Isolation and propagation of bovine blood-derived macrophages using a mixed culture with bovine endothelial B46 cells.

Macrophages are innate immune cells with multiple functions such as phagocytosis, cytokine production, and antigen presentation. Since macrophages play critical roles in some bacterial infectious diseases in cattle, including tuberculosis, paratuberculosis, and brucellosis, the in vitro culturing of bovine macrophages is useful for evaluating host-pathogen interactions at the cellular and molecular levels. We have previously reported the establishment of two immortalized bovine liver sinusoidal cell lines, endothelial B46 cells and myofibroblast-like A26 cells (Cell Biology International, 40, 1372-1379, 2016). In this study, we investigated the use of these cell lines as feeder cells that support the proliferation of bovine blood-derived macrophages (BBMs). Notably, the B46 cell line efficiently acts as feeder cells for the propagation of BBMs. Compared with primary cultured vascular endothelial cells, the infinite proliferation ability of B46 cells is more beneficial for preparing confluent feeder layers. In conclusion, this study provides a simple and efficient protocol for the isolation and propagation of BBMs using a primary mixed culture of bovine whole blood with B46 feeder cells. Isolated BBMs are expected to be useful for developing in vitro models for studying the interactions between bovine pathogens and host immune cells.

Direct effects of linoleic and linolenic acids on bovine uterine function using in vivo and in vitro studies.

The aim of the present study was to evaluate the effects of continuous administration of linoleic acid or linolenic acid into the intra-uterine horn, ipsilateral to the corpus luteum, on the duration of the estrous cycle and plasma progesterone (P4) concentration. The effects of linoleic and linolenic acids on bovine uterine and luteal functions were also studied using a tissue culture system. Intra-uterine administration of linoleic or linolenic acid (5 mg/10 ml of each per day) in cows, between days 12 and 21, resulted in a prolonged estrous cycle compared to the average duration of the last one to three estrous cycles before administration in each group (P < 0.05). Moreover, plasma P4 concentration in cows treated with linoleic or linolenic acid was high between days 19 and 21 (linoleic acid), or on day 20 (linolenic acid), compared to that of the control cows (saline administration; P < 0.05 or lower). Both linoleic (500 µg/ml) and linolenic (5 and 500 µg/ml) acids stimulated prostaglandin (PG) E2 but inhibited PGF2α production by cultured endometrial tissue (P < 0.01), while P4 production by cultured luteal tissue was not affected. These findings suggest that both linoleic and linolenic acids support luteal P4 production by regulating endometrial PG production and, subsequently, prolonging the duration of the estrous cycle in cows.

Steroidal but not embryonic regulation of mucin 1 expression in bovine endometrium.

In cow herd management, inadequate embryo implantation leads to pregnancy loss and causes severe economic losses. Thus, it is crucial to understand the molecular mechanisms underlying endometrial receptivity and subsequent embryo implantation. Transmembrane glycocalyx mucin 1 (MUC1) has a large and highly glycosylated extracellular domain known to inhibit embryo implantation via steric hindrance. The role of MUC1 in the bovine endometrium remains to be explored. Herein, we used simple but reliable in vivo and in vitro experiments to investigate the expression and regulation of MUC1 in the bovine endometrium. MUC1 gene expression was analyzed in endometrial epithelial cells collected by the cytobrush technique using reverse transcription-quantitative polymerase chain reaction. MUC1 protein expression was evaluated by immunohistochemical analysis of endometrial samples collected from slaughtered cows. We used an in vitro cell culture model to study the regulation of MUC1 expression by treating cells with sex steroidal hormones or co-culturing cells with a blastocyst. The results revealed that MUC1 was expressed and localized to the apical surface of luminal epithelial cells in the bovine endometrium. MUC1 expression disappeared during the luteal phase of the estrous cycle and during pregnancy. 17ß-estradiol induced MUC1 expression, whereas progesterone inhibited its increase and co-culturing with blastocysts did not affect the expression. A long postpartum interval is a known risk factor for reduced fertility, and MUC1 expression was higher in this compromised condition. Our results demonstrated the MUC1 regulation by steroid hormones in bovine endometrium for embryo implantation, and we observed a negative correlation between MUC1 expression and fertility.

