Hormonal Prostaglandin F2α Mediates Behavioral Responsiveness to a Species-Specific Multi-component Male Hormonal Sex Pheromone in a Female Fish.

Although hormonally-derived female sex pheromones have been well described in approximately a dozen species of teleost fish, only a few male sex pheromones have been characterized and the neuroendocrine underpinnings of behavioral responsiveness to them is not understood. Herein, we describe a study that addresses this question using the goldfish, Carassius auratus, an important model species of how hormones drive behavior in egg-laying teleost fishes. Our study had four components. First, we examined behavioral responsiveness of female goldfish and found that when injected with prostaglandin F2α (PGF2α), a treatment that drives female sexual receptivity, and found that they became strongly and uniquely attracted to the odor of conspecific mature males, while non-PGF2α-treated goldfish did not discern males from females. Next, we characterized the complexity and specificity of the male pheromone by examining the responsiveness of PGF2α-treated females to the odor of either mature male conspecifics or male common carp odor, as well as their nonpolar and polar fractions. We found that the odor of male goldfish was more attractive than that of male common carp, and that its activity was attributable to both its nonpolar and polar fractions with the later conveying information on species-identity. Third, we hypothesized that androstenedione (AD), a 19-carbon sex steroid produced by all male fish might be the nonpolar fraction and tested whether PGF2α-treated goldfish were attracted to either AD alone or as part of a mixture in conspecific water. We found that while AD was inactive on its own, it became highly attractive when added to previously unattractive female conspecific water. Lastly, in a test of whether nonhormonal conspecific odor might determine species-specificity, we added AD to water of three species of fish and found that while AD made goldfish water strongly attractive, its effects on other species holding water were small. We conclude that circulating PGF2α produced at the time of ovulation induces behavioral sensitivity to a male sex pheromone in female goldfish and that this male pheromone is comprised of AD and a mixture of body metabolites. Because PGF2α commonly mediates ovulation and female sexual behavior in egg-laying fishes, and AD is universally produced by male fishes as a precursor to testosterone, we suggest that these two hormones may have similar roles mediating male-female behavior and communication in many species of fish.

Prostaglandin F2α stimulates adhesion, migration, invasion and proliferation of the human trophoblast cell line HTR-8/SVneo.

INTRODUCTION: The amount of prostaglandin F2α (PGF2α) in the uterine lumen increases during the window of implantation in many mammals, including humans. We hypothesized that PGF2α regulates processes related to human embryo implantation. METHODS: The effect of PGF2α was studied using an in vitro model of human extravillous trophoblast (EVT) cell line (HTR-8/SVneo). Adhesion, proliferation, invasion and migration assays, zymography for metalloproteinases (MMP) activity, and gene/protein expression analyses were applied. Doses of 100 nM and/or 1 µM of PGF2α and fluprostenol were used. PGF2α receptor (PTGFR), MMP9 and MMP2 proteins in the human first trimester placenta were localized by immunohistochemistry and immunofluorescence. RESULTS: This study is the first reporting the expression of PTGFR protein in the first trimester placenta, as well as in HTR-8/SVneo cells. PGF2α and fluprostenol increased HTR-8/SVneo cell proliferation and adhesion to extracellular matrix protein (P < 0.05). This effect was abolished by mitogen activated protein kinases (MAPK) inhibitor. PGF2α induced phosphorylation of focal adhesion kinase and MAPK1/3 (P < 0.05). PGF2α increased mRNA content and protein activity of MMP9, and gene and protein expression of interleukin-6 (P < 0.05). EVT cell migration and invasiveness were stimulated by PGF2α (P < 0.05). The PGF2α effect on cell invasion was reduced by inhibitors of MMP2, MMP9 and mTOR. In all experiments, the stimulatory effects of PGF2α were diminished by using a PTGFR antagonist. DISCUSSION: Our findings suggest a significant role for PGF2α in mechanisms associated with implantation. PGF2α acting by PTGFR in HTR-8/SVneo cells stimulates their adhesion and proliferation through the MAPK signaling pathway and increases invasiveness inducing MMP proteolytic activity and mTOR signaling.

Regulation of the porcine corpus luteum during pregnancy.

