Resveratrol treatment increases mitochondrial biogenesis and improves viability of porcine germinal-vesicle stage vitrified-warmed oocytes.

Vitrification induces mitochondrial dysfunction in warmed oocytes, and degeneration and biogenesis of mitochondria are crucial for maintaining oocyte quality. The present study addresses a hypothesis that treatment of vitrified-warmed oocytes with resveratrol could improve the viability of oocytes by activating mitochondrial biosynthesis. Cumulus oocyte complexes (COCs) collected from gilt ovaries were vitrified, warmed, and cultured in a medium containing vehicle or 1 µM resveratrol. Resveratrol treatment improved survival, maturation, and mitochondrial membrane potential of vitrified-warmed oocytes, but did not improve the development into blastocysts after parthenogenetic activation. Resveratrol treatment increased mitochondrial synthesis, as determined by the expression levels of TOMM20 and mitochondrial DNA copy number, in vitrified-warmed oocytes, but not in non-vitrified oocytes. In addition, the effect of resveratrol treatment on mitochondrial synthesis was reduced by EX527, a SIRT1 inhibitor. Resveratrol treatment of vitrified-warmed oocytes increased the expression levels of genes involved in mitochondrial synthesis (TFAM, POLG, and PGC1α) and increased nuclear translocation of NRF2. Furthermore, vitrification induced mitophagy, as confirmed by a reduction in TOMM20 expression and decreased p62 aggregation, whereas resveratrol treatment did not affect these mitophagic features. In conclusion, vitrification induced mitochondrial clearance in oocytes, whereas activation of SIRT1 by resveratrol treatment facilitated the recovery of vitrified-warmed oocytes through the activation of mitochondrial synthesis.

Mitochondrial biogenesis and degradation are induced by CCCP treatment of porcine oocytes.

In this study, we investigated the mitochondrial quality control system in porcine oocytes during meiotic maturation. Cumulus cell oocyte complexes (COCs) collected from gilt ovaries were treated with 10  µM carbonyl cyanide-m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) for 2  h. The CCCP treatment was found to significantly reduce ATP content, increase the amount of phosphorylated AMP-activated protein kinase and elevate reactive oxygen species levels in oocytes. When the CCCP-treated COCs were cultured further for 44  h in maturation medium, the ATP levels were restored and the parthenogenetic developmental rate of oocytes to the blastocyst stage was comparable with that of untreated COCs. To examine the effects of CCCP treatment of oocytes on the kinetics of mitochondrial DNA copy number (Mt number), COCs treated with 0 or 10  µM CCCP were cultured for 44  h, after which the Mt number was determined by RT-PCR. CCCP treatment was found to increase the Mt number in the modified maturation medium in which mitochondrial degradation was inhibited by MG132, whereas CCCP treatment did not affect the Mt number in the maturation medium lacking MG132. The relative gene expression of TFAM was furthermore shown to be significantly higher in CCCP-treated oocytes than in untreated oocytes. Taken together, the finding presented here suggest that when the mitochondria are injured, mitochondrial biogenesis and degradation are induced, and that these processes may contribute to the recuperation of oocytes.

Resveratrol improves the quality of pig oocytes derived from early antral follicles through sirtuin 1 activation.

