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Although hormonal induction of parturition in cattle results in the successful delivery of healthy calves, the risk of retained fetal membrane is significantly increased. In a previous study, a combination of the long-acting glucocorticoid, triamcinolone acetonide, with a high dose of betamethasone partially normalized the placentomal gene expression during parturition; however, the incidence of retained fetal membrane remained high. This study further explored placentomal dysfunction and aimed to elucidate the mechanism of retained fetal membrane in parturition-induced cows. In this study, transcriptome analysis revealed that enhanced glucocorticoid exposure normalized the expression of a substantial fraction of genes in the cotyledons. In contrast, a significant reduction in the multiple signaling pathway activities, including interferon signaling, was found in the caruncles during induced parturition. Real-time PCR showed that the expression of interferon-tau in the caruncles, but not interferon-alpha or interferon-gamma, was significantly lower in induced parturition than spontaneous parturition. Interferon-stimulated gene expression was also significantly decreased in the caruncles during induced parturition. These results indicate that interferon signaling could be important for immunological control in placentomes during parturition. Additionally, this suggests that interferon-tau might be a pivotal ligand for interferon receptors in the caruncles. This study revealed that peripheral blood leukocytes in prepartum cows transcribed interferon-tau. Macrophage infiltration in the placentome is known to participate in the detachment of the fetal membrane from the caruncle. Thus, this study raised the possibility that immune cells migrating into the caruncles at parturition may act as a source of ligands that activate interferon signaling.

Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this "on-gel" culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency-related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways-related genes to facilitate the functional maturation required for feto-maternal interaction. Quantitative PCR analysis of TE cells derived from on-gel culture further confirmed time-dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species-specific strategies for mammalian preimplantation development.

Although in vitro maturation (IVM) of oocytes is important for assisted reproduction, the rate of development of embryos from IVM oocytes is lower than from their in vivo counterparts. It has been shown that an artificial increase of intracellular cAMP before culture significantly improves oocyte developmental competence in cattle and mice. Here, we revealed that forskolin and 3-isobutyl-1-methylxanthine treatment of prophase-stage oocytes induced the expression of genes required for glycolysis, fatty acid degradation, and the mitochondrial electron transport system and improved mitochondrial functions and ATP levels in oocytes without involving nuclear maturation. We propose the existence of a comprehensive energy-supply system in oocytes under follicle-stimulating hormone stimulation as a potential explanation of how oocytes acquire developmental competence.

The length and density of rumen papillae starts to increase during weaning and growth of ruminants. This significant development increases the intraruminal surface area and the efficiency of VFA (acetate, propionate, butyrate, etc.) uptake. Thus, it is important to investigate the factors controlling the growth and development of rumen papillae during weaning. This study aimed to compare the transcriptomes of rumen papillae in suckling and weaned calves. Total RNA was extracted from the rumen papillae of 10 male Japanese Black calves (5 suckling calves, 5 wk old; 5 weaned calves, 15 wk old) and used in RNA-sequencing. Transcript abundance was estimated and differentially expressed genes were identified and these data were then used in Ingenuity Pathway Analysis (IPA) to predict the major canonical pathways and upstream regulators. Among the 871 differentially expressed genes screened by IPA, 466 genes were upregulated and 405 were downregulated in the weaned group. Canonical pathway analysis showed that "atherosclerosis" was the most significant pathway, and "tretinoin," a derivative of vitamin A, was predicted as the most active upstream regulator during weaning. Analyses also predicted IgG, lipopolysaccharides, and tumor-necrosis factor-α as regulators of the microbe-epithelium interaction that activates rumen-related immune responses. The functional category and the up-regulators found in this study provide a valuable resource for studying new candidate genes related to the proliferation and development of rumen papillae from suckling to weaning Japanese Black calves.

Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor “EX527”, and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.