The new corpora lutea (CLs) in pigs are formed from the preovulatory follicles after the luteinizing hormone (LH) surge. However, total autonomy and independence of CLs from LH up to Day 12 of cycle has recently been questioned. Transformation of estrous cycle CL to CL of pregnancy initiated by embryonic signals requires not only the cessation of prostaglandin F2 (PGF2α) supply to the luteal tissue but also needs the CL to overcome luteolytic acquisition and/or changing its sensitivity to PGF2α during Days 12-14 of pregnancy. The luteolytic cascade is prevented by inhibition of lymphocyte infiltration and leucocyte recruitment, limitation of cell apoptosis, upregulation of pregnancy-associated genes and an enhanced antiluteolytic role of PGE2 Our 'two-signal switch hypothesis' highlights the importance of post PGF2α and PGE2 receptor signaling pathways activation in CLs during luteolysis and rescue. The 'luteolytic switch' involves increased expression of many regression mediators and activation of the post PTGFR signaling pathway. The 'rescue switch' initiated by embryonic signals - estradiol 17ß and PGE2 - induces post PTGER2/4 pathway, turning the 'luteolytic switch' off and triggering activity of genes responsible for CL maintenance. In mid and late pregnancy, CLs are maintained by LH and the synergistic action of metabolic hormones. This paper provides an outline of recent views on CL regression, rescue and maintenance during pregnancy in pigs that conflict with previous paradigms and highlights new findings regarding the actions of prostaglandins, role of microRNAs (miRNA) and immune system and signaling pathways governing the life cycle of porcine CL.

Differential contribution of COX-1 and COX-2 derived prostanoids to cortical spreading depression-Evoked cerebral oligemia.

Cortical spreading depression (CSD) is considered a significant phenomenon for human neurological conditions and one of its key signatures is the development of persistent cortical oligemia. The factors underlying this reduction in cerebral blood flow (CBF) remain incompletely understood but may involve locally elaborated vasoconstricting eicosanoids. We employed laser Doppler flowmetry in urethane-anesthetized rats, together with a local pharmacological blockade approach, to test the relative contribution of cyclooxygenase (COX)-derived prostanoids to the oligemic response following CSD. Administration of the non-selective COX inhibitor naproxen completely inhibited the oligemic response. Selective inhibition of COX-1 with SC-560 preferentially reduced the early reduction in CBF while selective COX-2 inhibition with NS-398 affected only the later response. Blocking the action of thromboxane A2 (TXA2), using the selective thromboxane synthase inhibitor ozagrel, reduced only the initial CBF decrease, while inhibition of prostaglandin F2alpha action, using the selective FP receptor antagonist AL-8810, blocked the later phase of the oligemia. Our results suggest that the long-lasting oligemia following CSD consists of at least two distinct temporal phases, mediated by preferential actions of COX-1- and COX-2-derived prostanoids: an initial phase mediated by COX-1 that involves TXA2 followed by a later phase, mediated by COX-2 and PGF2alpha.

Luteotropic and luteolytic factors regulate mRNA and protein expression of progesterone receptor isoforms A and B in the bovine endometrium.

The aim of the present study was to examine the effects of luteotropic and luteolytic factors on the mRNA and protein levels of progesterone receptor isoforms A (PGRA) and B (PGRB) in the bovine endometrium. Endometrial slices from Days 6-10 and 17-20 of the oestrous cycle were treated with LH (100ngmL-1), oestradiol (E2; 1×10-8M), prostaglandin (PG) E2 (1×10-6M) and PGF2α (1×10-6M) and the nitric oxide donor NONOate (1×10-4M); these treatments lasted for 6h for mRNA expression analysis and 24h for protein expression analysis. On Days 6-10 of the oestrous cycle PGRAB (PGRAB; the entire PGRA mRNA sequence is common to the PGRB mRNA sequence) mRNA expression in endometrial slices was enhanced by E2 treatment (P<0.001), whereas PGRB mRNA expression was increased by LH (P<0.001), E2 (P<0.05) and NONOate (P<0.05) treatment. On Days 17-20, PGRAB mRNA expression increased after E2 (P<0.001) and PGE2 (P<0.05) treatment; PGRB mRNA expression was increased by PGE2 (P<0.05) and PGF2α (P<0.01) treatment, but decreased by LH (P<0.05). On Days 6-10 protein levels of PGRA were stimulated by E2 (P<0.01), whereas PGRB protein levels were increased by LH (P<0.05) and E2 (P<0.05). On Days 17-20 of the oestrous cycle, PGRA protein levels were enhanced by E2 (P<0.05) and PGF2α (P<0.05), whereas PGRB protein levels were stimulated by PGE2 (P<0.05) and PGF2α (P<0.001). These data suggest that luteotropic and luteolytic factors affect PGRA and PGRB mRNA and protein levels, and this may regulate the effects of progesterone on endometrial cells.