During oocyte growth, the number of mitochondria drastically increases and mitochondrial function profoundly affects the oocyte competence. Resveratrol is a well-known activator of sirtuin 1 (SIRT1), which has a role in cellular energy homeostasis and mitochondrial biogenesis. The main aim of the present study was to examine the effect of supplementation of culture media with resveratrol on oocyte development and mitochondrial number and functions. Lipid contents and developmental ability of the oocytes grown in vitro were also examined. Oocyte-granulosa cell complexes were collected from early antral follicles of gilt ovaries and were cultured in medium containing 0 or 2 µM resveratrol for 14 days. Immunostaining revealed that resveratrol enhanced SIRT1 expression in oocytes. Antrum formation during the culture period and survivability of the granulosa cells surrounding the developed oocytes did not differ between the two concentrations of resveratrol. In addition, the ability of oocytes to complete meiotic maturation did not differ between the two concentrations of resveratrol, whereas the ability of oocytes to develop to the blastocyst stage was improved significantly by resveratrol (7.4% vs. 1.6%; P < 0.05). Resveratrol upregulated the ATP content in oocytes grown in vitro, and the addition of 2 µM of the SIRT1 inhibitor 6-Chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX527) diminished this effect although EX527 alone had no effect on ATP content. The mitochondrial DNA copy number in oocytes determined by quantitative real-time polymerase chain reaction increased during in vitro oocyte development, but resveratrol did not affect the kinetics of the mitochondrial DNA copy number. We found that resveratrol also increased the expression level of phospho-5'-adenosine monophosphate-activated protein kinase in oocytes but decreased the lipid content in oocytes grown in vitro. These results suggest that resveratrol increased the ATP content in oocytes via energy homeostasis and improved the developmental ability of oocytes grown in vitro.

CELL BIOLOGY SYMPOSIUM: perspectives: possible roles of polymorphonuclear neutrophils in angiogenesis and lymphangiogenesis in the corpus luteum during development and early pregnancy in ruminants.

The establishment of pregnancy requires well-balanced regulation of the endocrine and immune systems and involves interactions among the conceptus, oviduct-uterus, and corpus luteum (CL). In particular, a rapid increase in plasma progesterone during the first week after ovulation is critical for the growth of the conceptus and successful pregnancy in cattle. Events involved in maternal recognition of pregnancy (MRP) may commence within 1 wk from AI, when interferon-stimulated gene expression in circulating polymorphonuclear neutrophils (PMN) increases in pregnant cows. To regulate optimal endocrine conditions within this time, the CL must develop rapidly, with active angiogenesis and lymphangiogenesis. The major angiogenic factors, vascular endothelial growth factor and fibroblast growth factor 2, contribute to the development of the CL but may also act as chemoattractants for PMN. Indeed, the number of PMN is greatest in the new CL, where PMN together with IL-8 induce active angiogenesis and lymphangiogenesis. During MRP, the conceptus secretes interferon tau (IFNT), which prevents CL regression by inhibiting luteolytic release of PGF2α from uterine endometrium. In addition, IFNT and PGE2 reach the CL and may contribute to desensitizing the CL to the luteolytic effects of PGF2α. In the bovine CL, lymphangiogenesis, stimulated by IFNT, may occur during MRP, and thus a shift of local immunity might occur at this timing. The aforementioned evidence supports the possible involvement of PMN in the establishment of pregnancy via CL regulation. Further investigation could expand our understanding of the communication between zygotes, PMN, and reproductive organs during early pregnancy. This should provide new insight into the contribution of neutrophils to CL function and immune tolerance during early pregnancy in ruminants.

Possible involvement of cytokines, chemokines and chemokine receptors in the initiation and progression of chronic GVHD.

Chronic GVHD (cGVHD) after allogeneic hematopoietic SCT (HSCT) is characterized by an infiltration of T cells into target organs including the oral mucosa and salivary glands. This study was designed to clarify the molecular mechanism of the local accumulation of pathogenic T cells in cGVHD. The expression of cytokines, chemokines and chemokine receptors in the buccal mucosa (BM), labial salivary glands (LSG) and PBMC from 16 patients with cGVHD after allogeneic HSCT was examined. The mRNA expression of T helper 1 (Th1) and Th2 cytokines, and several chemokines and chemokine receptors was significantly increased in the BM and LSG from cGVHD patients, in comparison with both those in the BM and LSG from controls, respectively, and also with those in the PBMC from cGVHD patients. Furthermore, the mRNA expression of Th2 cytokines, macrophage-derived chemokine and CC chemokine receptor 4 was closely associated with a strong T-cell infiltration in the BM and LSG from cGVHD patients. These results suggest that cGVHD might be initiated and/or maintained by Th1/Th0 cells and thereafter progresses in association with Th2 cell accumulation via the interaction of particular chemokine and chemokine receptors.

