METTL3 promotes proliferation of goat endometrial epithelial cells by regulating CTGF in an m6A-dependent manner†.

N6-methyladenosine (m6A), an epigenetic modification on RNAs, plays an important role in many physiological and pathological processes. However, the involvement of m6A in goat uterus during early pregnancy remains largely unknown. In this study, we found that the total m6A level was increasing in goat uterus as early pregnancy progressed. Methyltransferase-like 3 (METTL3) is a core catalytic subunit of the m6A methyltransferase. We thus determined the expression and regulation of METTL3 in goat uterus. METTL3 was highly expressed in the luminal and glandular epithelia from day 16 (D16) to D25 of pregnancy, and it could be up-regulated by estrogen and progesterone in goat uterus and primary endometrial epithelial cells (EECs). In EECs, knockdown or overexpression of METTL3 resulted in a significant decrease or increase of cell proliferation, respectively. METTL3 knockdown reduced the m6A level of not only total RNA but also connective tissue growth factor (CTGF) mRNA. Luciferase assay suggested that METTL3 might target the potential m6A sites in the 3'untranslated region (3'UTR) of CTGF mRNA. Moreover, METTL3 positively regulated CTGF expression, and CTGF knockdown significantly counteracted the promoting effect of METTL3 overexpression on EEC proliferation. Collectively, METTL3 is dynamically expressed in goat uterus and can affect EEC proliferation by regulating CTGF in an m6A-dependent manner. Our results will lay a foundation for further studying the crucial mechanism of METTL3-mediated m6A modification in goat uterus during early pregnancy.

Peri-implantation expression and regulation of ITGB8 in goat uterus.

Ruminants have a superficial implantation pattern. The extended conceptus attaches to the receptive endometrium to form the cotyledonary placenta. During the attachment, a large number of events occur at the maternal-fetal interface. However, the related molecular mechanisms have not been fully understood. Integrin beta8 (ITGB8) is a subunit of integrin beta involved in embryo implantation. In this study, we determined peri-implantation expression and regulation of ITGB8 in goat uterus. The mRNA and protein levels of ITGB8 were both high in goat endometrial luminal epithelium (LE) and superficial glandular epithelium (sGE) during the adhesion period (Days 16-19 of pregnancy). Such expression profile was opposite to that of microRNA-187 (miR-187). Then, we validated that miR-187 targeted the 3' untranslated region (UTR) of ITGB8 in primary goat endometrial epithelial cells (EECs). In EECs, inhibition of miR-187 resulted in not only up-regulated ITGB8 level but also reduced cell proliferation and focal adhesion kinase (FAK) activity. Moreover, ITGB8 and miR-187 were regulated by interferon tau (IFNT). Altogether, in goat, the miR-187/ITGB8 axis may be involved in conceptus attachment and is downstream of IFNT. Our results will help us better understand the mechanisms of ruminant implantation and may provide a useful tool to improve the reproduction ratio for ruminants.

Evolution of Potato virus X.

Potato virus X (PVX) is the type potexvirus of economic significance. The pathogen is distributed worldwide, threatening solanaceous plants in particular. Based on the coat protein (CP) gene, PVX isolates are classified into two major genotypes (I and II). To gain more insights into the molecular epidemiology and evolution of PVX, recombination analyses were conducted and significant signals were detected. Bayesian coalescent method was then applied to the time-stamped entire CP sequences. According to the estimates, the global subtype I-1 went into expansion in the 20th century and was evolving at a moderate rate. Based on the CP phylogenies, a divergence scenario was proposed for PVX. Surveys of codon usage variation showed that PVX genes had additional bias independent of compositional constraint. In codon preference, PVX was both similar to and different from the three major hosts, potato (Solanum tuberosum), tobacco (Nicotiana tabacum), and tomato (S. lycopersicum). Moreover, the suppression of CpG and UpA dinucleotide frequencies was observed in PVX.

Novirhabdoviruses versus fish innate immunity: A review.