Pregnancy-associated changes of peroxisome proliferator-activated receptor delta (PPARD) and cytochrome P450 family 21 subfamily A member 2 (CYP21A2) expression in the bovine corpus luteum.

We investigated gene expression profiles of the corpus luteum (CL) at the time of maternal recognition to evaluate the functional changes of the CL during early pregnancy in cows and help improve reproductive efficiency and avoid defective fetuses. Microarray analyses using a 15 K bovine oligo DNA microarray detected 30 differentially expressed genes and 266 differentially expressed genes (e.g., PPARD and CYP21A2) in the CL on pregnancy days 15 (P15) and 18 (P18), respectively, compared with the CL on day 15 (NP15) of non-pregnancy (n = 4 for each group). PPARD expression was the highest while the CYP21A2 expression was the lowest in P15 and P18 compared with that of NP15. These microarray results were validated by quantitative real-time PCR analysis. The addition of interferon-τ and supernatants derived from homogenized fetal trophoblast increased ISG15 and MX1 expressions in the cultured luteal tissue (P < 0.01), but did not affect PPARD and CYP21A2 expressions. PPARD expression in the luteal tissue was stimulated (P < 0.05) by GW0742, known as a selective PPARD agonist, and PPARD ligands (i.e., arachidonic, linoleic and linolenic acids). In contrast, CYP21A2 mRNA expression was not affected by both agonist and ligands. The concentration of prostaglandin (PG) E2 and PGF2α decreased after GW0742 stimulation and increased after arachidonic acid stimulation (P < 0.05). The addition of GW0742 and arachidonic acid increased progesterone (P4) concentration. Collectively, these findings suggest that high expression levels of PPARD and low expression levels of CYP21A2 in the CL during early pregnancy may support P4 production by bovine luteal cells.

Characterisation of bovine embryos following prolonged culture in embryonic stem cell medium containing leukaemia inhibitory factor.

In order to help elucidate the process of epiblast and trophoblast cell differentiation in bovine embryos invitro, we attempted to develop a suitable culture medium to allow extended embryo culture. Day 7 bovine blastocysts developed in conventional medium were cultured further in embryonic stem cell medium with or without leukaemia inhibitory factor (LIF) until Day 23. At Day 14, the expression of octamer-binding transcription factor 3/4 (OCT3/4) and VIMENTIN was significantly higher in embryos cultured with than without LIF, but embryonic disc formation was not observed. Although expression of SRY (sex determining region Y)-box 17 (SOX17) mRNA was significantly lower in Day 14 embryos cultured with and without LIF than in invivo embryos, hypoblast cells formed just inside the trophoblast cells of the invitro-cultured embryos. On Day 23, expression of placental lactogen (PL) and prolactin-related protein 1 (PRP1) was not affected by LIF in invitro-cultured embryos, levels of both genes were significantly lower in the invitro than invivo embryos. Similar to invivo embryos, binucleate cell clusters seen in Day 23invitro-cultured embryos were composed of PL-negative and -positive cells. These results suggest that our culture system partially reproduced the differentiation process of trophoblast cells invivo.

Involvement of interferon-tau in the induction of apoptotic, pyroptotic, and autophagic cell death-related signaling pathways in the bovine uterine endometrium during early pregnancy.

