Interleukin-6 supplementation improves bovine conceptus elongation and transcriptomic indicators of developmental competence†.

A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine interleukin-6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n = 5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from interleukin-6-treated embryos. The interleukin-6 treatment generated 591 differentially expressed genes in conceptuses (n = 9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few differentially expressed genes were associated with extraembryonic development, but several differentially expressed genes were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that interleukin-6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy.

Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells.

BACKGROUND: Cytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence. METHODS: Here, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes. RESULTS: The BCB positive oocytes were twice as likely to produce a blastocyst in vitro compared to BCB- oocytes (P < 0.01). We determined that BCB negative oocytes have 1.3-fold more mtDNA copies than BCB positive oocytes (P = 0.004). There was no differential transcript abundance of genes expressed in oocytes, however, 172 genes were identified in cumulus cells with differential transcript abundance (FDR < 0.05) based on the BCB staining of their oocyte. Co-expression analysis between oocytes and their surrounding cumulus cells revealed a subset of genes whose co-expression in BCB positive oocytes (n = 75) and their surrounding cumulus cells (n = 108) compose a unique profile of the cumulus-oocyte complex. CONCLUSIONS: If oocytes transition from BCB negative to BCB positive, there is a greater likelihood of producing a blastocyst, and a reduction of mtDNA copies, but there is no systematic variation of transcript abundance. Cumulus cells present changes in transcript abundance, which reflects in a dynamic co-expression between the oocyte and cumulus cells.

Interleukin-6 promotes primitive endoderm development in bovine blastocysts.

BACKGROUND: Interleukin-6 (IL6) was recently identified as an embryotrophic factor in bovine embryos, where it acts primarily to mediate inner cell mass (ICM) size. This work explored whether IL6 affects epiblast (EPI) and primitive endoderm (PE) development, the two embryonic lineages generated from the ICM after its formation. Nuclear markers for EPI (NANOG) and PE (GATA6) were used to differentiate the two cell types. RESULTS: Increases (P < 0.05) in total ICM cell numbers and PE cell numbers were detected in bovine blastocysts at day 8 and 9 post-fertilization after exposure to 100 ng/ml recombinant bovine IL6. Also, IL6 increased (P < 0.05) the number of undifferentiated ICM cells (cells containing both PE and EPI markers). The effects of IL6 on EPI cell numbers were inconsistent. Studies were also completed to explore the importance of Janus kinase 2 (JAK2)-dependent signaling in bovine PE cells. Definitive activation of STAT3, a downstream target for JAK2, was observed in PE cells. Also, pharmacological inhibition of JAK2 decreased (P < 0.05) PE cell numbers. CONCLUSIONS: To conclude, IL6 manipulates ICM development after EPI/PE cell fates are established. The PE cells are the target for IL6, where a JAK-dependent signal is used to regulate PE numbers.

Physical parameters of bovine activated oocytes and zygotes as predictors of development success.

The worldwide production of in vitro-produced embryos in livestock species continues to grow. The current gold standard for selecting quality oocytes and embryos is morphologic assessment, yet this method is subjective and varies based on experience. There is a need for a non-invasive, objective method of selecting viable oocytes and embryos. The aim of this study was to determine if ooplasm area, diameter including zona pellucida (ZP), and ZP thickness of artificially activated oocytes and in vitro fertilized (IVF) zygotes are indicative of development success in vitro and correlated with embryo quality, as assessed by total blastomere number. Diameter affected the probability of development to the blastocyst stage in activated oocytes on day 7 (P < 0.01) and day 8 (P < 0.001), and had a tendency to affect IVF zygotes on day 8 (P = 0.08). Zona pellucida thickness affected the probability of development on day 7 (P < 0.01) and day 8 (P < 0.001) in activated oocytes, and day 8 for IVF zygotes (P < 0.05). An interaction between ZP thickness and diameter was observed on days 7 and 8 (P < 0.05) in IVF zygotes. Area did not significantly affect the probability of development, but was positively correlated with blastomere number on day 8 for IVF zygotes (P = 0.01, conditional R2 = 0.09). Physical parameters of bovine zygotes have the potential for use as a non-invasive, objective selection method. Upon further development, methods used in this study could be integrated into embryo production systems to improve IVF success.

Interleukin-6 increases inner cell mass numbers in bovine embryos.

