Genome activation in equine in vitro-produced embryos.

Embryonic genome activation is a critical event in embryo development, in which the transcriptional program of the embryo is initiated. The timing and regulation of this process are species-specific. In vitro embryo production is becoming an important clinical and research tool in the horse; however, very little is known about genome activation in this species. The objective of this work was to identify the timing of genome activation, and the transcriptional networks involved, in in vitro-produced horse embryos. RNA-Seq was performed on oocytes and embryos at eight stages of development (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst; n = 6 per stage, 2 from each of 3 mares). Transcription of seven genes was initiated at the 2-cell stage. The first substantial increase in gene expression occurred at the 4-cell stage (minor activation), followed by massive gene upregulation and downregulation at the 8-cell stage (major activation). An increase in intronic nucleotides, indicative of transcription initiation, was also observed at the 4-cell stage. Co-expression network analyses identified groups of genes that appeared to be regulated by common mechanisms. Investigation of hub genes and binding motifs enriched in the promoters of co-expressed genes implicated several transcription factors. This work represents, to the best of our knowledge, the first genomic evaluation of embryonic genome activation in horse embryos.

Allele-specific expression analysis reveals conserved and unique features of preimplantation development in equine ICSI embryos†.

Embryonic genome activation and dosage compensation are major genetic events in early development. Combined analysis of single embryo RNA-seq data and parental genome sequencing was used to evaluate parental contributions to early development and investigate X-chromosome dynamics. In addition, we evaluated dimorphism in gene expression between male and female embryos. Evaluation of parent-specific gene expression revealed a minor increase in paternal expression at the 4-cell stage that increased at the 8-cell stage. We also detected eight genes with allelic expression bias that may have an important role in early development, notably NANOGNB. The main actor in X-chromosome inactivation, XIST, was significantly upregulated at the 8-cell, morula, and blastocyst stages in female embryos, with high expression at the latter. Sexual dimorphism in gene expression was identified at all stages, with strong representation of the X-chromosome in females from the 16-cell to the blastocyst stage. Female embryos showed biparental X-chromosome expression at all stages after the 4-cell stage, demonstrating the absence of imprinted X-inactivation at the embryo level. The analysis of gene dosage showed incomplete dosage compensation (0.5 < X:A < 1) in MII oocytes and embryos up to the 4-cell stage, an increase of the X:A ratio at the 16-cell and morula stages after genome activation, and a decrease of the X:A ratio at the blastocyst stage, which might be associated with the beginning of X-chromosome inactivation. This study represents the first critical analysis of parent- and sex-specific gene expression in early equine embryos produced in vitro.

Arylic versus Alkylic-Hydrophobic Linkers Determine the Supramolecular Structure and Optoelectronic Properties of Tripodal Amphiphilic Push-Pull Thiazoles.

The supramolecular structures and their constituents essentially determine the optoelectronic properties of thin films. The introduction of amphiphilicity to the constituents and interface assembly is one established technique to control supramolecular structures and resulting material properties. To yield amphiphilicity, rather hydrophobic chromophores are linked to hydrophilic head groups via flexible alkyl chains. In the present work, we investigate whether replacement of the alkyl linkers by a phenylene linker, that is, replacing an electrically isolating moiety with a potentially semiconducting one, increases the conductivity through the resulting layers. After investigating the influence of the linker on molecular properties of the 2-(4- N, N-dimethylaminophenyl)-4-hydroxy-5-nitrophenyl-1,3 thiazoles exemplarily used in this work, we produce supramolecular structures by means of the Langmuir-Blodgett (LB) technique. Atomic force microscopy (AFM) and UV-vis absorption spectroscopy reveal that thin films made from the more rigid thiazole bearing the arylic linker feature a more homogeneous and stable supramolecular structure as compared to those made from the thiazole dye containing the flexible alkylic linker. Finally, conductive AFM (cAFM) results disclose that the LB films made from the thiazole bearing the π-conjugated arylic linker are less conductive than their counterparts based on the alkylic linkers. In the latter layers, the alkylic linkers provide sufficient motional degrees of freedom to allow for supramolecular rearrangement upon electrical operation during cAFM measurements, hence yielding supramolecular structures featuring increased conductivity with successive cAFM measurements. This work highlights the importance of supramolecular structures for optoelectronic properties by presenting a case where supramolecular effects excel the property changes introduced by molecular modifications.

Equine blastocyst production under different incubation temperatures and different CO2 concentrations during early cleavage.

