Dynamics of extracellular vesicle uptake by mural granulosa cells in mice.

Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte-derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence-labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin- and caveolae-mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin- and caveolae-mediated endocytosis and that oocytes may promote caveolae-mediated endocytosis to facilitate the uptake of EVs.

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.

New Roles for EVs, miRNA and lncRNA in Bovine Embryo Implantation.

The sine qua non of new life is fertilization. However, approximately 50% of fertilized eggs/blastocysts in cattle and up to 75% of those from human assisted reproductive procedures fail during the first 3 to 4 weeks of pregnancy, including peri-implantation periods. In these periods, blastocyst hatching and implantation to the maternal endometrium proceeds, during which physiological events such as epithelial-mesenchymal transition (EMT) and trophoblast cell fusion occur. Quite recently, extracellular vesicles (EVs) with micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been found to play a pivotal role for the establishment of the proper uterine environment required for peri-implantation processes to proceed. New findings of EVs, miRNA, and lncRNAs will be described and discussed to elucidate their connections with conceptus implantation to the maternal endometrium.

Characterization of Serum Metabolome and Proteome Profiles Identifies SNX5 Specific for Pregnancy Failure in Holstein Heifers.

Pregnancy loss predominantly occurs during the first 3-4 weeks due to fertilization failure or early embryonic losses in cattle. Insufficient biochemical communication between conceptus (embryo plus extraembryonic membranes) and endometrium has been suspected as the primary cause for early embryonic losses. If molecules regulating this communication were identified, molecular mechanisms associated with early pregnancy losses could be better understood. To identify candidate molecules as detection markers of non-pregnant or females undergoing embryonic loss, peripheral blood from embryo-transferred heifers on day 7 (day 0 = day of estrus) were collected on days 17 (pre-attachment), 20 (during attachment), and 22 (post-attachment), which were subjected to metabolome and global proteome iTRAQ analyses. The metabolome analysis partly divided serum components into pregnant or not. In the iTRAQ analysis, heatmap analysis with top 25 proteins was separated into pregnant or not on day 20 or 22. Furthermore, receiver operating characteristic curve (ROC) analysis identified five candidate proteins detecting non-pregnant heifers, of which SNX5 in day 22 serum had the highest area under the curve (AUC): 0.983. We also detected SNX5 in day 22 serum from non-pregnant heifers using western blotting. These results suggest that high SNX5 in day 22 serum could predict early pregnancy loss in heifers.

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.

Effects of oocyte-derived paracrine factors on release of extracellular vesicles by murine mural granulosa cells in vitro.

Both oocytes and extracellular vesicles (EV) have emerged as critical regulators of mammalian follicular development; however, the possible interaction between the oocyte-derived paracrine factor (ODPF) and EV signals has never been examined. Therefore, to explore the possibility of an interaction between oocyte and EV signals, the effects of ODPFs on the biogenesis of EVs as well as the expression levels of transcripts related to EV biogenesis in mural granulosa cells (MGCs) were examined using mice. The results showed that, while oocyte coculture has some effects on the expression levels of transcripts related to EV biogenesis, the number of EV particles present in the conditioned medium were not significantly different between ODPF-treated and non-treated MGCs. Therefore, oocytes have no effects on the EV biogenesis by MGCs, at least with respect to the numbers of EV particles.

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.

Effects of exosome-like vesicles on cumulus expansion in pigs in vitro.

