a transcellular pathway for Cl- flux. Furthermore, the role of cAMP in blastocoele formation has been investigated in mouse embryos treated with forskolin and cholera toxin. Early cavitating embryos treated with these agents displayed an increase in the rate of fluid accumulation, providing evidence that cAMP may have a physiological role in blastocoele formation (Manejwala et al., 1986). Whether the increased rate of blastocoele formation stimulated by cAMP could be mediated in part by CFTR channels remains to be elucidated.
In humans, we observed that mRNA for the CFTR gene was maternally inherited via storage of this transcript in the unfertilized oocytes. Similarly, Xenopus oocytes and early cleavage stage embryos express CFTR mRNA as detected by RT–PCR and by RNase protection assays (Tucker et al., 1992), suggesting conservation of CFTR biological function during early embryonic stages. Interestingly, no expression of the CFTR transcript could be observed in rodent ovaries using in-situ hybridization (Trezise et al., 1993), either reflecting differences in species or in the sensitivity of techniques used to detect expression. Species differences in the abundance and pattern of CFTR expression have been previously described in rodents and humans (Tizzano et al., 1993; Trezise et al., 1993; White et al., 1998). In humans, no information is available about localization of CFTR protein during early stages of embryonic development. In our study, CFTR protein could be detected from the 8-cell stage onwards, and appeared to localize to the cytoplasm of blastomeres. However, later in development, staining was observed in both membranes and cytoplasmic compartments. Using patch clamp electrophysiology, Cl- channels could easily be detected on the apical membrane of trophectodermal cells, confirming localization observed by immunocytochemistry. The properties of the detected channels are consistent with the activities of CFTR, confirming that CFTR is present during blastocyst formation and its protein functions as a cAMP-regulated Cl- channel.
At the early-expanded blastocyst stage, CFTR protein appeared to be localized to all trophectodermal cells. In contrast, fully expanded hatching blastocysts only showed expression within the ICM and the proliferative cells that overlie the ICM—the polar trophectoderm. Down-regulation of expression was observed in cells that were in direct contact with the blastocoele cavity—the mural trophectoderm. Also, in preliminary patch clamp studies, we were not able to detect any cAMP-activated Cl- channel activities in trophectoderm of hatched blastocysts, unlike in earlier blastocyst stages or morulae. These observations suggest that early embryonic expression reflects an involvement of CFTR protein in cavitation and blastocyst formation. Once the blastocyst is formed, the most differentiated cells of the blastocyst, the mural trophectoderm (Ziomek, 1987), seem to down-regulate their CFTR expression, most probably as a part of their differentiation pathway into primary giant cells (Cross, 2000). Thus, it is likely that activity of these Cl- channels is no longer required within these cells.
In contrast to the mural trophectoderm, cells of the ICM and polar trophectoderm—cells that give rise to the embryo proper, extra-embryonic and placental tissues—maintain CFTR protein expression. This observation correlates with the presence of CFTR transcript in a variety of embryonic tissues at 10 weeks gestation (Tizzano et al., 1993). We were also able to detect CFTR transcript by RT–PCR in first trimester placenta (8 weeks gestation). However, the expression is likely to be not very strong, since Northern blot analysis as well as in-situ hybridization experiments have failed to detect the CFTR transcript in this tissue (Riordan et al., 1989; E.Tizziano, personal communication). Consistent with this observation, chloride transport occurs in the syncytiotrophoblast membrane in both first and third trimester placenta via several mechanisms (Doughty et al., 1998).
