Noncanonical Role of Telomerase in Regulation of Microvascular Redox Environment With Implications for Coronary Artery Disease.

Telomerase reverse transcriptase (TERT) (catalytic subunit of telomerase) is linked to the development of coronary artery disease (CAD); however, whether the role of nuclear vs. mitchondrial actions of TERT is involved is not determined. Dominant-negative TERT splice variants contribute to decreased mitochondrial integrity and promote elevated reactive oxygen species production. We hypothesize that a decrease in mitochondrial TERT would increase mtDNA damage, promoting a pro-oxidative redox environment. The goal of this study is to define whether mitochondrial TERT is sufficient to maintain nitric oxide as the underlying mechanism of flow-mediated dilation by preserving mtDNA integrity.Immunoblots and quantitative polymerase chain reaction were used to show elevated levels of splice variants α- and ß-deletion TERT tissue from subjects with and without CAD. Genetic, pharmacological, and molecular tools were used to manipulate TERT localization. Isolated vessel preparations and fluorescence-based quantification of mtH2O2 and NO showed that reduction of TERT in the nucleus increased flow induced NO and decreased mtH2O2 levels, while prevention of mitochondrial import of TERT augmented pathological effects. Further elevated mtDNA damage was observed in tissue from subjects with CAD and initiation of mtDNA repair mechanisms was sufficient to restore NO-mediated dilation in vessels from patients with CAD. The work presented is the first evidence that catalytically active mitochondrial TERT, independent of its nuclear functions, plays a critical physiological role in preserving NO-mediated vasodilation and the balance of mitochondrial to nuclear TERT is fundamentally altered in states of human disease that are driven by increased expression of dominant negative splice variants.

Analysis of Mitochondrial Dimensions and Cristae Structure in Pluripotent Stem Cells Using Transmission Electron Microscopy.

Dynamic alterations to mitochondrial structure and function regulate cell fate decisions and underlie multiple age-related and genetic diseases that are modeled using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Transmission electron microscopy (TEM) can be used to obtain high-resolution micrographs of mitochondria, but mitochondrial ultrastructure is easily distorted during specimen processing. This unit describes a method that preserves mitochondrial membrane structure from adherent ESC cultures for TEM sample preparation. This procedure is useful for assessing ultrastructural changes to mitochondria during differentiation, reprogramming, or experimental manipulation of ESC metabolism. We provide comprehensive protocols for: (1) preparation of ESC cultures for TEM; (2) retrieval of thin sections from individual ESCs; and (3) contrast staining and morphometric analysis of mitochondria and cristae. This unit also describes an alternative procedure for samples with low cell numbers and a supporting protocol for morphometric image analysis. Collectively, these protocols allow for the observation and quantitative analysis of mitochondria in ESCs. © 2018 by John Wiley & Sons, Inc.

Metabolic plasticity during transition to naïve-like pluripotency in canine embryo-derived stem cells.

Pluripotent stem cells (PSCs) have been described in naïve or primed pluripotent states. Domestic dogs are useful translational models in regenerative medicine, but their embryonic stem cells (cESCs) remain narrowly investigated. Primed-like cESCs expanded in the presence of leukemia inhibitory factor and fibroblast growth factor 2 (LIF-FGF2) acquire features of naïve pluripotency when exposed to chemical inhibitors and LIF (2iL). However, proliferation of cESCs is influenced by the pluripotent state and is comparatively slower than human or mouse PSCs. We propose that different metabolic pathway activities support ATP generation and biomass accumulation necessary for LIF-FGF2 and 2iL cESC proliferation. We found that 2iL cESCs have greater respiratory capacity, altered mitochondrial chain complex stoichiometry and elevated mitochondrial polarization state. Yet, 2iL-enriched cESCs exhibited immature ultrastructure, including previously unrecognized changes to cristae organization. Enhanced ATP level in 2iL cESCs is associated with altered retrograde signalling, whereas LIF-FGF2 cESCs exhibit a lipogenic phenotype. Inhibition of oxidative phosphorylation impaired proliferation and ATP production in 2iL cESCs but not LIF-FGF2 cESCs, which remained sensitive to glycolysis inhibition. Our study reveals distinct bioenergetic mechanisms contributing to steady-state expansion of distinct canine pluripotent states that can be exploited to improve derivation and culture of canine PSCs.

Early embryonic development, assisted reproductive technologies, and pluripotent stem cell biology in domestic mammals.

