3D Tortuosity computation as a shape descriptor and its application to brain structure analysis.

In this study, we propose a novel method for quantifying tortuosity in 3D voxelized objects. As a shape characteristic, tortuosity has been widely recognized as a valuable feature in image analysis, particularly in the field of medical imaging. Our proposed method extends the two-dimensional approach of the Slope Chain Code (SCC) which creates a one-dimensional representation of curves. The utility of 3D tortuosity ( τ 3 D ) as a shape descriptor was investigated by characterizing brain structures. The results of the τ 3 D computation on the central sulcus and the main lobes revealed significant differences between Alzheimer's disease (AD) patients and control subjects, suggesting its potential as a biomarker for AD. We found a p < 0.05 for the left central sulcus and the four brain lobes.

The Jurassic rise of squamates as supported by lepidosaur disparity and evolutionary rates.

The squamates (lizards, snakes, and relatives) today comprise more than 10,000 species, and yet their sister group, the Rhynchocephalia, is represented by a single species today, the tuatara. The explosion in squamate diversity has been tracked back to the Cretaceous Terrestrial Revolution, 100 million years ago (Ma), the time when flowering plants began their takeover of terrestrial ecosystems, associated with diversification of coevolving insects and insect-eating predators such as lizards, birds, and mammals. Squamates arose much earlier, but their long pre-Cretaceous history of some 150 million years (Myr) is documented by sparse fossils. Here, we provide evidence for an initial radiation of squamate morphology in the Middle and Late Jurassic (174-145 Ma), and show that they established their key ecological roles much earlier than had been assumed, and they have not changed them much since.

Cortical Thickness Estimation: A Comparison of FreeSurfer and Three Voxel-Based Methods in a Test-Retest Analysis and a Clinical Application.

The cortical thickness has been used as a biomarker to assess different cerebral conditions and to detect alterations in the cortical mantle. In this work, we compare methods from the FreeSurfer software, the Computational Anatomy Toolbox (CAT12), a Laplacian approach and a new method here proposed, based on the Euclidean Distance Transform (EDT), and its corresponding computational phantom designed to validate the calculation algorithm. At region of interest (ROI) level, within- and inter-method comparisons were carried out with a test-retest analysis, in a subset comprising 21 healthy subjects taken from the Multi-Modal MRI Reproducibility Resource (MMRR) dataset. From the Minimal Interval Resonance Imaging in Alzheimer's Disease (MIRIAD) data, classification methods were compared in their performance to detect cortical thickness differences between 23 healthy controls (HC) and 45 subjects with Alzheimer's disease (AD). The validation of the proposed EDT-based method showed a more accurate and precise distance measurement as voxel resolution increased. For the within-method comparisons, mean test-retest measures (percentages differences/intraclass correlation/Pearson correlation) were similar for FreeSurfer (1.80%/0.90/0.95), CAT12 (1.91%/0.83/0.91), Laplacian (1.27%/0.89/0.95) and EDT (2.20%/0.88/0.94). Inter-method correlations showed moderate to strong values (R > 0.77) and, in the AD comparison study, all methods were able to detect cortical alterations between groups. Surface- and voxel-based methods have advantages and drawbacks regarding computational demands and measurement precision, while thickness definition was mainly associated to the cortical thickness absolute differences among methods. However, for each method, measurements were reliable, followed similar trends along the cortex and allowed detection of cortical atrophies between HC and patients with AD.

Ecomorphological diversification of squamates in the Cretaceous.

Squamates (lizards and snakes) are highly successful modern vertebrates, with over 10 000 species. Squamates have a long history, dating back to at least 240 million years ago (Ma), and showing increasing species richness in the Late Cretaceous (84 Ma) and Early Palaeogene (66-55 Ma). We confirm that the major expansion of dietary functional morphology happened before these diversifications, in the mid-Cretaceous, 110-90 Ma. Until that time, squamates had relatively uniform tooth types, which then diversified substantially and ecomorphospace expanded to modern levels. This coincides with the Cretaceous Terrestrial Revolution, when angiosperms began to take over terrestrial ecosystems, providing new roles for plant-eating and pollinating insects, which were, in turn, new sources of food for herbivorous and insectivorous squamates. There was also an early Late Cretaceous (95-90 Ma) rise in jaw size disparity, driven by the diversification of marine squamates, particularly early mosasaurs. These events established modern levels of squamate feeding ecomorphology before the major steps in species diversification, confirming decoupling of diversity and disparity. In fact, squamate feeding ecomorphospace had been partially explored in the Late Jurassic and Early Cretaceous, and jaw innovation in Late Cretaceous squamates involved expansions at the extremes of morphospace.

