Identification and mechanistic investigation of ellagitannins from Osbeckia octandra that attenuate liver fibrosis via the TGF-ß/SMAD signaling pathway.

Fibrosis is a major problem in chronic liver disease with limited treatment options due to its complex nature. Herbal medicines are often used as an alternative. The aim of this study was to investigate the therapeutic potential of Osbeckia octandra and to identify its active compounds and regulatory pathways. The effects of crude leaf suspension and boiled leaf extract were investigated in an animal model, and the extract was found to be the more effective treatment. Three major bioactive compounds, pedunculagin, casuarinin, and gallic acid, were isolated from the extract using the hepatic stellate cell line, LX-2-based antifibrotic effect evaluation system. The results showed that all these compounds ameliorated LX-2 in fibrotic state. This inhibitory mechanism was confirmed through the TGF-ß/SMAD signaling pathway. Collectively, the presence of these compounds in O. octandra suggests its potential as a treatment for liver fibrosis.

Anti-Fibrotic and Anti-Angiogenic Activities of Osbeckia octandra Leaf Extracts in Thioacetamide-Induced Experimental Liver Cirrhosis.

Chronic liver inflammation has become a major global health concern. In the absence of clinical surrogate markers to diagnose inflammatory liver disease, the intervention with effective drugs in modern medicine tends to be late. In Sri Lanka, traditional medical practitioners prescribe herbal preparations from Osbeckia octandra for the prevention and treatment of liver disorders. To test the efficacy of such treatments, we have administered thioacetamide (TAA) to male Wistar rats to induce chronic liver damage (disease control; DC) and examined how various leaf extracts: crude leaf suspension (CLS), boiled leaf extract (BLE), sonicated leaf extract (SLE), methanol leaf extract (MLE) and hexane leaf extract (HLE) of O. octandra ameliorate TAA-induced liver disease. The CLS, BLE and SLE treatments in cirrhotic rats significantly attenuated disease-related changes, such as liver weight and hepato-enzymes. The mRNA levels of Tnf-α were significantly decreased by 3.6, 10 and 3.9 times in CLS, BLE and SLE compared to DC. The same treatments resulted in significantly lower (19.5, 4.2 and 2.4 times) α-Sma levels compared to DC. In addition, Tgf-ß1 and Vegf-R2 mRNA expressions were significantly lower with the treatments. Moreover, BLE expressed a strong anti-angiogenic effect. We conclude that CLS, BLE and SLE from O. octandra have potent hepatic anti-fibrotic effects in TAA-induced liver cirrhosis.

In vitro and in vivo impairment of embryo implantation by commonly used fungicide Mancozeb.

The fungicide Mancozeb is an endocrine-disrupting chemical and the mode of action of Mancozeb on embryo implantation is largely unknown. Mancozeb (1 and 3 µg/ml) significantly reduced Jeg-3 trophoblastic spheroids attachment to endometrial epithelial Ishikawa cells. Mancozeb treatment from gestation day (GD) 1 to GD8 or from GD4 to GD8 significantly lowered the number of implantation sites with higher incidence of morphological abnormalities in the reproductive tissues. However, these were not seen in the treatment from GD1 to GD4. Mancozeb at 30 mg/kg BW/d did not alter the expression of p53, COX-2, or PGFS transcripts in the uterus, but down-regulated the PGES transcript and protein. Mancozeb treatment in human endometrial stromal cells did not alter the decidualization response, but the morphological transformation was impaired. Taken together, exposure to Mancozeb affected embryo implantation probably through the modulation of decidualization and to delineate the exact mode of action needs further investigations.

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.

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.

Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress.

Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival.

Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.

This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E2 (PGE2), which dose-dependently (10(-6), 10(-7), and 10(-8) M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP2 receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE2 or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE2 released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP2 receptors. Thus, the bovine oviduct provides a PGE2-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1.

Prostaglandin F2alpha increases endothelial nitric oxide synthase in the periphery of the bovine corpus luteum: the possible regulation of blood flow at an early stage of luteolysis.