The mare as a model for luteinized unruptured follicle syndrome: intrafollicular endocrine milieu.

Luteinized unruptured follicle (LUF) syndrome is a recurrent anovulatory dysfunction that affects up to 23% of women with normal menstrual cycles and up to 73% with endometriosis. Mechanisms underlying the development of LUF syndrome in mares were studied to provide a potential model for human anovulation. The effect of extended increase in circulating LH achieved by administration of recombinant equine LH (reLH) or a short surge of LH and decrease in progesterone induced by prostaglandin F2α (PGF2α) on LUF formation (Experiment 1), identification of an optimal dose of COX-2 inhibitor (flunixin meglumine, FM; to block the effect of prostaglandins) for inducing LUFs (Experiment 2), and evaluation of intrafollicular endocrine milieu in LUFs (Experiment 3) were investigated. In Experiment 1, mares were treated with reLH from Day 7 to Day 15 (Day 0=ovulation), PGF2α on Day 7, or in combination. In Experiment 2, FM at doses of 2.0 or 3.0 mg/kg every 12 h and human chorionic gonadotropin (hCG) (1500 IU) were administered after a follicle ≥32 mm was detected. In Experiment 3, FM at a dose of 2.0 mg/kg every 12 h plus hCG was used to induce LUFs and investigate the intrafollicular endocrine milieu. No LUFs were induced by reLH or PGF2α treatment; however, LUFs were induced in 100% of mares using FM. Intrafollicular PGF2α metabolite, PGF2α, and PGE2 were lower and the ratio of PGE2:PGF2α was higher in the induced LUF group. Higher levels of intrafollicular E2 and total primary sex steroids were observed in the induced LUF group along with a tendency for higher levels of GH, cortisol, and T; however, LH, PRL, VEGF-A, and NO did not differ between groups. In conclusion, this study reveals part of the intrafollicular endocrine milieu and the association of prostaglandins in LUF formation, and indicates that the mare might be an appropriate model for studying the poorly understood LUF syndrome.

Oxidative Stress and IgG Antibody Modify Periodontitis-CRP Association.

In a previous report, we demonstrated the inverse association of high serum 8-isoprostane levels, a marker for oxidative stress, with decreased serum IgG antibodies to oral bacteria. The association between increased serum IgG with increased plaque and periodontitis (increased probing depths) was attenuated by high systemic oxidative stress. Other investigations have reported a role for systemic oxidative stress as a stimulus of hepatic C-reactive protein (CRP) response. These observations led us to hypothesize that the reported relationship of periodontitis to elevated serum CRP, a systemic inflammatory marker, may be modified by oxidative stress and that the levels of serum antibodies to oral bacteria might be an intermediary explanatory variable linking the association of systemic oxidative stress, periodontal disease, and levels of CRP. This hypothesis was explored as a secondary analysis of the Dental ARIC (Atherosclerosis Risk in Communities) study using serum levels of CRP, serum IgG levels to 16 oral organisms, serum levels of 8-isoprostane, and periodontal status. The findings indicate periodontitis is associated with high CRP in the presence of elevated oxidative stress that serves to suppress the IgG response. Only within the highest 8-isoprostane quartile was periodontitis (pocket depth) associated with increased serum CRP levels (P = 0.0003). Increased serum IgG antibody levels to oral bacteria were associated with lowered serum CRP levels. Thus, systemic oxidative stress, which has been demonstrated to be associated with increased levels of CRP in other studies, appears to be associated with the suppression of bacterial-specific IgG levels, which in the presence of periodontal disease can result in an enhanced systemic CRP response. Conversely, individuals with increased serum IgG antibodies to plaque bacteria exhibit lowered serum CRP levels. These 2 factors, oxidative stress and the serum IgG response, appear to function in opposing directions to modify serum levels of CRP and the association with periodontitis.

