Low proviral load in the Kumamoto strain of Japanese Brown cattle infected with the bovine leukemia virus.

BACKGROUND: The Kumamoto strain of Japanese Brown (JBRK) cattle is a sub-breed of Wagyu and has a different genetic background than that of Japanese Black (JB) cattle. Bovine leukemia virus (BLV) is the pathogen causing enzootic bovine leukosis (EBL), the predominant type of bovine leukosis (BL). EBL is one of the most common bovine infectious diseases in dairy countries, including Japan. Some host genetic factors, including the bovine leukocyte antigen (BoLA)-DRB3 gene, have been associated with the proviral load (PVL) of BLV and/or onset of EBL. Here, we determined the number of BL cases by analyzing prefectural case records in detail. We measured the PVL of BLV-infected JBRK cattle and compared it with that obtained for other major breeds, JB and Holstein-Friesian (HF) cattle. Finally, the relationship between PVL levels and BoLA-DRB3 haplotypes was investigated in BLV-infected JBRK cattle. RESULTS: We determined the number of BL cases recorded over the past ten years in Kumamoto Prefecture by cattle breed. A limited number of BL cases was observed in JBRK cattle. The proportion of BL cases in the JBRK was lower than that in JB and HF. The PVL was significantly lower in BLV-infected JBRK cattle than that in the JB and HF breeds. Finally, in BLV-infected JBRK cattle, the PVL was not significantly affected by BoLA-DRB3 alleles and haplotypes. BoLA-DRB3 allelic frequency did not differ between BLV-infected JBRK cattle with low PVL and high PVL. CONCLUSIONS: To our knowledge, this is the first report showing that BL occurred less in the JBRK population of Kumamoto Prefecture. After BLV-infection, the PVL was significantly lower in JBRK cattle than that in JB and HF breeds. The genetic factors implicated in maintaining a low PVL have yet to be elucidated, but the BoLA-DRB3 haplotypes are likely not involved.

Expression of NFIL3 and CEBPA regulated by IFNT induced-PGE2 in bovine endometrial stromal cells during the pre-implantation period.

Prostaglandin E2 (PGE2) is considered as a luteoprotective factor, influencing the corpus luteum during the early pregnant period in the bovine species. Cyclic AMP (cAMP) is activated in response to PGE2 and plays a role in many physiological processes. The maternal recognition signal, interferon τ (IFNT), induces PGE2 secretion from the endometrial epithelial cells, the function of which in stroma cells has not been completely understood. In this study, PGE2 was found to activate cAMP in the bovine endometrial stromal cells (STRs). STRs were then treated with forskolin to activate the cAMP signaling, from which RNA extracted was subjected to global expression analysis. Transcripts related to transcription regulatory region nucleic acid binding of molecular function, nucleus of cellular component, and mitotic spindle organization of biological processes were up-regulated in cAMP-activated bovine STRs. An increase in the transcription factors, NFIL3, CEBPA, and HIF1A via the cAMP/PKA/CREB signaling pathway in the bovine STRs was also found by qPCR. Knockdown of NFIL3, CEBPA, or HIF1A blocked forskolin-induced PTGS1/2 and IGFBP1/3 expression. Moreover, NFIL3 and CEBPA were localized in endometrial stroma on pregnant day 17 (day 0 = estrous cycle), but not on cyclic day 17. These observations indicated that uterine PGE2 induced by conceptus IFNT is involved in the early pregnancy-related gene expression in endometrial stromal cells, which could facilitate pregnancy establishment in the bovine.

Epithelial-mesenchymal transition and bi- and multi-nucleated trophoblast cell formation in ovine conceptuses during the peri-implantation period.

Epithelial-mesenchymal transition (EMT), which is common in cancer metastasis, is also observed during developmental processes such as embryo implantation into the maternal endometrium in humans and rodents. However, this process has not been well characterized in the non-invasive type of implantation that occurs in ruminants. To understand whether EMT occurs in ruminant ungulates, ovine conceptuses (embryo plus extraembryonic membranes) from days 15 (P15: pre-attachment), 17 (P17: during attachment), and 21 (P21: post-attachment, day 0 = day of estrus) were evaluated. RNA-seq analysis revealed that the expression of EMT-related transcripts increased on P21. Real-time PCR and western blotting analyses indicated that levels of transcripts and proteins indicative of mesenchyme-related molecules increased on P21, but a minor expression of epithelium-related molecules remained. Immunohistochemical analysis revealed that E-cadherin (CDH1) was localized in the elongated trophectoderm on P15 and P17. On P21, CDH1 was localized to the trophectoderm and on the conceptus cells undergoing differentiation. Vimentin (VIM) was localized in the uterine stroma on P15 and P17, and its expression was observed at the edge of elongating trophoblast on P21. Further, it was found that some bi-nucleated trophoblast cells were present on P17; however, numerous bi- and multi-nucleated trophoblast cells on the uterine epithelium or next to the uterine stroma were found on P21. A minor expression of pregnancy-associated glycoprotein (PAG) transcripts was found on P15 and P17, but a definitive expression of PAGs, transcripts, and proteins was found on P21. Although further investigation is required, these observations indicate that bi-nucleated trophoblast cell formation begins on the day conceptus implantation to the maternal endometrium is initiated, followed by EMT in trophoblast cells. These results suggest that these sequential events are required if pregnancy is to be established in ruminants.

