Calponin Expression in Renal Tubulointerstitial Fibrosis Induced in Rats by Cisplatin

Renal tubulointerstitial fibrosis is the common feature of chronic renal failure, regardless of its etiology. Myofibroblasts play important roles in progression of the fibrosis and are characterized by expressions of various cytoskeletons such as vimentin, desmin and α-smooth muscle actin (α-SMA). To pursue the characteristics of the cells, we immunohistochemically investigated the relationship between calponin (a marker of terminal smooth muscles) expression and myofibroblasts in cisplatin-induced rat renal tubulointerstitial fibrosis. Calponin-expressing interstitial cells increased with fibrosis and reacted simultaneously to vimentin or α-SMA (a marker of well-differentiated myofibroblasts) but not desmin or Thy-1 (a marker of myofibroblasts at the early stage). The present study shows that calponin may be expressed transiently in relatively well-developed myofibroblasts in rat renal fibrosis. Calponin could become a marker for myofibroblast development in chronic renal toxicity in rats.     

Involvements of Estrogen Receptor,Proliferating Cell Nuclear Antigen and p53 in Endometrial Adenocarcinoma Development in Donryu Rats

Involvements of estrogen receptor (ER)α, proliferating cell nuclear antigen (PCNA) and p53 in the uterine carcinogenesis process in Donryu rats, a high yield strain of the uterine cancer were investigated immunohistochemically. ERα was expressed in atypical endometrial hyperplasia, accepted as a precancerous lesion of the uterine tumors, as well as well- and in moderately-differentiated endometrial adenocarcinomas, and the intensities of expression were similar to those in the luminal epithelial cells of the atrophic uterus at 15 months of age. The expression, however, was negative in the tumor cells of poorly differentiated type. Good growth of implanted grafts of the poorly-differentiated adenocarcinomas in both sexes with or without gonadectomy supported the estrogen independency of tumor progression to malignancy. PCNA labeling indices were increased with tumor development from atypical hyperplasia to adenocarcinoma. The tumor cells in poorly-differentiated adenocarcinomas were positive for p53 positive but negative for p21 expression, suggesting accumulation of mutated p53. These results indicate that the consistent ERα expression is involved in initiation and promotion steps of uterine carcinogenesis, but not progression. In addition, PCNA is related to tumor development and the expression of mutated p53 might be a late event during endometrial carcinogenesis.     

Simultaneous quantification of vitamin E and vitamin E metabolites in equine plasma and serum using LC-MS/MS

Vitamin E deficiencies can impact normal growth and development in humans and animals, and assessment of circulating levels of vitamin E and its metabolites may be an important endpoint for evaluation. Development of a sensitive method to detect and quantify low concentrations of vitamin E and metabolites in biological specimens allows for a proper diagnosis for patients and animals that are deficient. We developed a method to simultaneously extract, detect, and quantify the vitamin E compounds alpha-tocopherol (α-TP), gamma-tocopherol (γ-TP), alpha-tocotrienol (α-TT), and gamma-tocotrienol (γ-TT), and the corresponding metabolites formed after β-oxidation of α-TP and γ-TP, alpha-carboxymethylbutyl hydroxychroman (α-CMBHC) and alpha- or gamma-carboxyethyl hydroxychroman (α- or γ-CEHC), respectively, from equine plasma and serum. Quantification was achieved through liquid chromatography–tandem mass spectrometry. We applied a 96-well high-throughput format using a Phenomenex Phree plate to analyze plasma and serum. Compounds were separated by using a Waters ACQUITY UPLC BEH C18 column with a reverse-phase gradient. The limits of detection for the metabolites and vitamin E compounds were 8–330 pg/mL. To validate the method, intra-day and inter-day accuracy and precision were evaluated along with limits of detection and quantification. The method was then applied to determine concentrations of these analytes in plasma and serum of horses. Alpha-TP levels were 3–6 µg/mL of matrix; the metabolites were found at much lower levels, 0.2–1.0 ng/mL of matrix.     