Desarrollo tecnológico de una solución oftálmica de latanoprost para el tratamiento del glaucoma / Technological development of latanoprost eye solution for treatment of glaucoma

Introducción: el latanoprost, análogo de la prostaglandina F2α, es un agonista selectivo del receptor FP prostanoide que reduce la presión intraocular por incremento del flujo del humor acuoso, se clasifica como un antiglaucomoso y se indica en el tratamiento del glaucoma de ángulo abierto y la hipertensión ocular. Objetivo: se diseñó una formulación de uso oftálmico conteniendo latanoprost como sustancia activa, a una concentración de 500 μg/mL, que cumpla con los índices de control de calidad para esta forma farmacéutica y proporcione el efecto terapéutico deseado. Métodos: se realizaron los estudios de formulación y se ensayaron nueve variantes tecnológicas, seleccionándose la composición y procedimiento tecnológico más adecuados para su posterior escalado en la industria. Se ajustó el pH empleando la trometamina y la isotonicidad, con manitol, según las exigencias de una formulación oftálmica. Se desarrolló y validó una técnica analítica por Cromatografía Líquida de Alta Resolución para estudiar la estabilidad, determinar su fecha de vencimiento y para efectuar el control de la calidad de la formulación. Resultados: el desarrollo tecnológico, resultó satisfactorio, se obtiene una formulación que cumple con todas las especificaciones descritas en la técnica desarrollada por el fabricante, para el control de la calidad del producto. Se comprobó que la preparación mantiene sus propiedades físicas, químicas y microbiológicas inalterables por un período de 24 meses, almacenada a una temperatura controlada entre 2 y 8 °C y protegido de la luz. El escalado a nivel piloto no reveló problemas tecnológicos y resultó no irritante, según el criterio establecido por la Unión Europea para la irritabilidad oftálmica. Conclusiones: se obtuvo una formulación de colirio de latanoprost, útil en el tratamiento del glaucoma, con todas las especificaciones de calidad para este tipo de forma farmacéutica, con lo que pudiera aumentarse el arsenal terapéutico de Cuba(AU)

Introduction: latanoprost, F2α prostaglandine analogue, is a selective FP receptor Prostanoide agonist that reduces the intraocular pressure due to increase of aqueous humor; it is classified as an anti-glaucoma drug and indicated for the treatment of open angle glaucoma and eye hypertension. Objective: aformula for ophthalmic uses was designed, which contains latanoprost as active ingredient at concentration of 500 µg/mL and complies with the quality control indexes for this pharmaceutical form and provides the desired therapeutic effect. Methods: the formulation studies were conducted and nine technological variants were tested; the most adequate composicion and technological procedure was selected for further industry scaling. The pH index and isotonicity were then adjusted using trometamin and manitol, respectively according to the demands of the eye formulation. A high resolution liquid chromatography-based analytical technique was developed and validated to study the stability, to determine the expiry date and to make the quality control of the formulation. Results: the technological development proved to satisfactory since this formulation complies with all the specifications described in the manufacturer's technique for the quality control of the product. It was confirmed that this preparation keeps its physical, chemical and microbiological properties unchanged for 24 months if stored at 2-8 ºC and protected from light. Pilot scale-up did not show either technological problem or irritating effect according to the European Union criteria for eye irritability. Conclusions: there was attained a latanoprost eye drop formulation for the treatment of glaucoma, which complies with all the quality specifications for this type of pharmaceutical form and could increase the therapeutic arsenal in Cuba(AU)

Promising alternative clinical uses of prostaglandin F2α analogs: beyond the eyelashes.

Prostaglandin F2α analogs, commonly prescribed for glaucoma treatment, have been shown to induce side effects such as cutaneous hypertrichosis and hyperpigmentation. Therefore, these medications have theoretic applications in the treatment of alopecia and disorders of hypopigmentation. We reviewed the literature to find original studies assessing the use of prostaglandin F2α analogs in these settings. Studies and reports were analyzed in regards to androgenic alopecia, alopecia areata, chemotherapy-induced alopecia, vitiligo, and hypopigmented scarring. Based on the results of these studies, and consideration of pathophysiologic mechanism, the most promising applications for prostaglandin F2α analogs include androgenic alopecia, chemotherapy-induced alopecia, and alopecia areata concurrently treated with corticosteroids.

Modulation of AD neuropathology and memory impairments by the isoprostane F2α is mediated by the thromboxane receptor.