Characterization of lncRNA functioning in ovine conceptuses and endometria during the peri-implantation period.

In ruminants, RNA-sequence analyses have revealed many characteristics of transcripts expressed in conceptuses (embryo and extraembryonic membrane) during peri-implantation periods; however, lncRNA profiles are yet characterized. In this study, we aimed to characterize the lncRNA expression profile in conceptuses during peri-implantation periods in sheep. We analyzed the RNA-sequence data of ovine conceptuses and endometria obtained from pregnant animals on days 15, 17, 19 and 21 (day 0 = day of estrus, n = 3 or 4/day). We predicted the protein coding ability of the assembled transcripts to identify the lncRNA candidates. This analysis identified 8808 lncRNAs, 3423 of which were novel lncRNAs. Gene ontology analysis revealed that lncRNA target genes were enriched for biological processes involved in the respiratory electron transport chain (RETC). qPCR analysis demonstrated that the expression levels on transcripts encoding RETC such as mitochondrially encoded cytochrome c oxidase II (MTCO2) and mitochondria DNA copy number in conceptuses were not increased on P21, although western blotting analysis and immunohistochemistry demonstrated that MTCO2 protein in conceptuses was increased on P21. NAD/NADH assay revealed that NADH level in conceptuses was increased on P21. These results indicate that lncRNAs could regulate the RETC through post-transcriptional levels in the conceptuses. Therefore, lncRNA is a potential new regulator in ovine conceptus development during peri-implantation periods.

Formation of fibrin at sights of conceptus adhesion in the ewe.

In ruminants, various molecules are involved in regulating conceptus attachment and adhesion; however, molecules that maintain the conceptus adhesion have not been well characterized. We hypothesized that conceptus must produce a molecule(s), yet uncharacterized or overlooked, which maintain conceptus adhesion to the uterine epithelium. In this study, we aimed to identify new candidate(s) in conceptus secretory proteins responsible for maintaining conceptus adhesion in sheep. We performed RNA-sequence analysis with ovine conceptuses, followed by endometria obtained from pregnant animals on day 15 (P15: pre-attachment), 17 (P17: right after attachment), and 21 (P21: post-attachment; adhesion) and iTRAQ analysis of uterine flushing on P15 and P17. To identify the proteins secreted from conceptuses, we cross-referenced the transcriptome and proteome data. These analyses identified 16 and 26 proteins as conceptus secretory proteins on P15 and P17, respectively. Gene ontology analysis revealed that the conceptus secretory proteins were enriched in those categorized to fibrinolysis and coagulation. RT-qPCR analysis verified that the expression levels of transcripts in conceptuses encoding coagulation factors, fibrinogen subunits, and fibrinolysis factors were significantly higher on P21 than on P15 or P17, which were supported by those through in situ hybridization, Western blotting and immunohistochemistry. Histology analysis confirmed that fibrin protein was present at the conceptus adhesion region on P21. These results suggest that in addition to the numerous adhesion molecules so far characterized, fibrin is a new candidate molecule for maintaining conceptus adhesion for pregnancy continuation in ruminants.

Peptidoglycan disrupts early embryo-maternal crosstalk via suppression of ISGs expression induced by interferon-tau in the bovine endometrium.

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure.

Characterization of mRNA profiles of the exosome-like vesicles in porcine follicular fluid.

Extracellular vesicles such as exosomes contain several types of transcripts, including mRNAs and micro RNAs (miRNAs), and have emerged as important mediators of cell-to-cell communication. Exosome-like vesicles were identified in the ovarian follicles of several mammalian species. Although the miRNA contents have been extensively characterized, the detailed investigation of their mRNA profiles is lacking. Here, we characterize the mRNA profiles of exosome-like vesicles in ovarian follicles in a pig model. The mRNA contents of the exosome-like vesicles isolated from porcine follicular fluid were analyzed and compared with those from mural granulosa cells (MGCs) using the Illumina HiSeq platform. Bioinformatics studies suggested that the exosomal mRNAs are enriched in those encoding proteins involved in metabolic, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) -protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) pathways. While the mRNA profile of the exosome-like vesicles resembled that of MGCs, the vesicles contained mRNAs barely detectable in MGCs. Thus, while the majority of the vesicles are likely to be secreted from MGCs, some may originate from other cell types, including theca cells and oocytes, as well as the cells of non-ovarian organs/tissues. Therefore, the mRNA profiles unveiled several novel characteristics of the exosome-like vesicles in ovarian follicles.