Some basic parameters for equine invitro embryo production have not yet been established, including the optimum temperature for maturation and embryo culture, and the optimum CO2 concentration and pH during early embryo development. To explore this, we first performed cultures in incubators set at 37.2°C, 37.7°C or 38.2°C. At these temperatures, the corresponding maturation rates were 33%, 38% and 42%; cleavage rates were 84%, 86% and 88%; and blastocyst rates were 35%, 44% and 44% per injected oocyte. These rates did not differ significantly (P>0.2). We then evaluated three different CO2 concentrations (6%, 6.5% or 7% CO2) in 5% O2 for culture over Days 0-5 after intracytoplasmic sperm injection, using a commercial human embryo medium with added serum, at 38.2°C. The pH values of these media were 7.36, 7.33 and 7.29 respectively. In the presence of 6%, 6.5% or 7% CO2, cleavage rates were 68%, 80% and 70% respectively, and blastocyst rates per injected oocyte were 42%, 54% and 27% respectively. The blastocyst rate for the 7% CO2 treatment was significantly lower than that for the 6.5% CO2 treatment (P<0.05). We conclude that equine invitro embryo production is equally effective within the range of 37.2-38.2°C, but that equine early cleavage stage development is sensitive to small changes in CO2 atmosphere and/or medium pH.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection.

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22-27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

Application of embryo biopsy and sex determination via polymerase chain reaction in a commercial equine embryo transfer program in Argentina.

Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller embryos (70-85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive embryos were confirmed as male and 78% of Y-DNA-negative embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%.

Intrafollicular oocyte transfer in the horse: effect of autologous vs. allogeneic transfer and time of administration of ovulatory stimulus before transfer.

PURPOSE: To assess meiotic and developmental competence after transfer of immature cumulus-oocyte complexes (COCs) to the preovulatory follicles of mares (intrafollicular oocyte transfer (IFOT)). METHODS: In Experiment 1, mares received an ovulatory stimulus at IFOT. Thirty hours later, COCs were recovered from the follicle, and mature oocytes underwent ICSI and embryo culture. In Experiments 2 and 3, autologous vs. allogeneic COCs were used. The mares were inseminated and embryos were recovered. In Experiment 3, the ovulatory stimulus was administered 9 h (autologous) and 15 h (allogeneic) before IFOT. In Experiment 4, only allogeneic COCs were used; the ovulatory stimulus was administered 9 or 15 h before IFOT. Excess embryos (autologous) and parentage-verified embryos (allogeneic) were considered IFOT-derived. RESULTS: In Experiment 1, 36/54 IFOT oocytes (67%) were recovered, of which 56% were mature, vs. 49% of in vitro matured oocytes (P > 0.1). After ICSI, blastocyst rates were 25% and 18%, respectively (P > 0.1). In Experiment 2, 0/6 autologous and 2/6 allogeneic IFOT yielded IFOT-derived embryos. In Experiment 3, 0/7 autologous and 2/5 allogeneic IFOT yielded IFOT-derived embryos. The proportion of mares yielding IFOT-derived embryos was lower after autologous vs. allogeneic IFOT (0/13 vs. 4/11; P < 0.05). In Experiment 4, 1/8 9-h and 1/7 15-h IFOT yielded IFOT-derived embryos. CONCLUSIONS: Transferred oocytes mature within the follicle and can maintain developmental competence. Allogeneic IFOT was more efficient than was autologous IFOT. The time of ovulatory stimulation did not affect embryo yield. The IFOT procedure is still not repeatable enough to be recommended for clinical use.

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm.

Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA. Bicarbonate induced protein tyrosine phosphorylation (PY), even in the presence of calcium or BSA. Incubation at high pH enhanced PY but did not increase ROS production. Notably, no combination of these factors was associated with significant cholesterol efflux, as assessed by fluorescent quantitative cholesterol assay and confirmed by filipin staining. By contrast, sperm treated with methyl-ß-cyclodextrin showed a significant reduction in cholesterol levels, but no significant increase in PY or ROS. Presence of BSA increased sperm binding to bovine zonae pellucidae in all three stallions. These results show that presence of serum albumin is not associated with a reduction in membrane cholesterol levels in equine sperm, highlighting the failure of equine sperm to exhibit core capacitation-related changes in a standard capacitating medium. These data indicate an atypical relationship among cholesterol efflux, ROS production, and PY in equine sperm. Our findings may help to elucidate factors affecting failure of equine IVF under standard conditions.

Accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts.

Preimplantation genetic diagnosis has great potential in the horse, but information on evaluation of equine embryo biopsy samples is limited. Blastocysts were biopsied using a Piezo drill and methods for whole-genome amplification (WGA) investigated. Results for 33 genetic loci were then compared between biopsy samples from in vitro-produced (IVP) and in vivo-recovered (VIV) blastocysts. Under the experimental conditions described, WGA using the Qiagen Repli-g Midi kit was more accurate than that using the Illustra Genomiphi V2 kit (98.2% vs 25.8%, respectively). Using WGA with the Qiagen kit, three biopsy samples were evaluated from each of eight IVP and 19 VIV blastocysts, some produced using semen from stallions carrying the genetic mutations associated with the diseases hereditary equine regional dermal asthenia (HERDA), hyperkalemic periodic paralysis (HYPP) or polysaccharide storage myopathy 1 (PSSM1). Three of 81 biopsy samples (3.7%) returned 95% overall accuracy in IVP and VIV embryos, and this technique is suitable for use in a clinical setting.

Bacterial symbionts of Bathymodiolus mussels and Escarpia tubeworms from Chapopote, an asphalt seep in the Southern Gulf of Mexico.

Chemosynthetic life was recently discovered at Chapopote, an asphalt hydrocarbon seep in the southern Gulf of Mexico. Preliminary morphological analyses indicated that one tubeworm and two mussel species colonize Chapopote. Our molecular analyses identified the tubeworm as Escarpia sp., and the mussels as Bathymodiolus heckerae and B. brooksi. Comparative 16S rRNA analysis and FISH showed that all three species harbour intracellular sulfur-oxidizing symbionts highly similar or identical to those found in the same host species from northern Gulf of Mexico (nGoM). The mussels also harbour methane-oxidizing symbionts, and these shared highly similar to identical 16S rRNA sequences to their nGoM conspecifics. We discovered a novel symbiont in B. heckerae, which is closely related to hydrocarbon-degrading bacteria of the genus Cycloclasticus. In B. heckerae, we found key genes for the use of aromatic compounds, and its stable carbon isotope values were consistently higher than B. brooksi, indicating that the novel symbiont might use isotopically heavy aromatic hydrocarbons from the asphalt seep. This discovery is particularly intriguing because until now only methane and reduced sulfur compounds have been shown to power cold-seep chemosynthetic symbioses. The abundant hydrocarbons available at Chapopote would provide these mussel symbioses with a rich source of nutrition.

Evaluation of new geological reference materials for uranium-series measurements: Chinese Geological Standard Glasses (CGSG) and macusanite obsidian.

Recent advances in high-resolution, rapid, in situ microanalytical techniques present numerous opportunities for the analytical community, provided accurately characterized reference materials are available. Here, we present multicollector thermal ionization mass spectrometry (MC-TIMS) and multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) uranium and thorium concentration and isotopic data obtained by isotope dilution for a suite of newly available Chinese Geological Standard Glasses (CGSG) designed for microanalysis. These glasses exhibit a range of compositions including basalt, syenite, andesite, and a soil. Uranium concentrations for these glasses range from ∼2 to 14 µg g(-1), Th/U weight ratios range from ∼4 to 6, (234)U/(238)U activity ratios range from 0.93 to 1.02, and (230)Th/(238)U activity ratios range from 0.98 to 1.12. Uranium and thorium concentration and isotopic data are also presented for a rhyolitic obsidian from Macusani, SE Peru (macusanite). This glass can also be used as a rhyolitic reference material, has a very low Th/U weight ratio (around 0.077), and is approximately in (238)U-(234)U-(230)Th secular equilibrium. The U-Th concentration data agree with but are significantly more precise than those previously measured. U-Th concentration and isotopic data agree within estimated errors for the two measurement techniques, providing validation of the two methods. The large (238)U-(234)U-(230)Th disequilibria for some of the glasses, along with the wide range in their chemical compositions and Th/U ratios should provide useful reference points for the U-series analytical community.

Calcium-calmodulin and pH regulate protein tyrosine phosphorylation in stallion sperm.

