[Show abstract][Hide abstract] ABSTRACT: The corpus luteum (CL) is a temporary endocrine gland producing a large amount of progesterone, which is essential for the establishment and maintenance of pregnancy. Galectin-1 is a β-galactose-binding protein that can modify functions of membrane glycoproteins and is expressed in the CL of mice and women. However, the physiological role of galectin-1 in the CL is unclear. In the present study, we investigated the expression and localization of galectin-1 in the bovine CL and the effect of galectin-1 on cultured luteal steroidogenic cells (LSCs) with special reference to its binding to the glycans on vascular endothelial growth factor receptor-2 (VEGFR-2). Galectin-1 protein was highly expressed at the mid and late luteal stages in the membrane fraction of bovine CL tissue and was localized to the surface of LSCs in a carbohydrate-dependent manner. Galectin-1 increased the viability in cultured LSCs. However, the viability of LSCs was decreased by addition of β-lactose, a competitive carbohydrate inhibitor of galectin-1 binding activity. VEGFR-2 protein, like galectin-1, is also highly expressed in the mid CL, and it was modified by multi-antennary glycans, which can be recognized by galectin-1. An overlay assay using biotinylated galectin-1 revealed that galectin-1 directly binds to asparagine-linked glycans (N-glycans) on VEGFR-2. Enhancement of LSC viability by galectin-1 was suppressed by a selective inhibitor of VEGFR-2. The overall findings suggest that galectin-1 plays a role as a survival factor in the bovine CL, possibly by binding to N-glycans on VEGFR-2.
Journal of Reproduction and Development 07/2015; 61(5). DOI:10.1262/jrd.2015-056 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Galectins, beta-galactoside binding lectins, are involved in various physiological and pathological events. The corpus luteum (CL) is a transient endocrine tissue that produces large amounts of progesterone, which is essential for a successful pregnancy, and stage-specifically expresses galectin-1 and galectin-3. We herein summarized current knowledge on galectins in the CL of mice, cows, and women in order to clarify the expression profiles, regulatory mechanisms, and possible roles of galectins in the CL of different species. The regressing CL of mice contained both galectin-1 and galectin-3, suggesting the involvement of galectins in the regulation of luteolysis in mice. On the other hand, the healthy functional CL of cows and women abundantly expressed galectin-1, whereas galectin-3 was increased in the regressing CL. The expression of galectin in luteal cells is differentially regulated by known endocrine and paracrine molecules such as prolactin, luteinizing hormone, human chorionic gonadotropin, and prostaglandins E and F. Interestingly, 2,6-sialylation, which inhibit galectin-1 binding and are catalyzed by ST6GAL1, were increased in the regressing CL of all animals. These findings suggest that a “galectin switch”, coordinated changes in glycans and galectins in association with luteal function, represents a conserved mechanism in the regulation of luteal function beyond species.
Trends in Glycoscience and Glycotechnology 07/2015; · 0.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A successful pregnancy depends on the blastocyst's implantation to the maternal endometrium; however, the initial interaction between blastocyst and uterine epithelium has not been well characterized. The objectives of this study were to determine if selectins and their ligands were expressed in the bovine conceptus and/or uterus during the peri-attachment period, and to study whether selectins were associated with conceptus attachment to the uterine epithelium. Through the RNA-seq analysis of bovine conceptuses on Days 17, 20, and 22 (Day 0 = day of estrus), only the L-selectin (SELL) ligand, podocalyxin (PODXL) and P-selectin (SELP) ligand, SELPLG were found. Quantitative PCR analysis confirmed the presence of PODXL and SELPLG in these conceptuses and revealed that SELL, mRNA and protein, detected in the uterine epithelium but not in conceptuses, increased during the peri-attachment period. In the cultured endometrial epithelial cells (EECs), SELL transcript was up-regulated when uterine flushings from Day 20 pregnant animals were placed onto these cells. SELL was also up-regulated when cultured EECs were treated with progesterone, EGF or bFGF, but not with IFNT. In the co-culture system with EECs and bovine trophoblast CT-1 cells, SELL expression in EECs was effectively reduced by its siRNA, however, IFNT, a marker for CT-1 cell attachment to EECs, was not reduced, nor was a transcription factor of IFNT, CDX2. These observations suggest that the conceptus could attach to the uterine epithelium through the use of endometrial SELL and embryonic selectin ligands, possibly initiating the conceptus attachment process in the bovine species.