Interferon-tau (IFNT), a type I interferon (IFN), is known as pregnancy recognition signaling molecule secreted from the ruminant conceptus during the preimplantation period. Type I IFNs, such as IFN-alpha and IFN-beta, are known to activate cell-death pathways as well as induce apoptosis. In cows, induction of apoptosis with DNA fragmentation is induced by IFNT in cultured bovine endometrial epithelial cells. However, the status of cell-death pathways in the bovine endometrium during the preimplantation period still remains unclear. In the present study, we investigated the different cell-death pathways, including apoptosis, pyroptosis, and autophagy, in uterine tissue obtained from pregnant cows and in vitro cultured endometrial epithelial cells with IFNT stimulation. The expression of CASP7, 8, and FADD (apoptosis-related genes) was significantly higher in pregnant day 18 uterine tissue in comparison to non-pregnant day 18 tissue. The expression of CASP4, 11, and NLRP3 (pyroptosis-related genes) was significantly higher in the pregnant uterus in comparison to non-pregnant uterus. In contrast, autophagy-related genes were not affected by pregnancy. We also investigated the effect of IFNT on the expression of cell-death pathway-related genes, as well as DNA fragmentation in cultured endometrial epithelial cells. Similar to its effects in pregnant uterine tissue, IFNT affected the increase of apoptosis-related (CASP8) and pyroptosis-related genes (CASP11), but did not affect autophagy-related gene expression. IFNT also increased γH2AX-positive cells, which is a marker of DNA fragmentation. These results suggest that apoptosis- and pyroptosis-related genes are induced by IFNT in the pregnant bovine endometrial epithelial cells.

Differential gene expression profiling of endometrium during the mid-luteal phase of the estrous cycle between a repeat breeder (RB) and non-RB cows.

BACKGROUND: Repeat breeding directly affects reproductive efficiency in cattle due to an increase in services per conception and calving interval. This study aimed to investigate whether changes in endometrial gene expression profile are involved in repeat breeding in cows. Differential gene expression profiles of the endometrium were investigated during the mid-luteal phase of the estrous cycle between repeat breeder (RB) and non-RB cows using microarray analysis. METHODS: The caruncular (CAR) and intercaruncular (ICAR) endometrium of both ipsilateral and contralateral uterine horns to the corpus luteum were collected from RB (inseminated at least three times but not pregnant) and non-RB cows on Day 15 of the estrous cycle (4 cows/group). Global gene expression profiles of these endometrial samples were analyzed with a 15 K custom-made oligo-microarray for cattle. Immunohistochemistry was performed to investigate the cellular localization of proteins of three identified transcripts in the endometrium. RESULTS: Microarray analysis revealed that 405 and 397 genes were differentially expressed in the CAR and ICAR of the ipsilateral uterine horn of RB, respectively when compared with non-RB cows. In the contralateral uterine horn, 443 and 257 differentially expressed genes were identified in the CAR and ICAR of RB, respectively when compared with non-RB cows. Gene ontology analysis revealed that genes involved in development and morphogenesis were mainly up-regulated in the CAR of RB cows. In the ICAR of both the ipsilateral and contralateral uterine horns, genes related to the metabolic process were predominantly enriched in the RB cows when compared with non-RB cows. In the analysis of the whole uterus (combining the data above four endometrial compartments), RB cows showed up-regulation of 37 genes including PRSS2, GSTA3 and PIPOX and down-regulation of 39 genes including CHGA, KRT35 and THBS4 when compared with non-RB cows. Immunohistochemistry revealed that CHGA, GSTA3 and PRSS2 proteins were localized in luminal and glandular epithelial cells and stroma of the endometrium. CONCLUSION: The present study showed that endometrial gene expression profiles are different between RB and non-RB cows. The identified candidate endometrial genes and functions in each endometrial compartment may contribute to bovine reproductive performance.

Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy.

The aim of the present study was to determine the possible roles of chemokines in regulating bovine endometrial function during early pregnancy. The expression of six chemokines, including CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10, was higher in the endometrium at 15 and 18 days of pregnancy than at the same days in non-pregnant animals. Immunohistochemical staining showed that chemokine receptors (CCR1, CCR2, CCR3, and CXCR3) were expressed in the epithelial cells and glandular epithelial cells of the bovine endometrium as well as in the fetal trophoblast obtained from a cow on day 18 of pregnancy. The addition of interferon-τ (IFNT) to an endometrial tissue culture system increased CCL8 and CXCL10 expression in the tissues, but did not affect CCL2, CCL11, and CCL16 expression. CCL14 expression by these tissues was inhibited by IFNT. CCL16, but not other chemokines, clearly stimulated interferon-stimulated gene 15 (ISG15) and myxovirus-resistance gene 1 (MX1) expression in these tissues. Cyclooxygenase 2 (COX2) expression decreased after stimulation with CCL8 and CCL14, and oxytocin receptor (OTR) expression was decreased by CCL2, CCL8, CCL14, and CXCL10. Collectively, the expression of chemokine genes is increased in the endometrium during early pregnancy. These genes may contribute to the regulation of endometrial function by inhibiting COX2 and OTR expression, subsequently decreasing prostaglandin production and preventing luteolysis in cows.

