Biomedical subjects

L C Barcroft

Publications and source records attributed to L C Barcroft.

9 recordsLinked to original sources

PubMed2004

Molecular regulation of blastocyst formation.

Preimplantation development encompasses the interval from insemination until embryo implantation and thus includes the 'freeliving' period of oviduct and uterine development. Formation of the blastocyst is required for implantation and establishment of pregnancy, and is a principal determinant of embryo quality prior to embryo transfer. Development through this period is regulated by the expression of specific gene families that encode for cell polarity, cell junctional, cytoskeletal, ion transporter, and water channel gene products that direct the acquisition of cell polarity and differentiation of the outer cells of the early embryo. This results in the formation of the trophectoderm, which is the first epithelium of development. This review considers the roles of each of these gene families in trophectoderm differentiation and blastocyst formation. The principal hypothesis under investigation is that blastocyst formation is regulated by a Na/K-ATPase-generated trans-trophectoderm ion gradient that promotes the accumulation of water across the epithelium. This, combined with the formation of the tight junction seal controlling paracellular movement of water between adjacent trophectoderm cells, results in the formation of a fluid-filled blastocyst cavity and the expansion of the blastocyst. Results from recent experiments, however, have cast some doubt on the role of Na/K-ATPase in mediating these events and have defined water channels or Aquaporins (AQPs) as physiological mediators of fluid movement across the trophectoderm. In addition, studies have now implicated mitogen-activated protein kinase (MAPK) signaling as an important mediator of development to the blastocyst stage. Such studies define the physiology of blastocyst formation and serve to support the application of assisted reproductive technologies (ART) to both human and animal species.

Animals↗

PubMed2004

Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo.

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the predominant isozyme of the Na(+)-pump that has previously been localized to the basolateral membranes of mammalian trophectoderm cells. The presence of multiple isoforms of Na/K-ATPase alpha- and beta-subunits at the time of cavitation suggests that there may be a degree of genetic redundancy amongst isoforms of the catalytic alpha-subunit that allows blastocyst formation to progress in the absence of the alpha1-subunit.

Animals↗

PubMed2002

The gamma-subunit of the Na-K-ATPase as a potential regulator of apical and basolateral Na+-pump isozymes during development of bovine pre-attachment embryos.

Expression and activity of the Na-K-ATPase within the basolateral membrane domains of the trophectoderm epithelium provide the driving force for accumulation of Na(+) and Cl(-) across the nascent epithelium, mediating fluid movement into the forming blastocoel. Within the trophectoderm of the bovine blastocyst, multiple isozymes of the Na-K-ATPase are expressed. Immunolocalization has demonstrated that the alpha1-isozyme localizes within the basolateral membrane, whereas the alpha 3-isozyme localizes to the apical cell margins. Gene-specific RT-PCR and wholemount indirect immunofluorescence confocal laser scanning microscopy were used to examine expression of the Na-K-ATPase gamma-subunit (a regulatory subunit of the Na-K-ATPase) throughout development of bovine preattachment embryos in vitro. Expression of mRNA transcripts for the gamma-subunit was detected throughout bovine pre-attachment development from the fertilized one-cell embryo to the blastocyst stage. A similar pattern of expression was also observed for gamma-subunit protein, and immunofluorescence was detected within the membranes of embryonic blastomeres at all stages of development. In contrast to the expression patterns observed for the alpha-subunits, gamma-subunit proteins were detected in both the basolateral and apical cell margins of the trophectoderm, and surrounding all cells of the inner cell mass. Co-localization studies demonstrated that gamma-subunit peptides are co-expressed with the alpha1-subunit in the basolateral domains of the trophectoderm. These results indicate a role for the gamma-subunit of the Na-K-ATPase in modulating Na(+)-pump activity in both apical and basolateral margins of the trophectoderm during formation and expansion of the bovine blastocyst, and adds a further level of complexity to Na(+)-pump regulation of cavitation.

Animals↗

PubMed2001

Regulation of blastocyst formation.

