Katsuo Koshi

Fematrin-1 Is Involved in Fetomaternal Cell-to-Cell Fusion in Bovinae Placenta and Has Contributed to Diversity of Ruminant Placentation

ABSTRACT During placentation, mammals employ different strategies for nourishing and supporting fetuses. Members of the Bovidae family, consisting of cloven-hoofed ruminants, utilize multiple maternal attachment points on the placenta, known as cotyledons, and hybrid cells, named trinucleate cells or syncytial plaques, made up of a fusion of fetal trophoblasts and maternal endometrial cells to provide essential hormones and maintain long gestation periods. These hybrid cells are unique to the Bovidae, as fetomaternal borders are clearly separated by syncytiotrophoblasts or epithelial cells in the placenta of other mammals. Recently, it was reported that Syncytin-Rum1 was inserted into ruminant genomes, including cattle and sheep, and was possibly involved in fetomaternal cell-to-cell fusion in both species. However, Syncytin-Rum1 alone is insufficient to explain the morphological diversity of the fetomaternal hybrids between Bovinae and Caprinae (i.e., trinucleate cells in Bovinae and syncytial plaques in Caprinae). Here we report that the bovine endogenous retrovirus K1 (BERV-K1) envelope, which we term Fematrin-1, was specifically expressed in binucleated trophoblasts throughout gestation in cattle and induced fusion with bovine endometrial cells in vitro at a significantly higher level than Syncytin-Rum1 under physiological conditions. Fematrin-1 was found to be integrated into intron 18 of FAT tumor suppressor homolog 2 (FAT2) about 18.3 to 25.4 million years ago and has been subject to purifying selection through the evolution of Bovinae. Phylogenetically, Fematrin-1 is distinct from Syncytin genes found in other mammalian species that form syncytiotrophoblasts. Our results suggest that the newly acquired endogenous retroelement has contributed to generating placentation diversity through ruminant evolution.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

BackgroundExtracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow.MethodsIn this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis.ResultsEMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2) and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium.ConclusionEMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14 suggesting its crucial role in adhesion and fusion of embryo to luminal epithelium by directly itself through physiological tissues remodeling and developmental process, and/or stimulating MMPs to compensate endometrial functions.

Expression of endogenous retrovirus-like transcripts in bovine trophoblastic cells.

UNLABELLED Endogenous retrovirus envelope elements are considered to participate in trophoblastic cell fusion and multinucleate cell formation in humans, mice, and sheep. However, there is limited information about their roles in the ruminant placenta. OBJECTIVES We explore and identify the endogenous retrovirus envelope element genes expressed in bovine trophoblasts. METHODS The NCBI UniGene database (Build #97 Bos taurus) was screened by in silico analysis. After cloning endogenous retrovirus envelope element-like transcript (ERVE), expression profiles were analyzed with quantitative RT-PCR and in situ hybrizaidation. RESULTS Two UniGene clusters, UniGene ID: Bt.68042 and Bt.85243, were detected, and ERVE-A gene was cloned. Weak expression of this gene was first detected on Day 20 of gestation, and the intensity of its expression increased up to Day 70 of gestation. The intensity of its expression was maintained throughout gestation in the placenta, and its specific expression in trophoblastic binucleate cells was confirmed by in situ hybridization. CONCLUSIONS bERVE-A has a similar sequence to human syncytin-1, although it lacks an intact envelope sequence, and is specifically expressed in binucleate cells. This is the first evidence that endogenous retrovirus envelope element genes are expressed in bovine binucleate cells.