Gene expression profiles in the bovine corpus luteum (CL) during the estrous cycle and pregnancy: possible roles of chemokines in regulating CL function during pregnancy.

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.

Comparison of the global gene expression profiles in the bovine endometrium between summer and autumn.

Heat stress compromises fertility during summer in dairy and beef cows by causing nutritional, physiological and reproductive damages. To examine the difference in endometrial conditions in cows between summer and autumn, gene expression profiles were compared using a 15 K bovine oligo DNA microarray. The trial was conducted in the summer (early in September) and autumn (mid-November) seasons of two consecutive years (2013-2014) in Morioka, Japan. Endometrial samples were collected from the cows using a biopsy technique. The expressions of 268 genes were significantly higher in the endometrium collected in summer than those collected in autumn, whereas the expressions of 369 genes were lower (P<0.05 or lower). Messenger RNA expressions of glycoprotein 2 (GP2), neurotensin (NTS),E-cadherin (CDH1) and heat shock 105kDa/110kDa protein 1 (HSPH1) were validated by quantitative real-time PCR. Transcripts of GP2 and NTS were more abundant in the endometrium from summer than in the endometrium from autumn (P < 0.05). In contrast, the mRNA expressions of CDH1 were lower (P < 0.05) and those of HSPH1 tended to be low (P = 0.09) in the endometrium from summer. Immunohistochemical staining showed that GP2, NTS and HSPH1 were expressed in the endometrial epithelial or glandular epithelial cells. The serum concentrations of NTS collected from the cows in summer were higher than those collected from cows in autumn (P < 0.05). Collectively, the different gene expression profiles may contribute to functional differences in the endometrium between summer and autumn, and the increases in GP2 and NTS may have a relationship with the endometrial deficiency that causes infertility of cows in summer.

Temporo-spatial expression of adrenomedullin and its receptors in the bovine placenta.

BACKGROUND: Adrenomedullin (AM) is a potent vasodilator peptide and is also involved in various physiological activities. In humans and rodents, AM is found in the uteroplacental unit and may be responsible for fetal development and maintenance of placental function. This study investigated 1) the mRNA expression patterns of AM and its receptor components (calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) 2 and RAMP3) during pregnancy and 2) mRNA and protein localization of AM, CRLR and RAMPs in the bovine placentome. METHODS: For real-time quantitative RT-PCR, bovine uteroplacental tissues were collected from Day 25, 60, 100, 150, 200 and 250 of gestation and separated into uterine caruncle (CAR), intercaruncular endometrium (ICAR), extra-embryonic membranes on Day 25 and cotyledonary villous after Day 60 (EEM-COT) and intercotyledonary chorion (ICOT). In situ hybridization and immunohistochemistry was performed to investigate the cellular localization of mRNA and protein of AM, CRLR, RAMP2 and RAMP3 in the placentome on Day 56, 150 and 230 of gestation and interplacentomal tissues on Day 56 of gestation. RESULTS: AM mRNA was highly expressed on Day 200 in EEM-COT, CAR and ICAR. CRLR mRNA was highly expressed on Day 60 in all portions. RAMP2 mRNA was also highly expressed on Day 60 in ICOT and ICAR. In EEM-COT, mRNA expression of CRLR and RAMP2 decreased from Day 150 to 250. RAMP3 mRNA was highly expressed on Day 150 in EEM-COT, ICOT and ICAR. A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells. In interplacentomal tissues, AM was detected in BNCs of fetal membrane and a small part of luminal epithelium, endothelial lineage of blood vessels and glandular epithelium of the endometrium. Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium. CONCLUSIONS: Our results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period. Locally produced AM in the BNCs may play a crucial role in regulation of placental vascular and cellular functions during pregnancy.