Novirhabdoviruses belong to the Rhabdoviridae family of RNA viruses. All of the four members are pathogenic for bony fish. Particularly, Infectious hematopoietic necrosis virus (IHNV) and Viral hemorrhagic septicemia virus (VHSV) often cause mass animal deaths and huge economic losses, representing major obstacles to fish farming industry worldwide. The interactions between fish and novirhabdoviruses are becoming better understood. In this review, we will present our current knowledge of fish innate immunity, particularly type I interferon (IFN-I) response, against novirhabdoviral infection, and the evasion strategies exploited by novirhabdoviruses. Members of Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) appear to be involved in novirhabdovirus surveillance. NF-κB activation and IFN-I induction are primarily triggered for antiviral defense. Autophagy can also be induced by viral glycoprotein (G). Although sensitive to IFN-I, novirhabdoviruses have nucleoprotein (N), matrix protein (M), and non-virion protein (NV) to interfere with host signal transduction and gene expression steps toward antiviral state establishment. Moreover, novirhabdoviruses may exploit some microRNAs for immunosuppression.

[Expression and regulation of XBP1 in mouse uterus during early pregnancy].

The transcription factor X-box binding protein-1 (XBP1) plays a key role in unfolded protein reaction. This study was aimed to investigate the expression pattern and regulation of XBP1 in the mouse uterus during early pregnancy. The methods of immunohistochemistry (IHC) and real time quantitative RT-PCR were used to test XBP1 expression in early pregnancy, artificial decidualization, oestrous cycle and hormone-regulated mouse models. The results showed that XBP1 was spatiotemporally expressed in mouse uterus during early pregnancy. The XBP1 protein was mainly detected in the luminal and glandular epithelia on days 1-4 of pregnancy, and was strongly detected in the decidual area on days 5-8 of pregnancy. Similarly, XBP1 expression was also mainly expressed in decidual cells following artificial decidualization. During the oestrous cycle, Xbp1, Xbp1u, and Xbp1s mRNA was predominantly present in proestrus. In the ovariectomized uterus, the expression of XBP1 in luminal and glandular epithelia was up-regulated after estrogen treatment. These results suggest that XBP1 is associated with embryo implantation and decidualization during early pregnancy in mice, and the expression of XBP1 in luminal and glandular epithelia may be regulated by estrogen.

Connective tissue growth factor gene expression in goat endometrium during estrous cycle and early pregnancy.

Embryo implantation is crucial for a successful pregnancy. Although many essential molecular modulators and pathways have been identified, the precise mechanisms of the process in goat remain largely unknown. CCN2 is a connective tissue growth factor participating in many biological processes; however, its presence or function in goat uterus has not yet been reported. In this study, we determined the expression and regulation of CCN2 in goat uterus. CCN2 was not detected by in situ hybridization at ED0 (Day 0 of the estrous cycle), but at ED6 (metestrus), ED12 (dioestrus), and ED16 (proestrus), with high signals in luminal epithelium, superficial glands, and caruncula matrix. During early pregnancy, CCN2 was also detected in these locations on D0 and D6 (pre-receptive uterus). The signals significantly increased on D16 and D19 (receptive uterus), and remained at high levels on D25 and D30. Similarly, the RT-qPCR assays showed that the mRNA level of CCN2 was relatively low on D0 and D6, increased on D16, peaked on D19, and kept high thereafter. Moreover, CCN2 was up-regulated not only in ovariectomized ewes subcutaneously injected with 17ß-estradiol and progesterone (separately or together), but also in cultured goat uterine epithelial cells treated with the two hormones or interferon tau (IFNτ). In conclusion, CCN2 expression may be induced by 17ß-estradiol, progesterone, and IFNτ in the luminal epithelium of goat receptive uterus, suggesting that CCN2 is involved in goat embryo adhesion during early pregnancy.

Cellular prion protein is involved in decidualization of mouse uterus.