Copyright 2015 by The Society for the Study of Reproduction.
Biology of Reproduction 07/2015; 93(2). DOI:10.1095/biolreprod.115.128652 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is increasing interest in the role of oxygen conditions in the microenvironment of organs because of the discovery of a hypoxia-specific transcription factor, namely hypoxia-inducible factor (HIF) 1. Ovarian function has several phases that change day by day, including ovulation, follicular growth and corpus luteum formation and regression. These phases are regulated by many factors, including pituitary hormones and local hormones, such as steroids, peptides and cytokines, as well as oxygen conditions. Hypoxia strongly induces angiogenesis because transcription of the potent angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF1. Follicular development and luteal formation are accompanied by a marked increase in angiogenesis assisted by HIF1-VEGF signalling. Hypoxia is also one of the factors that induces luteolysis by suppressing progesterone synthesis and by promoting apoptosis of luteal cells. The present review focuses on recent studies of hypoxic conditions, as well as HIF1-regulated genes and proteins, in the regulation of ovarian function.
Reproduction Fertility and Development 05/2015; DOI:10.1071/RD15010 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8-12 after ovulation) were cultured with PGF (0.01, 0.1, 1 μM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 μM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One μM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.
Journal of Reproduction and Development 04/2015; 61(4). DOI:10.1262/jrd.2014-150 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Progesterone (P4) acts through different actuating pathways called genomic and non-genomic pathways. Here we investigated whether P4 regulates prostaglandin (PG) F2? (PGF) and PGE2 production in bovine endometrium through different pathways. Cultured endometrial cells were exposed to P4 for a short time (5-20min) or bovine serum albumin (BSA)-conjugated P4 (P4-BSA) for 24h. Progesterone treatment for 24h stimulated PGE2 production in epithelial cells, but suppressed both PGF and PGE2 production and the expression of PG-metabolising enzymes including phospholipase A2 (PLA2) and cyclooxygenase-2 (COX2) in stromal cells. Short-term (5-20min) P4 treatment did not affect PLA2 or COX2 transcript levels in either cell type. P4-BSA increased PGF and PGE2 production only in epithelial cells. Nuclear P4 receptor mRNA expression in endometrium was higher at the follicular phase than at the early- to mid-luteal stages, whereas membrane P4 receptor mRNA expression did not change throughout the oestrous cycle. The overall results suggest that P4 controls PG production by inhibiting enzymes via a genomic pathway and by stimulating signal transduction via a non-genomic pathway. Consequently, P4 may protect the corpus luteum by attenuating PGF production in stromal cells and by increasing PGE2 secretion from epithelial cells.
Reproduction Fertility and Development 04/2015; DOI:10.1071/RD14490 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3α, LC3β, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL.