If CFTR plays a role in blastocoele formation, how can embryos homozygous for CFTR mutations develop to term and be delivered? Our results suggest that mothers heterozygous for the CFTR mutation accumulate mRNA in the oocyte. Once the embryonic genome is activated, these maternally-derived CFTR transcripts may be translated into protein, which functions to produce at least some functional Cl- channels and to rescue the embryo by promoting blastocoele formation. This same `stockpile' mechanism has been proposed to be responsible for rescue of embryos from death caused by several targeted mutations during mouse preimplantation embryo development. One such example is disruption of ß-catenin, where maternally-derived proteins persisted until the expanded blastocyst stage (Haegel et al., 1995). On the other hand, when a homozygous affected mother conceives a heterozygous baby, the functional paternal allele may allow for sufficient production of CFTR protein. Reduced fertility of CF women (Kotloff et al., 1992) has always been attributed to pathological changes in the secretions of the uterus, cervix and Fallopian tubes (Tizzano et al., 1993; Trezise et al., 1993), rather than to abnormal embryo development. We are not aware of any report in the literature where a CFTR homozygous-affected mother has given birth to a CF-affected child, suggesting that CFTR protein may be required for proper embryo cavitation in humans.
Alternatively, as previously indicated, other biologically redundant mechanisms may play a role in proper water and Cl- transport during formation of the blastocoele. Other Cl- channels may be present on the apical membranes of trophectodermal cells. These channels may be able to participate in blastocoele development in the absence of functional CFTR protein in CF defective embryos or might even be up-regulated to compensate for lack of CFTR function. Alternatively, trophectodermal transepithelial Cl- entry may not be carrier-mediated and may occur through a paracellular route between the trophectodermal cells (Manejwala et al., 1989). In this case, CFTR may provide an additional pathway for transepithelial Cl- flux, or it may be involved in other functions that do not affect early embryogenesis.
In conclusion, the CFTR transcript is abundantly stored in human oocytes, but it appears to be translated only from the 8-cell stage onwards. CFTR mRNA and its protein product is expressed at highest levels during blastocyst formation and functions as a regulated Cl- channel in the apical membrane of trophectodermal cells at these embryonic stages. Our data suggest that the CFTR channel may contribute to blastocoele cavity formation in the normal human embryo and that its expression is tightly linked with the formation of the blastocyst.
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Acknowledgements |
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We thank the team of the IVF laboratory of Mount Sinai Hospital for their valuable help and support. We would also like to thank Dr N.Kartner for donation of M3A7 CFTR-specific and IgG non-specific antibodies. The technical assistance of Dr D.Mak and comments from Dr M.Buchwald during the preparation of this manuscript were greatly appreciated. The study was supported by grants from the Medical Research Council of Canada (R.F.C.), the Canadian Cystic Fibrosis Foundation (CCFF), and NIH grant (J.K.F). A.J. was supported by the Genesis Research Foundation, A.B. was supported by a fellowship grant from The Schiff Family Foundation and E.P. was a fellow of the CCFF.
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Notes |
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5 To whom correspondence should be addressed at: 600 University Ave, Room 876, Toronto, Ontario M5G 1X5, Canada. E-mail: r.casper{at}utoronto.ca
* These authors contributed equally to this work.
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References |
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|---|
Bear, C.E., Li, C.H., Kartner, N., Bridges, R.J., Jensen, T.J., Ramjeesingh, M. and Riordan, J.R. (1992) Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell, 68, 809–818.[ISI][Medline]
Benos, D.J. (1981) Ouabain binding to preimplantation rabbit blastocysts. Dev. Biol., 83, 69–78.[ISI][Medline]
Benos, D.J. and Biggers, J.D. (1983) Sodium and chloride co-transport by preimplantation rabbit blastocysts. J. Physiol, 342, 23–33.
Biggers, J.D., Bell, J.E. and Benos, D.J. (1988) Mammalian blastocyst: transport functions in a developing epithelium. Am. J. Physiol., 255, C419–C432.
