THE EFFECT OF OVULATION-INDUCING FACTOR (OIF) IN BOVINE SEMINAL PLASMA ON OVARIAN FUNCTION IN CATTLE

Three experiments were designed to gain an understanding of the role of ovulation-inducing factor (OIF) present in bovine seminal plasma. Within species, seminal plasma was pooled from 1 to 4 ejaculates per male (n=160 bulls, n=4 llamas in Experiments 1 and 2, and n=95 bulls in Experiment 3). The volume of seminal plasma used for treatment was adjusted to a total dose of 250 µg of OIF. Experiment 1 was done to verify the bioactivity of OIF in bovine seminal plasma. Mature female llamas were assigned randomly to be treated intramuscularly (i.m.) with either 10 ml of phosphate buffered saline (PBS, negative control, n=5), 50 µg GnRH (positive control, n=5), 6 ml of llama seminal plasma (n=6) or 12 ml of bull seminal plasma (n=6). Experiment 2 was done to determine the effect of OIF in bovine seminal plasma on LH-induced ovulation and luteal development. Beef heifers with a CL and a growing follicle ≥10 mm were given a luteolytic dose of prostaglandin followed by 25 mg pLH 12 h later. Heifers were assigned randomly to three groups and given 10 ml bovine seminal plasma i.m. 12 h after pLH treatment (n=10), bovine seminal plasma i.m. within 4 h after ovulation (n=9), or no further treatment (control, n=10).Experiment 3 was done to determine the effect of OIF in bovine seminal plasma on LH release, ovulation and luteal development. Ovulation in beef heifers was synchronized using a protocol with progesterone and estradiol. Six days after ovulation, , when a mature CL and a dominant follicle of 11-13 mm diameter were expected to be present, heifers were assigned randomly to four groups (n=8 per group) using a 2-by-2 design and treated with either pLH or phosphate-buffered saline i.m., followed 12 h later by treatment with either 10 ml bovine seminal plasma or phosphate-buffered saline i.m.; i.e., LH+PBS, LH+SP, PBS+SP, and PBS+PBS groups. In all experiments, ovulation and CL development were monitored by transrectal ultrasonography. In Experiment 1, llamas were scanned daily from treatment to Day 6 after treatment, while in the other two experiments ovulations were monitored every 4 h and CL development was monitored daily until the next ovulation. Ovulation rates were compared among groups by Fisher’s exact test, and continuous data were compared among groups by ANOVA for repeated measures. Single point data were compared by ANOVA. In Experiment 1, ovulation was detected in 0/5, 4/5, 4/6, 4/6 in PBS, GnRH, llama seminal plasma, and bovine seminal plasma groups, respectively (P<0.05). No difference was detected among groups in luteal development. In Experiment 2, all ovulations in the pre-ovulation treatment group occurred within a 4 h period, while the range for other groups was 22 h (P<0.0001). No difference was detected among groups in luteal development; however, plasma progesterone concentrations tended to be greater in the heifers treated with seminal plasma post-ovulation compared to the other two groups (treatment-by-day interaction, P=0.1). In Experiment 3, ovulations were detected in 5/8, 4/8, 0/7, 0/8 in pLH, pLH+SP, SP and control groups, respectively (P<0.05). Corpora lutea present at the time of treatment took longer to decrease significantly in size from the time they reached maximum size in heifers treated with seminal plasma (p=0.04), but, plasma progesterone concentrations did not differ among groups during this same period. Nevertheless, there was a more rapid increase in plasma progesterone concentration by 24 h after seminal plasma treatment than those not treated with seminal plasma (P=0.03). Results confirm the presence of bioactive OIF in bull seminal plasma and showed that bovine and llama seminal plasma have similar ovulatory and luteotrophic effects using a llama bioassay. Moreover, treatment of sexually mature heifers with OIF from bovine seminal plasma influenced the timing of ovulation and the duration of luteal function.

