Toru Takahashi

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 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.

Pregnancy prediction on the day of embryo transfer (Day 7) and Day 14 by measuring luteal blood flow in dairy cows

This study aimed to assess the suitability of luteal blood flow analyses measured by color Doppler ultrasonography (CDUS), to predict pregnancy at pre- and post-embryo transfer (ET) in dairy cows, and to compare with the established criterion like luteal size and plasma progesterone (P4) concentrations. Lactating Holstein cows (nxa0=xa065) with spontaneous (nxa0=xa034) or synchronized estrus (nxa0=xa031) were examined. Cows with a CL greater than or equal to 20xa0mm in diameter (nxa0=xa058) received embryo transfer on Day 7 (Day 0xa0=xa0estrus). Brightness mode images were captured for calculation of the CL area, luteal cavity area, and dominant follicle area on Days 3, 5, 7, and 14. Color Doppler ultrasonography examinations were conducted to determine the blood flow area (BFA) within the CL at the maximum diameter and the time-averaged maximum velocity (TAMV) of the base of the spiral artery on the same days. Plasma P4 concentrations were determined from blood samples collected at each ultrasound examination. Pregnancy was diagnosed by an ultrasound on Day 30. There was no significant difference in the proportion of cows received embryo (91.2% vs. 87.1%, Pxa0=xa00.70) and pregnancy rate (58.1% vs. 59.3%, Pxa0=xa01.00) between the spontaneous estrus and synchronized groups. The BFA values of the pregnant group (nxa0=xa034) were approximately 1.42 and 1.54 times higher than those of the nonpregnant group (nxa0=xa024) on Days 7 (0.54xa0±xa00.04xa0cm(2) vs. 0.38xa0±xa00.02xa0cm(2); Pxa0<xa00.01) and 14 (0.80xa0±xa00.23xa0cm(2) vs. 0.52xa0±xa00.22xa0cm(2); Pxa0<xa00.01), respectively. The TAMV of the pregnant group was approximately 1.45 times higher than that of the nonpregnant group on Day 14 (57.8xa0±xa03.5xa0cm/s vs. 40.0xa0±xa03.3xa0cm/s; Pxa0<xa00.01). However, no differences were found in the CL area, CL tissue area, dominant follicle area, and plasma P4 concentrations among these groups. In addition, the best logistic regression model to predict pregnancy included scores for BFA on Day 7, BFA and TAMV on Day 14. Setting the cutoff value of BFA at 0.43xa0cm(2) yielded the highest sensitivity (79.4%) and specificity (75.0%) on Day 7, indicating the effectiveness of using BFA data for predicting pregnancy on Day 7. Furthermore, setting the cutoff value at one obtained from a sample with BFA 0.63xa0cm(2) and TAMV 50.60xa0cm/s yielded the highest sensitivity (85.3%) and specificity (91.7%) on Day 14. In conclusion, the evaluations of BFA on Day 7, and paired BFA and TAMV on Day 14 represent reliable predictors of pregnancy in the cow.

Cleaved bovine prolactin-related protein-I stimulates vascular endothelial cell proliferation.

Prolactin-related protein-I (PRP1) is a member of a non-classical prolactin (PRL)/growth hormone family in cattle. However, its function is still unknown. PRL, when cleaved by cathepsin D and matrix metalloproteinases (MMPs), resulted in cleaved N-terminal 16kDa fragments (16K-PRL) that have antiangiogenetic properties in human and rodents. We examined the possibility of similar activity of bovine PRP1. PRP1 (normally 33kDa) was cleaved by cathepsins (CTSs), MMPs, and bovine cotyledonary-conditioned medium (BCCM), and generated mainly 26kDa N-terminal fragments. Two specific enzyme families, CTSs and MMPs cleaved intact PRP1, and BCCM also contained PRP1 cleavage activity. Bioactivity for pro- or anti-angiogenesis of the cleaved PRP1 was examined in a cell proliferation assay using bovine brain vascular endothelial cells. The cleaved PRP1 proliferated the endothelial cells in vitro. The endothelial cell proliferation activity of cleaved PRP1 may be shared in specific bovine placentomal angiogenesis.

A predictive threshold value for the diagnosis of early pregnancy in cows using interferon-stimulated genes in granulocytes

Interferon tau plays an important role in establishing bovine pregnancy. Interferon-stimulated genes (ISGs) have been examined to identify a suitable indicator for the diagnosis of early gestation in cows. Although ISGs can be specifically detected in peripheral white blood cells during early gestation, its reliability remains to be validated. In the current study, a predictive threshold level of ISGs to determine pregnancy in cows during Days 20-22 of gestation was verified by analyzing the expression of ISGs in granulocytes and peripheral blood leucocytes (a total of 57 cows were used, 28 of which were pregnant and 29 were non-pregnant). Four genes, interferon-stimulated gene 15 ubiquitin-like modifier (ISG15), MX dynamin like GTPase (MX) 1, MX2, and 2-5-oligoadenylate synthetase 1 (OAS1), were analyzed via quantitative RT-PCR and a receiver operating characteristic (ROC) curve was produced to visualize diagnostic accuracy measures. The expression values of the four ISGs during the estrous cycle (100 collection points from 65 cattle) were used to determine a pregnancy prediction cutoff value. Pregnancy status was determined using these cutoff values and then confirmed by ultrasonography. ROC analysis was then applied to confirm the accuracy of the pregnancy statuses (positive and negative) statistically. The statistical evaluation of the diagnostic accuracy measurements suggested that the average values of ISG15 and MX2 in granulocytes were reliable indicators of pregnancy within the three weeks after insemination with 80% accuracy. Average ISG15 and MX2 levels during the estrous cycle were more reliable biomarkers for the prediction of gestation. They predicted negative and positive pregnancies efficiently within three weeks after artificial insemination.