Differential expression of pro- and anti-angiogenic factors in the endometrium between repeat breeder and normally fertile cows.

In cattle, the establishment of appropriate endometrial vasculature during the estrous cycle is required for preparing a receptive endometrium. This study aimed to investigate 1) mRNA expression of potent pro- and anti-angiogenic factors, 2) protein localization of the anti-angiogenic factor thrombospondin (TSP), and 3) vascularity in the endometrium of repeat breeder (RB) and normally fertile (non-RB) cows. Caruncular and intercaruncular endometrium was collected from RB and non-RB cows during the luteal phase of the estrous cycle. RB cows had greater mRNA expression levels of TSP ligands (TSP1 and TSP2) and receptors (CD36 and CD47) than non-RB cows. Although the mRNA expression levels of most angiogenic factors did not change by repeat breeding, RB cows had greater mRNA expression of fibroblast growth factor receptor 1 (FGFR1), angiopoietin 1 (ANGPT1), and ANGPT2 and a less mRNA expression of vascular endothelial growth factor B (VEGFB) than non-RB cows. By immunohistochemistry, TSP1, TSP2, CD36, and CD47 were detected in the luminal epithelium, glandular epithelium, stromal cells, and blood vessels of the endometrium. Two indexes of vascularity, the number of blood vessels and the percentage of area stained positive for the von Willebrand factor, were lower in the endometrium of RB than in that of non-RB cows. These results demonstrate that RB cows have a greater expression of both ligands and receptors for the anti-angiogenic factor TSP and a reduced vascular distribution in the endometrium compared with non-RB cows, suggesting suppressed endometrial angiogenesis.

Possible roles of bone morphogenetic protein 4 in regulating endometrial function in cows.

This study investigated the expression dynamics of bone morphogenetic protein 4 (BMP4) and its receptors (BMPR1A, BMPR1B, and BMPR2) in bovine endometrium and examined the physiological function and regulatory mechanism of BMP4 expression. The messenger RNA (mRNA) expression of BMP4 and its receptors was detected in bovine endometrium of both ipsilateral (corpus luteum [CL]-side) and contralateral (non-CL-side) uterine horns during the estrous cycle and early pregnancy. BMP4 protein levels were higher in the endometrial tissues obtained from those cows in early pregnancy than in the estrous cycle. Immunohistochemical analysis showed that BMP4 and its receptors were localized in endometrial epithelial cells. The addition of BMP4 to cultured endometrial epithelial cells did not affect caspase-3/-8 mRNA expression, whereas it significantly inhibited cell proliferation. Both prostaglandin (PG) E2 and PGF2α concentrations in the culture supernatant were decreased when stimulated by BMP4. Furthermore, BMP4 mRNA expression was increased by stimulation with tumor necrosis factor-α (TNF) and interferon-γ (IFNG). In conclusion, BMP4 is produced in bovine endometrial epithelial cells and may contribute to the regulation of cell proliferation and suppression of PG secretion through autocrine or paracrine mechanisms. BMP4 expression in the bovine endometrium may be regulated by TNF and IFNG.

Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia.