Effects of lipopolysaccharide (LPS) and peptidoglycan (PGN) on estradiol production in bovine granulosa cells from small and large follicles.

In cows, postpartum uterine infection due to bacteria that produce lipopolysaccharide (LPS) or peptidoglycan (PGN) leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS and/or PGN on estradiol production from granulosa cells from small and large follicles in the bovine ovary. Granulosa cells from small and large ovarian follicles were exposed to LPS and/or PGN in vitro. LPS inhibited the expression of TLR4, CD14, MD2 and NOD1 genes in FSH-treated granulosa cells from small follicles. LPS suppressed estradiol (E2) production in granulosa cells from small and large follicles, while PGN inhibited E2 production in granulosa cells from large follicles. LPS or PGN did not affect granulosa cell survival. Although LPS alone suppressed E2 production in granulosa cells from small and large follicles, E2 production was not further suppressed when PGN was added to culture medium with LPS alone. Our data demonstrated that susceptibility to LPS or PGN in granulosa cells depends on the follicle developmental stage. The results of the present study suggest that ovarian dysfunction in cows with postpartum uterine infection may be caused by inhibitory effects of LPS and PGN on E2 production in granulosa cells.

Escherichia coli lipopolysaccharide administration transiently suppresses luteal structure and function in diestrous cows.

The objective was to characterize the effects of Escherichia coli lipopolysaccharide (LPS) endotoxin (given i.v.) on luteal structure and function. Seven nonlactating German Holstein cows, 5.1 ± 0.8 years old (mean ± s.e.m.), were given 10  ml saline on day 10 (ovulation=day 1) of a control estrous cycle. On day 10 of a subsequent cycle, they were given 0.5 µg/kg LPS. Luteal size decreased (from 5.2 to 3.8 cm², P≤0.05) within 24 h after LPS treatment and remained smaller throughout the remainder of the cycle. Luteal blood flow decreased by 34% (P≤0.05) within 3 h after LPS and remained lower for 72 h. Plasma progesterone (P4) concentrations increased (P≤0.05) within the first 3 h after LPS but subsequently declined. Following LPS treatment, plasma prostaglandin (PG) F metabolites concentrations were approximately tenfold higher in LPS-treated compared with control cows (9.2 vs 0.8 ng/ml, P≤0.05) within 30 min, whereas plasma PGE concentrations were nearly double (P≤0.05) at 1 h after LPS. At 12 h after treatment, levels of mRNA encoding Caspase-3 in biopsies of the corpus luteum (CL) were increased (P≤0.05), whereas those encoding StAR were decreased (P≤0.05) in cattle given LPS vs saline. The CASP3 protein was localized in the cytoplasm and/or nuclei of luteal cells, whereas StAR was detected in the cytosol of luteal cells. In the estrous cycle following treatment with either saline or LPS, there were no significant differences between groups on luteal size, plasma P4 concentrations, or gene expression. In conclusion, LPS treatment of diestrus cows transiently suppressed both the structure and function of the CL.

Vascular and immune regulation of corpus luteum development, maintenance, and regression in the cow.

The bovine corpus luteum (CL) is a unique, transient organ with well-coordinated mechanisms by which its development, maintenance, and regression are effectively controlled. Angiogenic factors, such as vascular endothelial growth factor A and basic fibroblast growth factor, play an essential role in promoting progesterone secretion, cell proliferation, and angiogenesis. These processes are critically regulated, through both angiogenic and immune systems, by the specific immune cells, including macrophages, eosinophils, and neutrophils, that are recruited into the developing CL. The bovine luteolytic cascade appears to be similar to that of general acute inflammation in terms of time-dependent infiltration by immune cells (neutrophils, macrophages, and T lymphocytes) and drastic changes in vascular tonus and blood flow, which are regulated by luteal nitric oxide and the vasoconstrictive factors endothelin-1 and angiotensin II. Over the period of maternal recognition of pregnancy, the maternal immune system should be well controlled to accept the semiallograft fetus. The information on the presence of the developing embryo in the genital tract is suggested to be transmitted to the ovary by both the endocrine system and the circulating immune cells. In the bovine CL, the lymphatic system, but not the blood vascular system, is reconstituted during early pregnancy, and interferon tau from the embryo could trigger this novel phenomenon. Collectively, the angiogenic and vasoactive factors produced by luteal cells and the time-dependently recruited immune cells within the CL and their interactions appear to play critical roles in regulating luteal functions throughout the life span of the CL.