Over many decades assisted reproductive technologies, including artificial insemination, embryo transfer, in vitro production (IVP) of embryos, cloning by somatic cell nuclear transfer (SCNT), and stem cell culture, have been developed with the aim of refining breeding strategies for improved production and health in animal husbandry. More recently, biomedical applications of these technologies, in particular, SCNT and stem cell culture, have been pursued in domestic mammals in order to create models for human disease and therapy. The following review focuses on presenting important aspects of pre-implantation development in cattle, pigs, horses, and dogs. Biological aspects and impact of assisted reproductive technologies including IVP, SCNT, and culture of pluripotent stem cells are also addressed.

Low levels of X-inactive specific transcript in somatic cell nuclear transfer embryos derived from female bovine freemartin donor cells.

The present study compared developmental potential, telomerase activity and transcript levels of X-linked genes (HPRT, MECP2, RPS4X, SLC25A6, XIAP, XIST and ZFX) in bovine somatic cell nuclear transfer (SCNT) embryos reconstructed with cells derived from a freemartin (female with a male co-twin) or from normal female cattle (control). The rates of cleavage, development to blastocyst and hatched blastocyst stage, and the mean numbers of total and inner cell mass cells in the freemartin SCNT embryos were not significantly different from those of control SCNT embryos (p > 0.05). The levels of telomerase activity analyzed by RQ-TRAP in the freemartin SCNT embryos were also similar to those of the normal SCNT embryos. Transcript levels of HPRT, MECP2, RPS4X and XIAP, measured by quantitative real-time RT-PCR, were not significantly different between the control and freemartin SCNT embryos (p > 0.05). However, the transcript levels of SLC25A6, XIST and ZFX were significantly decreased in the freemartin SCNT embryos compared to control SCNT embryos (p < 0.05). Transfer of 71 freemartin SCNT embryos to 22 recipient cows resulted in 4 (18%) pregnancies, which were lost between days 28 and 90 of gestation. Taken together, the present study demonstrates that the transcript levels of several X-linked genes, especially XIST, showed an aberrant pattern in the freemartin SCNT embryos, suggesting aberrant X inactivation in freemartin clones which may affect embryo survival.

Osteogenic differentiation of equine cord blood multipotent mesenchymal stromal cells within coralline hydroxyapatite scaffolds in vitro.

OBJECTIVE: To investigate the osteogenic differentiation potential of equine umbilical cord blood-derived multipotent mesenchymal stromal cells (CB-MSC) within coralline hydroxyapatite scaffolds cultured in osteogenic induction culture medium. METHODS: Scaffolds seeded with equine CB-MSC were cultured in cell expansion culture medium (control) or osteogenic induction medium (treatment). Cell viability and distribution were confirmed by the MTT cell viability assay and DAPI nuclear fluorescence staining, respectively. Osteogenic differentiation was evaluated after 10 days using reverse transcription polymerase chain reaction, alkaline phosphatase activity, and secreted osteocalcin concentration. Cell morphology and matrix deposition were assessed by scanning electron microscopy (SEM) after 14 days in culture. RESULTS: Cells showed viability and adequate distribution within the scaffold. Successful osteogenic differentiation within the scaffolds was demonstrated by the increased expression of osteogenic markers such as Runx2, osteopontin, osteonectin, collagen IA; increased levels of alkaline phosphatase activity; increased osteocalcin protein secretion and bone-like matrix presence in the scaffold pores upon SEM evaluation. CLINICAL SIGNIFICANCE: These results demonstrate that equine CB-MSC maintain viability and exhibit osteogenic potential in coralline hydroxyapatite scaffolds when induced in vitro . Equine CB-MSC scaffold constructs deserve further investigation for their potential role as biologically active fillers to enhance bone-gap repair in the horse.

In vitro developmental potential of nuclear transfer embryos cloned with enucleation methods using pre-denuded bovine oocytes.