Journal of Reproduction and Development 03/2015; 61(3). DOI:10.1262/jrd.2014-135 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activin (ACV) is known as a local regulator of several reproductive functions including follicular development and implantation in mammals. ACVA is a glycopeptide belonging to the transforming growth factor β superfamily, and is a homodimer of inhibin ßA (INHBA) subunits. Follistatin (FST), an ACV-specific binding protein, inhibits ligand-receptor binding. ACV receptor (ACVR) is a hetero-tetramer consisting of 2 kinds of protein, ACVR1 or ACVR1B and ACVR2A or ACVR2B. The oviduct provides an optimal environment for sperm capacitation, fertilization, and early embryonic development. Previous reports have demonstrated that ACVRs were expressed in bovine oocytes and embryos, and that early embryonic development is facilitated by ACVA in vitro. ACVA produced by the bovine oviduct may affect gametes and embryos as well as oviductal cells as a local regulator in cow. Bovine oviductal samples were classified into 6 stages of the oestrous cycle (day of ovulation; Days 2-3 after ovulation; Days 5-6; Days 8-12; Days 15-17; Days 19-21). We examined (1) protein expression of ACVA and FST in oviductal fluid collected from the ampulla and isthmus, (2) mRNA expression of INHBA and FST in the ampullary and isthmic oviductal tissues during the oestrous cycle, (3) the effects of oestradiol-17β (E2: 0.1, 1, 10nM) and progesterone (P4: 1, 10, 100nM) on mRNA expressions of INHBA and FST in cultured oviductal epithelial cells isolated from the ampulla and isthmus, and (4) mRNA expression of ACVRs in tissues and in cultured epithelial and stromal cells. The main findings were as follows: (1) Both ACVA and FST were detected throughout the oestrous cycle in the oviductal fluid of the ampulla and isthmus. (2) INHBA expression was higher in the isthmus than in the ampulla. FST expression in the ampulla was lowest at peri-ovulation, INHBA expression in the isthmus was highest on the day of ovulation and FST in the isthmus during Days 2-6 was highest. Because an increase of ACVA production and a decrease of FST production raise ACVA bioactivity, ACVA seems to be most active at peri-ovulation in both the ampulla and isthmus. (3) In the cultured isthmic oviductal epithelial cells, 10nM E2 significantly stimulated INHBA expression, but tended to suppress FST expression. Therefore, the bioactivity of ACVA seems to be controlled by E2 during the oestrous cycle in the isthmus. (4) The expression of ACVR1B and ACVR2A was clearly detected in the tissues as well as in cultured epithelial and stromal cells. The overall findings suggest that ACVA secreted into oviductal fluid plays an important role in oviductal functions, including fertilization in the ampulla and sperm motility and viability in the isthmus. It is also suggested that ACVA acts on both epithelial and stromal cells as a local regulator of cellular functions, such as cellular proliferation and secretion in the cow.
Reproduction Fertility and Development 12/2014; 27(1):145. DOI:10.1071/RDv27n1Ab104 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The oviduct is an essential organ for successful pregnancy in mammals. The transport of gametes and early embryos is mainly induced by contraction and relaxation of smooth muscle. The contraction and relaxation of bovine oviductal smooth muscle are induced by prostaglandin (PG) F2α and PGE2, respectively. Lysophosphatidic acid (LPA), a type of phospholipid, is involved in various physiological actions such as promoting inflammation and cellular proliferation in various organs. LPA acts through at least 6 G protein-coupled receptors. Both LPA and LPA receptors are expressed in endometrium and, moreover, LPA affects PG production by the endometrium in cow. Based on the above findings, we hypothesised that LPA is locally involved in PG production by oviductal cells to promote motility of oviductal smooth muscle in cow. Oviductal samples ipsilateral to a corpus luteum or a dominant follicle at peri-ovulation (0-6 and 19-21 days after ovulation) were collected in abattoir. Messenger RNA expression of LPA receptors (LPAR1-6) and LPA-producing enzymes (ATX, PLA1α, PLA1β) was examined in ampullary and isthmic tissues. Expression in cultured epithelial and stromal cells isolated from the bovine oviduct were also examined to determine the cells possessing LPA receptors and LPA-producing enzymes. In addition, the effect of LPA (0.1, 1, 10μM) on the expression of cyclooxygenase (COX)-1 and COX-2 (PG-synthesising enzymes) and on PGE2 and PGF2α production by cultured epithelial and stromal cells was investigated. The significant differences (P<0.05) were determined by Student's t-test for 2 groups, and by one-way ANOVA followed by Tukey's multiple comparison test for more than 3 groups. LPAR1-6, ATX, PLA1α, and PLA1β were expressed in both ampullary and isthmic tissues as well as in both cultured epithelial and stromal cells. The expression of LPAR1-3 was significantly lower in the isthmic tissues than in the ampullary tissues, whereas the expression of LPAR4-6 was significantly higher in the isthmic tissues than in the ampullary tissues. The expression of COX-2 was significantly stimulated by 10μM LPA in cultured isthmic stromal cells. In addition, LPA significantly stimulated both PGE2 and PGF2α production by cultured isthmic stromal cells. In the isthmus of the oviduct, LPA produced by epithelial and stromal cells may stimulate the expression of COX-2 in the stromal cells, followed by increased PG production. Because the mRNA expression of LPAR4-6 is higher in the isthmus than in the ampulla, those effects of LPA might be mediated by activation of LPAR4-6. The overall findings suggest that LPA is one of the regulating factors for transport of gametes and early embryos by controlling the motility of smooth muscle in the bovine oviduct.