Borland, R.M., Biggers, J.D. and Lechene, C.P. (1976) Kinetic aspects of rabbit blastocoele fluid accumulation: an application of electron probe microanalysis. Dev. Biol., 50, 201–211.[ISI][Medline]
Brady, G. and Iscove, N.N. (1993) Construction of cDNA libraries from single cells. Methods Enzymol., 225, 611–623.[ISI][Medline]
Braude, P., Bolton, V. and Moore, S. (1988) Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature, 332, 459–461.[Medline]
Brison, D.R. and Leese, H.J. (1993) Role of chloride transport in the development of the rat blastocyst. Biol. Reprod., 48, 692–702.[Abstract]
Cross, J.C. (2000) Genetic insights into trophoblast differentiation and placental morphogenesis. Semin. Cell Dev. Biol., 11, 105–113.[ISI][Medline]
Cross, M.H. (1973) Active sodium and chloride transport across the rabbit blastocoele wall. Biol. Reprod., 8, 566–575.[Abstract]
Daniel, J.C. Jr (1970) Culture of rabbit embryo in circulating medium. Nature, 225, 193–194.[Medline]
DiZio, S.M. and Tasca, R.J. (1977) Sodium-dependent amino acid transport in preimplantation mouse embryos. III. Na+-K+-atpase-linked mechanism in blastocysts. Dev. Biol., 59, 198–205.[ISI][Medline]
Doughty, I.M., Glazier, J.D., Powell, T.L., Jansson, T. and Sibley, C.P. (1998) Chloride transport across syncytiotrophoblast microvillous membrane of first trimester human placenta. Pediatr. Res., 44, 226–232.[ISI][Medline]
Ducibella, T., Albertini, D.F., Anderson, E. and Biggers, J.D. (1975) The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development. Dev. Biol., 45, 231–250.[ISI][Medline]
Fleming, T.P. and Johnson, M.H. (1988) From egg to epithelium. Ann. Rev. Cell. Biol., 4, 459–485.[ISI]
Gamow, E.I. and Prescott, D.M. (1970) The cell life cycle during early embryogenesis of the mouse. Exp. Cell Res., 59, 117–123.[ISI][Medline]
Haegel, H., Larue, L., Ohsugi, M., Fedorov, L., Herrenknecht, K. and Kemler, R. (1995) Lack of beta-catenin affects mouse development at gastrulation. Development, 121, 3529–3537.[Abstract]
Harris, A., Chalkley, G., Goodman, S. and Coleman, L. (1991) Expression of the cystic fibrosis gene in human development. Development, 113, 305–310.[Abstract]
Jurisicova, A., Casper, R.F., MacLusky, N.J., Mills, G.B. and Librach, C.L. (1996) HLA-G expression during preimplantation human embryo development. Proc. Natl Acad. Sci. USA, 93, 161–165.
Kartner, N., Augustinas, O., Jensen, T.J., Naismith, A.L. and Riordan, J.R. (1992) Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland. Nature Genet., 1, 321–327.[ISI][Medline]
Kolajova, M., Hammer, M.A., Collins, J.L. and Baltz, J.M. (2001) Developmentally regulated cell cycle dependence of swelling-activated anion channel activity in the mouse embryo. Development, 128, 3427–3434.
Kotloff, R.M., FitzSimmons, S.C. and Fiel, S.B. (1992) Fertility and pregnancy in patients with cystic fibrosis. Clin. Chest Med., 13, 623–635.[ISI][Medline]
Manejwala, F., Kaji, E. and Schultz, R.M. (1986) Development of activatable adenylate cyclase in the preimplantation mouse embryo and a role for cyclic AMP in blastocoel formation. Cell, 46, 95–103.[ISI][Medline]
Manejwala, F.M., Cragoe, E.J. Jr and Schultz, R.M. (1989) Blastocoel expansion in the preimplantation mouse embryo: role of extracellular sodium and chloride and possible apical routes of their entry. Dev. Biol., 133, 210–220.[ISI][Medline]
McCray, P.B. Jr, Reenstra, W.W., Louie, E., Johnson, J., Bettencourt, J.D. and Bastacky, J. (1992) Expression of CFTR and presence of cAMP-mediated fluid secretion in human fetal lung. Am. J. Physiol., 262, L472–L481.