BACKGROUND: Work in other species suggests that interleukin-6 (IL6) promotes early embryo development. It was unclear whether IL6 serves as an embryokine in cultured bovine embryos. This work was undertaken to elucidate the role of IL6 during in vitro bovine embryo production. RESULTS: Transcripts for IL6 and its two cognate receptor subunits (IL6R, IL6ST) were confirmed in bovine embryos from the 1-cell to blastocyst stages. Supplementing 100 ng/ml recombinant bovine IL6 to in vitro-produced bovine embryos at day 1, 3 or 5 increased (P < 0.05) inner cell mass (ICM) cell number and the ICM:trophectoderm (TE) ratio but not TE cell number. No increase in ICM or TE cell number was observed after supplementation of 1 or 10 ng/ml IL6 beginning at either day 1 or 5. Sequential supplementation with 100 ng/ml IL6 at both day 1 and 5 (for a total of 200 ng/ml IL6) increased (P < 0.05) ICM cell number to a greater extent than supplementing IL6 at a single time period in one study but not a second study. Additionally, providing 200 ng/ml IL6 beginning at day 1 or 5 yielded no further increase on ICM cell numbers when compared to supplementing with 100 ng/ml IL6. IL6 treatment had no effect on cleavage or blastocyst formation in group culture. However, IL6 supplementation increased cleavage and day 8 blastocyst formation when bovine embryos were cultured individually. CONCLUSIONS: These results implicate IL6 as an embryokine that specifically increases ICM cell numbers in bovine embryos and facilitates bovine blastocyst development in embryos cultured individually.

Interleukin-6 requires JAK to stimulate inner cell mass expansion in bovine embryos.

Supplementing interleukin-6 (IL6) to in vitro-produced bovine embryos increases inner cell mass (ICM) cell numbers in blastocysts. A series of studies were completed to further dissect this effect. Treatment with IL6 increased ICM cell numbers in early, regular and expanded blastocysts but had no effect on morulae total cell number. Treatment with IL6 for 30 min induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation in all blastomeres in early morulae and specifically within the ICM in blastocysts. Also, IL6 supplementation increased SOCS3 mRNA abundance, a STAT3-responsive gene, in blastocysts. Chemical inhibition of Janus kinase (JAK) activity from day 5 to day 8 prevented STAT3 activation and the IL6-induced ICM cell number increase. Global transcriptome analysis of blastocysts found that transcripts for IL6 and its receptor subunits (IL6R and IL6ST) were the most abundantly expressed IL6 family ligand and receptors. These results indicate that IL6 increases ICM cell numbers as the ICM lineage emerges at the early blastocyst stage through a STAT3-dependent mechanism. Also, IL6 appears to be the primary IL6 cytokine family member utilized by bovine blastocysts to control ICM cell numbers.

Symposium review: Predicting pregnancy loss in dairy cattle.

Several tools exist to diagnose pregnancy in dairy cattle. However, substantial pregnancy loss occurs within the first 60 d of gestation in cattle, and these losses have a profound adverse economic impact on the dairy and beef cattle industries. Detecting these impending pregnancy losses could offer producers an opportunity to reduce costs associated with this source of reproductive inefficiency. Several of the pregnancy diagnostic tools currently available and new technologies are being examined for their ability to predict pregnancies at risk for failing in early pregnancy. This review provides a synopsis of work undertaken recently to predict pregnancy losses in cattle. Currently, opportunities to predict pregnancy loss include (1) using transrectal ultrasonography to detect loss of the fetal heartbeat, floating debris within the placental fluids, and reductions in fetal size; (2) observing reductions in circulating progesterone concentrations; (3) detecting reductions in concentrations of circulating placental products; namely, pregnancy-associated glycoproteins and microRNAs; and (4) detecting reductions in the early pregnancy-dependent increase in interferon-stimulatory gene expression in peripheral blood leukocytes. An achievable goal may be to identify markers of embryo mortality so that researchers and clinicians can focus their efforts on developing intervention strategies for cows identified to be at risk for pregnancy failure.

Exposure to maternal obesity alters gene expression in the preimplantation ovine conceptus.