Expression of prostaglandin F2α (PGF2α) receptor and its isoforms in the bovine corpus luteum during the estrous cycle and PGF2α-induced luteolysis.

Prostaglandin F2α (PGF2α) induces luteolysis via a specific receptor, PTGFR. Although PTGFR mRNA expression in the bovine corpus luteum (CL) has been studied previously, changes in PTGFR protein and its localization are not fully understood during the life span of the CL. In addition to full-length PTGFR, several types of PTGFR isoforms, such as PTGFRα (type I) and PTGFRζ (type II), were reported in the bovine CL, suggesting isoform-specific luteal action. Full-length PTGFR mRNA in the bovine CL increased from the early to the mid-luteal phase and decreased during luteolysis, whereas PTGFR protein remained stable. PTGFR protein was localized to both luteal and endothelial cells and was expressed similarly during the life span of the CL. Like full-length PTGFR mRNA, PTGFRα and PTGFRζ mRNA also increased from the early to mid-luteal phases, and mRNA of PTGFRζ, but not PTGFRα, decreased in the regressing CL. During PGF2α-induced luteolysis, the mRNAs of full-length PTGFR, PTGFR,α and PTGFRζ decreased rapidly (from 5 or 15 min after PGF2α injection), but PTGFR protein decreased only 12 h later. Silencing full-length PTGFR using small interfering RNA prevented PGF2α-stimulated cyclooxygenase-2 (PTGS2) mRNA induction. By contrast, PGF2α could stimulate vascular endothelial growth factor A (VEGFA) mRNA even when full-length PTGFR was knocked down, thus suggesting that PGF2α may stimulate PTGS2 via full-length PTGFR, whereas VEGFA is stimulated via other PTGFR isoforms. Collectively, PTGFR protein was expressed continually in the bovine CL during the estrous cycle, implying that PGF2α could function throughout this period. Additionally, the bovine CL expresses different PTGFR isoforms, and thus PGF2α may have different effects when acting via full-length PTGFR or via PTGFR isoforms.

Regulation of corpus luteum development and maintenance: specific roles of angiogenesis and action of prostaglandin F2alpha.

Development of the corpus luteum (CL) in ruminants occurs in a rapid and time-dependent manner within 1 week after ovulation, with morphologic and biochemical changes in the cells of the theca interna and granulosa cells of the preovulatory follicle. These changes involve luteinisation of steroidogenic cells and angiogenesis to establish normal luteal function (progesterone secretion). The CL is composed of a large number of vascular endothelial cells, large and small steroidogenic luteal cells, smooth muscle cells, pericytes, fibrocytes and immune cells, indicating that the CL is a heterogeneous tissue. Moreover, the CL produces and secretes growth factors (fibroblast growth factor, vascular endothelial growth factor and insulin-like growth factor), vasoactive factors (nitric oxide, angiotensin II and endothelin-1), steroids (progesterone is important for its own production), oxytocin and prostaglandins (PGF2alpha and PGE2) to regulate luteal formation and development. Clearly, the main function of the CL is to produce progesterone, which is a prerequisite for survival of the embryo, implantation and maintenance of pregnancy. Inadequate luteinisation and angiogenesis during the early luteal phase results in poor progesterone secretion and causes compromised embryo development and reduced fertility. Secretion of adequate amounts of progesterone during luteal development requires "precise luteinisation" of theca and granulosa cells to form luteal cells, neovascularization, and the establishment of a blood supply (angiogenesis). PGF2alpha in the developing CL acts as a local regulator to enhance progesterone secretion directly and indirectly by stimulating angiogenic factors, VEGF and FGF2. The preceding role of PGF2alpha may explain why the developing CL does not acquire luteolytic capacity until several days following ovulation. The balance between luteotrophic and luteolytic factors as well as stimulation and inhibition of angiogenic factors during luteal formation, development and maintenance can have a profound effect on the fate of the CL.