Beside amyloid-ß plaques and neurofibrillary tangles, brain oxidative damage has been constantly implicated in Alzheimer's disease (AD) pathogenesis. Numerous studies demonstrated that F2-isoprostanes, markers of in vivo lipid peroxidation, are elevated in AD patients and mouse models of the disease. Previously, we showed that the 8-isoprostaneF2α, (8ISO) increases brain amyloid-ß levels and deposition in the Tg2576 mice. However, no data are available on its effects on behavior and tau metabolism. To this end, we characterize the behavioral, biochemical, and neuropathologic effects of 8ISO in the triple transgenic mouse model. Compared with controls, mice receiving 8ISO showed significant memory deficits, increase in tau phosphorylation, activation of the cyclin kinase 5 pathway, and neuroinflammation. All these effects were blocked by pharmacologic blockade of the thromboxane receptor. Our findings establish the novel functional role that oxidative stress via the formation of this isoprostane plays in the development of cognitive impairments and AD-related tau neuropathology. It provides important preclinical support to the neurobiological importance of the thromboxane receptor as an active player in the pathogenesis of AD.

Auto-amplification system for prostaglandin F2α in bovine corpus luteum.

The bovine corpus luteum (CL) is hypothesized to utilize a local auto-amplification system for prostaglandin (PG) F2α production. The objective of the present study was to determine if such a PGF2α auto-amplification system exists in the bovine CL, and if so, which factors regulate it. PGF2α significantly stimulated intra-luteal PGF2α production in all luteal phases, but did not affect PGE2 production. The stimulatory effect of exogenous PGF2α on CL PGF2α production was lower at the early luteal phase. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthase (PTGS), significantly suppressed the PGF2α-stimulated PGF2α production by luteal tissue, indicating that the PGF2α in the medium was of luteal origin. Consistent with these secreted-PGF2α profiles, PGF2α receptor (PTGFR) protein expression was higher during the mid and late luteal phases than at early and developing luteal phases. Treatment of cultured bovine luteal cells obtained from the mid-luteal phase with PGF2α (1 µM) significantly increased the expressions of PTGS2, PGF synthase (PGFS), and carbonyl reductase1 (CBR1) at 24 hr post-treatment. Together, these results suggest the presence of a local auto-amplification system for PGF2α mediated by PTGS2, PGFS, and CBR1 in the bovine CL, which may play an important role in luteolysis.

Mechanisms of intracellular calcium homeostasis in developing and mature bovine corpora lutea.

Although calcium (Ca(2+)) is accepted as an intracellular mediator of prostaglandin F2 alpha (PGF2alpha) actions on luteal cells, studies defining mechanisms of Ca(2+) homeostasis in bovine corpora lutea (CL) are lacking. The increase in intracellular Ca(2+) concentration ([Ca(2+)]i) induced by PGF2alpha in steroidogenic cells from mature CL is greater than in those isolated from developing CL. Our hypothesis is that differences in signal transduction associated with developing and mature CL contribute to the increased efficacy of PGF2alpha to induce a Ca(2+) signal capable of inducing regression in mature CL. To test this hypothesis, major genes participating in Ca(2+) homeostasis in the bovine CL were identified, and expression of mRNA, protein, or activity, in the case of phospholipase Cbeta (PLCbeta), in developing and mature bovine CL was compared. In addition, we examined the contribution of external and internal Ca(2+) to the PGF2alpha stimulated rise in [Ca(2+)]i in LLCs isolated from developing and mature bovine CL. Three differences were identified in mechanisms of calcium homeostasis between developing and mature CL, which could account for the lesser increase in [Ca(2+)]i in response to PGF2alpha in developing than in mature CL. First, there were lower concentrations of inositol 1,4,5-trisphosphate (IP3) after similar PGF2alpha challenge, indicating reduced phospholipase C beta (PLCbeta) activity, in developing than mature CL. Second, there was an increased expression of sorcin (SRI) in developing than in mature CL. This cytoplasmic Ca(2+) binding protein modulates the endoplasmic reticulum (ER) Ca(2+) release channel, ryanodine receptor (RyR), to be in the closed configuration. Third, there was greater expression of ATP2A2 or SERCA, which causes calcium reuptake into the ER, in developing than in mature CL. Developmental differences in expression detected in whole CL were confirmed by Western blots using protein samples from steroidogenic cells isolated from developing and mature CL. Localization of these genes in steroidogenic luteal cells was confirmed by immunohistochemistry. Therefore, it is concluded that the cellular mechanisms that allow PGF2alpha to induce a calcium signal of greater magnitude in mature than in developing CL involve 1) greater PLCbeta activity with enhanced generation of IP3, 2) an enhanced Ca(2+) release from the ER via unrestrained RYR2 due to a decrease in SRI expression, and 3) a reduction in calcium reuptake to the ER due to lower expression of ATP2A2. Accordingly, the increase in [Ca(2+)]i induced by PGF2alpha in mature large steroidogenic cells had less dependency from extracellular calcium than in those isolated from immature CL.