Prostaglandin F(2)(alpha) (PGF(2)(alpha)) released from the uterus causes alterations in luteal blood flow, reduces progesterone secretion, and induces luteolysis in the bovine corpus luteum (CL). We have recently discovered that luteal blood flow in the periphery of the mature CL acutely increases coincidently with pulsatile increases in a metabolite of PGF(2)(alpha) (PGFM). In this study, we characterized changes in regional luteal blood flow together with regional alterations in endothelial nitric oxide synthase (eNOS) expression during spontaneous luteolysis and in response to PGF(2)(alpha). Smooth muscle actin-positive blood vessels larger than 20 microm were observed mainly in the periphery of mature CL. PGF(2)(alpha) receptor was localized to luteal cells and large blood vessels in the periphery of mid-CL. PGF(2)(alpha) acutely stimulated eNOS expression in the periphery but not in the center of mature CL. Injection of the NO donor S-nitroso-N-acetylpenicillamine into CL induced an acute increase in luteal blood flow and shortened the estrous cycle. In contrast, injection of the NOS inhibitor l-NAME into CL completely suppressed the acute increase in luteal blood flow induced by PGF(2)(alpha) and delayed the onset of luteolysis. In conclusion, PGF(2)(alpha) has a site-restricted action depending on not only luteal phase but also the region in the CL. PGF(2)(alpha) stimulates eNOS expression, vasodilation of blood vessels, and increased luteal blood flow in periphery of mature CL. Furthermore, the increased blood flow is mediated by NO, suggesting that the acute increase in peripheral blood flow to CL is one of the first physiological indicators of NO action in response to PGF(2)(alpha).

Spermatozoa stimulate prostaglandin synthesis and secretion in bovine oviductal epithelial cells.

The dynamic action of oviductal secretory compounds on spermatozoa function is well documented. In contrast, the effect of spermatozoa on oviductal function remains poorly characterized. Previously, our lab and others have shown that prostaglandin (PG), together with other vasoactive peptides, plays major roles in oviductal contractions and sperm transport. We therefore examined the effect of spermatozoa on the production of PG by cow oviductal epithelial cells (OEC). A bovine spermatozoa-OEC co-culture system was utilized for this purpose. OECs in the second passage were co-cultured for 0, 1, 3, 6, 12, and 24 h with six doses of motile, killed, or truncated spermatozoa heads (control; without spermatozoa, 10(2)-10(6) spermatozoa/ml medium). The levels of PGE(2) and PGF(2alpha) in the medium were measured using enzyme immunoassays. Messenger RNA expression of cyclooxygenase-2, PGF synthase (PGFS), and PGE synthase (PGES) was investigated using real-time RT-PCR. The results indicated that motile spermatozoa increased the secretion of PG by OEC as well as cellular expression of mRNA for cyclooxygenase, PGES, and PGFS in a dose- and time-dependent manner. A maximum three- to fivefold increased secretion of PG was observed with a dose of 10(5) spermatozoa/ml after a 12-h co-incubation. Neither killed spermatozoa nor truncated spermatozoa heads stimulated oviductal biosynthesis and secretion of PG at any dose or time point observed. The results provide the first evidence that live spermatozoa in the oviduct up-regulate the local PG system, and thereby, enhance oviductal contractions. Thus, spermatozoa may bear a role in accelerating their own transport into the fertilization site.

Vascular endothelial growth factor system in the cow oviduct: a possible involvement in the regulation of oviductal motility and embryo transport.