Local regulation of corpus luteum development and regression in the cow: Impact of angiogenic and vasoactive factors.

The corpus luteum (CL) of the estrous cycle in the cow is a dynamic organ which has a life time of approximately 17-18 days. The main function of the CL is to secrete a large amount of progesterone (P) thereby supporting the achievement of pregnancy. As the CL matures, the steroidogenic cells establish contact with many capillaries and the matured CL is composed of many vascular endothelial cells that account for up to 50% of all CL cells. The bovine CL produces several major angiogenic and vasoactive foctors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and -2 (ANPT-1 and -2), prostaglandin F(2alpha) (PGF(2alpha)), endothelin-1 (EDN1), angiotensin II (Ang II) and nitric oxide (NO). These factors regulate P secretion directly and/or indirectly within the CL. Moreover, different actions of PGF(2alpha) in the early cycle CL (non-luteolytic) and the mid cycle CL (luteolytic) may provide insight into the luteolysis cascade in the cow. The aim of the present review is to describe the current concepts of the local mechanisms for the cascade of development and regression of the bovine CL as regulated by luteal angiogenic and vasoactive factors.

Expression of keratinocyte growth factor and its receptor in odontogenic keratocysts.

BACKGROUND: The mitotic activity of the epithelial cells of odontogenic keratocysts (OKCs) is greater than that of other odontogenic jaw cysts, and the mitotic activity of the epithelial cells decreases after marsupialization. Keratinocyte growth factor (KGF) interacts with its specific receptor (KGFR), and elicits the proliferation and/or differentiation of the various types of epithelial cells. The aim of this study was to investigate the expression of KGF/KGFR in OKCs before and after marsupialization. METHODS: The expression of KGF was immunohistochemically detected in the specimens of 16 OKCs and 11 dentigerous cysts before and after marsupialization. The expression of KGF mRNA was measured in the fibroblasts isolated from OKCs by real-time PCR. RESULTS: KGF was expressed in the epithelial cells and fibroblasts of 12 and seven of 16 OKC specimens, respectively. The intensity of the KGF expression in both the epithelial cells and the fibroblasts significantly decreased after marsupialization. KGFR was expressed throughout the epithelium in 15 of 16 OKC specimens, but the intensity of the KGFR expression did not change after marsupialization. The expression of KGF was detected in the epithelium of two of 11 dentigerous cyst specimens, but not in the fibroblasts before marsupialization. Real-time PCR revealed that recombinant human interleukin (IL)-1alpha increased the expression of KGF mRNA in the fibroblasts isolated from OKCs. CONCLUSION: KGF/KGFR signaling may play a crucial role in the epithelial cells of OKCs. Furthermore, the expression of KGF in the fibroblasts of OKCs is regulated by IL-1alpha.

Prostaglandin F 2 alpha stimulates endothelial nitric oxide synthase depending on the existence of bovine granulosa cells: analysis by co-culture system of endothelial cells, smooth muscle cells and granulosa cells.