Preimplantation or pre-attachment development encompasses the "free"-living period of mammalian embryogenesis, which directs development of the zygote through to the blastocyst stage. Blastocyst formation is essential for implantation, establishment of pregnancy and is a principal determinant of embryo quality prior to embryo transfer. Cavitation (blastocyst formation) is driven by the expression of specific sets of gene products that direct the acquisition of cell polarity within the trophectoderm, which is both the first epithelium of development and the outer cell layer encircling the inner cell mass of the blastocyst. Critical gene families controlling these events include: the E-cadherin-catenin cell adhesion family, the tight junction gene family, the Na/K-ATPase gene family and perhaps the aquaporin gene family. This review will update the roles of each of these gene families in trophectoderm differentiation and blastocyst formation. The current principal hypothesis under investigation is that blastocyst formation is mediated by a trans-trophectoderm ion gradient(s) established, in part, by Na/K-ATPase, which drives the movement of water through aquaporins (AQPs) across the epithelium into the extracellular space of the blastocyst to form the fluid-filled blastocoel. The trophectoderm tight junctional permeability seal regulates the leakage of blastocoel fluid, and also assists in the maintenance of a polarized Na/K-ATPase distribution to the basolateral plasma membrane domain of the mural trophectoderm. The cell-to-cell adhesion provided by the E-cadherin-catenin gene families is required for the establishment of the tight junction seal and the maintenance of the polarized Na/K-ATPase distribution. Blastocyst formation is therefore directly linked with trophectoderm cell differentiation, which arises through fundamental cell biological processes that are associated with the establishment of cell polarity.

Animals↗

PubMed2000

mRNAs encoding aquaporins are present during murine preimplantation development.

The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) were applied to murine embryo cDNA samples. PCR reactions were conducted for up to 40 cycles involving denaturation of DNA hybrids at 95 degrees C, primer annealing at 52-60 degrees C and extension at 72 degrees C. PCR products were separated on 2% agarose gels and were stained with ethidium bromide. AQP PCR product identity was confirmed by sequence analysis. mRNAs encoding AQPs 1, 3, 5, 6, 7, and 9 were detected in murine embryos from the one-cell stage up to the blastocyst stage. AQP 8 mRNAs were not detected in early cleavage stages but were present in morula and blastocyst stage embryos. The results were confirmed in experimental replicates applied to separate embryo pools of each embryo stage. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development. These findings predict that AQPs may function as conduits for trophectoderm fluid transport during blastocyst formation.

Animals↗

PubMed2000

Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis.

The objectives were 1) to investigate the effects of oocyte maturation in serum-free and amino acid-supplemented defined media on oocyte transcript levels, blastocyst cell number, and apoptosis; 2) to investigate the influence of oocyte maturation culture atmosphere on blastocyst development, total cell number, and apoptosis; and 3) to examine the influence of epidermal growth factor (EGF) during oocyte maturation on blastocyst cell number and apoptosis. The results demonstrate that blastocysts derived from in vitro maturation, fertilization, and embryo culture protocols undergo apoptosis but that apoptotic levels are not greatly influenced by the oocyte maturation environment. Amino acid supplementation of oocyte maturation media was associated with enhanced developmental frequencies, increased blastocyst cell number, and elevated oocyte maternal mRNA levels. Oocyte maturation with supplemented synthetic oviduct fluid medium (cSOFMaa) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199 + newborn calf serum. Blastocyst development was reduced following oocyte maturation under a 5% CO(2), 7% O(2), 88% N(2) culture atmosphere. EGF supplementation of oocyte maturation medium resulted in a concentration-dependent increase in blastocyst development but did not influence blastocyst total cell number or apoptosis. Our findings indicate that cSOFMaa medium is an effective base medium for bovine oocyte maturation.

Amino Acids↗

PubMed1999

Gene expression regulating blastocyst formation.

Development of embryos to the blastocyst stage is a critical event in the early lives of all eutherian mammalian species. Blastocyst formation is essential for implantation and is the principal morphological determinant of embryo quality prior to embryo transfer. The physiological events and roles of specific gene families that regulate blastocyst formation are subjects of intense research Recent findings have demonstrated that bovine embryos express multiple members of the Na/K-ATPase ion transporter gene family. Two members of this family have been co-localized to bovine trophectoderm, but each becomes largely confined to opposing cell membrane margins. Bovine blastocysts display a greater sensitivity to ouabain (potent inhibitor of the Na/K-ATPase) than murine blastocysts, and enzyme activity (ouabain sensitive 86Rb+ uptake) undergoes a 9-fold increase from the bovine morula to the blastocyst stage. Disruption of Na/K-ATPase gene expression by antisense oligodeoxynucleotide inhibition abolishes blastocyst formation. These results have implicated the Na/K-ATPase as a key regulator of bovine blastocyst formation and have provided insights necessary for the production of healthy bovine embryos by the application of in vitro maturation, in vitro fertilization and in vitro culture methods.