Enucleation of a recipient oocyte is an important essential process in the procedure of somatic cell nuclear transfer (SCNT). The present study investigated a method for the improvement of enucleation efficiency. Oocytes were denuded of cumulus cells before the completion of nuclear maturation (pre-denuded) after 12 h of culture at MI stage and subsequently cultured for additional 6 h until the completion of nuclear maturation and extrusion of the first polar body (PB1). The extrusion rate of PB1 was not significantly different in the pre-denuded oocyte group, compared with control oocyte group matured for 18 h. However, the number of oocytes showing the metaphase II (MII) located just underneath the PB1 was significantly higher (p<0.05) in the pre-denuded oocyte group than those in control oocyte group. To test the effect of pre-denuding on the enucleation rate and developmental potential of embryos to blastocyst stage, subsequent somatic cell nuclear transfer comparisons were made with three different methods of enucleation at MII stage using vital dyes (demicoline and Hoescht) or the PB1 (blind enucleation) to localize the chromosome plate. Enucleation rate of the oocytes with demicoline, Hoechst and pre-denuding enucleation groups were significantly higher (p<0.05) than those of blind enucleation groups. However, cleavage rate to two-cell stage and, developmental rate to blastocyst and hatched blastocyst stage, the mean numbers of total and ICM cells in the SCNT embryos with Hoechst enucleation groups were significantly decreased (p<0.05), compared to those of blind, demicoline and pre-denuding enucleation groups. Moreover, the level of telomerase activity was also significantly (p<0.05) decreased in SCNT blastocysts of Hoechst enucleation group, compared to those of blind, demicoline and pre-denuding enucleation groups. Taken together, pre-denuding enucleation group using pre-denuded oocytes was a useful and simple enucleation method for bovine SCNT embryos.

Permanent embryo arrest: molecular and cellular concepts.

Developmental arrest is one of the mechanisms responsible for the elevated levels of embryo demise during the first week of in vitro development. Approximately 10-15% of IVF embryos permanently arrest in mitosis at the 2- to 4-cell cleavage stage showing no indication of apoptosis. Reactive oxygen species (ROS) are implicated in this process and must be controlled in order to optimize embryo production. A stress sensor that can provide a key understanding of permanent cell cycle arrest and link ROS with cellular signaling pathway(s) is p66Shc, an adaptor protein for apoptotic-response to oxidative stress. Deletion of the p66Shc gene in mice results in extended lifespan, which is linked to their enhanced resistance to oxidative stress and reduced levels of apoptosis. p66Shc has been shown to generate mitochondrial H(2)O(2) to trigger apoptosis, but may also serve as an integration point for many signaling pathways that affect mitochondrial function. We have detected elevated levels of p66Shc and ROS within arrested embryos and believe that p66Shc plays a central role in regulating permanent embryo arrest. In this paper, we review the cellular and molecular aspects of permanent embryo arrest and speculate on the mechanism(s) and etiology of this method of embryo demise.

Alternative splicing and expression analysis of bovine DNA methyltransferase 1.

Methylation of specific CG residues in the mammalian genome results in tissue-specific patterns of gene expression, which are critical for cell differentiation. Embryos that fail to establish and maintain proper DNA methylation patterns show severe developmental abnormalities as is the case of DNA methyltransferase 1 (Dnmt1) -deficient embryos. Dnmt1 is the main maintenance methyltransferase in the mouse and its expression is regulated by a splicing mechanism that dictates the expression of stage-specific isoforms. Little is known about Dnmt1 expression in the cow and isoforms of Dnmt1 are yet unknown in this species. Here we demonstrate that the previously described bovine Dnmt1 transcript is ubiquitously expressed in embryos and fetal tissue. In addition, we report the identification of a splice variant of the bovine Dnmt1, which shows a ubiquitous expression pattern. This new transcript was detected using 5'RACE and genomic mapping and its expression pattern was shown to be consistent with a tissue-specific mode of regulation. Furthermore, our analysis shows that the expression of an oocyte-specific isoform of Dnmt1 is unlikely to occur in cattle. The newly reported isoform of Dnmt1 was demonstrated to be, similarly to Dnmt1a, polyadenylated and if translated possess the functional domains necessary for maintenance and de novo methyltransferase activity.

Early pregnancy diagnosis by serum progesterone and ultrasound in sheep carrying somatic cell nuclear transfer-derived pregnancies.

Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)-derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus-synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post-estrus (day 7 sample) and 19 days post-estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.

Genomic stability and physiological assessments of live offspring sired by a bull clone, Starbuck II.

It appears that overt phenotypic abnormalities observed in some domestic animal clones are not transmitted to their progeny. The current study monitored Holstein heifers sired by a bull clone, Starbuck II, from weaning to puberty. Genomic stability was assessed by telomere length status and chromosomal analysis. Growth parameters, blood profiles, physical exams and reproductive parameters were assessed for 12 months (and compared to age-matched control heifers). Progeny sired by the clone bull did not differ (P>0.05) in weight, length and height compared to controls. However, progeny had lower heart rates (HR) (P=0.009), respiratory rates (RR) (P=0.007) and body temperature (P=0.03). Hematological profiles were within normal ranges and did not differ (P>0.05) between both groups. External and internal genitalia were normal and both groups reached puberty at expected ages. Progeny had two or three ovarian follicular waves per estrous cycle and serum progesterone concentrations were similar (P=0.99) to controls. Telomere lengths of sperm and blood cells from Starbuck II were not different (P>0.05) than those of non-cloned cattle; telomere lengths of progeny were not different (P>0.05) from age-matched controls. In addition, progeny had normal karyotypes in peripheral blood leukocytes compared to controls (89.1% versus 86.3% diploid, respectively). In summary, heifers sired by a bull clone had normal chromosomal stability, growth, physical, hematological and reproductive parameters, compared to normal heifers. Furthermore, they had moderate stress responses to routine handling and restraint.