Influencia de la suplementación del medio de cultivo con ácido linoleico en la supervivencia a la congelación de embriones bovinos

Studies report that the addition of linoleic acid to bovine embryo production media in vitro improves its resistance to conventional freezing. In this study the effect on survival of in vitro bovine embryos under freezing was evaluated by the addition of linoleic acid (LA) in the culture medium on days 3 or 5. A total of 1452 oocytes were recovered and assigned to 4 experimental groups and a control group. Six replicates were made to obtain approximately 100 freeze-thawed embryos per group. Group 1: 100 M on Day 3, group 2: 50 M on Day 3, group 3: 100 M on Day 5, group 4: 50 M on Day 5. The embryos were frozen and one week after they were thawed to evaluate the percentages of re-expansion (24 h) and hatching (72 h). The results were analyzed by ANOVA and when the differences were significant, the means between the groups were compared by Fischer's LSD Test (p <0.05). It was observed that the percentage of re-expansion was significantly improved with the addition of 50 μM LA on Day 5 (54.94 ± 28.8 vs. 19.19 ± 28.4, p = 0.001) and the percentage of hatching with the addition of 50 μM of LA at day 3 (37.9 ± 18.3 vs. 9.02 ± 12.0, p = 0.014), when them were compared with the control group. In conclusion, the addition of linoleic acid improves the survival to freezing of bovine embryos produced by in vitro techniquesEstudios reportan que la adición de ácido linoleico en los medios de producción de embriones bovinos in vitro, mejora su resistencia a la congelación convencional. En este estudio se evaluó el efecto en la supervivencia de embriones bovinos in vitro a la congelación, mediante la adición de ácido linoleico (LA) en el medio de cultivo los días 3 o 5. Un total de 1452 oocitos fueron recuperados y asignados a 4 grupos experimentales y un grupo control. Se realizaron seis réplicas, para obtener aproximadamente 100 embriones congelables por grupo. Grupo 1: 100 M en Día 3, grupo 2: 50 M en Día 3, grupo 3: 100 M en Día 5, grupo 4: 50 M en Día 5. Los embriones se congelaron y a la semana fueron descongelados para evaluar los porcentajes de re-expansión (24 h) y de eclosión (72 h). Los resultados fueron analizados mediante ANOVA y cuando las diferencias fueron significativas, se compararon las medias entre los grupos por el Test LSD de Fischer (p < 0.05). Se observó que el porcentaje de re-expansión mejoró significativamente con la adición de 50 M de LA en el Día 5 (54.94 ± 28.8 vs. 19.19 ± 28.4, p = 0.001) y el porcentaje de eclosión con la adición de 50 M de LA en el Día 3 (37.9 ± 18.3 vs. 9.02 ± 12.0, p = 0.014), cuando se comparó con el grupo control. En conclusión, la adición de ácido linoleico mejora la supervivencia a la congelación de los embriones bovinos producidos mediante técnicas in vitrFil: Bernal Ballesteros, Beatriz H.. Instituto de Reproducción Animal Córdoba; Argentina. Vitrogen Colombia S.A.S; ColombiaFil: Tribulo, Humberto Elias. Instituto de Reproducción Animal Córdoba; ArgentinaFil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Bo, Gabriel Amilcar. Instituto de Reproducción Animal Córdoba; Argentina. Universidad Nacional de Villa María; Argentin

Draxin acts as a molecular rheostat of canonical Wnt signaling to control cranial neural crest EMT

Neural crest cells undergo a spatiotemporally regulated epithelial-to-mesenchymal transition (EMT) that proceeds head to tailward to exit from the neural tube. In this study, we show that the secreted molecule Draxin is expressed in a transient rostrocaudal wave that mirrors this emigration pattern, initiating after neural crest specification and being down-regulated just before delamination. Functional experiments reveal that Draxin regulates the timing of cranial neural crest EMT by transiently inhibiting canonical Wnt signaling. Ectopic maintenance of Draxin in the cranial neural tube blocks full EMT; while cells delaminate, they fail to become mesenchymal and migratory. Loss of Draxin results in premature delamination but also in failure to mesenchymalize. These results suggest that a pulse of intermediate Wnt signaling triggers EMT and is necessary for its completion. Taken together, these data show that transient secreted Draxin mediates proper levels of canonical Wnt signaling required to regulate the precise timing of initiation and completion of cranial neural crest EMT

