The newly formed corpus luteum (CL) develops rapidly and has the features of active vascularization and mitosis of steroidogenic cells. Such local mechanisms must be strictly regulated by the complex relationship between angiogenic growth factors and vasoactive peptides such as angiotensin (Ang) II, atrial natriuretic peptide (ANP), and endothelin (ET)-1. Thus, the objective of the present study was to determine 1) the changes in vasoactive peptides and progesterone (P) concentrations within the developing CL, along with the changes in concentration in ovarian venous plasma (OVP) and jugular venous plasma (JVP) in the cow, 2) the effects of CL exposure to vasoactive peptides on Ang II and P secretion, and 3) the expression of mRNA for ANP type C receptor in the bovine CL and endothelial cells (ETC) from bovine developing CL. A microdialysis system (MDS) was surgically implanted into multiple CL of six cows on Day 3 after a GnRH injection that induced superovulation, and a catheter was simultaneously inserted into the ovarian vein. The Ang II concentration in OVP was higher than that in JVP throughout the experiment, while the intraluteal release of Ang II was stable. During the experimental period, the concentrations of other vasoactive peptides (ANP and ET-1) showed no clear changes in plasma and were below detectable levels in the MDS perfusate. Exposure of CL to Ang II using the MDS stimulated P release, while exposure to ANP enhanced Ang II release within the developing CL. However, ET-1 had no effect on either P or Ang II release. The expression of mRNA for ANP type C receptor was mainly observed in early CL and ETC. The results suggest that the ET-Ang-ANP system in the preovulatory follicle switches to an Ang-ANP system to enhance both the angiogenesis and steroidogenesis that are actively occurring in developing CL.
Recent evidence suggests the presence of a functional endothelin-angiotensin-atrial natriuretic peptide system at the ovarian level. This study aimed to investigate 1) the local interrelationships among angiotensin II (Ang II), endothelin-1 (ET-1), and atrial natriuretic peptide (ANP); 2) the possible effect of each vasoactive peptide on the secretion of steroid hormones and prostaglandins (PGs) in isolated bovine mature follicles; and 3) the expression of mRNAs for Ang II, ET-1, and ANP receptors in the theca layer of follicles at different developmental stages.
The presence of gonadotropin-releasing hormone (GnRH)-like protein has been previously demonstrated in the bovine follicle. This GnRH-like protein was purified and concluded to be histone H2A. However, neither GnRH peptide nor specific GnRH-immunoreactivity (GnRH-IR) has been demonstrated in the bovine follicle so far. Thus, this study focused on the detection of specific GnRH-IR using the second-antibody enzyme immunoassay in isolated bovine mature follicles, and on an examination of the direct effect of luteinizing hormone (LH), endothelin-1 (ET-1), and cytokines on the GnRH-IR using an in vitro microdialysis system (MDS). We further examined a cross-reactivity of the GnRH antibody with bovine histone H2A. GnRH-IR was detected in microdialyzed perfusate from isolated bovine mature follicles at 4.40 ± 0.35 pg/ml (mean ± SEM). Bovine histone H2A showed no GnRH-IR at all. Heat treatment of the extract (100 C for 10 min) did not affect the GnRH-IR. Single infusion of LH, ET-1, or cytokines into the MDS did not affect the GnRH-IR. However, infusion of ET-1 after LH exposure increased the GnRH-IR. These results demonstrate the presence of specific GnRH-IR, that is different from histone H2A, in microdialyzed perfusate of isolated bovine mature follicles in vitro, suggesting that the GnRH-IR may reflect a role of GnRH-like peptide or some peptide structurally similar to GnRH in the local regulation of mature follicles.
Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype.A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically.Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays.The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.