Animals↗

PubMed1998

Na/K-ATPase-mediated 86Rb+ uptake and asymmetrical trophectoderm localization of alpha1 and alpha3 Na/K-ATPase isoforms during bovine preattachment development.

This study evaluated Na/K-ATPase alpha 1- and alpha 3-subunit isoform polypeptide expression and localization during bovine preattachment development. Na/K-ATPase cation transport activity from the one-cell to blastocyst stage was also determined by measuring ouabain-sensitive 86Rb+ uptake. Both alpha1- and alpha 3-subunit polypeptides were detected by immunofluorescence to encircle the entire cell margins of each blastomere of inseminated zygotes, cleavage stage embryos, and morulae. Immunofluorescent localization of alpha1-subunit polypeptide in bovine blastocysts revealed an alpha1 immunofluorescence signal confined to the basolateral membrane margins of the trophectoderm and encircling the cell periphery of each inner cell mass (ICM) cell. In contrast, alpha 3-subunit polypeptide immunofluorescence was localized primarily to the apical cell surfaces of the trophectoderm with a reduced signal present in basolateral trophectoderm regions. There was no apparent alpha 3-subunit signal in the ICM. Analysis of 86Rb+ transport in vitro demonstrated ouabain-sensitive activity throughout development from the one-cell to the six- to eight-cell stage of bovine development. 86Rb+ uptake by morulae (day 6 postinsemination) did not vary significantly from uptake detected in cleavage stage embryos; however, a significant increase was measured at the blastocyst stage (P < 0.05). Treatment of embryos with cytochalasin D (5 micrograms/ml) did not influence 86Rb+ uptake in cleavage stage embryos. Cytochalasin D treatment however was associated with a significant rise in ion transport in morulae and blastocysts (13.49 and 61.57 fmol/embryo/min, respectively) compared to untreated controls (2.65 and 22.83 fmol/embryo/min, respectively). Our results, for the first time, demonstrate that multiple Na/K-ATPase alpha-subunit isoforms are distributed throughout the first week of mammalian development and raise the possibility that multiple isozymes of the Na/K-ATPase contribute to blastocyst formation.

Animals↗

PubMed1998

Trophectoderm differentiation in the bovine embryo: characterization of a polarized epithelium.

Blastocytst formation is dependent on the differentiation of a transporting epithelium, the trophectoderm, which is coordinated by the embryonic expression and cell adhesive properties of E-cadherin. The trophectoderm shares differentiative characteristics with all epithelial tissues, including E-cadherin-mediated cell adhesion, tight junction formation, and polarized distribution of intramembrane proteins, including the Na-K ATPase. The present study was conducted to characterize the mRNA expression and distribution of polypeptides encoding E-cadherin, beta-catenin, and the tight junction associated protein, zonula occludens protein 1, in pre-attachment bovine embryos, in vitro. Immunocytochemistry and gene specific reverse transcription--polymerase chain reaction methods were used. Transcripts for E-cadherin and beta-catenin were detected in embryos of all stages throughout pre-attachment development. Immunocytochemistry revealed E-cadherin and beta-catenin polypeptides evenly distributed around the cell margins of one-cell zygotes and cleavage stage embryos. In the morula, detection of these proteins diminished in the free apical surface of outer blastomeres. E-cadherin and beta-catenin became restricted to the basolateral membranes of trophectoderm cells of the blastocyst, while maintaining apolar distributions in the inner cell mass. Zonula occludens protein 1 immunoreactivity was undetectable until the morula stage and first appeared as punctate points between the outer cells. In the blastocyst, zonula occludens protein 1 was localized as a continuous ring at the apical points of trophectoderm cell contact and was undetectable in the inner cell mass. These results illustrate that the gene products encoding E-cadherin, beta-catenin and zonula occludens protein 1 are expressed and maintain cellular distribution patterns consistent with their predicted roles in mediating trophectoderm differentiation in in vitro produced bovine embryos.

Animals↗