Prostaglandin F(2 alpha) (PGF(2 alpha)) induces luteolysis in the mid but not in the early luteal phase; despite this, both the early and the mid corpus luteum (CL) have PGF(2 alpha) receptor (FPr). We previously indicated that the luteal blood flow surrounding the CL drastically increases prior to a decrease of progesterone (P) in the cows, suggesting that an acute increase of luteal blood flow may be an early sign of luteolysis in response to PGF(2 alpha) and that this may be induced by a vasorelaxant nitric oxide (NO). The aim of this study was to investigate the luteal stage-dependent and the site-restricted effect of PGF(2 alpha) and NO on the mRNA expressions and P secretion. To mimic the local luteal region both of peripheral and central areas of the CL, we utilized co-cultures using bovine aorta endothelial cells (EC), smooth muscle cells (SMC) and luteinizing granulosa cells (GC) or fully-luteinized GC. PGF(2 alpha) stimulated the expression of endothelial NO synthase (eNOS) mRNA at 0.5 h in mix-cultures of EC and SMC with fully-luteinized GC but not with luteinizing GC. The expression of eNOS mRNA in EC was increased by PGF(2 alpha) at 1 h only when EC was cultured together with fully-luteinized GC but not with luteinizing GC. In all co-cultures, PGF(2 alpha) did not affect the mRNA expression of FPr. Treatment of NO donor inhibited P secretion at 0.5 h. In conclusion, the present study suggests that the coexistence of the mature luteal cells (fully-luteinized GC) with EC/SMC may be crucial for acquiring functional NO synthesis induced by PGF(2 alpha).

Bovine endothelial cells interact with fully-luteinized, but not luteinizing, granulosa cells in the mRNA expression of endothelin-1 system in response to prostaglandin F(2alpha).

The corpus luteum (CL) undergoes regression by prostaglandin (PG)F(2alpha) from uterus and endothelin-1 (ET-1) plays an important role during luteolysis as a local mediator of PGF(2alpha) in the cow. Endothelial cells (EC) and luteal cells are main cell types making up the CL and their interactions are vital for CL function. We aimed to examine the relevance of interactions between EC and luteal cells on stimulation of genes which involved ET-1 synthesis by PGF(2alpha). We further focused the impact of maturity of luteal cells on the stimulation of the genes. To make a microenvironment which resembles the CL, we used bovine aortic endothelial cells (BAEC) and luteinizing or fully-luteinized granulosa cells (GC) and evaluated the effect of PGF(2alpha) on the expression for mRNA of ET-1 system by using real-time RT-PCR. PGF(2alpha) stimulated the expression of preproET-1 and endothelin converting enzyme-1 mRNA only in the co-cultures of BAEC with fully-luteinized GC, but not with luteinizing GC. The data suggest that interactions between BAEC and fully-luteinized GC enhance the capability of BAEC to produce ET-1 in response to PGF(2alpha). This mechanism may contribute to the local induction of luteolytic action of PGF(2alpha) which is dependent on the age/maturation of the CL.

Signaling pathways regulating IL-1alpha-induced COX-2 expression.

Interleukin-1alpha(IL-1alpha) stimulates the production of prostaglandin E(2) (PGE(2)) in odontogenic keratocyst fibroblasts. However, the signaling pathways remain obscure. In this study, we investigated IL-1alphasignaling pathways that regulate cyclooxygenase-2 (COX-2) expression in odontogenic keratocyst fibroblasts. IL-1alphaincreased the expression of COX-2 mRNA and protein, and PGE(2) secretion in the fibroblasts. IL-1alphaincreased the phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). PD-98059, SB-203580, SP-600125, and PDTC-which are inhibitors of ERK1/2, p38, JNK, and nuclear factor-kappaB (NF-kappaB), respectively-attenuated the IL-1alpha-induced COX-2 mRNA expression and activated protein kinase C PGE(2) secretion. IL-1alpha(PKC), and PKC inhibitor staurosporine inhibited IL-1alpha-induced phosphorylation of ERK1/2, p38, and JNK, and decreased IL-1alpha-induced COX-2 mRNA expression. Thus, in odontogenic keratocyst fibroblasts, IL-1alphamay stimulate COX-2 expression both through the PKC-dependent activation of ERK1/2, p38, and JNK signaling pathways, and through the NF-kappaB cascade.

Expression of tumor-associated antigen RCAS1 and its possible involvement in immune evasion in oral squamous cell carcinoma.