The Effect of Single Nucleotide Polymorphisms in the Tumor Necrosis Factor-α Gene on Reproductive Performance and Immune Function in Dairy Cattle

The present study aimed to assess the effect of polymorphisms in the tumor necrosis factor α (TNF-α) promoter (A/A, A/G and G/G) and exons (T/T, T/C and C/C) on immune function and reproductive performance in dairy cows. The occurrence of the first postpartum ovulation within 3 weeks in the cows with the TNF-α promoter A/G and G/G genotypes was higher than in the A/A group. Among the different TNF-α exon genotypes, the occurrence of early first postpartum ovulation was higher in the T/C and C/C genotype groups than in the T/T group. Single nucleotide polymorphisms (SNPs) in the TNF-α gene did not affect the rate of artificial insemination (AI) or duration from parturition to next conception (days open). The apoptosis rate of polymorphonuclear leukocytes (PMNs) did not differ among the TNF-α promoter genotypes, but the PMN transmigration rate was significantly higher for the A/A and A/G genotypes than for the G/G genotype. Interleukin 8 (IL-8) mRNA expression in PMNs and peripheral blood mononuclear cells (PBMCs) before culture was significantly higher for the A/A genotype compared with the G/G genotype. There were no significant differences between the genotypes in the mRNA expression of TNF-α, IL-6, IL-1β, and toll-like receptor 4 (TLR4) in PMNs and PBMCs before and 4 h after culture. IL-8 and IL-1β production by PBMCs cultured for 4 h was significantly higher for the animals with the A/A genotype than for those with the G/G genotype. On the other hand, no significant difference was observed in IL-8 and IL-1β production by PMNs among different TNF-α genotypes. Taken together, these results suggest that SNP in the TNF-α gene affects immune function and reproductive performance in dairy cows.     

Expression of Glucocorticoid Receptor α and Its Regulation in the Bovine Endometrium: Possible Role in Cyclic Prostaglandin F2α Production

Cortisol (Cr), the most important glucocorticoid (GC), is well known to suppress uterine prostaglandin F2α (PGF) production. However, the details of the regulatory mechanisms controlling the cyclic changes in endometrial PGF production remain unclear. Here we investigated the expression of the GC receptor (GC-Rα), the actions of cortisol throughout the estrous cycle and the regulatory mechanism of GC-Rα in the bovine endometrium. The levels of GC-Rα protein were greater at the mid-luteal stage (Days 8–12) than at the other stages. Cr more strongly suppressed PGF production at the mid-luteal stage than at the follicular stage. GC-Rα expression was increased by progesterone (P4) but decreased by estradiol-17β (E2) in cultured endometrial stromal cells. The overall results suggest that ovarian steroid hormones control the cyclic changes in endometrial PGF production by regulating GC-Rα expression in bovine endometrial stromal cells.     

Characterization of β-adrenergic receptors in bovine intramuscular and subcutaneous adipose tissue: comparison of lubabegron fumarate with β-adrenergic receptor agonists and antagonists

Chinese hamster ovary cell constructs expressing either the β ₁-, β ₂- or β ₃-adrenergic receptor (AR) were used to determine whether a novel β-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific β-AR subtype. EC₅₀ values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the β ₃-AR subtype, with an EC₅₀ of 6 × 10^–9 M, with no detectible agonistic activity at the β ₂-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the β-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for β ₁-, β₂-, and β ₃-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; β-AR pan-agonist), dobutamine hydrochloride (DOB; specific β ₁-AA), salbutamol sulfate (SAL; specific β ₂-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific β ₃-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for β ₁-, β ₂-, and β ₃-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant β-AR mRNA in both adipose tissues was the β ₂-AR (P < 0.05), with the β ₁- and β ₃-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these β-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the β ₁- and β ₂-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to β-adrenergic ligands, especially those that are agonists at the β ₁- and β₃-receptor subtypes. The minimal mRNA expression of the β ₁- and β ₃ subtypes in i.m. adipose tissue likely limits the response potential to agonists for these β-AR subtypes.     