Oestradiol and prostaglandin F2α regulate sexual displays in females of a sex-role reversed fish.

The mechanisms regulating sexual behaviours in female vertebrates are still poorly understood, mainly because in most species sexual displays in females are more subtle and less frequent than displays in males. In a sex-role reversed population of a teleost fish, the peacock blenny Salaria pavo, an external fertilizer, females are the courting sex and their sexual displays are conspicuous and unambiguous. We took advantage of this to investigate the role of ovarian-synthesized hormones in the induction of sexual displays in females. In particular, the effects of the sex steroids oestradiol (E2) and testosterone (T) and of the prostaglandin F2α (PGF2α) were tested. Females were ovariectomized and their sexual behaviour tested 7 days (sex steroids and PGF2α) and 14 days (sex steroids) after ovariectomy by presenting females to an established nesting male. Ovariectomy reduced the expression of sexual behaviours, although a significant proportion of females still courted the male 14 days after the ovary removal. Administration of PGF2α to ovariectomized females recovered the frequency of approaches to the male's nest and of courtship displays towards the nesting male. However, E2 also had a positive effect on sexual behaviour, particularly on the frequency of approaches to the male's nest. T administration failed to recover sexual behaviours in ovariectomized females. These results suggest that the increase in E2 levels postulated to occur during the breeding season facilitates female mate-searching and assessment behaviours, whereas PGF2α acts as a short-latency endogenous signal informing the brain that oocytes are mature and ready to be spawned. In the light of these results, the classical view for female fishes, that sex steroids maintain sexual behaviour in internal fertilizers and that prostaglandins activate spawning behaviours in external fertilizers, needs to be reviewed.

Pharmacologic characterization of human male urethral smooth muscle: an in vitro approach.

OBJECTIVE: To elucidate the functional responses of isolated human urethral smooth muscle to various agents known to exert smooth muscle contraction or relaxation. METHODS: Specimens of penile urethra were obtained from male patients who had undergone male-to-female gender reassignment surgery. Using the tissue bath technique, the contraction induced by increasing concentrations (1 nM-10 µM) of norepinephrine, phenylephrine, acetylcholine, carbachol, prostaglandin F2α, endothelin 1, angiotensin II, and oxytocin was measured. In another set-up, the effects of C-type natriuretic peptide (0.1 nM-1 µM), sodium nitroprusside, sildenafil, forskolin, alpha2-antagonist delquamine, and acetylcholine (1 nM/10 nM-10 µM) on the tension induced by norepinephrine were investigated. The production of cyclic guanosine monophosphate (GMP) and cyclic adenosine monophosphate (AMP) was measured by means of specific radioimmunoassays. RESULTS: Endothelin 1, oxytocin, prostaglandin F2α, norepinephrine, and phenylephrine induced dose-dependent contraction of the isolated urethral tissue, whereas acetylcholine, carbachol, and angiotensin II had no or only minor contractile effects. The contraction induced by norepinephrine was reversed by the drugs with the following rank order of efficacy: sodium nitroprusside > delquamine > sildenafil > C-type natriuretic peptide > forskolin > acetylcholine. The maximal reversion of tension ranged from 68% (sodium nitroprusside) to 22% (acetylcholine). The relaxing effects of the drugs were paralleled by a several-fold increase in tissue levels of cyclic GMP and cyclic adenosine monophosphate. CONCLUSION: The results provide evidence that urethral smooth muscle is under the control of endogenous compounds, such as adrenergic agonists (norepinephrine and phenylephrine), vasoactive peptides, prostagladins, NO/cyclic GMP, and acetylcholine, assumed to influence micturition at the peripheral level.

Isolation and characterization of luteal cells in buffalo (Bubalus bubalis).

The objective of the study was to evaluate the morphological and functional characteristics of luteal cells isolated from buffalo ovary. Luteal cells exhibited columnar morphology and contact inhibition at the stage of confluence. Protein concentrations increased linearly from Day 3 to Day 7 of culture. DNA concentrations increased from Day 3 to Day 5 and then declined to Day 7 of culture. PGF2alpha concentrations decreased progressively from Day 3 to Day 7 of culture. It was concluded that buffalo luteal cells could serve as an excellent model for studying the specific role of PGF2alpha in maternal recognition of pregnancy and implantation.