Prion protein (PrP) is encoded by a single copy gene Prnp in many cell and tissue types. PrP is very famous for its infectious conformers (PrPSC) resulting in transmissible spongiform encephalopathies. At present, physiological functions of its cellular isoform (PrPC) remain ambiguous. Although PrPC expression has been found in uterus, whether it functions in maternal-fetal dialogue during early pregnant is unknown. In this study, we examined PrPC mRNA and protein in the uterus of peri-implantation mice, and found that they were expressed with a spatiotemporal dynamic pattern. Interestingly, PrPC was significantly increased in the decidual zones around the implanting embryos at the implantation window stage. To further demonstrate that PrPC is involved in the decidualization of mouse uterus during embryo implantation, we constructed the artificial decidualization models and the delayed implantation models. Once the pseudopregnant mice were artificially induced to decidualization, the PrPC expression then increased significantly in the decidua zone. And also, if the delayed implantation embryos were allowed to implant, PrPC protein was also simultaneously improved in stromal cells surrounding the implanting embryos. Moreover, PrPC expression can be inhibited by progesterone but upregulated by estrogen in mouse uterus. These results suggest that PrPC may play an important role in embryo implantation and decidualization.

A permanent host shift of rabies virus from Chiroptera to Carnivora associated with recombination.

Bat virus host shifts can result in the spread of diseases with significant effects. The rabies virus (RABV) is able to infect almost all mammals and is therefore a useful model for the study of host shift mechanisms. Carnivore RABVs originated from two historical host shifts from bat viruses. To reveal the genetic pathways by which bat RABVs changed their host tropism from bats to carnivores, we investigated the second permanent bat-to-carnivore shift resulting in two carnivore variants, known as raccoon RABV (RRV) and south-central skunk RABV (SCSKV). We found that their glycoprotein (G) genes are the result of recombination between an American bat virus and a carnivore virus. This recombination allowed the bat RABV to acquire the head of the G-protein ectodomain of the carnivore virus. This region is involved in receptor recognition and binding, response to changes in the pH microenvironment, trimerization of G proteins, and cell-to-cell transmission during the viral infection. Therefore, this recombination event may have significantly improved the variant's adaptability to carnivores, altering its host tropism and thus leading to large-scale epidemics in striped skunk and raccoon.

Dating the divergence of the infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is an economically significant rhabdovirus that can cause severe disease to most salmonid fish. Phylogenetic analyses of worldwide IHNV isolates have defined five major genogroups. Herein, to further the understanding of the molecular epidemiology and evolution of IHNV, Bayesian coalescent analyses were conducted to the time-stamped coding sequences of the N, G and NV genes. The nucleotide substitution rates and the divergence times were assessed. Among the three genes, NV, the smallest one coding for a non-virion protein, was conferred the highest mean rate. Estimates for the G subsets based upon the five genogroups indicated that L and U evolved much slower than the others. Age calculations suggested that the first diversification event of the IHNV isolates analyzed might have happened before the notification of the disease during the early 1950s. Selection analyses suggested that the three genes were mainly subject to purifying selection. In addition, surveys of codon usage variation showed that the three genes had influences other than mutational bias.

Natural recombination between tobacco and tomato mosaic viruses.

Tobacco mosaic virus (TMV) is a positive-sense plant RNA virus which has a wide host range and a worldwide distribution. Other than a troublesome pathogen, TMV is regarded as a model system pioneering biologic research for a century. The tomato strain of TMV has been recognized to be a distinct tobamovirus as the tomato mosaic virus (ToMV). Recombination has been increasingly recognized as an important factor generating genetic diversity in many RNA viruses. However, it is still unclear whether recombination can function in driving the evolution of tobamoviruses. Herein, based on sequence comparison, we found three recombinants involving each viral gene, all of which might be derived from homologous or aberrant homologous recombination between TMV and ToMV. The study provided evidence that recombination did contribute to the genetic diversity of tobamoviruses.

Peroxisome proliferator-activated receptor delta expression and regulation in mouse uterus during embryo implantation and decidualization.