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.

Different prostaglandin F2 α secretion in response to oxytocin injection between pregnant and non-pregnant cows: effect of the day of oxytocin challenge test for determining the difference

The present study was conducted to determine the difference in plasma prostaglandin F2 α metabolite concentrations following oxytocin (OT) challenge between pregnant and non-pregnant cows. Experiment 1: cows were subjected to the OT challenge test on days 12, 14 or 16 (day of estrusxa0=xa0day 0) with or without prior insemination and plasma 13,14-dihydro-15-keto prostaglandin F2 α (PGFM) concentrations were measured from -30 to 180xa0min after OT injection. On day 16, the increment of plasma PGFM concentrations in response to OT injection was significantly smaller in pregnant than that in cyclic cows. On days 12 and 14, there was little OT-induced PGFM secretion and no difference in PGFM increase between the pregnant and cyclic cows. Experiment 2: cows were inseminated on day 0 and subjected to the OT challenge test on day 16. Cows were classified into non-pregnant/early embryonic death (NP/EED), late embryonic death (LED) and pregnant (PREG) groups. The increment of PGFM concentrations in response to OT injection was less in both PREG and LED groups than that in the NP/EED group. In conclusion, plasma PGFM secretion induced by OT is suggested as the base of pregnancy diagnosis prior to returning estrus in cows.

Evaluation of interferon-stimulated genes in peripheral blood granulocytes as sensitive responders to bovine early conceptus signals

Early detection of gestation is important in the bovine industry. New methods have been developed to detect gene expression in leucocytes induced by interferon-tau (IFNT) as gestation biomarkers. However, it is debatable which blood cell is suitable for detecting gene expression. This study was aimed at confirming whether granulocytes respond to IFNT specifically. Granulocytes and mononuclear cells (MNCs) from cows, and several types of bovine cultured cells, were treated with recombinant (r) IFNT and gene expression was analysed by quantitative real-time reverse transcriptase (RT)-PCR and microarray analysis. Expression levels of IFN receptors (R1 and R2) were approximately 30- to 900-fold higher in granulocytes than in other cultured cells, and 1.5- to 2.5-fold higher in MNCs than in granulocytes. Microarray analysis following a 2h recombinant IFNT (rIFNT) treatment revealed expression changes for 900 genes in granulocytes. Genes with expression changes included known IFN-stimulated genes (ISGs; ISG15, OAS1, MX1, and MX2). Eighteen genes were selected following granulocyte microarray analysis and their expression changes were confirmed in early gestation, which revealed that nine genes had significantly higher expression levels in pregnant than in non-pregnant animals. In conclusion, granulocytes specifically responded to rIFNT treatment and the resulting gene expression changes correlated with those in vivo. Microarray analysis indicated that various genes showed expression changes in rIFNT-treated granulocytes, which may result in the identification of alternate candidate genes for the early detection of gestation. These results strongly indicate that gene expression in granulocytes is a suitable tool to determine pregnancy status.

Administration of gonadotropin-releasing hormone agonist on day 5 increases luteal blood flow and improves pregnancy prediction accuracy on day 14 in recipient holstein cows

This study assessed the effects of gonadotropin-releasing hormone (GnRH) treatment on Day 5 (Day 0 = estrus) on luteal blood flow and accuracy of pregnancy prediction in recipient cows. On Day 5, 120 lactating Holstein cows were randomly assigned to a control group (n = 63) or GnRH group treated with 100 μg of GnRH agonist (n = 57). On Days 3, 5, 7, and 14, each cow underwent ultrasound examination to measure the blood flow area (BFA) and time-averaged maximum velocity (TAMV) at the spiral arteries at the base of the corpus luteum using color Doppler ultrasonography. Cows with a corpus luteum diameter ≥ 20 mm (n = 120) received embryo transfers on Day 7. The BFA values in the GnRH group were significantly higher than those in the control group on Days 7 and 14. TAMV did not differ between these groups. According to receiver operating characteristic analyses to predict pregnancy, a BFA cutoff of 0.52 cm2 yielded the highest sensitivity (83.3%) and specificity (90.5%) on Day 7, and BFA and TAMV values of 0.94 cm2 and 44.93 cm/s, respectively, yielded the highest sensitivity (97.1%) and specificity (100%) on Day 14 in the GnRH group. The areas under the curve for the paired BFA and TAMV in the GnRH group were 0.058 higher than those in the control group (0.996 and 0.938, respectively; P < 0.05). In conclusion, GnRH treatment on Day 5 increased the luteal BFA in recipient cows on Days 7 and 14, and improved the accuracy of pregnancy prediction on Day 14.

Development of a programmable piggyback syringe pump and four-times-a-day injection regimen for superovulation in non-lactating Holstein cows

The objectives of the present study were to develop a programmable piggyback syringe pump for bovine superovulation and to evaluate the effects of a four-times-a-day injection regimen using the pump. Non-lactating Holstein cows were treated with a total of 30 armour units of porcine FSH by injection four times a day with the pump (study, n = 9) or injection twice a day manually (control, n = 9) for four consecutive days from D10 of the estrous cycle. The pump-driven program successfully induced superovulation in all cows tested. The numbers of small (3– < 5 mm in diameter) and large (≥ 10 mm in diameter) follicles were greater in the study group on D11-13 and D14, respectively. There were fewer unovulated follicles detected on D21 (7 days after estrus) in the study group than in the control group (1.2 ± 0.4 and 3.2 ± 0.6, respectively).