Vascular endothelial growth factor (VEGF) is a potent angiogenic and permeability enhancing factor, which shows the highest activity in the oviduct during the periovulatory period of the estrous cycle in cattle. It has also been shown that the contraction activity of oviduct is highest during the periovulatory period. The present study therefore focused on the possible involvement of VEGF in the regulation of biosynthesis and secretion of contraction-relaxation-related substances in the cow oviduct. Possible autonomous VEGF system in the oviduct as well as its endocrine control was also studied. Bovine oviductal epithelial cells (BOEC) in the second passage were cultured with VEGF (1 ng/ml) alone or with luteinizing hormone (LH; 10 ng/ml), estradiol 17-beta (E2; 1 ng/ml), and/or progesterone (P4; 1 ng/ml). The levels of prostaglandins (PGs), endothelin-1 (ET-1), and angiotensin II (Ang II) in the medium were measured using second antibody enzymeimmunoassay (EIA). The mRNA expressions for cycloxygenase-2 (Cox-2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), prepro-ET-1, endothelin converting enzyme-1 (Ece-1), angiotensin converting enzyme-1 (Ace-1), VEGF and its receptors were investigated using real-time RT-PCR. The results indicate that, (1) VEGF dose-dependently stimulated the release of prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2alpha), and ET-1, but not Ang II. VEGF and VEGF with LH, E2, and P4 upregulated mRNA expression for biosynthesis cascade of PG, ET-1 as well as their release. However, only the combination of VEGF with LH, E2, and P4 upregulated mRNA for Ace-1 and Ang II release, but not VEGF alone. (2) Treatments of LH, with E2 and/or P4 increased the mRNA expression for VEGF, Flk-1 and Flt-1, and (3) VEGF itself downregulated the expression of mRNA for VEGF, and LH, E2, and P4 enhanced this downregulatory effect. The results of the present study provide the first evidence that (1) VEGF directly stimulates the biosynthesis and release of PGE2, PGF2alpha, and ET-1 in the bovine oviduct, (2) LH stimulates the oviductal VEGF system, and (3) VEGF downregulates the oviductal VEGF system and this downregulation was further intensified in the presence of LH. The data suggest that the preovulatory LH-surge, together with increasing E2 secretion from the Graffian follicle and basal P4 levels from the regressing corpus luteum (CL), upregulates the oviductal VEGF system, inducing the maximum oviductal production of contraction-relaxation-related substances for active oviduct contraction and rapid transport of gametes to the fertilization site. However, the oviductal VEGF elevation caused by the LH-surge, appears to downregulate the oviductal VEGF system immediately after ovulation thereby may contribute to suppress oviductal contraction to secure slow transport of the embryo to the uterus at the optimal time.

N-acetylglucosaminyltransferase I activity of bovine oviduct epithelial cells: stimulation by luteinizing hormone, vascular endothelial growth factor and tumor necrosis factor alpha.

N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101), which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins, was found in media cultured with bovine oviduct epithelial cells (BOEC) obtained from non-pregnant cows during the follicular phase. Combined treatment with specific hormones increased GnT I release from BOEC. Luteinizing hormone (LH; 10 ng/ml) alone slightly, but together with 17beta-estradiol (E2; 1 ng/ml), synergistically increased GnT I activity. Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF) alpha, which have been shown to have their highest activities in the bovine oviduct during the periovulatory period, also increased in GnT I activity. This study provides the first evidence of an increase of GnT I release from BOEC in vitro, and shows that endocrine as well as local factors such as LH, VEGF and TNFalpha increase this activity. The results suggest that GnT I activity in the bovine oviduct may contribute to the induction of glycosylation and thereby contributing to the provision of the optimal microenvironment for fertilization and early development of the embryos.

Cooperative expression of monocyte chemoattractant protein 1 within the bovine corpus luteum: evidence of immune cell-endothelial cell interactions in a coculture system.

Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF 2alpha together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF 2alpha for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P < 0.05). Basal secretion of EDN1 by EC was substantial (approximately 2 ng/ml), but was not affected by coculture with PBMC (P > 0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P < 0.05), but again, EDN1 secretion was unchanged (P > 0.05). Interestingly, PGF 2alpha did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P > 0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF 2alpha (P > 0.05), but MLC produced less progesterone in the presence of ActPBMC (P < 0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF 2alpha. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.

A female pseudohermaphrodite Holstein heifer with gonadal mosaicism.

An 11-month-old Holstein heifer diagnosed as a female pseudohermaphrodite (PH) was subjected to clinical, hormonal, histological, and cytogenetic examinations. The urogenital sinus and external genitalia of the heifer were virilized and no vulval orifice was present. A male urethra-like tubular structure was palpable in the perineal area; urination occurred through a urethral fossa at the distal end of the urethra (cranial aspect of the mammary tissue). Bilateral ovaries and uterine horns, ovarian follicular development, ovulation and CL formation (with increasing blood P4 concentration) were similar to a normal heifer. Testosterone (T) concentrations of all samples collected both before and after hCG challenge were <0.1 ng/mL (absence of a responsive endogenous androgen source). Histologically, the ovary, uterus, oviduct and vagina resembled the normal female reproductive tract. Although the corpus cavernosum was present, the penile urethra and corpus spongiosum penis were hypoplastic. A normal female karyotype (60, XX) was detected in metaphase spreads obtained from cultured peripheral lymphocytes. Moreover, Y-chromosome-specific sequences were not detected in genomic DNA from lymphocytes. By PCR analysis of genomic DNA (from the tissues of urogenital organs), Y-chromosome-specific sequences (both SRY and Amelogenin (Amel) locus) were detected. In conclusion, this was a female PH heifer due to gonadal mosaicism, with normal female genitalia and ovarian activity. This is the first report of an SRY-positive, mosaic female PH heifer.

