Novel Approach for Evaluating Pregnancy-Associated Glycoprotein and Inflammation Markers during the Postpartum Period in Holstein Friesian Cows.

We evaluated the relationship between decreased pregnancy-associated glycoprotein (PAG) levels, inflammatory parameters (serum amyloid A [SAA] and milk amyloid A [MAA]), postpartum inflammatory conditions (mastitis, ketosis, and follicular cysts), and the FOXP3 gene. Nineteen Holstein-Friesian cows were included in this study. Up to approximately eight weeks after delivery, weekly health examinations were performed for mastitis and ketosis, and reproductive organ ultrasonography was performed. The decreasing PAG rate was negatively correlated with SAA concentration (r = -0.493, p = 0.032). Cows with mastitis exhibited a slower trend of PAG decrease (p = 0.095), and a greater percentage of these cows had MAA concentrations above 12 µg/mL (p = 0.074) compared with those without mastitis. A negative correlation, although nonsignificant (r = -0.263, p = 0.385), was observed between the day-open period and decreased PAG rate. The day-open period was correlated with the presence or absence of follicular cysts (p = 0.046). Four cows that developed follicular cysts were homozygous for the G allele of the FOXP3 gene related to repeat breeders. These results indicate a relationship between a decreased PAG rate and inflammatory status during the postpartum period. Thus, suppressing inflammation during the perinatal period may improve reproductive efficiency in the dairy industry.

Zearalenone interferes with the sperm-triggered inflammation in the bovine uterus in vitro: Negative impact on sperm motility and survival.

Zearalenone (ZEN)-contaminated diets induce detrimental effects on the bovine reproduction. Recently, we reported that active sperm induce pro-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. This study aimed to investigate the impact of presence of ZEN on the sperm-uterine crosstalk in vitro. BEECs monolayers were stimulated by ZEN (10, 100, and 1000 ng/mL) for 0, 3, 6, 12, or 24 h and gene expressions were analyzed by real-time PCR. Moreover, BEECs were pre-exposed to ZEN (10, 100, and 1000 ng/mL) for 24 h then, co-incubated with sperm for 6 h. Conditioned media (CM) from a sperm-BEECs co-culture, after pre-exposure to ZEN, were harvested and exploited to challenge either polymorphonuclear cells (PMNs) or sperm. Both PMNs phagocytic activity toward sperm and sperm motility parameters were then assessed. Results showed that ZEN alone induced pro-inflammatory responses in BEECs through the induction of mRNA expressions of pro-inflammatory cytokines (TNFA and IL1B) and PGES1 at different time points. Pre-exposure of BEECs to ZEN, amplified the sperm-triggered upregulation of pro-inflammatory cytokines (TNFA and IL1B) and chemokine IL8 mRNA abundance in BEECs. Sperm-BEECs conditioned media, primed by ZEN, stimulated the PMNs phagocytosis for sperm whereas suppressed sperm motility parameters. Taken together, these findings indicate that the presence of ZEN augments the pro-inflammatory cascade triggered by sperm in BEECs, provokes PMNs phagocytosis for sperm, and reduces sperm motility parameters. Such immunological reactions may create a hostile environment for sperm competence and survival in the bovine uterus, thus impair fertility.

Comparative Effects of Different Dosages of hCG on Follicular Development in Postpartum Dairy Cows With Cystic Ovarian Follicles.

The objective of this study was to determine the effects of different intramuscular dosages of human chorionic gonadotropin (hCG) on ovarian follicular development of dairy cows diagnosed with refractory cystic ovarian follicles (COFs). Cows diagnosed with COFs (≥25 mm in diameter) were allocated to four treatment groups: hCG-1 (n = 3), a single dose of 4,500 IU on day 1; hCG-2 (n = 3), 2,250 IU on days 1 and 3; hCG-3 (n = 3), 1,500 IU on days 1, 3, and 5; and hCG-C (n = 3) received saline on day 1. Blood sampling and ovarian ultrasonographic (US) examinations were performed on days 1, 3, 5, 7, and 14. A progesterone (P4) value < 1 ng/ml was used as an indicator of absence of a functional CL. A significant increase (P < 0.05) in the number of follicles < 4 mm in diameter was observed in the hCG-2 group on day 5. Additionally, there was a significant difference in the number of follicles < 4 mm (P < 0.05) between both the hCG-2 and hCG-3 groups compared to the hCG-C group on day 5, and a tendency (P = 0.08) toward a difference in the number of 5-9 mm follicles in groups hCG-3, hCG-2, and hCG-1, compared with the hCG-C group on day 7. The proportion of cows on days 7 and 14 with P4 > 1 ng/ml was 100% (3/3) and 100% (3/3) in group hCG-1; 100% (3/3) and 67% (2/3) in group hCG-2; 67% (2/3) and 100% (3/3) in group hCG-3; and 33% (1/3) and 33% (1/3) in group hCG-C, respectively. Strong tendencies of P4 increases in group hCG-1 (P = 0.054) and hCG-2 (P = 0.051) were measured after hCG administration. Additionally, P4 values tended to be higher (P = 0.07) for group hCG-1 compared to group hCG-C on day 5. The preliminary findings of this study suggest that multiple smaller doses of hCG might be equally effective as a single large dose of hCG in modulating ovarian follicular development in dairy cows with COFs.