Bovine trophoblastic cell differentiation and binucleation involves enhanced endogenous retrovirus element expression

BackgroundEndogenous retrovirus (ERV) envelope (env) genes are involved in the differentiation of trophoblastic cells in humans and mice. However, there is limited information about their roles in ruminant trophoblastic cells. Thus, we attempted to explore the possible roles of ERV elements in the binucleation of bovine trophoblastic cells using in vitro bovine trophoblastic (BT) cell lines.MethodsIn this study, blastocysts and elongated embryos were obtained from Japanese Black cows, and endometrial and fetal membrane tissues were collected from day 17 to 37 of gestation. The gene expression levels of four ERV elements, bERVE (bovine endogenous retrovirus envelope element-like transcript) -A, bERVE–B, BERV (bovine endogenous retrovirus) -K1 env, and BERV-K2 env, were analyzed in the fetal and endometrial tissue and cultured BT cell lines using quantitative RT-PCR. On-Matrigel gel and on-collagen gel culturing were used to induce binucleate cell (BNC) formation in the BT cell lines. How the culture conditions affected the expression of BNC-specific genes and ERV elements was examined by quantitative RT-PCR and immunocytochemistry.ResultsbERVE-A, bERVE–B, BERV-K1 env, and BERV-K2 env were expressed in almost all BT cell lines; however, only bERVE-A and BERV-K1 env were detected in trophoblastic tissues during the peri-implantation period. In the on-Matrigel cultures, the expression levels of BNC-specific genes and molecules were enhanced in the BT cells. The expression levels of bERVE-A and BERV-K1 env were also increased in the BT cells during on-Matrigel culturing. The BT cell expression levels of these ERV elements were consistent with those of BNC-specific genes during on-Matrigel culturing (P < 0.01).ConclusionsThese results suggest that bERVE-A and BERV-K1 env are involved in the expression of BNC-specific genes and the progression of bovine trophoblastic cell binucleation, as their expression levels increased during periods of increased BNC-specific molecule expression, which is strongly suggestive of the development of BNC from mononucleate trophoblastic cells. The on-Matrigel culture system is a convenient in vitro tool for studying bovine trophoblastic cell lineages.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation.

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta.

Bone morphogenetic protein-4 accelerates the establishment of bovine trophoblastic cell lines

Trophoblastic cells play a crucial role in implantation and placentogenesis. A large proportion of the failures of conception in cows occur in the peri-implantation period, which are known as early embryo losses. In exploring this critical phenomenon, trophoblastic cell lines can provide substantial information. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stock in the long term. In this study, 12 new cell lines were established using bone morphogenetic protein 4 (BMP4). BMP4 stimulated embryonic cells to enter the trophoblastic cell lineage but there were no significant differences between intact and BMP4-treated groups. Only one out of 49 embryos developed trophoblastic cells in the intact group. Finally, 12 cell lines were maintained for around 30 passages, and they retained trophoblastic characteristics and expressed bovine trophoblastic genes: placental lactogen, interferon-τ, pregnancy-associated glycoprotein 1, and prolactin-related protein 1. Although the gene expression patterns were different among cell lines and depended on the cells, there was no significant relationship between the expression intensities of genes and the treatment dose of BMP4. All of them expressed bovine POU domain class 5 transcription factor 1 and caudal-type homeobox 2. The expression of these genes was confirmed by quantitative RT-PCR and immunohistochemical detection. These results suggest that BMP4 is involved in the raising of trophoblast cell lines from early embryonic cells and the newly developed cell lines can provide different types of bovine trophoblastic cells with different cell lineages. This may constitute a significant new tool for the examination of trophoblastic differentiation.

Expression profiles of perforin, granzyme B and granulysin genes during the estrous cycle and gestation in the bovine endometrium.

The conceptus is susceptible to destruction by maternal cytotoxic lymphocytes, which have cytotoxic potential. Therefore, it is expected that mechanisms for regulating cytotoxic lymphocytes exist, but little is known about the expression of cytotoxic genes in the endometrium. In the present study, we examined the spatial and temporal expression patterns of the cytotoxic genes perforin, granzyme B, and granulysin during the estrous cycle and gestation in the bovine endometrium. Endometrial tissues were collected from cows during the estrous cycle and gestation. The gene expression patterns of the three cytotoxic genes were examined using quantitative polymerase chain reaction and in situ hybridization, and cytotoxic lymphocyte subsets were characterized using immunohistochemistry. During mid- to late gestation in the intercaruncular (ICAR), granulysin expression was significantly increased, and a large number of granulysin-expressing cells were localized in the luminal epithelium. Perforin and granzyme B displayed similar expression profiles and were highly expressed in the peri-implantation endometrium, but few cells expressing these genes were found in the endometrial stroma. In conclusion, these findings suggest that in the ICAR epithelium granulysin may play important roles in the establishment and maintenance of gestation during normal pregnancy.