Real-time relationships in intraluteal release among Prostaglandin F2alpha, endothelin-1, and angiotensin II during spontaneous luteolysis in the cow.

It is well known that prostaglandin F(2alpha) (PGF(2alpha)) is a physiological luteolysine, and that its pulsatile release from the endometrium is a luteolytic signal in many species. There is now clear evidence that the vasoactive peptides endothelin-1 (ET-1) and angiotensin II (Ang II) interact with PGF(2alpha) in the luteolytic cascade during PGF(2alpha)-induced luteolysis in the cow. Thus, we investigated the local secretion of PGF(2alpha), ET-1, and Ang II in the corpus luteum (CL) and their real-time relationships during spontaneous luteolysis in the cow. For this purpose, an in vivo microdialysis system (MDS) implanted in the CL was utilized to observe local secretion changes within the CL microenvironment. Each CL of cyclic Holstein cows (n = 6) was surgically implanted with MDS capillary membranes (18 lines/6 cows) on Day 15 (estrus = Day 0) of the estrous cycle. The concentrations of PGF(2alpha), ET-1, Ang II, and progesterone (P) in the MDS samples were determined by enzyme immunoassays. The intraluteal PGF(2alpha) secretion slightly increased from 12 h after the onset of luteolysis (0 h) and drastically increased (by about 300%) from 24 h. Intraluteal ET-1 secretion increased from 12 h. Intraluteal Ang II secretion was elevated from 0 h and was maintained at high levels (about 180%) toward estrus. In each MDS lines (in the same microenvironment) within the regressing CL, the local releasing profiles of PGF(2alpha), ET-1, and Ang II CL positively correlated with each other (P < 0.05) at high proportions in 18 MDS lines (PGF(2alpha) vs. ET-1, 44.4%; PGF(2alpha) vs. Ang II, 55.6%; ET-1 vs. Ang II, 38.9%). In contrast, there was no clear relationship among these substances released into different MDS lines implanted in the same CL (with different microenvironments). In conclusion, we propose that the increase of PGF(2alpha), ET-1, and Ang II within the CL during luteolysis is a common phenomenon for both PGF(2alpha)-induced and spontaneous luteolysis. Moreover, this study illustrated the in vivo relationships in intraluteal release among PGF(2alpha), ET-1, and Ang II during spontaneous luteolysis in the cow. The data suggest that these vasoactive substances may interact with each other in a local positive feedback manner to activate their secretion in the regressing CL, thus accelerating and completing luteolysis.

Real-time dynamics of prostaglandin F2alpha release from uterus and corpus luteum during spontaneous luteolysis in the cow.

Prostaglandin (PG) F2alpha released from the uterus in a pulsatile fashion is essential to induce regression of the corpus luteum (CL) in the cow. In addition to the uterus, the CL has also been recognized as a site of PGF(2alpha) production. Therefore, this study aimed to determine the detailed dynamics of the releasing profile of CL-derived PGF2alpha together with uterus-derived PGF2alpha during spontaneous luteolysis in the cow. Non-lactating Holstein cows (n = 6) were surgically implanted with a microdialysis system (MDS) on day 15 (oestrus = day 0) of the oestrous cycle. Simultaneously, catheters were implanted to collect ovarian venous plasma ipsilateral to the CL as well as jugular venous plasma. The concentrations of PGF2alpha, 13,14-dihydro-15-keto-PGF2alpha (PGFM) and progesterone in the MDS and plasma samples were determined by enzyme immunoassays. The intra-luteal PGF2alpha secretion slightly increased after the onset of luteolysis (0 h) and drastically increased from 24 h, and was maintained at high levels towards the following oestrus. Furthermore, PGF2alpha was released from the CL into the ovarian vein in a pulsatile manner during spontaneous luteolysis. Also, the fact that intra-luteal secretion of PGF2alpha and PGFM showed a positive correlation indicates the existence of a local metabolic pathway for PGF2alpha in the CL. In conclusion, the present study clarified the real-time dynamics of uterus-derived PGF2alpha and CL-derived PGF2alpha during spontaneous luteolysis in the cow, and gives the first in vivo evidence that the CL releases PGF2alpha during spontaneous luteolysis in the cow. Although the physiological relevance of CL-derived PGF2alpha appears to be restricted to a local role as an autocrine/paracrine factor in the CL, overall results support the concept that the local release of PGF2alpha within the regressing CL amplifies the luteolytic action of PGF2alpha from the uterus.