A novel voxel-based method to estimate cortical sulci width and its application to compare patients with Alzheimer's disease to controls.

A voxel-based method for measuring sulcal width was developed, validated and applied to a database. This method (EDT-based LM) employs the 3D Euclidean Distance Transform (EDT) of the pial surface and a Local Maxima labeling algorithm. A computational phantom was designed to test method performance; results revealed the method's inaccuracy δ, to range between 0.1 and 0.5 voxels, for a width that varied between 1 and 7 voxels. Two morphological descriptors were computed to characterize each defined sulcus: mean sulcal width (MSW) and mean absolute deviation (MAD). The former is the average width for all available width measurements within the sulcus, and the latter is the deviation of these measurements. The EDT-based LM method was applied to the Minimal Interval Resonance Imaging in the Alzheimer's Disease (MIRIAD) database, for a set of high-resolution Magnetic Resonance (MR) images of 66 subjects: 43 patients with Alzheimer Disease (AD) and 23 control subjects. AD causes significant gray matter loss; hence, some sulci were expected to broaden. Methodological results concurred with this hypothesis. After a Wilcoxon test, MSW was grater in the case of all sulci pertaining to AD patients, (p < 0.05, FDR corrected), whereas MAD showed significant differences in 8 sulci (p < 0.05, FDR corrected). This work presents a novel voxel-based method for measuring sulcal width and extracting descriptors to characterize and compare the sulci within and across subjects.

Virtual reality simulation of robotic transsphenoidal brain tumor resection: Evaluating dynamic motion scaling in a master-slave system.

BACKGROUND: Integrating simulators with robotic surgical procedures could assist in designing and testing of novel robotic control algorithms and further enhance patient-specific pre-operative planning and training for robotic surgeries. METHODS: A virtual reality simulator, developed to perform the transsphenoidal resection of pituitary gland tumours, tested the usability of robotic interfaces and control algorithms. It used position-based dynamics to allow soft-tissue deformation and resection with haptic feedback; dynamic motion scaling control was also incorporated into the simulator. RESULTS: Neurosurgeons and residents performed the surgery under constant and dynamic motion scaling conditions (CMS vs DMS). DMS increased dexterity and reduced the risk of damage to healthy brain tissue. Post-experimental questionnaires indicated that the system was well-evaluated by experts. CONCLUSION: The simulator was intuitively and realistically operated. It increased the safety and accuracy of the procedure without affecting intervention time. Future research can investigate incorporating this simulation into a real micro-surgical robotic system.

Acute Variceal Bleeding: Does Octreotide Improve Outcomes in Patients with Different Functional Hepatic Reserve?