Dose-dependent effects of rumen-protected choline on hepatic metabolism during induction of fatty liver in dry pregnant dairy cows

Objectives were to determine the effects of supplementing increasing amounts of choline ion on hepatic composition and mRNA abundance in pregnant dry cows subjected to a fatty liver induction protocol. Holstein cows (35 primiparous and 41 multiparous) at mean (± standard deviation) of 211 ± 9.9 days of gestation were blocked by body condition (3.59 ± 0.33) andassigned to receive 0, 6.45, 12.90, 19.35, and 25.80 g/day of choline ion as rumen-protected choline (RPC) as a top-dress for 14 days. Cows were fed for ad libitum intake on days 1 to 5andrestricted to 30% of the required net energy for lactation from days 6 to 14 of the experiment. Hepatic tissue was sampled on days 5 and 14 and analyzed for concentrations of triacylglycerol and glycogen, and mRNA abundance was investigated. Orthogonal contrasts evaluated the effects of supplementing RPC (0 g/day vs. rest), and the linear, quadratic, and cubic effects of increasing intake of choline ion from 6.45 to 25.80 g/day. Results are depicted in sequence of treatments from 0 to 25.8. During feed restriction, RPC reduced the concentration of hepatic triacylglycerol by 28.5% and increased that of glycogen by 26.1%, and the effect of increasing RPC intake on triacylglycerol was linear (6.67 vs. 5.45 vs. 4.68 vs. 5.13 vs. 3.81 ± 0.92% wet-basis). Feeding RPC during feed restriction increased abundance of transcripts involved in choline metabolism (CHKA, PLD1), synthesis of apolipoprotein-B100 (APOB100), and antioxidant activity (GPX3), and decreased the abundance of transcripts involved in hepatic lipogenesis (DGAT2, SREBF1) and acute phase response (SAA3). Most effects were linear with amount of choline fed. Changes in hepatic mRNA abundance followed a pattern of reduced lipogenesis and enhanced lipids export, which help explain the reduced hepatic triacylglycerol content in cows fed RPC. Choline exerts lipotropic effects in dairy cows by altering transcript pathways linked to hepatic lipids metabolism.Fil: Arshad, Usman. University of Florida; Estados UnidosFil: Zenobi, Marcos G.. Universidad Nacional de Córdoba; ArgentinaFil: Tribulo, Paula. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Staples, Charles R.. University of Florida; Estados UnidosFil: Santos, José E. P.. University of Florida; Estados Unido

SLUG transcription factor : a pro-survival and prognostic factor in gastrointestinal stromal tumour

Background: The SLUG transcription factor has been linked with the KIT signalling pathway that is important for gastrointestinal stromal tumour (GIST) tumourigenesis. Its clinical significance in GIST is unknown. Methods: Influence of SLUG expression on cell proliferation and viability were investigated in GIST48 and GIST882 cell lines. The association between tumour SLUG expression in immunohistochemistry and recurrence-free survival (RFS) was studied in two clinical GIST series, one with 187 patients treated with surgery alone, and another one with 313 patients treated with surgery and adjuvant imatinib. Results: SLUG downregulation inhibited cell proliferation, induced cell death in both cell lines, and sensitised GIST882 cells to lower imatinib concentrations. SLUG was expressed in 125 (25.0%) of the 500 clinical GISTs evaluated, and expression was associated with several factors linked with unfavourable prognosis. SLUG expression was associated with unfavourable RFS both when patients were treated with surgery alone (HR = 3.40, 95% CI = 1.67-6.89, P = 0.001) and when treated with surgery plus adjuvant imatinib (HR = 1.83, 95% CI = 1.29-2.60, P = 0.001). Conclusions: GIST patients with high tumour SLUG expression have unfavourable RFS. SLUG may mediate pro-survival signalling in GISTs.Peer reviewe