BACKGROUND: SERPINs (serine protease inhibitors) regulate proteases involving fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation and apoptosis. This study aimed to investigate differentially expressed genes of members of the SERPIN superfamily between healthy and atretic follicles using a combination of microarray and quantitative real-time PCR (QPCR) analysis. In addition, we further determined mRNA and protein localization of identified SERPINs in estradiol (E2)-active and E2-inactive follicles by in situ hybridization and immunohistochemistry. METHODS: We performed microarray analysis of healthy (10.7 +/- 0.7 mm) and atretic (7.8 +/- 0.2 mm) follicles using a custom-made bovine oligonucleotide microarray to screen differentially expressed genes encoding SERPIN superfamily members between groups. The expression profiles of six identified SERPIN genes were further confirmed by QPCR analysis. In addition, mRNA and protein localization of four SERPINs was investigated in E2-active and E2-inactive follicles using in situ hybridization and immunohistochemistry. RESULTS: We have identified 11 SERPIN genes expressed in healthy and atretic follicles by microarray analysis. QPCR analysis confirmed that mRNA expression of four SERPINs (SERPINA5, SERPINB6, SERPINE2 and SERPINF2) was greater in healthy than in atretic follicles, while two SERPINs (SERPINE1 and SERPING1) had greater expression in atretic than in healthy follicles. In situ hybridization showed that SERPINA5, SERPINB6 and SERPINF2 mRNA were localized in GCs of E2-active follicles and weakly expressed in GCs of E2-inactive follicles. SERPING1 mRNA was localized in both GCs and the theca layer (TL) of E2-inactive follicles and a weak hybridization signal was also detected in both GCs and TL of E2-active follicles. Immunohistochemistry showed that SERPINA5, SERPINB6 and SERPINF2 were detected in GCs of E2-active and E2-inactive follicles. SERPING1 protein was localized in both GCs and the TL of E2-active and E2-inactive follicles. CONCLUSIONS: Our results demonstrate a characteristic expression of SERPIN superfamily member genes in bovine healthy and atretic follicles. The cell-type-and stage-specific expression of SERPINs may be associated with bovine follicular growth and atresia.

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries.

BACKGROUND: It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes. METHODS: Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR) and in situ hybridization. Immunohistochemical analysis was also performed. RESULTS: The expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry. CONCLUSIONS: Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles.

BACKGROUND: Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes. METHODS: Global gene expression profiles of F1 (10.7 +/- 0.7 mm) and F2 (7.8 +/- 0.2 mm) were analyzed by hierarchical cluster analysis and expression profiles of 16 representative genes were confirmed by QPCR analysis. In addition, localization of six identified transcripts was investigated in healthy and atretic follicles using in situ hybridization. The healthy or atretic condition of examined follicles was classified by progesterone and estradiol concentrations in follicular fluid. RESULTS: Hierarchical cluster analysis of microarray data classified the follicles into two clusters. Cluster A was composed of only F2 and was characterized by high expression of 31 genes including IGFBP5, whereas cluster B contained only F1 and predominantly expressed 45 genes including CYP19 and FSHR. QPCR analysis confirmed AMH, CYP19, FSHR, GPX3, PlGF, PLA2G1B, SCD and TRB2 were greater in F1 than F2, while CCL2, GADD45A, IGFBP5, PLAUR, SELP, SPP1, TIMP1 and TSP2 were greater in F2 than in F1. In situ hybridization showed that AMH and CYP19 were detected in granulosa cells (GC) of healthy as well as atretic follicles. PlGF was localized in GC and in the theca layer (TL) of healthy follicles. IGFBP5 was detected in both GC and TL of atretic follicles. GADD45A and TSP2 were localized in both GC and TL of atretic follicles, whereas healthy follicles expressed them only in GC. CONCLUSION: We demonstrated that global gene expression profiling of F1 and F2 clearly reflected a difference in their follicular status. Expression of stage-specific genes in follicles may be closely associated with their growth or atresia. Several genes identified in this study will provide intriguing candidates for the determination of follicular growth.

The chemokines CCL2 and CXCL10 produced by bovine endometrial epithelial cells induce migration of bovine B lymphocytes, contributing to transuterine transmission of BLV infection.

Bovine leukemia virus (BLV) causes a lymphoproliferative disease in cattle and is transmitted horizontally and vertically via infected lymphocytes. Although transplacental infection is considered the predominant route of vertical transmission of BLV, the molecular mechanisms of this process remain to be elucidated. Notably, how BLV passes through the blood-placental barrier remains unclear, given that BLV is transmitted primarily by cell-to-cell contact. One hypothesis is that B cell migration to the placenta may be induced by certain endometrium-expressed chemokines. To test this hypothesis, we performed an in vitro cell migration assay using bovine B cell lines and endometrial epithelial cells. Cell migration assays showed that two bovine B cell lines, BL2M3 and BL3.1 cells, were attracted to the supernatant of bovine endometrial epithelial cells (BEnEpCs). Quantitative real-time RT-PCR showed that expression levels of mRNAs encoding the chemokines CCL2 and CXCL10 were higher in BEnEpCs than in MDBK cells. Additionally, an inhibition assay using immune serum against CCL2 and CXCL10 showed suppression of migration of bovine B cell lines. A syncytium assay showed that cells expressing BLV envelope (Env) protein fused with BEnEpCs. Here we found that bovine B cells are attracted by chemokines produced in the endometrium and that cells expressing BLV Env protein fused with endometrium epithelial cells. These results explain part of the molecular mechanism of transplacental transmission during BLV infection, although further analysis will be required. Advances in these areas are expected to contribute to controlling the spread of BLV.