Abstract Background Prostaglandin F2alpha (PGF) induces luteolysis in cow by inducing a rapid reduction in progesterone production (functional luteolysis) followed by tissue degeneration (structural luteolysis). However the mechanisms of action of PGF remain unclear. Reactive oxygen species (ROS) play important roles in regulating the luteolytic action of PGF. The local concentration of ROS is controlled by superoxide dismutase (SOD), the main enzyme involved in the control of intraluteal ROS. Thus SOD seems to be involved in luteolysis process induced by PGF in cow. Methods To determine the dynamic relationship between PGF and ROS in bovine corpus luteum (CL) during luteolysis, we determined the time-dependent change of Copper/Zinc SOD (SOD1) in CL tissues after PGF treatment in vivo . We also investigated whether PGF and hydrogen peroxide (H2O2) modulates SOD1 expression and SOD activity in cultured bovine luteal endothelial cells (LECs) in vitro. Results Following administration of a luteolytic dose of PGF analogue (0 h) to cows at the mid-luteal stage, the expression of SOD1 mRNA and protein, and total SOD activity in CL tissues increased between 0.5 and 2 h, but fell below the initial (0 h) level at 24 h post-treatment. In cultured LECs, the expression of SOD1 mRNA was stimulated by PGF (1–10 microM) and H2O2 (10–100 microM) at 2 h (P<0.05). PGF and H2O2 increased SOD1 protein expression and total SOD activity at 2 h (P<0.05), whereas PGF and H2O2 inhibited SOD1 protein expressions and total SOD activity at 24 h (P<0.05). In addition, H2O2 stimulated PGF biosynthesis at 2 and 24 h in bovine LECs. Overall results indicate that, SOD is regulated by PGF and ROS in bovine LECs. SOD may play a role in controlling intraluteal PGF and ROS action during functional and structural luteolysis in cows.
Estrogen (E) exerts its function by binding to two intracellular estrogen receptors, ERα and ERβ. Although ERs have been reported to be expressed in the bovine corpus luteum (CL), the mechanisms that control ER expression in the bovine CL are not fully understood. To determine the possible regulatory mechanisms of ERα and ERβ that meditate distinct E functions, we examined 1) the changes in the protein expressions of ERs in the CL throughout the luteal phase and 2) the effects of prostaglandin (PG) F2α, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) on the expressions of ERs in cultured bovine luteal cells. Western blot analyses revealed that ERα and ERβ proteins were expressed throughout the luteal phase. The ERα protein level was high at the early luteal (Days 2-3 after ovulation) and mid-luteal stages (Days 8-12) and was extremely low at the regressed luteal stage (Days 19-21). The ERβ protein level increased from the early to developing luteal stage, remained at the same level at the mid-luteal stage and decreased thereafter. The ratio of ERβ to ERα was higher in the regressed stage than in the other stages. Luteal cells obtained from mid-stage CLs (Days 8-12) were incubated with PGF2α (0.01-1 μM), TNFα (0.0145-1.45 nM) or IFNγ (0.0125-1.25 nM) for 24 h. PGF2α and TNFα inhibited ERa and ERβ mRNA expressions. IFNγ suppressed ERβ mRNA expression but did not affect the expression of ERα mRNA. However, the ERα and ERβ protein levels were not affected by any of the above treatments. These data indicate that PGF2α, TNFα and IFNγ regulate ERα and ERβ mRNA expressions in bovine luteal cells. Moreover, the changes in the ERβ/ERα ratio throughout the luteal phase suggest that ERα is associated with luteal maintenance. Therefore, a dramatic decrease in ERα at the regressed luteal stage could result in progression of structural luteolysis in the bovine CL.
Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.
To determine whether glucocorticoids (GCs) play a role in regulating uterine function in cow, the present study examined the expression of mRNA encoding GC receptor (GC-R) α, 11β-hydroxysteroid dehydrogenase (11-HSD) type 1 and type 2, and the activity of 11-HSD1 in bovine endometrial tissue throughout the estrous cycle. We also studied the effects of cortisol on basal, oxytocin (OT)- and tumor necrosis factor-α (TNFα)-stimulated prostaglandin (PG) production. A quantitative real-time PCR analysis revealed that GC-Rα mRNA was expressed more strongly in the mid-luteal stage (days 8–12) than in the other stages. In contrast to GC-Rα mRNA expression, 11-HSD1 mRNA expression was greater in the follicular stage than in the other stages, whereas 11-HSD2 mRNA expression was lowest in the follicular stage. The activity of 11-HSD1 was greater in the follicular stage and estrus than in the other stages and was lowest in the mid-luteal stage. Cortisone was dose-dependently converted to cortisol in the cultured endometrial tissue. Although cortisol did not affect either the basal or OT-stimulated production of PGs in the cultured epithelial cells, the production of PGs stimulated by TNFα in the stromal cells was suppressed by cortisol ( P < 0.05). Cortisol suppressed basal prostaglandin (PG)F2α without affecting basal PGE2 production in the stromal cells. The overall results suggest that the level of cortisol is locally regulated in bovine endometrium throughout the estrous cycle by 11-HSD1, and that cortisol could act as a luteoprotective factor by selectively suppressing luteolytic PGF2α production in bovine endometrium.
