Enhancement of maternal recognition of pregnancy with parthenogenetic embryos in bovine embryo transfer

This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in in vivo–fertilized and parthenogenetic bovine embryos and their interferon-τ (IFNT) secretion amounts during the elongation phase. We assessed the induction capability of maternal recognition of pregnancy by parthenogenetic embryos and attempted cotransfer of in vivo–fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in in vivo–fertilized embryos peaked on Day 18 after estrus, and the highest amount of uterine IFNT was observed on Day 20. Transfer of 10 parthenogenetic embryos produced a detectable amount of uterine IFNT. Transfer of one or three parthenogenetic embryos inhibited luteolysis. An increase in ISG15 mRNA levels in peripheral granulocytes was induced by the transfer of three parthenogenetic embryos. Cotransfer of three parthenogenetic embryos significantly improved the pregnancy rate on Day 40 in code 3 in vivo–fertilized embryos compared with single transfer without parthenogenetic embryos (65% vs. 35%). However, the pregnancy rate on Day 90 (35%) in cotransfer of code 3 in vivo–fertilized embryos did not differ from that upon single transfer (29%), because the cotransfer group had a higher incidence of pregnancy loss than with single transfer (47% vs. 17%) after Day 40. Cotransfer did not affect the pregnancy rate of code 2 in vivo–fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 in vivo–fertilized embryos than in single transfer (30% vs. 7%). In conclusion, parthenogenetic embryos in the elongation phase secreted IFNT, enabling induction of maternal recognition of pregnancy. The present study revealed that enhancement of the maternal recognition of pregnancy using parthenogenetic embryos promoted the viability of poor-quality embryos until Day 40 of gestation. However, the incidence of pregnancy loss increased after Day 40 in the cotransfer of parthenogenetic embryos. A technique for promoting the full-term survival of poor-quality embryos is needed.     

Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats

Understanding the survival of fecal indicator bacteria (FIB) and microbial source-tracking (MST) markers is critical to developing pathogen fate and transport models. Although pathogen survival in water microcosms and manure-amended soils is well documented, little is known about their survival in intact cow pats deposited on pastures. We conducted a study to determine decay rates of fecal indicator bacteria (Escherichia coli and enterococci) and bovine-associated MST markers (CowM3, Rum-2-bac, and GenBac) in 18 freshly deposited cattle feces from three farms in northern Georgia. Samples were randomly assigned to shaded or unshaded treatment in order to determine the effects of sunlight, moisture, and temperature on decay rates. A general linear model (GLM) framework was used to determine decay rates. Shading significantly decreased the decay rate of the E. coli population (P < 0.0001), with a rate of −0.176 day⁻¹ for the shaded treatment and −0.297 day⁻¹ for the unshaded treatment. Shading had no significant effect on decay rates of enterococci, CowM3, Rum-2-bac, and GenBac (P > 0.05). In addition, E. coli populations showed a significant growth rate (0.881 day⁻¹) in the unshaded samples during the first 5 days after deposition. UV-B was the most important parameter explaining the decay rate of E. coli populations. A comparison of the decay behaviors among all markers indicated that enterococcus concentrations exhibit a better correlation with the MST markers than E. coli concentrations. Our results indicate that bovine-associated MST markers can survive in cow pats for at least 1 month after excretion, and although their decay dynamic differs from the decay dynamic of E. coli populations, they seem to be reliable markers to use in combination with enterococci to monitor fecal pollution from pasture lands.     

Does conditioned taste aversion learning in the pond snail Lymnaea stagnalis produce conditioned fear?

In conditioned taste aversion (CTA) training performed on the pond snail Lymnaea stagnalis, a stimulus (the conditional stimulus, CS; e.g., sucrose) that elicits a feeding response is paired with an aversive stimulus (the unconditional stimulus, US) that elicits the whole-body withdrawal response and inhibits feeding. After CTA training and memory formation, the CS no longer elicits feeding. We hypothesize that one reason for this result is that after CTA training the CS now elicits a fear response. Consistent with this hypothesis, we predict the CS will cause (1) the heart to skip a beat and (2) a significant change in the heart rate. Such changes are seen in mammalian preparations exposed to fearful stimuli. We found that in snails exhibiting long-term memory for one-trial CTA (i.e., good learners) the CS significantly increased the probability of a skipped heartbeat, but did not significantly change the heart rate. The probability of a skipped heartbeat was unaltered in control snails given backward conditioning (US followed by CS) or in snails that did not acquire associative learning (i.e., poor learners) after the one-trial CTA training. These results suggest that as a consequence of acquiring CTA, the CS evokes conditioned fear in the conditioned snails, as evidenced by a change in the nervous system control of cardiac activity.     

Identification of a line of sheep carrying a putative autosomal gene increasing ovulation rate in sheep that does not appear to interact with mutations in the transforming growth factor beta superfamily

Sheep lines with mutations in single genes that have major effects on ovulation rate have been very useful in gaining a better understanding of pathways important in controlling follicular development and ovulation rate. To date however, all known mutations are in the transforming growth factor beta (TGFB) superfamily. Ovulation rates were measured in 720 progeny of 20 rams that were descendants of a single prolific ewe. Evaluation of ovulation rates of daughters of closely related sires suggests the presence of a segregating major gene Fecundity Davisdale (FECD) that increases ovulation rate between 0.4 and 0.8 in heterozygous daughters. Key features of mutations in genes of the TGFB superfamily pathway, such as synergistic interactions with other family members, infertility in homozygous carriers, and increased responsiveness to exogenous gonadotropins, were not observed in this line; thus, the mutation does not appear to be acting in the TGFB pathway. Hence, there is likely a novel mutation being carried in this line of sheep that alters ovulation rate. Future identification of the causative mutation may provide new insights into regulation of follicular development and ovulation rate.     

Comparative study of the cell wall composition of broccoli, carrot, and tomato: structural characterization of the extractable pectins and hemicelluloses

This study delivers a comparison of the pectic and hemicellulosic cell wall polysaccharides between the commonly used vegetables broccoli (stem and florets separately), carrot, and tomato. Alcohol-insoluble residues were prepared from the plant sources and sequentially extracted with water, cyclohexane-trans-1,2-diamine tetra-acetic acid, sodium carbonate, and potassium hydroxide solutions, to obtain individual fractions, each containing polysaccharides bound to the cell wall in a specific manner. Structural characterization of the polysaccharide fractions was conducted using colorimetric and chromatographic approaches. Sugar ratios were defined to ameliorate data interpretation. These ratios allowed gaining information concerning polysaccharide structure from sugar composition data. Structural analysis of broccoli revealed organ-specific characteristics: the pectin degree of methoxylation (DM) of stem and florets differed, the sugar composition data inferred differences in polymeric composition. On the other hand, the molar mass (MM) distribution profiles of the polysaccharide fractions were virtually identical for both organs. Carrot root displayed a different MM distribution for the polysaccharides solubilized by potassium hydroxide compared to broccoli and tomato, possibly due to the high contribution of branched pectins to this otherwise hemicellulose-enriched fraction. Tomato fruit showed the pectins with the broadest range in DM, the highest MM, the greatest overall linearity and the lowest extent of branching of rhamnogalacturonan I, pointing to particularly long, linear pectins in tomato compared with the other vegetable organs studied, suggesting possible implications toward functional behavior.     

Wound healing and blastema formation in regenerating digit tips of adult mice

Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation.     

Phosphorylation state regulates the localization of Scribble at adherens junctions and its association with E-cadherin-catenin complexes

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin–catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin–catenin system and may control cadherin-mediated cell–cell adhesion.