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As obese and overweight patients commonly display hyperlipidemia and are increasingly accessing fertility clinics for their conception needs, our studies are directed at understanding the effects of hyperlipidemia on early pregnancy. We have focused on investigating palmitic acid (PA) and oleic acid (OA) treatment alone and in combination from the mouse two-cell stage embryos as a model for understanding their effects on the mammalian preimplantation embryo. We recently reported that PA exerts a negative effect on mouse two-cell progression to the blastocyst stage, whereas OA co-treatment reverses that negative effect. In the present study, we hypothesized that PA treatment of mouse embryos would disrupt proper localization of cell fate determining and blastocyst formation gene products and that co-treatment with OA would reverse these effects. Our results demonstrate that PA treatment significantly (P < 0.05) reduces blastocyst development and cell number but did not prevent nuclear localization of YAP in outer cells. PA treatment significantly reduced the number of OCT4+ and CDX2+ nuclei. PA-treated embryos had lower expression of blastocyst formation proteins (E-cadherin, ZO-1 and Na/K-ATPase alpha1 subunit). Importantly, co-treatment of embryos with OA reversed PA-induced effects on blastocyst development and increased inner cell mass (ICM) and trophectoderm (TE) cell numbers and expression of blastocyst formation proteins. Our findings demonstrate that PA treatment does not impede cell fate gene localization but does disrupt proper blastocyst formation gene localization during mouse preimplantation development. OA treatment is protective and reverses PA's detrimental effects. The results advance our understanding of the impact of FFA exposure on mammalian preimplantation development.

Obesity is associated with altered fatty acid profiles, reduced fertility, and assisted reproductive technology (ART) success. The effects of palmitic acid (PA), oleic acid (OA), and their combination on mouse preimplantation development, endoplasmic reticulum (ER) stress pathway gene expression, lipid droplet formation, and mitochondrial reactive oxygen species (ROS) were characterized. Two-cell stage mouse embryos collected from superovulated and mated CD1 females were placed into culture with KSOMaa medium, or PA alone or in combination with OA for 46 h. PA significantly reduced blastocyst development in a concentration-dependent manner, which was prevented by co-treatment with OA. PA and OA levels in mouse reproductive tracts were assessed by liquid chromatography coupled to mass spectrometry (LC-MS). LC-MS indicated higher concentrations of PA in the mouse oviduct than the uterus. Transcript analysis revealed that PA alone groups had increased ER stress pathway (ATF3, CHOP, and XBP1 splicing) mRNAs, which was alleviated by OA co-treatment. OA co-treatment significantly increased lipid droplet accumulation and significantly decreased mitochondrial ROS from PA treatment alone. PA treatment for only 24 h significantly reduced its impact on blastocyst development from the 2-cell stage. Thus, PA affects ER stress pathway gene expression, lipid droplet accumulation, and mitochondrial ROS in treated preimplantation embryos. These mechanisms may serve to offset free fatty acid exposure effects on preimplantation development, but their protective ability may be overwhelmed by elevated PA.

Study Question: What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function?, Summary Answer: AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur., What Is Known Already: AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK., Study Design, Size, Duration: Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 μM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 μM AICAR., Participants/materials, Setting, Methods: Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR., Main Results and the Role of Chance: Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible., Large Scale Data: Not applicable., Limitations, Reasons for Caution: Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin., Wider Implications of the Findings: Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles., Study Funding and Competing Interest(s): Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

Preimplantation development shifts from a maternal to embryonic programme rapidly after fertilization. Although the majority of oogenetic products are lost during the maternal to embryonic transition (MET), several do survive this interval to contribute directly to supporting preimplantation development. Embryonic genome activation (EGA) is characterized by the transient expression of several genes that are necessary for MET, and while EGA represents the first major wave of gene expression, a second mid-preimplantation wave of transcription that supports development to the blastocyst stage has been discovered. The application of genomic approaches has greatly assisted in the discovery of stage specific gene expression patterns and the challenge now is to largely define gene function and regulation during preimplantation development. The basic mechanisms controlling compaction, lineage specification and blastocyst formation are defined. The requirement for embryo culture has revealed plasticity in the developmental programme that may exceed the adaptive capacity of the embryo and has fostered important research directions aimed at alleviating culture-induced changes in embryonic programming. New levels of regulation are emerging and greater insight into the roles played by RNA-binding proteins and miRNAs is required. All of this research is relevant due to the necessity to produce healthy preimplantation embryos for embryo transfer, to ensure that assisted reproductive technologies are applied in the most efficient and safest way possible.

Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase alpha and beta isoforms during mouse preimplantation development, only the alpha1 and beta1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/K-ATPase beta1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either Stealthtrade mark Na/K-ATPase beta1 subunit oligos or nonspecific Stealthtrade mark siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase beta1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase beta1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase beta1 subunit mRNAs and protein were not detectable in beta1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between beta1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase alpha1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase beta1 subunit siRNA-microinjected embryos. Our results demonstrate that the beta1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.

Efforts have intensified to successfully mature and inseminate oocytes in vitro and then culture ensuing embryos to transferable stages from a large number of mammalian species. Success varies, but generally even for the most successful species it is only possible to obtain a maximum of a 40 to 50% development of zygotes to the blastocyst stage. Reduced oocyte developmental competence is suggested as a primary reason for the reduced potential of in vitro-produced embryos. The vast majority of in vitro-matured oocytes are meiotically competent; however, many do not attain an optimal oocyte diameter before insemination. Variations in oocyte in vitro maturation media can influence embryo development, blastocyst cell number, and apoptosis. In addition, studies have indicated that cytoplasmic donation from so-called competent to incompetent oocytes can improve developmental outcomes. Oocyte cytoplasmic maturation includes those events that instill upon the oocyte a capacity to complete nuclear maturation, insemination, early embryogenesis and thus provide a foundation for implantation, initiation of pregnancy, and normal fetal development. Although we can define oocyte cytoplasmic maturation, we are only now beginning to understand the molecular steps that underlie this process. In general terms, oocyte cytoplasmic maturation involves the accumulation of mRNA, proteins, substrates, and nutrients that are required to achieve the oocyte developmental competence that fosters embryonic developmental competence. Collectively we are beginning to specify oocyte cytoplasmic maturation, and eventually a coherent understanding of this critical event in oocyte biology will emerge.

Research applied to the early embryo is required to effectively treat human infertility and to understand the primary mechanisms controlling development to the blastocyst stage. The present study investigated whether the Na(+)/K(+)-ATPase regulates tight junction formation and function during blastocyst formation. To investigate this hypothesis, three experimental series were conducted. The first experiments defined the optimal dose and treatment time intervals for ouabain (a potent and specific inhibitor of the Na(+)/K(+)-ATPase) treatment. The results demonstrated that mouse embryos maintained a normal development to the blastocyst stage following a 6-h ouabain treatment. The second experiments investigated the effects of ouabain treatment on the distribution of ZO-1 and occludin (tight junction associated proteins). Ouabain treatment (up to 6 h) or culture in K(+)-free medium (up to 6 h) resulted in the appearance of a discontinuous ZO-1 protein distribution and a loss of occludin immunofluorescence. The third set of experiments examined the influence of ouabain treatment on tight junction function. Ouabain treatment or culture in K(+)-free medium affected tight junction permeability as indicated by an increase in the proportion of treated embryos accumulating both 4 kDa and 40 kDa fluorescein isothiocyanate (FITC)-dextran into their blastocyst cavities. The results indicate that the Na(+)/K(+)-ATPase is a potent regulator of tight junction formation and function during mouse preimplantation development.

Blastocyst formation, as a critical period during development, is an effective indicator of embryonic health and reproductive efficiency. Out of a number of mechanisms underlying blastocyst formation, highly conserved mitogen-activated protein kinase (MAPK) signaling has emerged as a major mechanism involved in regulating murine preimplantation embryo development. The objective of our study was to ascertain the role of MAPK signaling in regulating bovine development to the blastocyst stage. Using reverse transcriptase PCR and immunohistochemical staining procedures we have demonstrated that mRNA transcripts and polypeptides encoding p38 MAPK pathway constituents are detectable in preimplantation bovine embryos from the one-cell to the blastocyst stage. Further, the effects on bovine embryo development following inhibition of p38 alpha/beta and extracellular signal-regulated kinase (ERK) signaling by treatment with SB220025 and U0126, respectively, were investigated. Eight-cell bovine embryos (50 per group; three replicates) were placed into treatments consisting of synthetic oviductal fluid (SOF) medium: SOF + SB202474 (inactive analogue), SOF + SB220025, SOF + U0124 (inactive analogue), SOF + U0126, and SOF + SB220025 + U0126. Inhibition of p38 MAPK or ERK signaling individually did not affect development to the blastocyst stage. However, when both pathways were blocked simultaneously there was a significant reduction (P < 0.05) in blastocyst formation, cell number and immunofluorescence of phosphorylated downstream pathway constituents. We have determined that, in variance to what was observed during murine preimplantation development, bovine early embryos progress at normal frequencies to the blastocyst stage in the presence of p38 MAPK inhibitors.