Methionine adenosyltransferase (MAT) is involved in folate-mediated one-carbon metabolism, which is essential for preimplantation embryos in terms of both short-term periconceptional development and long-term phenotypic programming beyond the periconceptional period. Here, our immunofluorescence analysis of bovine oocytes and preimplantation embryos revealed the consistent expression of MAT2A (the catalytic subunit of the ubiquitously expressed-type of MAT isozyme) during this period. Addition of the MAT2A inhibitor FIDAS to the culture media of bovine preimplantation embryos reduced their blastocyst development, revealing the particular importance of MAT2A in successful blastocyst development. Exploration of MAT2A-associated genomic regions in bovine blastocysts using chromatin immunoprecipitation and sequencing (ChIP-seq) identified candidate MAT2A-associated genes implicated not only in short-term periconceptional embryo development, but also in long-term phenotypic programming during this period in terms of growth, metabolism, and immune functions. These results suggest the critical involvement of MAT2A in the periconceptional period in life-long programming of health and disease as well as successful preimplantation development.

Background Endometrial cells secrete various cytokines and the dysfunction of endometrial cells may directly lead to infertility. Interferon tau (IFNT) secreted by trophoblast cells, a well-known pregnancy recognition signal in ruminants, acts on the uterus to prepare for pregnancy. Aging causes cellular and organ dysfunction, and advanced maternal age is associated with reduced fertility. However, few studies have investigated age-dependent changes in the uterus. Methods Using next generation sequencing and real-time PCR, we examined mRNA expression in bovine endometrial cells in vitro obtained from young (mean 45.2 months) and aged (mean 173.5 months) animals and the effects of IFNT depending on the age. Results We showed that inflammation-related (predicted molecules are IL1A, C1Qs, DDX58, NFKB, and CCL5) and interferon-signaling (predicted molecules are IRFs, IFITs, STATs, and IFNs) pathways were activated in endometrial cells obtained from aged compared to young cows. Also, the activation of “DNA damage checkpoint regulation” and the inhibition of “mitotic mechanisms” in endometrial cells obtained from aged cows were evident. Moreover, we showed lower cell viability levels in endometrial cells obtained from aged compared to young cows. Although treatment with IFNT upregulated various types of interferon stimulated genes both in endometrial cells obtained from young and aged cows, the rate of increase by IFNT stimulus was obviously lower in endometrial cells obtained from aged compared to young cows. Conclusions Endometrial cells obtained from aged cows exhibited higher levels of inflammatory- and IFN-signaling, and dysfunction of cell division compared with young cows. In addition, a high basal level of IFN-related genes in endometrial cells of aged cows is suggested a concept of “inflammaging”. Electronic supplementary material The online version of this article (doi:10.1186/s12958-017-0284-z) contains supplementary material, which is available to authorized users.

To assess the age-associated changes in oocytes and granulosa cells derived from early antral follicles (EAFs).Gene expression analysis of granulosa cells of the EAFs using a genome analyzer (Illumina) and in vitro culture of oocyte-granulosa cell complexes (OGCs) of EAFs (400-700 μm in diameter) collected from ovaries of aged (120 months) and young (50 months) cows.Gene expression profiles in granulosa cells of EAFs of aged cows, which included changes in genes that encode chaperone proteins and antioxidants. In vivo development of EAFs, as determined by oocyte diameter of EAFs and AFs (3-6 mm in diameter), appeared to be impaired in aged cows and the OGCs of aged cows contained low GSH compared to younger counterparts. When the OGCs were cultured in a medium containing low estradiol (E2, 0.1 μg/mL), the ratio of antrum formation was higher for OGCs from aged animals than that from young animals, while higher abnormal fertilization rate and lower total cell number of the blastocysts were observed in the OGCs of aged cows compared with those of young cows. On the contrary, when the OGCs were cultured in a medium containing 10 μg/mL E2, the ratio of antrum formation and fertilization outcome was comparable between the two age groups, whereas the total cell number of the blastocysts was still low in the aged group.Aging affects the gene expression profiles of the granulosa cells, and impairs in vitro developmental ability of OGCs collected from EAFs.