Reproduction Fertility and Development 12/2014; 27(1):145. DOI:10.1071/RDv27n1Ab105 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The corpus luteum (CL) forms after ovulation and acts as a temporary endocrine gland that produces progesterone (P4), a hormone that is essential for implantation and maintenance of pregnancy in mammals. In pregnant women, human chorionic gonadotropin (hCG) secreted by the conceptus prevents luteolysis. hCG also increases the survival of cultured human luteinized granulosa cells (hLGCs). To clarify the maintenance mechanism of the human CL, we investigated the effects of hCG and P4 receptor antagonists, onapristone (OP) and RU486, on the viability of hLGCs. With the patients’ consent, hLGCs were isolated from follicular aspirates for in vitro fertilization. The cells were cultured with hCG (0.1, 1, 10, 100 IU/ml), OP (10, 25, 50, 100 μM), RU486 (100 μM), P4 (1, 10, 25, 50 μM) or some combination of the four for 24 h. Cell viability was significantly increased by hCG (100 IU/ml) and significantly decreased by OP (100 μM) compared with the control. Cells
treated with hCG and OP together were significantly less viable than the control and OP-treated cells. The combined treatment also significantly increased CASP3 activity and cleaved CASP3 protein expression. Furthermore, P4 addition reversed the reduction in cell viability caused by the combination of hCG and OP treatment. The overall findings suggest that hCG cooperates with P4 to increase survival of hLGCs and to induce apoptosis when P4 action supported by hCG is attenuated in the human CL.
Journal of Reproduction and Development 11/2014; 61(1). DOI:10.1262/jrd.2014-115 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine functional differences between the corpus luteum (CL) of the estrous cycle and pregnancy in cows, gene expression profiles were compared using a 15 K bovine oligo DNA microarray. In the pregnant CL at days 20–25, 40–45 and 150–160, the expressions of 138, 265 and 455 genes differed by a factor of > 2-fold (P < 0.05) from their expressions in the cyclic CL (days 10–12 of the estrous cycle). Messenger RNA expressions of chemokines (eotaxin, lymphotactin and ENA-78) and their receptors (CCR3, XCR1 and CXCR2) were validated by quantitative real-time PCR. Transcripts of eotaxin were more abundant in the CL at days 40–45 and 150–160 of pregnancy than in the cyclic CL (P < 0.01). In contrast, the mRNA expressions of lymphotactin, ENA-78 and XCR1 were lower in the CL of pregnancy (P <
0.05). Messenger RNAs of CCR3 and CXCR2 were similarly detected both in the cyclic and pregnant CL. Tissue protein levels of eotaxin were significantly higher in the CL at days 150–160 of pregnancy than in the CL at other stages, whereas the lymphotactin protein levels in the CL at days 20–25 of pregnancy were lower (P < 0.05). Immunohistochemical staining showed that CCR3 was expressed in the luteal cells and that XCR1 was expressed in both the luteal cells and endothelial cells. Collectively, the different gene expression profiles may contribute to functional differences between the cyclic and pregnant CL, and chemokines including eotaxin and lymphotactin may regulate CL function during pregnancy in cows.
