Predicting of molecules mediating an interaction between bovine embryos and uterine epithelial cells.

Embryo-maternal reproductive tract interactions are pivotal for successful pregnancy. The present study predicted the molecules modulating embryo-uterine communication by comparing two sets of differentially expressed genes (DEGs): DEGs in uterine epithelial cells (UECs) collected from the uterus with and without blastocysts and DEGs between blastocysts developed in vivo and in vitro. Cows were subjected to super ovulation (SOV), followed by insemination or non-insemination at estrus (SOV + AI and SOV cows). Seven days after estrus, the uterus was flushed to collect UECs, and the presence of blastocysts in the uterus was confirmed. UECs were subjected to RNA-Sequencing (RNA-Seq) to identify DEGs. Publicly available RNA-Seq data of in vivo and in vitro developed bovine blastocysts were used to determine DEGs. Then, using ingenuity pathway analysis, activated- and inhibited-upstream regulators (USRs) for UECs in blastocysts were compared with those for blastocysts developed in vivo. RNA-Seq of UECs revealed that the DEGs were associated with immune response and cell adhesion pathways. The activated and inhibited USRs of UECs derived from SOV+ AI cows overlapped with the activated and inhibited USRs of blastocysts developed in vivo. Overlapping activated USRs include leukemia inhibitory factor, interleukin 6, fibroblast growth factor-2, transforming growth factor beta-1, and epidermal growth factor. In conclusion, the present study predicted the molecules that potentially mediate communication between the developing embryo and the uterus in vivo and prepare the uterus for pregnancy.

Molecular analysis of bovine leukemia virus in early epidemic phase in Japan using archived formalin fixed paraffin embedded histopathological specimens.

Bovine leukemia virus (BLV) is an important pathogen associated with enzootic bovine leukosis. In this study, we performed PCR and sequencing analysis to characterize BLVgp51 sequences from formalin-fixed paraffin-embedded (FFPE) specimens made from 1974 to 2000 and successfully obtained BLV proviral genome sequences from 94% of the analyzed samples. Furthermore, from these samples, we reconstructed eight full-length and nearly full-length BLVgp51 sequences. These sequences were classified as BLV genotype 1, implying that genotype1 has already been circulating in Japan since the 1970s. In our results, the proviral DNA was detected in the 1970s, 1980s, and 1990s in the same manner, indicating that the detection of BLV proviral genome depends on storage conditions rather than storage period. The sequences obtained in this study provide direct insights into BLV sequences before 2000, which serves as a good calibrator for inferring ancient BLV diversity.

Acetoacetate is a trigger of NLRP3 inflammasome activation in bovine peripheral blood mononuclear cells.

Repeat breeding, which is non-pregnancy following three or more breeding attempts, is a serious reproductive disorder in cattle. In the present study, metabolomic profiling was used to identify metabolites in the blood plasma of repeat breeder cows (RBCs) and non-RBCs. Metabolomic analysis showed that acetoacetate (AcAc), a ketone body, was detected in RBCs, but not in non-RBCs. In contrast, ß-hydroxybutyrate (BHB) was at similar levels in both RBCs and non-RBCs. We hypothesized that an imbalance of AcAc and BHB induces abnormal inflammatory conditions, especially the NLRP3 inflammasome, which regulates sterile inflammation to control interleukin (IL)-1ß secretion, and may be associated with repeat breeding in cattle. To investigate this hypothesis, blood samples were collected from both non-RBCs and RBCs on day 7 of the estrous cycle. The mRNA expression of IL1B in peripheral blood mononuclear cells (PBMCs) was observed to be higher in RBCs than in non-RBCs. To test the effects of AcAc and BHB on inflammatory responses, blood samples were collected from healthy cows and PBMCs were isolated. PBMCs were treated with AcAc and BHB to investigate the activation of the NLRP3 inflammasome (complex of NLRP3, ASC, and caspase-1) and IL-1ß secretion. AcAc treatment resulted in higher protein and/or mRNA expression of NLRP3 and IL-1ß in PBMCs. Moreover, AcAc increased the co-localization of NLRP3 and ASC and stimulated caspase-1 activation, indicating the formation of the platform of the NLRP3 inflammasome. Addition of specific NLRP3 inhibitor, MCC950, suppressed AcAc stimulation-induced IL-1ß secretion. Contrary to the effects of AcAc, BHB treatment suppressed the activation of NLRP3 inflammasome and IL-1ß secretion in response to AcAc and typical NLRP3 inflammasome triggers. These findings demonstrate that AcAc can potentially trigger NLRP3 inflammasome activation, resulting in IL-1ß secretion, and that these inflammatory responses are suppressed by BHB in bovine PBMCs. In addition, the imbalance between AcAc and BHB with higher levels of IL-1ß may be associated with repeat breeding in cattle.

Xanthan gum and locust bean gum substrate improves bovine embryo development.