BACKGROUND: Embryonic and fetal exposure to maternal obesity causes several maladaptive morphological and epigenetic changes in exposed offspring. The timing of these events is unclear, but changes can be observed even after a short exposure to maternal obesity around the time of conception. The hypothesis of this work is that maternal obesity influences the ovine preimplantation conceptus early in pregnancy, and this exposure will affect gene expression in embryonic and extraembryonic tissues. RESULTS: Obese and lean ewe groups were established by overfeeding or normal feeding, respectively. Ewes were then bred to genetically similar rams. Conceptuses were collected at day 14 of gestation. Morphological assessments were made, conceptuses were sexed by genomic PCR analysis, and samples underwent RNA-sequencing analysis. While no obvious morphological differences existed between conceptuses, differentially expressed genes (≥ 2-fold; ≥ 0.2 RPKM; ≤ 0.05 FDR) were detected based on maternal obesity exposure (n = 21). Also, differential effects of maternal obesity were noted on each conceptus sex (n = 347). A large portion of differentially expressed genes were associated with embryogenesis and placental development. CONCLUSIONS: Findings reveal that the preimplantation ovine conceptus genome responds to maternal obesity in a sex-dependent manner. The sexual dimorphism in response to the maternal environment coupled with changes in placental gene expression may explain aberrations in phenotype observed in offspring derived from obese females.

Reduced skeletal muscle fiber size following caloric restriction is associated with calpain-mediated proteolysis and attenuation of IGF-1 signaling.

Caloric restriction decreases skeletal muscle mass in mammals, principally due to a reduction in fiber size. The effect of suboptimal nutrient intake on skeletal muscle metabolic properties in neonatal calves was examined. The longissimus muscle (LM) was collected after a control (CON) or caloric restricted (CR) diet was cosnumed for 8 wk and muscle fiber size, gene expression, and metabolic signal transduction activity were measured. Results revealed that CR animals had smaller (P < 0.05) LM fiber cross-sectional area than CON, as expected. Western blot analysis detected equivalent amounts of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) but reduced (P < 0.05) amounts of the splice-variant, PGC1α-4 in CR LM. Expression of IGF-1, a PGC1α-4 target gene, was 40% less (P < 0.05) in CR than CON. Downstream mediators of autocrine IGF-1 signaling also are attenuated in CR by comparison with CON. The amount of phosphorylated AKT1 was less (P < 0.05) in CR than CON. The ratio of p4EBP1T37/46 to total 4EBP1, a downstream mediator of AKT1, did not differ between CON and CR. By contrast, protein lysates from CR LM contained less (P < 0.05) total glycogen synthase kinase-3ß (GSK3ß) and phosphorylated GSK3ß than CON LM, suggesting blunted protein synthesis. Smaller CR LM fiber size associates with increased (P < 0.05) calpain 1 (CAPN1) activity coupled with lower (P < 0.05) expression of calpastatin, the endogenous inhibitor of CAPN1. Atrogin-1 and MuRF expression and autophagy components were unaffected by CR. Thus CR suppresses the hypertrophic PGC1α-4/IGF-1/AKT1 pathway while promoting activation of the calpain system.

The evolution of interferon-tau.

Thirty years ago, a novel type I interferon (IFN) was identified by molecular cloning of cDNA libraries constructed from RNA extracted from ovine and bovine pre-implantation embryos. This protein was eventually designated as IFN-tau (IFNT) to highlight its trophoblast-dependent expression. IFNT function is not immune related. Instead, it interacts with the maternal system to initiate the establishment and maintenance of pregnancy. This activity is indispensable for the continuation of pregnancy. Our review will describe how IFNT evolved from other type I IFNs to function in this new capacity. IFNT genes have only been identified in pecoran ruminants within the Artiodactyla order (e.g. cattle, sheep, goats, deer, antelope, giraffe). The ancestral IFNT gene emerged approximately 36 million years ago most likely from rearrangement and/or insertion events that combined an ancestral IFN-omega (IFNW) gene with a trophoblast-specifying promoter/enhancer. Since then, IFNT genes have duplicated, likely through conversion events, and mutations have allowed them to adapt to their new function in concert with the emergence of different species. Multiple IFNT polymorphisms have been identified in cattle, sheep and goats. These genes and gene alleles encode proteins that do not display identical antiviral, antiproliferative and antiluteolytic activities. The need for multiple IFNT genes, numerous alleles and distinct activities remains debatable, but the consensus is that this complexity in IFNT expression and biological activity must be needed to provide the best opportunity for pregnancy to be recognized by the maternal system so that gestation may continue.