Zearalenone (ZEN) disrupts the anti-inflammatory response of bovine oviductal epithelial cells to sperm in vitro.

Dietary contamination by Zearalenone (ZEN) has a detrimental effect on bovine fertility. Recently, we showed a novel anti-inflammatory response of bovine oviductal epithelial cells (BOEC) to active sperm cells in vitro. The aim of the present study was to investigate the effect of ZEN exposure of BOEC on the immune-related cytokine expression in response to bovine sperm. At concentrations of 100 and 1000ng/mL, ZEN induced the expression of TNF and IL1B (pro-inflammatory cytokines) as well as IL8 (chemokine) in BOEC in a dose-dependent manner. Furthermore, ZEN induced PTGES expression and PGE2 secretion in BOEC. Sperm co-culture induced an anti-inflammatory response in BOEC with upregulation of TGFB, secretion of PGE2 and downregulation of TNF. Most importantly, ZEN at 1-1000ng/mL eliminated the response of BOEC to sperm. Estradiol-17ß (5ng/mL) treatment did not produce the same effects as ZEN, suggesting that the response of BOEC to ZEN is, at least in part, not mediated by estrogen receptors. Taken together, ZEN can produce inflammatory effects on BOEC by stimulating the expressions of pro-inflammatory cytokines and disrupt the normal interaction between sperm and BOEC at the level of cytokine expressions and PGE2 production. Thus, exposure of the bovine oviduct to ZEN may negatively affect sperm survival and reduce fertility.

Gas Chromatography-Mass Spectrometry for Metabolite Profiling of Japanese Black Cattle Naturally Contaminated with Zearalenone and Sterigmatocystin.

The objective of this study was to evaluate the metabolic profile of cattle fed with or without zearalenone (ZEN) and sterigmatocystin (STC)-contaminated diets using a gas chromatography-mass spectrometry metabolomics approach. Urinary samples were collected from individual animals (n = 6 per herd) from fattening female Japanese Black (JB) cattle herds (23 months old, 550-600 kg). Herd 1 had persistently high urinary ZEN and STC concentrations due to the presence of contaminated rice straw. Herd 2, the second female JB fattening herd (23 months old, 550-600 kg), received the same dietary feed as Herd 1, with non-contaminated rice straw. Urine samples were collected from Herd 1, two weeks after the contaminated rice straw was replaced with uncontaminated rice straw (Herd 1N). Identified metabolites were subjected to principal component analysis (PCA) and ANOVA. The PCA revealed that the effects on cattle metabolites depended on ZEN and STC concentrations. The contamination of cattle feed with multiple mycotoxins may alter systemic metabolic processes, including metabolites associated with ATP generation, amino acids, glycine-conjugates, organic acids, and purine bases. The results obtained from Herd 1N indicate that a two-week remedy period was not sufficient to improve the levels of urinary metabolites, suggesting that chronic contamination with mycotoxins may have long-term harmful effects on the systemic metabolism of cattle.

Lack of epithelial PPARγ causes cystic adenomatoid malformations in mouse fetal lung.

Peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in lipid and glucose metabolism. In this study, the function of PPARγ on lung development was investigated. Lung-specific Pparg conditional knockout mice (PpargΔLuEpC) were developed using Cre-Lox system. PpargΔLuEpC mice showed abnormal lung development with enlarged airspaces and followed by increase of apoptotic cells at E14.5 to E18.5. Gene analysis revealed that expression of Pmaip1, a gene related to apoptosis, was significantly increased while expression of Retnla, a gene related to anti-apoptosis, was dramatically decreased in the fetal lung (E14.5) of PpargΔLuEpC mice. In addition, expression of Pthlh, a gene phenotypically expressed in the congenital cystic adenomatoid malformation (CCAM), was increased at E14.5 to E18.5 in the lung of PpargΔLuEpC mice. Cell culture studies revealed that PPARγ could bind to promoter region of Pthlh gene as a repressor in the immortalized mouse lung epithelial cell line MLE-15. Surprisingly, phenotypic changes in MLE-15-shPparg cells, stably transfected with shPparg plasmid, were similar to the PpargΔLuEpC mice model. In addition, MLE-15-shPparg cells were easily detached from the cultured plate when cold phosphate buffered saline was applied. Furthermore, expression of Cdh1, a gene related to cell adhesion, was significantly reduced in the MLE-15-shPparg cells. Taken together, PPARγ may play an important role in fetal lung development via alveolar cell-to-cell adhesion system.