The native Japanese cattle Mishima-Ushi, a designated national natural treasure, are bred on a remote island, which has resulted in the conservation of their genealogy. We examined the genetic characteristics of 8 Mishima-Ushi individuals by using single nucleotide polymorphisms (SNPs), insertions, and deletions obtained by whole-genome sequencing. Mapping analysis with various criteria showed that predicted heterozygous SNPs were more prevalent than predicted homozygous SNPs in the exonic region, especially non-synonymous SNPs. From the identified 6.54million polymorphisms, we found 400 non-synonymous SNPs in 313 genes specific to each of the 8 Mishima-Ushi individuals. Additionally, 3,170,833 polymorphisms were found between the 8 Mishima-Ushi individuals. Phylogenetic analysis confirmed that the Mishima-Ushi population diverged from another strain of Japanese cattle. This study provides a framework for further genetic studies of Mishima-Ushi and research on the function of SNP-containing genes as well as understanding the genetic relationship between the domestic and native Japanese cattle breeds.

Antrum formation and estradiol (E2) secretion are specific features of oocyte and granulosa cell complexes (OGCs). This study investigates the effect of E2 on the in vitro development of bovine OGCs derived from early antral follicles as well as on the expression of genes in granulosa cells (GCs). The supplementation of culture medium with either E2 or androstenedione (A4) improved the in vitro development of OGCs and the nuclear maturation of enclosed oocytes. When OGCs were cultured in medium containing A4, developmentally competent OGCs secreted more E2 than OGCs that were not competent. In addition, fulvestrant inhibited the effect of both E2 and A4 on OGCs development. Comprehensive gene expression analysis using next-generation sequence technology was conducted for the following three types of GCs: i) GCs of OGCs cultured for 4 days with E2 (1 μg/ml; E2(+)), ii) GCs of OGCs cultured for 4 days without E2 (E2(−)) or iii) OGCs that formed clear antrum after 8 days of in vitro culture in medium containing E2 (1 μg/ml; AF group). GCs of the E2(+) group had a similar gene expression profile to the profile reported previously for the in vivo development of large follicles. This genetic profile included factors implicated in the up-regulation of E2 biosynthesis and down-regulation of cytoskeleton and extracellular matrices. In addition, a novel gene expression profile was found in the AF group. In conclusion, E2 impacts the gene expression profile of GCs to support the in vitro development of OGCs.

Background Because the Japanese native cattle Kuchinoshima-Ushi have been isolated in a small island and their lineage has been intensely protected, it has been assumed to date that numerous and valuable genomic variations are conserved in this cattle breed. Results In this study, we evaluated genetic features of this breed, including single nucleotide polymorphism (SNP) information, by whole-genome sequencing using a Genome Analyzer II. A total of 64.2 Gb of sequence was generated, of which 86% of the obtained reads were successfully mapped to the reference sequence (Btau 4.0) with BWA. On an average, 93% of the genome was covered by the reads and the number of mapped reads corresponded to 15.8-fold coverage across the covered region. From these data, we identified 6.3 million SNPs, of which more than 5.5 million (87%) were found to be new. Out of the SNPs annotated in the bovine sequence assembly, 20,432 were found in protein-coding regions containing 11,713 nonsynonymous SNPs in 4,643 genes. Furthermore, phylogenetic analysis using sequence data from 10 genes (more than 10 kbp) showed that Kuchinoshima-Ushi is clearly distinct from European domestic breeds of cattle. Conclusions These results provide a framework for further genetic studies in the Kuchinoshima-Ushi population and research on functions of SNP-containing genes, which would aid in understanding the molecular basis underlying phenotypic variation of economically important traits in cattle and in improving intrinsic defects in domestic cattle breeds.