The mechanisms leading to capacitation in stallion sperm are poorly understood. The objective of our study was to define factors associated with regulation of protein tyrosine phosphorylation in stallion sperm. Stallion sperm were incubated for 4 h in modified Whitten's media with or without bicarbonate, calcium, or BSA. When sperm were incubated in air at 30×106/ml at initial pH 7.25, protein tyrosine phosphorylation was detected only in medium containing 25 mM bicarbonate alone; calcium and BSA inhibited phosphorylation. Surprisingly, this inhibition did not occur when sperm were incubated at 10×106/ml. The final pH values after incubation at 30×106 and 10×106 sperm/ml were 7.43 ± 0.04 and 7.83 ± 0.07 (mean ± s.e.m.) respectively. Sperm were then incubated at initial pH values of 7.25, 7.90, or 8.50 in either air or 5% CO2. Protein tyrosine phosphorylation increased with increasing final medium pH, regardless of the addition of bicarbonate or BSA. An increase in environmental pH was observed when raw semen was instilled into the uteri of estrous mares and retrieved after 30 min (from 7.47 ± 0.10 to 7.85 ± 0.08), demonstrating a potential physiological role for pH regulation of capacitation. Sperm incubated in the presence of the calmodulin (CaM) inhibitor W-7 exhibited a dose-dependent increase in protein tyrosine phosphorylation, suggesting that the inhibitory effect of calcium was CaM mediated. These results show for the first time a major regulatory role of external pH, calcium, and CaM in stallion sperm protein tyrosine phosphorylation.

Rapid metabolism fosters microbial survival in the deep, hot subseafloor biosphere.

A fourth of the global seabed sediment volume is buried at depths where temperatures exceed 80 °C, a previously proposed thermal barrier for life in the subsurface. Here, we demonstrate, utilizing an extensive suite of radiotracer experiments, the prevalence of active methanogenic and sulfate-reducing populations in deeply buried marine sediment from the Nankai Trough subduction zone, heated to extreme temperature (up to ~120 °C). The small microbial community subsisted with high potential cell-specific rates of energy metabolism, which approach the rates of active surface sediments and laboratory cultures. Our discovery is in stark contrast to the extremely low metabolic rates otherwise observed in the deep subseafloor. As cells appear to invest most of their energy to repair thermal cell damage in the hot sediment, they are forced to balance delicately between subsistence near the upper temperature limit for life and a rich supply of substrates and energy from thermally driven reactions of the sedimentary organic matter.

Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse.

Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P < 0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sperm lyophilized from fresh or frozen-thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze-thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species.

Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis.

The equine embryo possesses a capsule that is considered essential for its survival. We assessed viability after breaching the capsule of early (Day 6) and expanded (Day 7 and 8) equine blastocysts by micromanipulation. The capsule was penetrated using a Piezo drill, and trophoblast biopsy samples were obtained for genetic analysis. Pregnancy rates for Day-6 embryos, which had intact zonae pellucidae at the time of recovery, were 3/3 for those biopsied immediately after recovery and 2/3 for those biopsied after being shipped overnight under warm (∼28 °C) conditions. The pregnancy rates for encapsulated Day-7 expanded blastocysts were 5/6 for those biopsied immediately and 5/6 for those biopsied after being shipped overnight warm. Two of four encapsulated Day-8 blastocysts, 790 and 1350 µm in diameter, established normal pregnancies after biopsy. Nine mares were allowed to maintain pregnancy, and they gave birth to nine normal foals. Biopsied cells from eight embryos that produced foals were subjected to whole-genome amplification. Sex was successfully determined from amplified DNA in 8/8 embryos. Identification of disease-causing mutations matched in the analyses of 6/6 samples for the sodium channel, voltage-gated, type IV, alpha subunit (SCN4A) gene and in 6/7 samples for the peptidylprolyl isomerase B (PPIB) gene, in embryo-foal pairs. Thus, the capsule of the equine embryo can be breached without impairing viability. Further work is needed to determine whether this breach is transient or permanent. These findings are relevant to the understanding of equine embryo development and to the establishment of methods for micromanipulation and embryo cryopreservation in this species.

In vitro production of equine embryos: state of the art.

CONTENTS: In vitro embryo production is possible in the horse both clinically and for research applications. Oocytes may be collected from excised ovaries post-mortem, or from either immature follicles or stimulated pre-ovulatory follicles in the live mare. In vitro maturation of immature oocytes typically yields approximately 60% mature oocytes. As standard in vitro fertilization is not yet repeatable in the horse, fertilization is performed by intracytoplasmic sperm injection. Embryo culture requires medium with high glucose, at least during blastocyst development, and rates of blastocyst development similar to those for cattle (25% to 35%) may be obtained. Pregnancy rates after transfer of in vitro-produced blastocysts are similar to those for embryos recovered ex vivo.