Detection of zearalenone and its metabolites in naturally contaminated follicular fluids by using LC/MS/MS and in vitro effects of zearalenone on oocyte maturation in cattle.

Zearalenone (Zen) and its metabolites are estrogenic and may be important factors involved in reproductive disorders in domestic animals. We aimed to (1) simultaneously detect Zen and its metabolites in bovine follicular fluids (FFs) by liquid chromatography-tandem mass spectrometry and (2) examine the in vitro effects of Zen on bovine oocytes. Zen and its metabolites were detected in 6 of 32 normal follicles and 7 of 20 cystic follicles. Bovine oocytes were cultured in a maturation media containing various Zen concentrations (0 [control], 1, 10, 100, and 1000microg/L), fertilized, and cultured further. Maturation rates decreased dose-dependently. Further, maturation of 62 (50%) of 124 oocytes examined in the 1000-microg/L group was arrested in metaphase I, without affecting the fertilization rate. Blastocyst-formation rates did not significantly differ among the groups. Zen and its metabolites were detectable in bovine FFs. High Zen concentration may adversely affect meiotic competence but not the fertilization and development rates.

Luteal lifespan and fertility after estrus synchronization in goats.

The present experiment aims to examine the efficiency of estrus synchronization using progesterone and equine chorionic gonadotrophin (eCG) and to look at luteal function. During the non-breeding and breeding season, 5 adult female Korean native goats were injected intramuscularly with 2.5 ml of physiological saline as the control. A progesterone impregnated intravaginal sponge was then kept in the same goats for 10 days followed, after a week, by an intramuscular injection of 500 IU eCG. Five adult female Nubian goats were mated with a fertile buck during the non-breeding season. During the non-breeding season 2 of the 5 goats showed a normal estrous cycle (ranging from 18 to 21 days) and 3 a short estrous cycle (ranging from 3 to 6 days). During the breeding season the equivalent figures were 1 and 2. The major axes of the corpus luteum (CL) were measured by means of calipers built into the ultrasonography system, and the concentrations of plasma progesterone (P(4)) were determined by double antibody radioimmunoassay. The mean major axes of the CL in goats showing the short cycle (6.1 +/- 0.5 mm) was significantly smaller than in those showing the normal cycle (8.9 +/- 0.5 mm; p < 0.01) and also the value of P(4) in goats showing the short cycle (4.2 +/- 2.1 ng/ml) was significantly lower than for those showing the normal cycle (10.3 +/- 4.3 ng/ml; p < 0.05) at day 3 following ovulation. Three out of 5 Nubian goats became pregnant but only one goat carried to full term. The present experiment indicated that a combination of progesterone and eCG was effective in inducing estrus, although it resulted in a high incidence of short luteal lifespan. The low kidding rate and high incidence of embryonic loss may be due to the instability of the luteal lifespan.

Peroxisome proliferator-activated receptor alpha regulates a microRNA-mediated signaling cascade responsible for hepatocellular proliferation.

Activation of peroxisome proliferator-activated receptor alpha (PPARalpha) leads to hepatocellular proliferation and liver carcinomas. The early events mediating these effects are unknown. A novel mechanism by which PPARalpha regulates gene expression and hepatocellular proliferation was uncovered. MicroRNA (miRNA) expression profiling demonstrated that activated PPARalpha was a major regulator of hepatic miRNA expression. Of particular interest, let-7C, an miRNA important in cell growth, was inhibited following 4-h treatment and 2-week and 11-month sustained treatment with the potent PPARalpha agonist Wy-14,643 in wild-type mice. let-7C was shown to target c-myc via direct interaction with the 3' untranslated region of c-myc. The PPARalpha-mediated induction of c-myc via let-7C subsequently increased expression of the oncogenic mir-17-92 cluster; these events did not occur in Pparalpha-null mice. Overexpression of let-7C decreased c-myc and mir-17 and suppressed the growth of Hepa-1 cells. Furthermore, using the human PPARalpha-expressing mouse model, which is responsive to Wy-14,643 effects on beta-oxidation and serum triglycerides but resistant to hepatocellular proliferation and tumorigenesis, we demonstrated a critical role for let-7C in liver oncogenesis. Wy-14,643 treatment did not inhibit let-7C or induce c-myc and mir-17 expression. These observations reveal a let-7C signaling cascade critical for PPARalpha agonist-induced liver proliferation and tumorigenesis.

Sterol Regulatory Element-Binding Protein-2 modulates human brain acyl-CoA hydrolase gene transcription.