Growth and regression in bovine corpora lutea: regulation by local survival and death pathways.

The bovine corpus luteum (CL) is a transient gland with a life span of only 18 days in the cyclic cow. Mechanisms controlling CL development and secretory function may involve factors produced both within and outside this gland. Although luteinizing hormone (LH) surge is the main trigger of ovulation and granulosa cells luteinization, many locally produced agents such as arachidonic acid (AA) metabolites, growth factors and cytokines were shown to complement gonadotropins action in the process of CL development. Bovine CL is a highly vascular gland, where the very rapid angiogenesis rate (until Day 5 of the cycle) results in the development of a capillary network, endowing this gland with one of the highest blood flow rate per unit mass in the body. Angiogenesis in the developing CL is later followed by either controlled regression of the microvascular tree in the non-fertile cycle or maintenance and stabilization of the blood vessels, as seen during pregnancy. Different luteal cell types (both steroidogenic and accessory luteal cells: immune cells, endothelial cells, pericytes and fibroblasts) are involved in the pro- and/or anti-angiogenic responses. The balance between pro- and anti-angiogenic responses to the main luteolysin - prostaglandin F2α (PGF2α) could be decisive in whether or not PGF2α induces CL regression. Fibroblast growth factor-2 (FGF2) may be one of the factors that modulate the angiogenic response to PGF2α. Manipulation of local production and action of FGF2 will provide new tools for reproductive management of dairy cattle. Luteolysis is characterized by a rapid decrease in progesterone production, followed by structural regression. Factors like endothelin-1, cytokines (tumour necrosis factorα, interferons) and nitric oxide were all shown to play critical roles in functional and structural regression of the CL by inhibiting steroidogenesis and inducting apoptosis.

Role of PGF2α in luteolysis based on inhibition of PGF2α synthesis in the mare.

The effects of inhibition of PGF2α synthesis on luteolysis in mares and on the incidence of prolonged luteal activity were studied in controls and in a group treated with flunixin meglumine (FM), a PGF2α inhibitor (n = 6/group). The FM was given every 8 hours (1.0 mg/kg) on each of Days 14.0 to 16.7. Concentration (pg/mL) of PGF2α metabolite averaged over 8 hours of hourly blood sampling at the beginning of each day, was lower in the FM group than in the controls on Day 14 after ovulation (6.7 ± 1.3 vs. 13.8 ± 2.9, P < 0.05), Day 15 (15.0 ± 3.9 vs. 35.2 ± 10.4, P < 0.10), and Day 16 (21.9 ± 5.7 vs. 54.7 ± 11.4, P < 0.03). Concentration (ng/mL) of progesterone (P4) was greater in the FM group than in the controls on Day 14 (10.1 ± 0.9 vs. 7.7 ± 0.9, P < 0.08), Day 15 (9.2 ± 1.0 vs. 4.3 ± 1.0, P < 0.008), and Day 16 (5.6 ± 1.6 vs. 1.2 ± 0.4, P < 0.02). The interval from ovulation to the beginning of a decrease in P4 and to the end of luteolysis (P4 < 1 ng/mL) was each delayed (P < 0.03) by ~1 day in the FM group. Intervals involving the luteal phase were long (statistical outliers, P < 0.05) in two mares in the FM group, indicating prolonged luteal activity. Results supported the hypotheses that (1) inhibition of PGF2α synthesis interferes with luteolysis in mares and (2) inhibition of PGF2α at the expected time of luteolysis may lead to prolonged luteal activity.

Prostaglandins and prostaglandin receptor antagonism in migraine.