The aim of this study was to examine the expression and regulation of peroxisome proliferator-activated receptor (PPAR) PPARdelta gene in mouse uterus during early pregnancy by in situ hybridization and immunohistochemistry. PPARdelta expression under pseudopregnancy, delayed implantation, hormonal treatment, and artificial decidualization was also investigated. There was a very low level of PPARdelta expression on days 1-4 of pregnancy. On day 5 when embryo implanted, PPARdelta expression was exclusively observed in the subluminal stroma surrounding the implanting blastocyst. No corresponding signals were seen in the uterus on day 5 of pregnancy. There was no detectable PPARdelta signal under delayed implantation. Once delayed implantation was terminated by estrogen treatment and embryo implanted, a strong level of PPARdelta expression was induced in the subluminal stroma surrounding the implanting blastocyst. Estrogen treatment induced a moderate level of PPARdelta expression in the glandular epithelium, while progesterone treatment had no effects in the ovariectomized mice. A strong level of PPARdelta expression was seen in the decidua on days 6-8 of pregnancy. PPARdelta expression was also induced under artificial decidualization. These data suggest that PPARdelta expression at implantation sites require the presence of an active blastocyst and may play an essential role for blastocyst implantation.

Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR.

Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 +/- 0.0282 in the oocyte, 0.0102 +/- 0.0036 in the zygote, 0.0007 +/- 0.0003 in the 2-cell embryo, 0.0031 +/- 0.0017 in the 4-cell embryo, 0.0084 +/- 0.0024 in the 8-cell embryo, 0.0537 +/- 0.0121 in the morula and 0.0392 +/- 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.

Basigin expression and hormonal regulation in mouse uterus during the peri-implantation period.

Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, has been shown to be essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of basigin gene in mouse uterus during the peri-implantation period. Basigin immunostaining and mRNA were strongly localized in luminal and glandular epithelium on day 1 of pregnancy and gradually decreased to a basal level from day 2-4 of pregnancy. Basigin mRNA expression in the sub-luminal stroma was first detected on day 3 of pregnancy and increased on day 4 of pregnancy. On day 5 of pregnancy, the expression of basigin protein and mRNA was only detected in the implanting embryos, and the luminal epithelium and sub-luminal stroma surrounding the embryos. A similar expression pattern of basigin was also induced in the delayed-implantation uterus which was activated by estrogen injection. On day 6-8 of pregnancy, although a basal level of basigin protein was detected in the secondary decidual zone, basigin mRNA expression was strongly seen in this location. Basigin mRNA was also highly expressed in the decidualized cells under artificial decidualization. Estrogen significantly stimulated basigin expression in the ovariectomized mouse uterus. A high level of basigin immunostaining and mRNA was also seen in proestrus and estrus uteri. These results suggest that basigin expression is closely related to mouse implantation and up-regulated by estrogen.

Expression of leukemia inhibitory factor receptor and gp130 in mouse uterus during early pregnancy.

Leukemia inhibitory factor (LIF) is essential for implantation of mouse embryos. The aim of this study was to assess the expression and regulation of LIF receptor (LIFR) and gp130 mRNA in mouse uterus during early pregnancy using in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). A strong level of LIFR mRNA signal was detected in the luminal epithelium on day 5 of pregnancy. On day 6-8 of pregnancy, LIFR signal was mainly localized in the decidua. LIFR signal in the luminal epithelium of ovariectomized mouse was slightly stimulated by a combination of progesterone and estrogen. By RT-PCR, LIFR mRNA signal was detected in all of the uteri from day 1-8 of pregnancy, being at the highest level on day 5. A low level of gp130 mRNA signal was seen in the glandular epithelium on day 3 of pregnancy and reached a high level on day 4 of pregnancy. gp130 signal in the decidua appeared on day 6 of pregnancy and reached a high level on day 7 and 8 of pregnancy. After the ovariectomized mouse was treated with a combination of progesterone and estrogen, a strong gp130 signal was observed in the glandular epithelium, while no signal was detected after progesterone or estrogen treatment alone. By RT-PCR analysis, gp130 mRNA was detected in all of the uteri from day 1-8 of pregnancy, being at the highest level on day 4 of pregnancy. Additionally, a high level of LIFR and gp130 mRNA signal was detected in the decidua under artificial decidualization compared to the control uninjected horn. The co-expression of a high level of LIFR and gp130 mRNA in the decidua may play a key role during the decidualization and placentation.