Endothelin-1 within the corpus luteum during spontaneous luteolysis in the cow: local interaction with prostaglandin F2alpha and angiotensin II.

It is generally accepted that endothelin-1 (ET-1) is involved in the regression of the corpus luteum (CL) in cows, but there are few in vivo data available on the local release of vasoconstrictors, including ET-1. Thus, we aimed to determine in detail the local secretion of ET-1, angiotensin II (Ang II) and prostaglandin F2alpha (PGF2alpha) within the CL during spontaneous luteolysis in the cow. To observe real-time dynamics of the releasing profile of CL-derived factors, a microdialysis system was surgically implanted in the CL on day 15 of the estrous cycle and continuously perfused with Ringer's solution. Local secretion of ET-1, Ang II and PGF2alpha increased immediately after the onset of luteolysis (the time point when progesterone release started to decrease within the CL) and was maintained at high levels. A positive relationship was observed in intra-luteal changes among ET-1, Ang II and PGF2alpha release. This is the first real-time and in vivo evidence that the secretion of ET-1 together with Ang II and PGF2alpha immediately increases within the CL after the onset of spontaneous luteolysis. Consequently, we suggest that the activation of a local positive feedback mechanism among ET-1, Ang II and PGF2alpha might play a functional role in the paracrine modulation of luteolytic cascade and, simultaneously, the elevated ET-1 with Ang II and PGF2alpha should induce a strong vasoconstriction, thereby reducing the blood supplying the CL during spontaneous luteolysis.

Tumor necrosis factor alpha in the bovine oviduct during the estrous cycle: messenger RNA expression and effect on secretion of prostaglandins, endothelin-1, and angiotensin II.

Tumor necrosis factor (TNF) alpha is an important physiological mediator of cell-to-cell communication. Recent observations suggest that TNFalpha is involved in the control of reproductive functions. The present study examined the role of TNFalpha in the secretion of factors involved in regulating smooth muscle contraction, such as prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2alpha), endothelin-1 (ET-1), and angiotensin II (Ang II), as it was in the original by the cow oviduct at different stages of the estrous cycle using an in vitro microdialysis system. Expression of mRNA for TNFalpha and its receptors (TNFalpha-R) was also evaluated. For microdialysis, the lumen of a portion (length, 10 cm) of the each oviductal segment was implanted with a dialysis capillary membrane, and TNFalpha (100 ng/ml) was infused for 4-8 h during a 16-h incubation period. The microdialysis system maintains cell-to-cell integrity and cell-to-cell communication, and it enables real-time observation of physiological changes in the luminal release of different substances. Concentrations of PG, ET-1, and Ang II in 4-h fractions were measured using second-antibody enzyme immunoassays. Infusion of TNFalpha stimulated oviductal secretion of PG, ET-1, and Ang II during the follicular and postovulatory stages, but not during the luteal stage. Expression of TNFalpha, TNFalpha-R type I, and TNFalpha-R type II mRNA was detected in the bovine oviduct by reverse transcription-polymerase chain reaction. High expression of both TNFalphaR types and ligands was detected during the follicular and postovulatory stages, whereas low expression was detected during the luteal stage. The results of the present study provide, to our knowledge, the first direct evidence that TNFalpha stimulates PG, ET-1, and Ang II secretion and that up-regulation of the TNFalpha system occurs in the cow oviduct during the periovulatory period. In conclusion, the TNFalpha system may optimize the release of contraction-related substances and modulate local contraction to regulate the oviductal transport of the gametes and embryo.