Human models of headache may contribute to understanding of prostaglandins' role in migraine pathogenesis. The current thesis investigated the migraine triggering effect of prostaglandin E2 (PGE2) in migraine patients without aura, the efficacy of a novel EP4 receptor antagonist, BGC20-1531, in prevention of PGE2-induced headache and the ability of prostaglandin F2α (PGF2α) to trigger headache without any vasodilatation in healthy volunteers. All studies were designed as double-blind, placebo-controlled, cross-over experiments, where PGE2/PGF2α or saline were infused over 20-25 min. In the study with EP4 receptor antagonist healthy volunteers were pre-treated with two different doses of BGC20-1531 or placebo followed by PGE2 infusion over 25 min. The headache data were collected during the whole study day, whereas the possible vascular changes were measured during the in-hospital phase of 1.5 h. The infusion of PGE2 caused the immediate migraine-like attacks and vasodilatation of the middle cerebral artery in migraine patients without aura. The highly specific and potent EP4 receptor antagonist, BGC20-1531, was not able to attenuate PGE2-induced headache and vasodilatation of both intra- and extra-cerebral arteries. The intravenous infusion of PGF2α did not induce headache or statistically significant vasoconstriction of cerebral arteries in healthy volunteers. Novel data on PGE2-provoked immediate migraine-like attacks suggest that PGE2 may be one of the important final products in the pathogenesis of migraine. The lack of efficacy of EP4 receptor antagonist suggests that a single receptor blockade is not sufficient to block PGE2 responses, hence EP2 receptor should be investigated as a potential drug target for the treatment of migraine. The absence of headache during the PGF2α infusion demonstrates that vasodilating properties are necessary for the induction of headache and migraine.

Biosynthesis and degradation of canine placental prostaglandins: prepartum changes in expression and function of prostaglandin F2α-synthase (PGFS, AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD).

There is no distinct explanation of the mechanism for the prepartal prostaglandin F2alpha (PGF2alpha) increase in pregnant dogs. Although the PGF2alpha-synthase (PGFS [AKR1C3]) mRNA expression and localization profiles have been previously investigated in canine utero/placental compartments, the availability and biochemical activity of the PGFS (AKR1C3) protein remain unknown. In order to better understand the regulation of canine uterine PGF2alpha availability and eventual prepartum release in luteolytic amounts in dogs, canine-specific PGFS (AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD) antibodies were generated and used to characterize the expression, cellular localization, and biochemical properties of PGFS (AKR1C3) and HPGD in the utero/placental compartments and corpus luteum throughout pregnancy and at prepartum luteolysis. PGFS (AKR1C3) expression was weak or absent in luteal samples. Uterine PGFS (AKR1C3) was up-regulated postimplantation and declined prepartum. The utero/placental expression of PGFS (AKR1C3) was identified in the superficial uterine glands throughout gestation and in the trophoblast cells within the feto-maternal contact zone during placentation, suggesting a possible role for PGFS (AKR1C3) in the trophoblast invasion. Utero-placental HPGD was up-regulated until postimplantation, lower at midgestation, and greatly suppressed at prepartum. Expression was routinely identified in the endometrial surface and glandular epithelia, and positive signals were also observed in the trophoblast cells at the feto-maternal contact zone. The biochemical activity of recombinant PGFS (AKR1C3) and HPGD was confirmed after its expression in a heterologous system. The colocalization of HPGD with PGFS (AKR1C3) expression suggests a modulatory role for HPGD as a gatekeeper of the supply of prostaglandin in the pregnant canine uterus.

Arachidonic acid supplementation enhances in vitro skeletal muscle cell growth via a COX-2-dependent pathway.

Arachidonic acid (AA) is the metabolic precursor to a diverse range of downstream bioactive lipid mediators. A positive or negative influence of individual eicosanoid species [e.g., prostaglandins (PGs), leukotrienes, and hydroxyeicosatetraenoic acids] has been implicated in skeletal muscle cell growth and development. The collective role of AA-derived metabolites in physiological states of skeletal muscle growth/atrophy remains unclear. The present study aimed to determine the direct effect of free AA supplementation and subsequent eicosanoid biosynthesis on skeletal myocyte growth in vitro. C2C12 (mouse) skeletal myocytes induced to differentiate with supplemental AA exhibited dose-dependent increases in the size, myonuclear content, and protein accretion of developing myotubes, independent of changes in cell density or the rate/extent of myogenic differentiation. Nonselective (indomethacin) or cyclooxygenase 2 (COX-2)-selective (NS-398) nonsteroidal anti-inflammatory drugs blunted basal myogenesis, an effect that was amplified in the presence of supplemental free AA substrate. The stimulatory effects of AA persisted in preexisting myotubes via a COX-2-dependent (NS-389-sensitive) pathway, specifically implying dependency on downstream PG biosynthesis. AA-stimulated growth was associated with markedly increased secretion of PGF(2α) and PGE(2); however, incubation of myocytes with PG-rich conditioned medium failed to mimic the effects of direct AA supplementation. In vitro AA supplementation stimulates PG release and skeletal muscle cell hypertrophy via a COX-2-dependent pathway.