One of the major difference between the in vivo and in vitro embryonic environments is the stiffness of the culture substrate. Xanthan gum (XG) and locust bean gum (LBG) are natural materials that are safe, inexpensive and easy to handle. In this study, we investigated the effects of using a polysaccharide culture substrate made from 1% XG and 1% LBG (XG-LBG gel) on bovine embryonic development. Oocytes collected from bovine ovaries were subjected to maturation, and fertilization to generate embryos at an early developmental stage (>4 cell stage). Cleaved embryos were further cultured in a well of 96-well cell culture plate coated with or without XG-LBG gel for 5 days. While the developmental rate up to the blastocyst stage did not differ between the two culture systems (control, 38.0 vs. gel, 38.6%), blastocysts developed on the XG-LBG gel produced significantly high cell numbers and ATP content. Embryos cultured on XG-LBG gels for 24 hr had high expression levels of F-actin and a highly even distribution of E-cadherin. In addition, embryos developed on XG-LBG gel demonstrated increased translocation of YAP to the nucleus and increased connective tissue growth factor (CTGF) protein levels (downstream of Hippo signalling). These findings suggest that soft culture substrates improve embryonic development by enhancing mechanotransduction, including YAP-CTGF signalling.

Comparison of gene expression and mitochondria number between bovine blastocysts obtained in vitro and in vivo.

Embryo transfer uses embryos developed in vivo or in vitro for cattle production, however there is a difference in the quality of the embryos obtained by the two methods. This study addresses the differences in gene expression between blastocysts developed in vitro and in vivo. In vivo blastocysts were flushed from the uteri of super-ovulated cows and blastocysts developed in vitro were derived from in vitro matured and fertilized embryos. The same batch of frozen bull sperm was used for insemination and in vitro fertilization. Blastocysts were then subjected to RNA sequencing. Differentially expressed genes upregulated in in vitro blastocysts were annotated to focal adhesion, extracellular matrix (ECM)-receptor interaction, and PI3K-Akt signaling and the genes that were upregulated in in vivo blastocysts were annotated to oxidation-reduction processes, mitochondrion organization, and mitochondrial translation. Although the total cell number of the two types of blastocysts was similar, the mitochondrial quantity (determined by mitochondrial DNA copy numbers and expression levels of TOMM20), and ATP content in the blastocysts were lower in in vivo blastocysts compared with those developed in vitro. In conclusion, RNAseq revealed differential molecular backgrounds between in vitro and in vivo developed blastocysts and mitochondrial number and function are responsible for these differences.

The transfer of parthenogenetic embryos following artificial insemination in cows can enhance pregnancy recognition via the secretion of interferon tau.

Repeat breeding is a reproductive disorder in cattle. Embryo transfer following artificial insemination (AI) improves pregnancy rate by replenishing interferon tau (IFNT), but it results in a notably higher rate of twin occurrence. This study hypothesized that parthenogenetic (PA) embryo transfer following AI (AI + PA) could improve the conception rate because that PA embryo become as a supplemental source of IFNT without twins. PA embryos showed higher IFNT mRNA expression than in vitro fertilization (IVF) embryos. An examination of the effect of the cultured conditioned media (CM) of PA or IVF embryos on Madin-Darby bovine kidney cells with stably introduced promoter-reporter constructs of interferon-stimulated gene 15 (ISG15, marker of IFN response) showed higher stimulation levels of ISG15 promoter activity with PA than with IVF embryo. We investigated in vivo the effect of AI + PA on healthy Japanese Black cattle. Cattle transferred with PA embryo alone were non-fertile, but those that underwent AI + PA showed a pregnancy rate of 53.3%, the similar as that with AI alone (60%). In pregnant cattle in AI + PA group, adding the PA embryo upregulated the expression of ISGs and plasma progesterone concentration. No twin were generated in AI only and AI + PA groups. Using repeat breeding Holstein cows that did not become pregnant with 4-9 times of AI, transfer of PA embryo following AI resulted in a higher pregnancy rate than that of control (AI only). We suggest that AI + PA may be beneficial for improving maternal pregnancy recognition in repeat breeder cattle while avoiding twin generation.

Improvement of fertility in repeat breeder dairy cattle by embryo transfer following artificial insemination: possibility of interferon tau replenishment effect.

Repeat breeder cattle do not become pregnant until after three or more breeding attempts; this represents a critical reproductive disorder. Embryo transfer (ET) following artificial insemination (AI) in repeat breeder cattle reportedly improves pregnancy rate, leading to speculation that interferon tau (IFNT) is associated with this phenomenon. However, the reason why the conception rate improves remains unknown. We investigated the effect of ET following AI on repeat breeder cattle in field tests, and determined whether adding an embryo affects the maternal immune cells detected by interferon-stimulated genes (ISGs), marker genes of IFN response. In total, 1122 repeat breeder cattle were implanted with in vitro fertilization (IVF) embryos after previous AI. ET following AI resulted in pregnancy rates of 46.9% in repeat breeder dairy cattle. In basic in vivo tests, to investigate the effect of adding embryos, ISGs mRNA expression levels were significantly higher in the AI + ET group than in the AI + sham group (transfer of only embryonic cryopreservation solution). Then, we examined the effect of cultured conditioned media (CM) of IVF embryos on splenic immune cells and Madin-Darby bovine kidney (MDBK) cells with stably introduced ISG15 promoter-reporter constructs. These cells exhibited a specific increase in ISG15 mRNA expression and promoter activity when treated with the CM of IVF embryos, suggesting that IVF embryos have the potential to produce and release IFNT. In conclusion, ET following AI is beneficial for improving conception in repeat breeder cattle. Added embryos may produce and secrete IFNT, resulting in the increased expression of ISGs.

Modification of mitochondrial function, cytoplasmic lipid content and cryosensitivity of bovine embryos by resveratrol.

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.

Abundant sequence divergence in the native Japanese cattle Mishima-Ushi (Bos taurus) detected using whole-genome sequencing.

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 6.54 million 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.