BACKGROUND: Current guidelines do not differentiate in the utilization of vasoactive drugs in patients with cirrhosis and acute variceal bleeding (AVB) depending on liver disease severity. MATERIAL AND METHODS: In this retrospective study, clinical outcomes in 100 patients receiving octreotide plus endoscopic therapy (ET) and 216 patients with ET alone were compared in terms of failure to control bleeding, in-hospital mortality, and transfusion requirements stratifying the results according to liver disease severity by Child-Pugh (CP) score and MELD. RESULTS: In patients with CP-A or those with MELD < 10 octreotide was not associated with a better outcome compared to ET alone in terms of hospital mortality (CP-A: 0.0 vs. 0.0%; MELD < 10: 0.0 vs. 2.9%, p = 1.00), failure to control bleeding (CP-A: 8.7 vs. 3.7%, p = 0.58; MELD < 10: 5.3 vs. 4.3%, p = 1.00) and need for transfusion (CP-A: 39.1 vs. 61.1%, p = 0.09; MELD < 10: 63.2 vs. 62.9%, p = 1.00). Those with severe liver dysfunction in the octreotide group showed better outcomes compared to the non-octreotide group in terms of hospital mortality (CP-B/C: 3.9 vs. 13.0%, p = 0.04; MELD ≥ 10: 3.9 vs. 13.3%, p = 0.03) and need for transfusion (CP-B/C: 58.4 vs. 71.6%, p = 0.05; MELD ≥ 10: 50.6 vs. 72.7%, p < 0.01). In multivariate analysis, octreotide was independently associated with in-hospital mortality (p = 0.028) and need for transfusion (p = 0.008) only in patients with severe liver dysfunction (CP-B/C or MELD ≥ 10). CONCLUSION: Patients with cirrhosis and AVB categorized as CP-A or MELD < 10 had similar clinical outcomes during hospitalization whether or not they received octreotide.

Reporte de caso: intoxicación por clorfenapir con desenlace fatal / Report a case: chlorfenapyr intoxication with a fatal ending

El clorfenapir es un insecticida de uso agrícola, cuya ingesta en las personas produce un envenenamiento que a veces es fatal. Se presenta un caso clínico de paciente de sexo masculino con ingesta por vía oral del clorfenapir, con presentación de un cuadro compatible con deterioro neurológico y rabdomiolisis con desenlace fatal a pesar del tratamiento de soporte. El mecanismo de acción de esta substancia es la inhibición de la fosforilación oxidativa en las mitocondrias y se postula que este sea el mecanismo condicionante de la mortalidad en las personas, con lesiones en órganos principales como SNC y musculo, reportados en casos clínicos alrededor del mundo.

Chlorfenapyr is an insecticide for agricultural use, whose ingestion in people produces a poisoning that is sometimes fatal. It is presented a clinical case of a male patient with oral intake of chlorfenapyr, presenting a likeness compatible with neurological deterioration and rhabdomyolysis with fatal outcome despite supportive treatment. The mechanism of action of this substance is the inhibition of oxidative phosphorylation in the mitochondria and it is postulated that this is the conditioning mechanism of mortality in people, with lesions in major organs such as CNS and muscle, reported in clinical cases around the world.

Motivos de atención en salas de emergencia en hospitales públicos de La Paz Bolivia / Statement of care in emergency rooms in public hospitals of La Paz Bolivia

INTRODUCCIÓN: El presente estudio tiene como finalidad determinar los principales motivos de atención en las salas de emergencias de los hospitales públicos de La Paz. OBJETIVO: Determinar los principales motivos de atención en las salas de emergencias en Hospitales Públicos de La Paz - Bolivia. MATERIAL Y MÉTODOS: Se realizó un estudio descriptico retrospectivo transversal, recabando datos a través de una hoja de recolección de datos que identificaba los 10 principales motivos de atención en emergencias en 5 hospitales públicos de La paz, en el periodo de enero 2015 a octubre del 2015. RESULTADOS: Las principales causas de atención médica fueron las urgencias médicas (clínicas) con 70%, y la atención del trauma fue del 30%. CONCLUSIONES. Los principales motivos de atención médica en los servicios de urgencias en Hospitales Públicos de La Paz, son urgencias médicas con un componente clínico. DISCUSIÓN: Los servicios de emergencias constituyen una extensión hacia la comunidad de los centros hospitalarios, es frecuentes la percepción de mala calidad hacia las salas de emergencias por parte de los usuarios por diferentes motivos (sobresaturación, falta de equipamiento, etc.), para afrontar la problemática de esta situación es necesario conocer el comportamiento de las principales patologías en las salas de emergencias, por lo que se presenta el presente estudio determinando los principales motivos de atención en las salas de emergencias de los hospitales públicos en La Paz.