The impact of oocyte maturation media on early bovine embryonic development.

Successful production of high quality blastocysts in vitro depends on the use of a culture system that ensures the acquisition of developmental competence by the maturing oocyte. It is now clear that the in vitro maturation environment has a major influence on the oocyte's ability to acquire the potential to develop into blastocysts. In this work we examine the impact of oocyte culture media on the quality of blastocysts by comparing developmental rates, cell number and their allocation to embryonic cell lineages, apoptosis, and expression of developmentally important genes. Higher total cell count and ICM:TCN ratio, which are indicative of embryo viability, were observed in embryos derived from oocyte maturation in TCM-199 supplemented with serum when compared to blastocysts derived from oocyte maturation in SOF BSA. Moreover, oocyte maturation in TCM-199 supplemented with serum-generated embryos of higher morphological quality and producing higher levels of Interferon Tau transcripts when compared to embryos derived from oocyte maturation in SOF BSA. In conclusion, the oocyte maturation regimen affected the morphological feature of blastocysts, including total cell count and allocation of cells to trophectoderm (TE) and inner cell mass (ICM) lineages and the expression profiles of genes involved in various embryo functions such as early embryonic growth, regulation of gene transcription, trophoblast differentiation and function, embryo-maternal communication, and stress response. Our results show that the oocyte culture media have strong impact on the quality of embryos produced in vitro and emphasize the need for more in depth evaluation of oocyte maturation protocols.

The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos.

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX- treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.

The impact of chromosomal alteration on embryo development.

Chromosome alterations, such as those affecting telomere erosion, predictably occur with each cell division, others, which involve changes to the expression and replication of the X-chromosome occur at particular stages of development, while those that involve loss or gain of chromosomes occur in a random and so far unpredictable manner. The production of embryos in vitro and by somatic cell nuclear transfer (SCNT) has been associated with altered expression of marker genes on the X-chromosome and an increased incidence of chromosomally abnormal cells during early development. In the case of SCNT embryos chromosome abnormalities may be associated with the nuclear donor cell. Telomere rebuilding subsequent to SCNT appears to vary according to species and type of donor cell used. It is speculated that the rate of telomere erosion and incidence of chromosome abnormalities affects developmental potential of early embryos and may be potential predictors of developmental outcome.

Different culture media requirements of IVF and nuclear transfer bovine embryos.

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.

p66shc, but not p53, is involved in early arrest of in vitro-produced bovine embryos.

High embryo loss occurs in the first week of bovine embryo development, with a high percentage of embryonic arrest. We hypothesized that arrested embryos enter a 'senescence-like state' and that both the cell cycle regulatory protein p53 and the stress-related protein p66(shc), which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest. In our in vitro production system, 13.5 +/- 0.5% of embryos arrest at the 2-4-cell stage. First cleavage occurs between 26 and 48 h post insemination (hpi), with early cleaving embryos showing only 0.6 +/- 0.3% arrest, with later cleaving embryos exhibiting up to 14.2 +/- 0.9% arrest. We compared 2-4-cell embryos collected at 28 hpi with those arrested at the 2-4-cell stage collected at day 8 post insemination. Quantification by real-time PCR and by semi-quantitative immunofluorescence showed significantly higher p66(shc) mRNA and protein levels in both arrested and late cleaving embryos versus 28 hpi embryos. By comparison, no significant changes in p53 mRNA, protein and phosphorylation levels were detected. Taken together, these results demonstrate that embryonic developmental potential is related to the time of first cleavage and that p66(shc), but not p53, is up-regulated in early arrested in vitro-produced bovine embryos.

Telomerase activity in clinically normal dogs and dogs with malignant lymphoma.