Metformin acts to suppress β-hydroxybutyric acid-mediated inflammatory responses through activation of AMPK signaling in bovine hepatocytes

The occurrence of bovine ketosis involves the accumulation of β-hydroxybutyric acid (BHBA), which contributes to the initiation and acceleration of hepatic metabolic stress and inflammation. Metformin has other beneficial effects apart from its medical intervention for diabetes, such as prevention of laminitis and hyper-triglyceridemic. AMPK maintains energy homeostasis and is the intracellular target of metformin action. This study aims to uncover the role of metformin in modulating BHBA-induced inflammatory responses through the activation of AMPK signaling. The hepatocytes were isolated from the liver tissue of mid-lactation multiparous Holstein cows (~160 d postpartum). Treatments were conducted as follows: treated with PBS for 18 h (control); pretreated with PBS for 12 h followed by treatment of 1.2 mM BHBA for 6 h (BHBA); pretreated with 1.5 mM or 3 mM metformin for 12 h followed by the BHBA treatment (1.2 mM) for 6 h (M(1.5)+B; M(3)+B). The inhibitor of AMPK, Compound C, at a concentration of 10 μM, was applied to substantiate the AMPK-dependent responses. RT-qPCR were applied for the mRNA expression while Western-blots and immunofluorescence were conducted for the target proteins expression. Among dose-dependent assays for BHBA, the concentration of BHBA at 1.2 mM activated NF-κB signaling by upregulating the expression of phosphorylated NF-κB and pro-inflammatory cytokines compared with the control cells (P < 0.05). Along with the upregulation of phosphorylated AMPKα and ACCα, metformin at 1.5 and 3 mM inactivated NF-κB signaling components (p65 and IκBα) and the inflammatory genes (TNFA, IL6, IL1B and COX-2) which were activated by BHBA. Additionally, BHBA inhibited cells staining intensity in EdU assay were increased by pretreatment with metformin. The activation of AMPK resulted in the increased gene and protein expression of SIRT1, along with the deacetylation of H3K9 and H3K14. However, the AMPK inhibitor compound C blocked this effect. Compared with BHBA treated cells, the protein expression of COX-2 and IL-1β were decreased by the pretreatment with metformin, and the inhibitory effect of metformin was released by compound C. The bound of NF-κB onto IL1B promoter displayed higher in BHBA group and this was suppressed by pretreatment with metformin (P < 0.05). Altogether, metformin attenuates the BHBA-induced inflammation through the inactivation of NF-κB as a target for AMPK/SIRT1 signaling in bovine hepatocytes.     

Expression of Aldo-keto Reductase 1C23 in the Equine Corpus Luteum in Different Luteal Phases

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P₄) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P₄ into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P₄ concentration and levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3β-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P₄ by AKR1C23 is one of the processes contributing to functional luteolysis in mares.     

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.     

Effect of Conceptus on Transforming Growth Factor (TGF) β1 mRNA Expression and Protein Concentration in the Porcine Endometrium—In Vivo and In Vitro Studies

Transforming growth factor (TGF) β and its receptors are expressed at the conceptus-maternal interface during early pregnancy in the pig. The present studies were conducted to examine: (1) the effect of conceptus products on TGFβ1 mRNA expression and protein concentration in the porcine endometrium using in vivo and in vitro models, and (2) the effect of TGFβ1 on proliferation of porcine trophoblast cells in vitro. During in vivo experiments, gilts with one surgically detached uterine horn were slaughtered on days 11 or 14 of the estrous cycle and pregnancy. For in vitro studies, endometrial explants and luminal epithelial (LE) cells co-cultured with stromal (ST) cells were treated with conceptus-exposed medium (CEM). Moreover, porcine trophoblast cells were treated with TGFβ1, and the number of viable cells was measured. On day 11, the presence of conceptuses had no effect on TGFβ1 mRNA expression, but decreased the TGFβ1 protein concentration in the connected uterine horn compared with the detached uterine horn. In contrast to day 11, on day 14 after estrus, TGFβ1 mRNA expression and protein content in the endometrium collected from the gravid uterine horn were greater when compared with the contralateral uterine horn. The treatment of endometrial slices with CEM resulted in greater TGFβ1 mRNA expression and protein secretion. LE cells responded to CEM with an increased TGFβ1 mRNA level. Moreover, TGFβ1 stimulated the proliferation of day 14 trophoblast cells. In summary, porcine conceptuses may regulate TGFβ1 synthesis in the endometrium at the time of implantation. TGFβ1, in turn, may promote conceptus development by increasing the proliferation of trophoblast cells.     