Consequences of endogenous and exogenous WNT signaling for development of the preimplantation bovine embryo

The specific role of WNT signaling during preimplantation development remains unclear. Here, we evaluated consequences of activation and inhibition of β-catenin (CTNNB1)-dependent and -independent WNT signaling in the bovine preimplantation embryo. Activation of CTNNB1- mediated WNT signaling by the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3- methoxyphenyl)pyrimidine (AMBMP) and a glycogen synthase kinase 3 inhibitor reduced development to the blastocyst stage. Moreover, the antagonist of WNT signaling, dickkopf-related protein 1 (DKK1), alleviated the negative effect of AMBMP on development via reduction of CTNNB1. Based on labeling for phospho c-Jun N-terminal kinase, there was no evidence that DKK1 activated the planar cell polarity (PCP) pathway. Inhibition of secretion of endogenous WNTs did not affect development but increased number of cells in the inner cell mass (ICM). In contrast, DKK1 did not affect number of ICM or trophectoderm (TE) cells, suggesting that embryo-derived WNTs regulate ICM proliferation through a mechanism independent of CTNNB1. In addition, DKK1 did not affect the number of cells positive for the transcription factor yes-associated protein 1 (YAP1) involved in TE formation. In fact, DKK1 decreased YAP1. In contrast, exposure of embryos to WNT family member 7A (WNT7A) improved blastocyst development, inhibited the PCP pathway, and did not affect amounts of CTNNB1. Results indicate that embryo-derived WNTs are dispensable for blastocyst formation but participate in regulation of ICM proliferation, likely through a mechanism independent of CTNNB1. The response to AMBMP and WNT7A leads to the hypothesis that maternally derived WNTs can play a positive or negative role in regulation of preimplantation development.Supplemental File S1. Information on antibodies used.The L.E. "Red" Larson Endowment, Agriculture and Food Research Initiative Competitive Grant no. 2011-67015-30688 from the United States Dept. of Agriculture National Institute of Food and Agriculture, and National Institutes of Health Grant R03 HD080855.http://www.biolreprod.orgam2017Animal and Wildlife Science

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The Bone Morphogenetic Protein (BMP) family reiteratively signals to direct disparate cellular fates throughout embryogenesis. In the developing dorsal spinal cord, multiple BMPs are required to specify sensory interneurons (INs). Previous studies suggested that the BMPs act as concentration-dependent morphogens to direct IN identity, analogous to the manner in which sonic hedgehog patterns the ventral spinal cord. However, it remains unresolved how multiple BMPs would cooperate to establish a unified morphogen gradient. Our studies support an alternative model: BMPs have signal-specific activities directing particular IN fates. Using chicken and mouse models, we show that the identity, not concentration, of the BMP ligand directs distinct dorsal identities. Individual BMPs promote progenitor patterning or neuronal differentiation by their activation of different type I BMP receptors and distinct modulations of the cell cycle. Together, this study shows that a 'mix and match' code of BMP signaling results in distinct classes of sensory INs

Analyses of zebrafish and Xenopus oocyte maturation reveal conserved and diverged features of translational regulation of maternal cyclin B1 mRNA