New insights into the early development of large mammals are becoming available through the measurement of differential mRNA levels in oocytes and preimplantation embryos. These advances in knowledge are rapidly picking up in pace, mainly owing to the advantages brought by new molecular biology approaches being developed. The possibility of amplifying the starting material and therefore making measurements in single embryo units is now feasible. With these tools, the evaluation of variations in gene expression patterns during the preimplantation period or the impact of culture on mRNA levels is now possible. However, it is important to keep in mind that these methods still have limitations associated with sample preparation or the use of the appropriate controls. Even proper methods of analysis are very important to achieve the full benefit of the application of these tools. The present paper describes some of the potential, as well as limitations, of mRNA level analysis in early embryos, especially for microarray analysis. We have generated a bovine cDNA array (>2000 clones) that contains expressed sequence tags (ESTs) collected from various preimplantation development stages. Using this chip, we have initiated the characterisation of global mRNA level patterns of several key developmental stages from the immature oocyte to the blastocyst stage. As expected, the hybridisation results indicate very different expression profiles involving hundreds of genes when comparing oocyte and blastocyst samples to a reference mRNA sample made from a pool of ESTs from pooled somatic tissues. Although this array is still in its preliminary stage and the EST bank has not been processed to contain only unigenes, it is already a very useful tool for discovering candidate genes that may play important roles during early embryonic life.

Mitogen-activated protein kinase (MAPK) pathways mediate some important cellular processes and are likely to also regulate preimplantation development. The role of p38 MAP kinase signaling during murine preimplantation development was investigated in the present study. p38 MAPK, p38-regulated or -activated kinase (PRAK; MK5), map kinase-activated protein kinase 2 (MK2), and heat shock protein 25 (hsp25) mRNAs and proteins were detected throughout preimplantation development. Two-cell stage embryos cultured in the presence of SB220025 and SB203580 (specific inhibitors of p38 MAPK alpha/beta), progressed to the eight-cell stage with the same frequency as controls; however, treated embryos halted their development at the 8- to 16-cell stage. In addition, embryos treated with p38 MAPK inhibitors displayed a complete loss of MK2 and hsp25 phosphorylation and also a complete loss of filamentous actin as indicated by the absence of rhodamine-phalloidin staining. In these inhibitor-treated groups, the embryos were composed of a mixture of compacting and noncompacting cells, and the embryos were one to two cell divisions behind controls. Treated embryos remained viable as the developmental blockade was rescued by removing embryos from the drug treatment and placing them in drug-free medium until they progressed to the blastocyst stage. This study demonstrates that p38 MAPK activity is required to support development through the murine preimplantation interval.

Adherens junction formation is fundamental for compaction and trophectoderm differentiation during mammalian preimplantation development. We recently isolated an IQGAP-2 cDNA from a differential display-polymerase chain reaction screen of bovine preimplantation developmental stages. IQGAP-1 and -2 proteins mediate E-cadherin-based cell-to-cell adhesion through interactions with beta-catenin and the Rho GTPases, rac1 and cdc42. Our study demonstrates IQGAP-1,-2, rac-1 and cdc42 mRNAs are present throughout murine preimplantation development. IQGAP-1 and rac-1 protein distribution changes from predominantly plasma membrane associated to predominantly cytoplasmic as the embryo progresses through cleavage divisions and compaction to the blastocyst stage.

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.

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.

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., (Copyright 1998 Academic Press.)

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGF alpha, bFGF, TGF beta 1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.

This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation-stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two-cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (greater than 32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one-cell and eight- to 16-cell bovine embryo stages but increased 4.4-fold between the eight- to 16-cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.