5α-Androstenone and Testosterone in Peripheral Plasma of the Boar During and Following Copulation

Copulation was generally followed by increases in peripheral plasma 5α-androstenone and testosterone levels lasting for periods of about 60 to 100 min. The effect of copulation on the plasma levels of these steroids did, however, vary between boars. In seven out of eight boars the maximum levels of 5α-andro-stenone in the period 60 to 100 min. after copulation were from 114 to 218 % (mean 150 %) of the levels in samples collected before copulation. The corresponding figures for testosterone were from 104 to 283 % (mean 190 %). One boar showed decreasing plasma steroid levels after copulation.The coefficient of correlation between the peripheral plasma levels of 5α-androstenone and testosterone was found to be + 0.61 (n = 2.03).     

Downregulation of the expression of inhibin α subunit and betaglycan in porcine cystic follicles

Inhibins, as members of the transforming growth factor beta (TGF-β) superfamily, downregulate the synthesis and secretion of follicle-stimulating hormone (FSH) in an endocrine manner. The role of inhibin/betaglycan in the ovary regulation recently gained attention. To date, no data exist on the function of inhibin α subunit and betaglycan in cystic follicles. In this study, the expressions of inhibin α subunit and betaglycan in cystic follicles were investigated using immunohistochemistry, real-time PCR and Western blot analysis. Both inhibin α subunit and betaglycan immunoreactivities were mainly localized in the granulosa cells of follicles. Expression of inhibin α subunit and betaglycan was inferior in cystic follicles compared with that in normal large follicles. However, the result of enzyme-linked immunosorbent assay showed no significant difference in the decreasing in concentration of inhibin α subunit in cystic follicular fluid compared with the control (P>0.05). In this study, we explored the effects of FSH on betaglycan expression in granulosa cells in vitro. As expected, a significant increase in the expressions of betaglycan mRNA and protein in granulosa cells was observed in response to exogenous FSH (30 ng/ml) (P<0.05) compared with the control. Consequently, this study provides evidence that the expressions of inhibin α subunit and betaglycan are inferior in cystic follicles, and this may be caused by the decrease in FSH in the presence of a cystic follicle.     

Immature T cell neoplasms in three young cattle

Immature T cell neoplasms in three young Holstein cattle with neoplastic involvement of the thymus are described. Case 1, with a precursor T lymphoblastic leukemia (calf form of leukosis), was an 86-day-old female calf. The leukemia was characterized by replacement of the bone marrow and spleen by leukemia cells, but preservation of epithelial frameworks throughout the thymus. The other two neoplasms were thymic γδ T cell lymphomas, which were observed in a 246-day-old steer (case 2) and a 16-month-old heifer (case 3). Histological examination revealed obliteration of the normal thymic architecture and stromal fibrosis, with the spleen and liver far less severely affected than in case 1. There were cytological differences bewteen the tumors in case 1 and cases 2 and 3. Additionally, WC1 and CD8 were expressed only in the latter. Thus, the leukemia and these lymphomas should be regarded as independent disease entities on the basis of histological and immunohistochemical characteristics.     

Calcium potentiates the effect of estradiol on PGF2α production in the bovine endometrium

Background

Estradiol (E₂) is required for luteolysis in cows and its injection stimulates prostaglandin F2α (PGF2α) release. The main goal of our study was to investigate the ability of endometrial explants and cells treated with E₂ and the calcium ionophore (CI) A23187 to synthesize PGF2α.

Results

Treatment with E₂in vivo resulted in a 48.4% increase of PGF2α production by endometrial explants treated in vitro with A23187. Production of PGF2α was better stimulated with A23187 at concentrations of 10^-6 and 10^-5 mol/L compared with other concentrations used. The concentration of PGF2α for untreated bovine endometrial cell cultures was 33.1 pg/mL, while for cultures treated with E₂, A23187, or a combination of E₂ and A23187, the PGF2α concentration was 32.5, 92.4 and 145.6 pg/mL, respectively.

Conclusions

Treatment with A23187 tended to stimulate PGF2α production. In the presence of E2, A23187 significantly stimulated PGF2α synthesis. It appears that A23187 potentiates the effects of E₂ with respect to synthesis of endometrial PGF2α in cattle.