BACKGROUND: RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is known to induce apoptosis in its receptor-positive cells. The authors investigated RCAS1 expression in oral squamous cell carcinoma (SCC) and its association with the apoptosis of tumor-infiltrating lymphocytes (TILs). METHODS: In 130 patients with oral SCC, the expression of RCAS1 in tumor cells was immunohistochemically examined and the apoptosis of TILs was examined by Terminal Deoxynucleotidyltransferase-mediated dUTP Nick End Labeling (TUNEL) staining. RESULTS: RCAS1 was detected both on the cytoplasm and the membrane of tumor cells in 41 of 130 cases (31.5%). Focusing on the expression at the invasive front interacting with host immune cells, RCAS1 was detected in 22 of 130 cases (16.9%). The percentage of TUNEL-positive TILs in cases with RCAS1-positive SCCs was significantly higher than in cases with RCAS1-negative SCCs (P < 0.0001). CONCLUSIONS: RCAS1 can be expressed on oral SCC cells and may be involved in the tumor escape from the host immune system by inducing the apoptosis of TILs.

A cooperative action of endothelin-1 with prostaglandin F(2alpha) on luteal function in the cow.

Prostaglandin F(2alpha) (PGF(2alpha)) is the primary luteolysin in the cow, and luteal endothelin-1 (ET-1) interacts with PGF(2alpha) during the process of luteolysis. In contrast, a developing corpus luteum (CL) is refractory to exogenous administration of PGF(2alpha). Thus, the present study was aimed to investigate the functional relationship between ET-1 and PGF(2alpha) in the mid-CL (PGF(2alpha)-sensitive) and early-CL (PGF(2alpha)-refractory). In the mid-CL model, cows (n = 6/treatment) were assigned to receive one of five types of treatments on day 10 of the estrous cycle: (1) an injection of saline; control, (2) a 500 microg of PGF(2alpha) analogue (sufficient dose to induce luteolytis); full-PG, (3) an intraluteal injection of 0.25 mg ET-1; ET-1, (4) a 125 micro g of PGF(2alpha) (insufficient dose to induce luteolytis); 1/4PG or (5) an intraluteal injection of 0.25 mg ET-1 after administration of a insufficient dose of PGF(2alpha) analogue; 1/4PG/ET. In the early-CL model, cows were assigned to receive one of two types of treatments on day 5 of the estrous cycle: (1) a sufficient dose of PGF(2alpha) analogue; PG (n = 5) or (2) an intraluteal injection ET-1 after a sufficient dose of PGF(2alpha); PG/ET (n = 7). In the mid-CL model, 1/4PG/ET resulted in a rapid reduction of progesterone (P) concentrations similar to that in full-PG from the next day. However, the levels of P in 1/4PG/ET (1.5-2.5 ng/ml) kept significantly higher than that in full-PG (< 0.5 ng/ml). ET-1 or 1/4PG did not decrease plasma P concentrations (4-6 ng/ml). The plasma ET-1 levels increased with the full-PG administration. In the early-CL model, both treatments had no effect on plasma P increase and ET-1 levels. The overall results indicate that the intraluteal ET-1 injection after administration of insufficient dose of PGF(2alpha) induces the depression of P secretion in vivo during the mid luteal phase in the cow, supporting the concept that ET-1 is one of a local mediator of functional luteolysis in the cow. The result further indicates that the early-CL is not only PG-refractory but also ET-1-refractory.

Effects of positive pressure in odontogenic keratocysts.