Temporal expression and localization of vascular endothelial growth factor family members in the bovine uterus during peri-implantation period.

The aim of this study was to determine endometrial mRNA expression patterns and uterine protein localizations of vascular endothelial growth factor (VEGF) ligands (VEGFA, VEGFB, VEGFC, and VEGFD) and their receptors (VEGFR1, soluble VEGFR1 (sVEGFR1), VEGFR2, and VEGFR3) during the peri-implantation period in cows. The number of blood and lymphatic vessels in the bovine uterus was also investigated. Bovine uterine tissues were collected from pregnant animals on days 15, 18, and 27 after artificial insemination and from non-pregnant animals on days 15 and 18 of the estrous cycle (day 0 = day of estrus). The mRNA expression level of VEGFA, VEGFR1, sVEGFR1, and VEGFR3 were higher on day 18 than on day 15 in the non-pregnant group. On day 18, the levels of mRNA expression of these genes were higher in the non-pregnant group than in the pregnant group. VEGFB mRNA expression levels was higher on day 15 than on days 18 and 27 of gestation and was higher in the pregnant group than in the non-pregnant group on day 15. Using immunohistochemistry, VEGF ligands and their receptors were found in luminal epithelium, glandular epithelium, stroma, and blood vessels of the endometrium. In addition, VEGFA, VEGFD, and VEGFR3 were also detected in the uterine myometrium. In the pregnant group, the number of blood vessels in the endometrium increased from day 15 to 18 and was greater than that of the non-pregnant group on day 18. Our results demonstrate that the VEGF family is expressed and regulated in the bovine uterus during the peri-implantation period, which may be associated with uterine functions, including vascular remodeling in maternal recognition of pregnancy and implantation.

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle.

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.

Gene expression of CCL8 and CXCL10 in peripheral blood leukocytes during early pregnancy in cows.

BACKGROUND: The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes (PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination (AI). Based on the day of return of estrus, cows were divided into three groups, pregnant (n = 5), early embryonic mortality (EEM; n = 5) and late embryonic mortality (LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR. RESULTS: The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d (P < 0.05), whereas no significant changes were observed both in EEM and LEM cows. Interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance gene (MX) 1 and MX2 mRNA expression in PBLs increased from 14 to 18 d which was significant on 18 d of pregnant cows as well as in LEM cows (P < 0.05), but no changes were observed in EEM cows. To determine whether the expression of CCL8 and CXCL10 in PBLs was regulated by pregnancy-related substances or not, expression level was assessed after exposure to interferon-τ (IFNT) and CCL16. Monocytes, granulocytes and lymphocytes were obtained using density-gradient centrifugation and flow cytometry. The addition of IFNT (100 ng/mL) and CCL16 (100 ng/mL) to cultured PBLs increased the expression of CCL8 and CXCL10 mRNA (P < 0.05). The expression of ISG15, MX1 and MX2 mRNA in PBLs was also stimulated by IFNT and CCL16 (P < 0.05). CONCLUSIONS: The expression of CCL8 and CXCL10 genes increased in PBLs during early pregnancy. Since IFNT stimulated CCL8 and CXCL10 expression in cultured PBLs, the increase of CCL8 and CXCL10 might be pregnancy-dependent events. The expression of both CCL8 and CXCL10 in PBLs was stimulated by CCL16 as well as IFNT, suggesting a chemokine interaction that at least includes CCL8, CXCL10 and CCL16, and may play a role in regulating maternal recognition in cows.