Expression of cystic fibrosis transmembrane conductance regulator during early human embryo development

Molecular Human Reproduction Vol.8, No.8 pp. 758–764, 2002 Expression of cystic fibrosis transmembrane conductance regulator during early human embryo development Avraham Ben-Chetrit1,*, Monica Antenos1,*, Andrea Jurisicova1, Eva A.Pasyk3, David Chitayat2, J.Kevin Foskett3,4 and Robert F.Casper1,5 1Division of Reproductive Sciences, Department of Obstetrics and Gynaecology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 2Prenatal Diagnosis Program, the Toronto Hospital General Division, 3Division of Cell Biology, 4Department of Biochemistry, The Hospital for Sick Children and 5Department of Physiology, University of Toronto, M5G 1X8, Ontario, Canada 5To whom correspondence should be addressed at: 600 University Ave, Room 876, Toronto, Ontario M5G 1X5, Canada. E-mail: 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. Key words: blastocoele/blastocyst/CFTR/cystic fibrosis/embryo cavitation Introduction 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 *These authors contributed equally to this work. 758 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 diisothiocyanatostilbene-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 © European Society of Human Reproduction and Embryology CFTR and early human embryo development 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. Materials and methods (...truncated)