Intracystic fluid pressure is thought to be involved in odontogenic cyst growth. In this study, we investigated the effects of positive pressure on the expression of interleukin-1alpha (IL-1alpha), matrix metalloproteinases (MMPs), and prostaglandin E2 (PGE2) in odontogenic keratocysts to determine whether this pressure stimulates inflammatory cytokine production and signaling of osteoclastogenic events. Positive pressure enhanced the expression of IL-1alpha mRNA and protein in odontogenic keratocyst epithelial cells, and increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in a co-culture of odontogenic keratocyst fibroblasts and the epithelial cells. The pressure-induced secretions were inhibited by an interleukin-1 receptor antagonist. Recombinant human interleukin-1alpha (rhIL-1alpha) increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in the fibroblasts. Furthermore, in the fibroblasts, rhIL-1alpha enhanced the expression of macrophage colony-stimulating factor (M-CSF) mRNA, and rhIL-1alpha-induced PGE2 increased the expression of nuclear factor kappaB ligand (RANKL) mRNA. Thus, positive pressure may play a crucial role in odontogenic keratocyst growth via stimulating the expression of IL-1alpha in epithelial cells.

Blood flow: a key regulatory component of corpus luteum function in the cow.

Prostaglandin F2alpha (PGF2alpha) is the primary luteolysin in the cow. During the early luteal phase, the corpus luteum (CL) is resistant to the luteolytic effect of PGF2alpha. Once mature, the CL becomes responsive to PGF2alpha and undergoes luteal regression. These actions of PGF2alpha coincide with changes in luteal blood flow (BF): PGF2alpha has no effect on BF in the early CL, but acutely increases BF in the peripheral vasculature of the mature CL within 30 min of PGF2alpha injection. During spontaneous luteolysis, luteal BF increases on Days 17-18 of the estrous cycle, prior to any decrease in plasma progesterone (P). The increase in luteal BF is synchronous with an increase in plasma PGFM levels, suggesting that pulsatile release of PGF2alpha from uterus stimulates the increase in luteal BF. Serial biopsies of these CL showed that mRNA expression for endothelial nitric oxide synthase (eNOS) together with endothelin-1 (ET-1) and angiotensin converting enzyme (ACE) increases on Days 17-18 when the luteal BF is elevated. On Day 19 when plasma P level firstly decreases, eNOS mRNA returns to the basal level whereas ET-1 and ACE mRNA remains elevated. Cyclooxygenase-2 (COX-2) mRNA expression increases on Day 19. In support of these data, an in vivo microdialysis study revealed that luteal ET-1 and angiotensin II (Ang II) secretion increases and precedes PGF2alpha secretion during spontaneous luteolysis. In conclusion, we show for the first time that an acute increase of BF occurs in the peripheral vasculature of the mature CL together with increases in eNOS expression and ET-1 and Ang II secretion in the CL during the early stages of luteolysis in the cow. We propose that the increase in luteal BF may be induced by NO from large arterioles surrounding the CL, and simultaneously uterine or exogenous PGF2alpha directly increases ET-1 and Ang II secretion from endothelial cells of microcapillary vessels within the CL, thereby suppressing P secretion by luteal cells. Taken together, our results indicate that an acute increase in luteal BF occurs as a first step of luteolysis in response to PGF2alpha. Therefore, local BF plays a key role to initiate luteal regression in the cow.

Severity of oral mucositis correlates with the response of oral cancer to preoperative radiochemotherapy.

Oral mucositis is a dose-limiting toxic effect of radiotherapy and chemotherapy on oral cancer. The purpose of the present study is to assess the relationship between tumor response and oral mucositis in preoperative radiochemotherapy for oral cancer retrospectively. Fifty-four cases of oral squamous cell carcinoma were treated with concurrent radiochemotherapy prior to surgery. When oral mucositis was evaluated with the WHO scale, severe oral mucositis (Grades 3 and 4) developed in 22 cases (41%). A more than 50% reduction in tumor size was clinically observed in 38 cases (70%). From histopathological analysis of the surgical specimens all tumor cells observed appeared to be non-viable in 16 cases (29%). The cases with Grade 1, Grade 2, Grade 3 and Grade 4 oral mucositis included 33%, 62%, 85% and 89% of clinical good-response cases and 0%, 24%, 31% and 55% of histopathological good-response cases, respectively. This retrospective study suggests that severe oral mucositis promises a good response of oral squamous cell carcinoma to radiochemotherapy.