Offenberg, H., Barcroft, L.C., Caveney, A., Viuff, D., Thomsen, P.D. and Watson, A.J. (2000) mRNAs encoding aquaporins are present during murine preimplantation development. Mol. Reprod. Dev., 57, 323–330.[ISI][Medline]
Overstrom, E.W., Benos, D.J. and Biggers, J.D. (1989) Synthesis of Na+/K+ ATPase by the preimplantation rabbit blastocyst. J. Reprod. Fertil., 85, 283–295.[Abstract]
Rambhatla, L., Patel, B., Dhanasekaran, N. and Latham, K.E. (1995) Analysis of G protein alpha subunit mRNA abundance in preimplantation mouse embryos using a rapid, quantitative RT–PCR approach. Mol. Reprod. Dev., 41, 314–324.[ISI][Medline]
Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.L. et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science, 245, 1066–1073.
Robinson, D.H., Bubien, J.K., Smith, P.R. and Benos, D.J. (1991) Epithelial sodium conductance in rabbit preimplantation trophectodermal cells. Dev. Biol., 147, 313–321.[ISI][Medline]
Smith, M.W. (1970) Active transport in the rabbit blastocyst. Experientia, 26, 736–738.[ISI][Medline]
Telford, N.A., Watson, A.J. and Schultz, G.A. (1990) Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol. Reprod. Dev., 26, 90–100.[ISI][Medline]
Tizzano, E.F., Chitayat, D. and Buchwald, M. (1993) Cell-specific localization of CFTR mRNA shows developmentally regulated expression in human fetal tissues. Hum. Mol. Genet., 2, 219–224.
Trezise, A.E., Linder, C.C., Grieger, D., Thompson, E.W., Meunier, H., Griswold, M.D. and Buchwald, M. (1993) CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents. Nature Genet., 3, 157–164.[ISI][Medline]
Tucker, S.J., Tannahill, D. and Higgins, C.F. (1992) Identification and developmental expression of the Xenopus laevis cystic fibrosis transmembrane conductance regulator gene. Hum. Mol. Genet., 1, 77–82.
Vestweber, D., Gossler, A., Boller, K. and Kemler, R. (1987) Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos. Dev. Biol., 124, 451–456.[ISI][Medline]
Watson, A.J. and Barcroft, L.C. (2001) Regulation of blastocyst formation. Front. Biosci., 6, D708–D730.[ISI][Medline]
Watson, A.J., Kidder, G.M. and Schultz, G.A. (1992) How to make a blastocyst. Biochem. Cell. Biol., 70, 849–855.[ISI][Medline]
Welsh, M.J., Anderson, M.P., Rich, D.P., Berger, H.A., Denning, G.M., Ostedgaard, L.S., Sheppard, D.N., Cheng, S.H., Gregory, R.J. and Smith, A.E. (1992) Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation. Neuron, 8, 821–829.[ISI][Medline]
White, N.L., Higgins, C.F. and Trezise, A.E. (1998) Tissue-specific in vivo transcription start sites of the human and murine cystic fibrosis genes. Hum. Mol. Genet., 7, 363–369.
Wiley, L.M. (1984) Cavitation in the mouse preimplantation embryo: Na/K-ATPase and the origin of nascent blastocoele fluid. Dev. Biol., 105, 330–342.[ISI][Medline]
Wiley, L.M., Kidder, G.M. and Watson, A.J. (1990) Cell polarity and development of the first epithelium. Bioessays, 12, 67–73.[ISI][Medline]
Zhao, Y. and Baltz, J.M. (1996) Bicarbonate/chloride exchange and intracellular pH throughout preimplantation mouse embryo development. Am. J. Physiol., 271, C1512–C1520.
Zhao, Y., Chauvet, P.J., Alper, S.L. and Baltz, J.M. (1995) Expression and function of bicarbonate/chloride exchangers in the preimplantation mouse embryo. J. Biol. Chem., 270, 24428–24434.
Zhao, Y., Doroshenko, P.A., Alper, S.L. and Baltz, J.M. (1997) Routes of Cl- transport across the trophectoderm of the mouse blastocyst. Dev. Biol., 189, 148–160.[ISI][Medline]
Ziomek, C.A. (1987) Cell polarity in the preimplantation mouse embryo. In Bavister B.D. (ed.) The Mammalian Preimplantation Embryo. Plenum Press, New York, USA, pp. 23–41.
Submitted on October 1, 2001; accepted on May 7, 2002.
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