Energy metabolism of the equine cumulus oocyte complex during in vitro maturation.

Horses are one of the few species, beside humans, in which assisted reproductive technology has important clinical applications. Furthermore, the horse can serve as a valuable model for the study of comparative reproductive biology. Here we present the first comprehensive characterisation of energy metabolism and mitochondrial efficiency in equine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as determined using a combination of non-invasive consumption and release assays and mitochondrial function analysis. These data reveal notable species-specific differences in the rate and kinetics of glucose consumption and glycolysis throughout IVM. Approximately 95% of glucose consumed was accounted for by lactate production; however, high concurrent oxygen consumption indicated a comparatively increased role for non-glycolytic oxidative phosphorylation. Up to 38% of equine COC oxygen consumption could be attributed to non-mitochondrial activities and there was a significant loss of spare respiratory capacity over the course of IVM. Notably, our data also revealed that current IVM protocols may be failing to satisfy the metabolic demands of the equine COC. Our findings constitute the first report on mitochondrial efficiency in the equine COC and provide new insight into comparative gamete biology as well as metabolism of the COC during in vitro maturation.

Equine fetal genotyping via aspiration of yolk-sac fluid at 22-28 days of gestation.

Fetal genotyping has important applications in the horse, but currently necessitates embryo recovery and biopsy. We investigated whether fetal genotyping could be performed on yolk-sac fluid recovered from pregnant mares via transvaginal aspiration. Fluid was collected before Day 30 to provide results before establishment of the endometrial cups (Day 37). Genotyping and assessment of maternal DNA contamination was performed by analyzing histograms of PCR results for 19 loci. In Exp. 1, mares underwent yolk-sac aspiration on Days 22-28 of gestation. Fluid (0.56-1.02 mL) was recovered from five of seven mares. Four of the five mares maintained pregnancy. One pregnancy was electively terminated at Day 75; the other three mares delivered healthy foals. Extraction of DNA from the fluid sample followed by direct PCR allowed the highest rate of determination of fetal alleles. Fetal genotype was correctly determined in three samples, and for 14/19 alleles in one sample. In Exp. 2, we evaluated whether recovery of more fluid (up to 1.6 mL), and fractionation of the sample, would minimize maternal DNA contamination. One of four mares maintained pregnancy. Evaluation at informative loci showed no difference in maternal contamination among fractions. We determined that mares can maintain pregnancy after aspiration of yolk-sac fluid, and that fetal genotype can be accurately determined from the sample obtained. Further work is needed on factors affecting maintenance of pregnancy after the procedure. The ability to access the yolk sac in early pregnancy opens the door to novel potential clinical and research applications.

The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts.

The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout the blastocysts. In contrast, in vivo-produced day-7 to -10 blastocysts showed greatly reduced trophoectodermal POU5F1 protein expression. To determine whether the uterine environment modulates POU5F1 expression, IVP blastocysts were transferred to the uteri of mares, then recovered 2-3 days later (IVP-ET embryos). These embryos showed similar POU5F1 expression as the in vivo-produced embryos. Levels of POU5F1, SOX2, and NANOG mRNA in IVP-ET blastocysts were significantly higher in the inner cell mass than in trophectoderm (TE) cells. These data suggest that the differentiation of equine TE, as indicated by loss of POU5F1 expression, is impaired during in vitro culture, but proceeds normally when the embryos are exposed to the uterine environment. Previously reported differences in trophectodermal expression of POU5F1 among species may thus be in part artifactual, i.e. related to in vitro culture. Failure for correction of such changes by the uterine environment is a potential factor in the placental abnormalities seen after transfer of cultured embryos in some species.

Methane-consuming archaea revealed by directly coupled isotopic and phylogenetic analysis.

Microorganisms living in anoxic marine sediments consume more than 80% of the methane produced in the world's oceans. In addition to single-species aggregates, consortia of metabolically interdependent bacteria and archaea are found in methane-rich sediments. A combination of fluorescence in situ hybridization and secondary ion mass spectrometry shows that cells belonging to one specific archaeal group associated with the Methanosarcinales were all highly depleted in 13C (to values of -96 per thousand). This depletion indicates assimilation of isotopically light methane into specific archaeal cells. Additional microbial species apparently use other carbon sources, as indicated by significantly higher 13C/12C ratios in their cell carbon. Our results demonstrate the feasibility of simultaneous determination of the identity and the metabolic activity of naturally occurring microorganisms.