Dynamics of CD3+ T-cell Distribution Throughout the Estrous Cycle and Gestation in the Bovine Endometrium

T cells are the dominant lymphocytes in the endometrium and are considered to play a crucial role in implantation and in the maintenance of gestation through cytokine production and immune regulation. The mechanisms underlying immunoregulation at the feto-maternal interface are still obscure for this complex system. Understanding the role of T cells is a key factor in understanding the endometrial immune system. In this study, the distribution of endometrial CD3+ T cells in bovines was examined by immunohistochemical analysis. The estrous cycle and gestation was divided into 4 stages, and the number of CD3+-positive T cells was counted in each stage. CD3+ cells were found in the endometrium in significant numbers throughout the estrous cycle and were mostly located in the subepithelial area. The number of CD3+ cells significantly increased in the early and mid-luteal phases but decreased after implantation with the progression of gestation. No T cells were found in the placentome or specifically in the tissues near the fetus, including the trophoblastic area. In addition, very few T cells were found in stromal regions close to the myometrium of the endometrium. These findings suggest that downregulation of bovine endometrial CD3+ T-cell functions is closely related to the successful maintenance of gestation in a spatiotemporal manner.

A cell-based interferon-tau assay with an interferon-stimulated gene 15 pro moter

Interferon-tau (IFNT) is known as an early pregnancy recognition signal in ruminants. An accurate and convenient IFNT detection system is desirable for the diagnosis of endometrial and trophoblastic functions, including gestation status, in cows. The aim of this study was to develop a new cell-based assay, which involved the stable introduction of an interferon-stimulated gene promoter to a luciferase reporter system. The reactivity of four interferon-stimulated genes to IFNT in Madin-Darby bovine kidney (MDBK) cells was confirmed using reverse transcription-quantitative PCR. The upstream region of the interferon-stimulated gene 15 ubiquitin-like modifier (ISG15) gene as the promoter of the reporter gene, which is more responsive to IFNT and other IFNs, was determined using the luciferase assay. The reporter gene with the ISG15 upstream region was stably transfected into MDBK cells using the PiggyBac vector system; this cell line responded to type I IFNs in a dose-dependent manner. Because of its convenience, this cell line is suitable for the quantification of IFNT as well as other type I IFNs activities.

Induction of ovine trophoblast cell fusion by fematrin-1 in vitro.

Endogenous retroviruses present in the genomes take a specific role in placental formation in various vertebrates, including bovine and sheep. Fematrin-1, which is the envelope (Env) protein of bovine endogenous retrovirus found in bovine placenta, is involved in the formation of fetomaternal hybrid cells in cattle placenta. This study was conducted to clarify whether fematrin-1 possesses fusogenic activity in trophoblast cells. Another question is whether Env proteins only have species-specific activity or not. For this, fematrin-1 gene was transfected in ovine trophoblast cells, and we examined fusogenic activity with Cos-7 cells. Although fematrin-1 fusogenic activity was detected in both neutral and acidic pH conditions, acidic condition significantly enhanced it. These activities were rather weaker than those of vesicular stomatitis virus G protein as a positive control. However, the ratio of fematrin-1 and vesicular stomatitis virus G protein fusion index was confirmed similar to those in the previous reports. Some fusion cells showed multinucleate cells. These results imply that fematrin-1 is involved in the formation of trophoblast hybrid cells even in different species trophoblastic cells.