The brain shows high catalyzing activity during hydrolysis of long-chain acyl-CoAs into fatty acids and CoA-SH. Brain acyl-CoA hydrolase (BACH) is responsible for most of the long-chain acyl-CoA hydrolyzing activity in the brain and is localized exclusively in neurons. We analyzed the human BACH gene promoter, focusing on transcriptional regulation by Sterol Regulatory Element-Binding Protein-2 (SREBP-2), which is a transcription factor that activates genes involved in cholesterol biosynthesis and uptake. When the nuclear form of SREBP-2 gene was transfected into human neuroblastoma cells, transcription of a BACH gene promoter-luciferase reporter gene was activated through a sterol regulatory element (SRE) motif. Moreover, a gel shift assay demonstrated that SREBP-2 specifically bound to the SRE motif. These results suggest that transcription of the BACH gene is activated by SREBP-2. This study also provides insights into BACH function in the interaction between the metabolism of acyl-CoAs and cholesterol in neurons.

A 50-kDa isoform of mouse brain acyl-CoA hydrolase: expression and molecular properties.

Brain acyl-CoA hydrolase (BACH) is responsible for most of the long-chain acyl-CoA hydrolyzing activity in the brain and is localized exclusively in neurons. There are two BACH isoforms: the major isoform, a 43-kDa BACH, and a lesser isoform, a 50-kDa BACH. In our previous work [Brain Res. Mol. Brain Res. 98 (2002) 81], a possibility was raised that these BACH isoforms might be generated from a single mRNA species via a mechanism of alternative use of translation start sites. However, the results obtained in the current study indicated that the 43-kDa BACH and 50-kDa BACH are not generated from a single mRNA species, but from distinct mRNA species transcribed by alternative use of transcription start sites. The molecular properties of the 50-kDa BACH were compared to those of the 43-kDa BACH. Palmitoyl-CoA hydrolase activity and protein stability were almost the same between both BACH isoforms. In addition, both 43-kDa BACH and 50-kDa BACH that were fused to green fluorescent protein showed cytosolic distribution. These results suggest that the 50-kDa BACH plays a similar role as the 43-kDa BACH. Therefore, since the 43-kDa BACH is expressed at higher levels than 50-kDa BACH, the 43-kDa BACH should largely contribute to understanding the physiological functions of the BACH gene in neurons.

Inducible expression of long-chain acyl-CoA hydrolase gene in cell cultures.

A long-chain acyl-CoA hydrolase, BACH, is markedly distributed in the brain and localized in neurons. However, the physiological significance of BACH is unclear. To study the gene function, we expressed the mouse BACH gene in C3H 10T1/2 fibroblastic cells using a mifepristone (RU486)-inducible gene expression system. A cell clone, 10T-S6/44, was generated by stable transfection of two plasmids encoding a mifepristone-dependent transactivator and an inducible transgene product, BACH with a C-terminal MYC-tag (BACH-MYC). The transgene expression in the 10T-S6/44 cells was tightly regulated by mifepristone. Induction of BACH-MYC and an increase in palmitoyl-CoA hydrolase activity were observed in the cells treated with 3 x 10(-11) M mifepristone and reached maximal levels at a concentration of 1 x 10(-9) M for 48 h. The growth rate of cells showing the maximal induction of BACH-MYC was reduced, whereas phospholipid synthesis was unchanged. These results suggested that BACH affects specific cellular systems and functions, but not all acyl-CoA-utilizing processes.

Effects of LH and PGF2alpha in equine dominant follicles observed by MDS.

Microdialysis System (MDS) is a novel technique used for investigation of molecule secretion between different cell populations. Local hormonal secretion at follicular wall has been still unclear. This MDS study was used to determine progesterone (P4), androstenedione (A4), estradiol-17beta (E2) and Prostaglandin F2alpha (PGF2alpha) release in mare pre-ovulatory follicles. Follicles larger than 30 mm were isolated from the ovary and follicular fluid aspirated for hormone assay. Follicular fluid collected from small, middle and large follicles were analyzed by EIA. The concentrations of P4 and PGF2alpha were similar among the different sizes of follicles. The release of A4 was observed in middle and large follicles. E2 concentration was observed in middle follicles and was higher in large follicles compared with middle follicles. Follicular wall was cut and incubated for MDS and when LH was infused, there was an increase in P4 and A4 release. PGF2alpha release was considerably high after LH infusion compared to the control group. Infusion of PGF2alpha increased P4 and A4 release but there was no change in E2 release. This results suggest that in pre-ovulatory follicles, LH stimulates theca interna cells and also PGF2alpha seemed to have a mediator role to induce steroid hormone production and luteinization of follicular cells. The nature of the mechanisms involved in selection of large follicles is still a perplexing research problem in reproduction.