<p>Abstract</p> <p>Background</p> <p>Vertebrate development relies on the regulated translation of stored maternal mRNAs, but how these regulatory mechanisms may have evolved to control translational efficiency of individual mRNAs is poorly understood. We compared the translational regulation and polyadenylation of the cyclin B1 mRNA during zebrafish and <it>Xenopus </it>oocyte maturation. Polyadenylation and translational activation of cyclin B1 mRNA is well characterized during <it>Xenopus </it>oocyte maturation. Specifically, <it>Xenopus </it>cyclin B1 mRNA is polyadenylated and translationally activated during oocyte maturation by proteins that recognize the conserved AAUAAA hexanucleotide and U-rich Cytoplasmic Polyadenylation Elements (CPEs) within cyclin B1 mRNA's 3'<b>U</b>n<b>T</b>ranslated <b>R</b>egion (3'<b>UTR</b>).</p> <p>Results</p> <p>The zebrafish cyclin B1 mRNA was polyadenylated during zebrafish oocyte maturation. Furthermore, the zebrafish cyclin B1 mRNA's 3'UTR was sufficient to stimulate translation of a reporter mRNA during zebrafish oocyte maturation. This stimulation required both AAUAAA and U-rich CPE-like sequences. However, in contrast to AAUAAA, the positions and sequences of the functionally defined CPEs were poorly conserved between <it>Xenopus </it>and zebrafish cyclin B1 mRNA 3'UTRs. To determine whether these differences were relevant to translation efficiency, we analyzed the translational activity of reporter mRNAs containing either the zebrafish or <it>Xenopus </it>cyclin B1 mRNA 3'UTRs during both zebrafish and <it>Xenopus </it>oocyte maturation. The zebrafish cyclin B1 3'UTR was quantitatively less effective at stimulating polyadenylation and translation compared to the <it>Xenopus </it>cyclin B1 3'UTR during both zebrafish and <it>Xenopus </it>oocyte maturation.</p> <p>Conclusion</p> <p>Although the factors that regulate translation of maternal mRNAs are highly conserved, the target sequences and overall sequence architecture within the 3'UTR of the cyclin B1 mRNA have diverged to affect translational efficiency, perhaps to optimize levels of cyclin B1 protein required by these different species during their earliest embryonic cell divisions.</p

Threshold-Dependent BMP-Mediated Repression: A Model for a Conserved Mechanism That Patterns the Neuroectoderm

Subdivision of the neuroectoderm into three rows of cells along the dorsal-ventral axis by neural identity genes is a highly conserved developmental process. While neural identity genes are expressed in remarkably similar patterns in vertebrates and invertebrates, previous work suggests that these patterns may be regulated by distinct upstream genetic pathways. Here we ask whether a potential conserved source of positional information provided by the BMP signaling contributes to patterning the neuroectoderm. We have addressed this question in two ways: First, we asked whether BMPs can act as bona fide morphogens to pattern the Drosophila neuroectoderm in a dose-dependent fashion, and second, we examined whether BMPs might act in a similar fashion in patterning the vertebrate neuroectoderm. In this study, we show that graded BMP signaling participates in organizing the neural axis in Drosophila by repressing expression of neural identity genes in a threshold-dependent fashion. We also provide evidence for a similar organizing activity of BMP signaling in chick neural plate explants, which may operate by the same double negative mechanism that acts earlier during neural induction. We propose that BMPs played an ancestral role in patterning the metazoan neuroectoderm by threshold-dependent repression of neural identity genes

An NF-κB and Slug Regulatory Loop Active in Early Vertebrate Mesoderm

BACKGROUND: In both Drosophila and the mouse, the zinc finger transcription factor Snail is required for mesoderm formation; its vertebrate paralog Slug (Snai2) appears to be required for neural crest formation in the chick and the clawed frog Xenopus laevis. Both Slug and Snail act to induce epithelial to mesenchymal transition (EMT) and to suppress apoptosis. METHODOLOGY & PRINCIPLE FINDINGS: Morpholino-based loss of function studies indicate that Slug is required for the normal expression of both mesodermal and neural crest markers in X. laevis. Both phenotypes are rescued by injection of RNA encoding the anti-apoptotic protein Bcl-xL; Bcl-xL's effects are dependent upon IκB kinase-mediated activation of the bipartite transcription factor NF-κB. NF-κB, in turn, directly up-regulates levels of Slug and Snail RNAs. Slug indirectly up-regulates levels of RNAs encoding the NF-κB subunit proteins RelA, Rel2, and Rel3, and directly down-regulates levels of the pro-apopotic Caspase-9 RNA. CONCLUSIONS/SIGNIFICANCE: These studies reveal a Slug/Snail–NF-κB regulatory circuit, analogous to that present in the early Drosophila embryo, active during mesodermal formation in Xenopus. This is a regulatory interaction of significance both in development and in the course of inflammatory and metastatic disease