Journal of Reproduction and Development 11/2014; 61(1). DOI:10.1262/jrd.2014-101 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelin (EDN) is a possible regulating factor of oviductal motility, which is important for the transport of gametes and embryo. To clarify the factors that control the secretion of EDN in the bovine oviduct, the expression of EDNs, EDN-converting enzymes (ECEs) and EDN receptors (EDNRs) were investigated. All isoforms of EDN (EDN1-3), ECE (ECE1 and ECE2) and EDNR (EDNRA and EDNRB) were immunolocalised in the epithelial cells of the ampulla and the isthmus. EDNRs were also immunolocalised in smooth-muscle cells. The mRNA expression of EDN2 and ECE2 was higher in cultured ampullary oviductal epithelial cells than in isthmic cells. The expression of EDN1, EDN2 and ECE2 in the ampullary tissue was highest on the day of ovulation. Oestradiol-17? increased EDN2 and ECE1 expression, while progesterone increased only ECE1 expression in cultured ampullary epithelial cells. These results indicate that EDNs are produced by epithelial cells and their target site is smooth-muscle and epithelial cells, and suggest that ovarian steroids are regulators of endothelin synthesis in ampullary oviductal epithelial cells.
Reproduction Fertility and Development 11/2014; DOI:10.1071/RD14076 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In many mammals, endometrial cells are remodeled by apoptosis and cell proliferation throughout the estrous cycle. Although apoptosis is known to be induced by various factors involving two major apoptotic pathways (the death receptor- and mitochondria-mediated pathways), how it is regulated in the bovine endometrium is unclear. We examined 1) the cyclic expressions of apoptosis-related factors, FAS, DcR3, BCL2 and BAX, in the bovine endometrium and 2) the effect of death ligands on the viability of, and FAS mRNA expression in, cultured bovine endometrial epithelial and stromal cells. FAS expression did not change during the estrous cycle, whereas DcR3 expression was higher at the mid and late luteal stages than at the early luteal and follicular stages. BCL2 expression was higher at the late luteal stage than at the early luteal and follicular stages, and the BAX/BCL2 ratio was higher at the early luteal stage than at the late luteal stage. Treatment or pretreatment with tumor necrosis factor-α (TNF) + interferon γ (IFNG) in combination with FAS ligand significantly reduced the viability of both epithelial and stromal cells. Furthermore, TNF + IFNG treatment significantly increased the expression of FAS mRNA in both types of endometrial cells. The overall results suggest that both extrinsic and intrinsic pathways are involved in remodeling the bovine endometrium throughout the estrous cycle, and that the death ligands produced by immune cells and the endometrium play important roles in inducing cyclic endometrial cell death.
[Show abstract][Hide abstract] ABSTRACT: Progesterone (P4) derivatives which are commonly used to block the cyclicity of domestic cats disturb the endocrine balance in the endometrium. The aims of this study were (i) to examine whether lipopolysaccharide (LPS) is responsible for enhancement of tumor necrosis factor-α (TNFα) secretion by the feline endometrial epithelial and stromal cells in vitro, (ii) to know whether immunolocalization of TNFα/TNFR1 and TNFR2 differs in cats at estrus or diestrus, receiving medroxyprogesterone acetate and suffering from pyometra, and (iii) to determine if TNFα-challenged prostaglandin secretion is stopped by prostaglandin synthases inhibitors. A total of 37 domestic adult cats in estrus or diestrus, receiving octane medroxyprogesterone or having clinical symptoms of pyometra, were enrolled in this study. The results obtained showed a distinct increase in LPS-challenged TNFα secretion in endometrial epithelial, but not stromal cells. TNFα augmented PG secretion was blocked by phospholipase A2 (PLA2) and cyclooxygeanase-2 (COX-2), but not by mitogen-activated protein kinase (MAPK) inhibitor. TNFα/TNFR1 and 2 protein expressions were limited mostly to the surface and glandular epithelium. TNFα/TNFRs protein was upregulated in the inflammatory uterus and hence may be involved in development of pathologic changes in the endometrial glands in cats receiving exogenous P4 as a hormonal contraceptive.