OBJECTIVES: To determine whether telomerase activity was present in lymph nodes, buffy coat, and serum samples from dogs with malignant lymphoma (ML) and in liver, lymph node, buffy coat, and serum samples from clinically normal dogs SAMPLE POPULATION: Tissue specimens and blood samples were obtained from 11 clinically normal adult dogs (age range, 1 to 4 years) and 14 client-owned dogs with ML. PROCEDURE: The telomere repeat amplification protocol assay was used to quantify telomerase activity in the tissues from clinically normal dogs and dogs with ML. RESULTS: Of 11 clinically normal dogs, 8 had lymph node samples, 5 had liver samples, and 1 had buffy coat samples with detectable telomerase activity. None of the serum samples from the clinically normal dogs had detectable telomerase activity. Of 14 dogs with ML, 9 had lymph node samples, 3 had buffy coat samples, and 1 had serum samples with measurable telomerase activity. CONCLUSIONS AND CLINICAL RELEVANCE: Telomerase activity was not specific to tumor cells and overlapped with that found in cells from clinically normal dogs. Telomerase activity in neoplastic lymph nodes was not substantially different from that found in lymph nodes from clinically normal dogs. The determination of telomerase activity cannot be used as a sole diagnostic test for cancer. Therapeutic modalities directed toward the telomerase enzyme may not be feasible in dogs, because somatic tissues from clinically normal dogs possess variable amounts of telomerase activity.

The effects of antibodies to heat shock protein 70 in fertilization and embryo development.

The role of heat shock proteins in shielding organisms from environmental stress is illustrated by the large-scale synthesis of these proteins by the organisms studied to date. However, recent evidence also suggests an important role for heat shock proteins in fertilization and early development of mammalian embryos. We found that the presence of anti-HSP70 antibody significantly reduced tight binding of spermatozoa to the zona pellucida of bovine oocytes and interrupted completion of meiosis II and pronuclear formation. Furthermore, the presence of anti-HSP70 in culture medium from day 3 to day 9 of development increased apoptosis and significantly reduced the number of embryos reaching the blastocyst stage. We further observed that the proportion of apoptotic cells in bovine blastocysts was significantly lower after in-vitro culture with a prior exposure to increased temperature. However, nuclear localization of the p53 protein, which is thought to be essential for the up-regulation of genes involved in apoptosis and cell cycle arrest, was detected in the majority of nuclei in blastocysts exposed to increased temperature, whereas in their untreated (control) counterparts, p53 protein was only detected in the cytoplasm. The decrease in apoptosis after exposure of blastocysts to increased temperature may be attributed to cell cycle arrest resulting from nuclear localization of the p53 protein and/or to an increase in heat shock protein synthesis. We propose that HSP70 plays a critical role in fertilization and early embryonic development.

Genetic regulation of embryo death and senescence.

The survival of the preimplantation mammalian embryo depends not only on providing the proper conditions for normal development but also on acquiring the mechanisms by which embryos cope with adversity. The ability of the early conceptus to resist stress as development proceeds may be regulated by diverse factors such as the attainment of a cell death program and protective mechanisms involving stress-induced genes and/or cell cycle modulators. This paper reviews the recent research on the genetic regulation of early embryo cell death and senescence focussing on the bovine species where possible. The different modes of cell death will be explained, clarifying the confusing cell death terminology, by advocating the recommendations set forth by the Cell Death Nomenclature Committee to extend to the embryology research field. Specific pro-death and anti-death genes will be discussed with reference to their expression patterns during early mammalian embryogenesis.

Differential involvement of Na(+),K(+)-ATPase isozymes in preimplantation development of the mouse.

Na(+),K(+)-ATPase plays an essential role in mammalian blastocoel formation (cavitation) by driving trans-epithelial sodium transport. Previously, the alpha1 and beta1 subunit isoforms of this enzyme were identified in preimplantation mouse embryos and were assumed to be responsible for this function. Here we show that mRNAs encoding an additional alpha subunit isoform (alpha3) and the remaining two beta subunit isoforms are also present in preimplantation embryos. Whereas alpha3 mRNA accumulates between the four-cell and the blastocyst stages and thus results from embryonic transcription, the same could not be demonstrated for beta2 and beta3 mRNAs. Immunoblot analyses confirmed that these subunits are present in cavitating embryos. Using confocal immunofluorescence microscopy we found that alpha1 and beta1 subunits are concentrated in the basolateral membranes of the trophectoderm while being equally distributed in plasma membranes of the inner cell mass. In contrast, alpha3, beta2, and beta3 subunits were not detected in plasma membranes. Our current assessment, therefore, is that as many as six isozymes of Na(+),K(+)-ATPase could be involved in preimplantation development although it is primarily the alpha1beta1 isozyme that is responsible for blastocoel formation. Our findings imply that the regulation of sodium transport within the preimplantation mouse embryo is more complex than had been appreciated.