Cell-secreted vesicles, such as exosomes, have recently been recognized as mediators of cell communication. A recent study in cattle showed the involvement of exosome-like vesicles in the control of cumulus expansion, a prerequisite process for normal ovulation; however, whether this is the case in other mammalian species is not known. Therefore, this study aimed to examine the presence of exosome-like vesicles in ovarian follicles and their effects on cumulus expansion in vitro in pigs. The presence of exosome-like vesicles in porcine follicular fluid (pFF) was confirmed by transmission electron microscopic observation, the detection of marker proteins, and RNA profiles specific to exosomes. Fluorescently labeled exosome-like vesicles isolated from pFF were incorporated into both cumulus and mural granulosa cells in vitro. Exosome-like vesicles were not capable of inducing cumulus expansion to a degree comparable to that induced by follicle-stimulating hormone (FSH). Moreover, exosome-like vesicles had no significant effects on the expression levels of transcripts required for the normal expansion process (HAS2, TNFAIP6, and PTGS2). Interestingly, FSH-induced expression of HAS2 and TNFAIP6 mRNA, but not of PTGS2 mRNA, was significantly increased by the presence of exosome-like vesicles; however, the degree of FSH-induced expansion was not affected. In addition, porcine exosome-like vesicles had no significant effects on the expansion of mouse cumulus-oocyte complexes. Collectively, the present results suggest that exosome-like vesicles are present in pFF, but they are not efficient in inducing cumulus expansion in pigs.

Mouse oocytes suppress miR-322-5p expression in ovarian granulosa cells.

This study tested the hypothesis that oocyte-derived paracrine factors (ODPFs) regulate miRNA expression in mouse granulosa cells. Expression of mmu-miR-322-5p (miR-322) was higher in mural granulosa cells (MGCs) than in cumulus cells of the Graafian follicles. The expression levels of miR-322 decreased when cumulus cells or MGCs were co-cultured with oocytes denuded of their cumulus cells. Inhibition of SMAD2/3 signaling by SB431542 increased miR-322 expression by cumulus-oocyte complexes (COCs). Moreover, the cumulus cells but not the MGCs in Bmp15(-/-)/Gdf9(+/-) (double-mutant) mice exhibited higher miR-322 expression than those of wild-type mice. Taken together, these results show that ODPFs suppress the expression of miR-322 in cumulus cells. Gene ontology analysis of putative miR-322 targets whose expression was detected in MGCs with RNA-sequencing suggested that multiple biological processes are affected by miR-322 in MGCs. These results demonstrate that ODPFs regulate miRNA expression in granulosa cells and that this regulation may participate in the differential control of cumulus cell versus MGC functions. Therefore, the ODPF-mediated regulation of cumulus cells takes place at both transcriptional and post-transcriptional levels.

Effects of porcine oocytes on the expression levels of transcripts encoding glycolytic enzymes in granulosa cells.

Oocytes play critical roles in regulating the expression of transcripts encoding the glycolytic enzymes phosphofructokinase, platelet (PFKP) and lactate dehydrogenase A (LDHA) in granulosa cells in mice, but whether this is the case in pigs or other mammals has not been adequately investigated. Therefore, the aim of this study was to determine whether porcine oocytes regulate the expression levels of these transcripts in granulosa cells in vitro. Porcine cumulus cells expressed higher levels of PFKP and LDHA transcripts than mural granulosa cells (MGCs). However, co-culturing with oocytes had no significant effect on the isolated cumulus cells. While murine oocytes promoted the expression of both Pfkp and Ldha transcripts by murine MGCs, porcine oocytes promoted the expression of only Pfkp, but not Ldha transcripts by murine MGCs. Neither murine nor porcine oocytes affected PFKP and LDHA expression by porcine MGCs. Moreover, in the presence of porcine follicular fluid, porcine oocytes maintained the expression of PFKP, but not LDHA by porcine cumulus cells. Therefore, porcine oocytes are capable of regulating the expression of PFKP but not LDHA in granulosa cells in coordination with unknown factor(s) present in the follicular fluid.

Experimental realization of a quantum phase transition of polaritonic excitations.

We report an experimental realization of the Jaynes-Cummings-Hubbard model using the internal and radial phonon states of two trapped ions. An adiabatic transfer corresponding to a quantum phase transition from a localized insulator ground state to a delocalized superfluid (SF) ground state is demonstrated. The SF phase of polaritonic excitations characteristic of the interconnected Jaynes-Cummings (JC) system is experimentally explored, where a polaritonic excitation refers to a combination of an atomic excitation and a phonon interchanged via a JC coupling.