Mediators of Inflammation 06/2014; 2014(20):689280. DOI:10.1155/2014/689280 · 3.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Luteolysis is characterized by a reduction in progesterone (P4) production and tissue degeneration in the corpus luteum (CL). One of major events during luteolysis is luteal cell death. Galectin-3, a ubiquitously expressed protein involved in many cellular processes, serves as an anti-apoptotic and/or pro-apoptotic factor in various cell types. Although galectin-3 is detected in the bovine CL, its role remains unclear. The expression of galectin-3 in the bovine CL was higher at the regressed stage than at the other luteal stages. Galectin-3 was localized on luteal steroidogenic cells (LSCs). When cultured LSCs were exposed to prostaglandin F2alpha (PGF) for 48 h, the expression and secretion of galectin-3 increased. When the cultured LSCs were treated with galectin-3 for 24 h, cleaved caspase-3 expression was increased and the cell viability was decreased, whereas P4 production did not change. Beta 1 integrin, a target protein of galectin-3, was expressed in bovine CL and possessed glycans which galectin-3 binds. Furthermore, galectin-3 bound to glycans of luteal beta 1 integrin. The decreased cell viability of cultured LSCs by galectin-3 was suppressed by beta1 integrin antibody. The overall findings suggest that the secreted galectin-3 stimulated by PGF plays a role in structural luteolysis by binding to beta 1 integrin.
Biology of Reproduction 05/2014; 91(1). DOI:10.1095/biolreprod.114.119057 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Following bidirectional communication, the conceptus and the uterine epithelium must establish the proper cell-cell interaction, resulting in the progression of implantation processes. To clarify the mechanism of conceptus attachment to the uterine endometrium, we studied if vascular cell adhesion molecule (VCAM-1) was expressed in bovine conceptuses or endometrium during the peri-attachment period. Uterine VCAM-1 expression was minimal in day 17 (day 0 = day of estrus) cyclic and pregnant animals, but increased between days 20 and 22 of pregnancy. In the intercaruncular regions, VCAM-1 protein was localized in the luminal and glandular epithelia whereas in the caruncular regions, VCAM-1 protein was detected in the stroma and endothelia of the uterine endometrium. In cultured endometrial epithelial cells (EECs), VCAM-1 expression was up-regulated when treated with uterine flushings or growth factor, and further increased when EECs were cocultured with bovine trophoblast CT-1 cells. VCAM-1 expression in CT-1 cells was also up-regulated with the use of uterine flushings, and further increased when these cells were cocultured with EECs. Expression of VCAM-1 receptor, integrin alpha 4 (ITGA4) mRNA, increased significantly in day 22 conceptuses. In day 22 pregnant uteri, VCAM-1 protein was found in both EECs and conceptuses, but ITGA4 was localized only in trophoblasts. These observations indicate that in the bovine species, cell-cell interactions between conceptuses and uterine epithelial cells are required for sufficient VCAM-1 and ITGA4 expressions, and suggest that uterine VCAM-1 and conceptus ITGA4 play a role in the establishment of conceptus adhesion to the uterine endometrium.
[Show abstract][Hide abstract] ABSTRACT: Bovine primary uterine endometrial epithelial cells (EECs) are not ideal for long-term studies, because primary EECs lose hormone responsiveness quickly, and/or they tend to have a short life span. The aims of this study were to establish immortalized bovine EECs and to characterize these cells following long-term cultures. Immortalized bovine EECs were established by transfecting retroviral vectors encoding human papillomavirus (HPV) E6 and E7, and human telomerase reverse transcriptase (hTERT) genes. Established bovine immortalized EECs (imEECs) showed the same morphology as primary EECs, and could be grown without any apparent changes for over 60 passages. In addition, imEECs have maintained the features as EECs, exhibiting oxytocin (OT) and interferon tau (IFNT) responsiveness. Therefore, these imEECs, even after numbers of passages, could be used as an in vitro model to investigate cellular and molecular mechanisms, by which the uterine epithelium responds to IFNT stimulation, the event required for the maternal recognition of pregnancy in the bovine species.