INTRODUCTION: This study aims to determine the main reasons for care in emergency rooms of public hospitals in La Paz. OBJECTIVE: To determine the main reasons for care in emergency rooms in public hospitals in La Paz - Bolivia MATERIAL AND METHODS: A retrospective cross-sectional descriptive study was conducted by collecting data through a data collection sheet identifying the top 10 reasons for emergency care in five public hospitals in La Paz, in the period January 2015 to October 2015. RESULTS: The main causes of medical care were medical emergencies (clinics) with 70% and trauma care was 30%. CONCLUSIONS: The main reasons for medical care in emergency departments in public hospitals in La Paz are medical emergencies with a clinical component. Discussion: Emergency services are an extension to the community hospitals poor quality to emergency rooms in often percieved by users for different reasons (supersaturation, lack of equipment, etc.) to meet the problems of this situation it is necessary to know the behavior of the main pathologies in emergency rooms, so this study is presented by determining the main reasons for care in emergency rooms of public hospitals in La Paz.

Activation of Adenosine Monophosphate-Activated Protein Kinase Is an Additional Mechanism That Participates in Mediating Inhibitory Actions of Prostaglandin F2Alpha in Mature, but Not Developing, Bovine Corpora Lutea.

Elevated cytosolic calcium and protein kinase C are well-established mediators of luteolytic actions of prostaglandin F2alpha (PGF2alpha). The objectives of this study were to determine 1) if calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) participates in mediating PGF2alpha actions in developing (Day [d]-4) and mature (d-10) bovine corpus luteum (CL), 2) distal targets of CAMKK2, 3) developmental expression of adenosine monophosphate-activated protein kinase (AMPK), and 4) effects of AMPK activation on progesterone (P4) production. Expression of AMPK increased as the CL matured. Activation of the prostaglandin receptor (FP) induced rapid phosphorylation of AMPK, which was blocked by a CAMKK2 inhibitor. Changes in basal P4 secretion in vitro were determined in response to AMPK activation via metformin (met) or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) in d-4 and d-10 CL. Production of P4 in d-10 CL decreased with met or AICAR compared to control, similar to activation by PGF2alpha. Therefore, potential distal targets of AMPK in d-10 CL were examined during induced functional regression via exogenous PGF2alpha. Serum and luteal P4 decreased at 2 and 4 h after administration of PGF2alpha. Protein expression of LDLR decreased at 2 and 4 h, while those of ACAT1 and STAR increased 4 h after PGF2alpha. During induced regression, alterations of cholesterol transport proteins contributed to decreased luteal and serum P4. Therefore, developmental differences in signal transduction associated with FP, specifically CAMKK2 and AMPK, partially contribute to differences in the ability of PGF2alpha to induce regression in mature, but not developing, bovine CL. Multiple cholesterol transport proteins, including LDLR, were altered by PGF2alpha and could be potential AMPK targets.

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.

Differential gene expression in the bovine corpus luteum during transition from early phase to midphase and its potential role in acquisition of luteolytic sensitivity to prostaglandin F2 alpha.

Prostaglandin F2 alpha (PGF(2alpha)) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF(2alpha) is used in beef and dairy cattle to synchronize estrus. A limitation of this protocol is insensitivity of the early CL to luteolytic actions of PGF(2alpha). The mechanisms underlying this differential luteal sensitivity are poorly understood. The developing CL has a maximum number of PGF(2alpha) receptors; therefore, differences in signaling events may be responsible for luteal insensitivity. Hence, differential gene expression at two developmental stages of CL, Day 4 (D-4) and D-10 after estrus, might account for differences in signal transduction pathways associated with luteal sensitivity. This possibility was examined in these studies. Microarray analysis (n = 3 cows per stage) identified 167 genes that were differentially expressed (P < 0.05). These were categorized into genes involved in protein biosynthesis and modification (18.5%), transcription regulation and DNA biosynthesis (18.5%), miscellaneous (17.0%), cell signaling (12.0%), steroidogenesis and metabolism (10.2%), extracellular matrix and cytoskeletal proteins (9.5%), unknown functions (6.0%), protein degradation (5.3%), and antioxidant property (3.0%). Real-time PCR confirmed the differential expression of nine selected genes, including tyrosine 3-monooxygenase/tryptophan 5-monooxygense activation protein zeta polypeptide (YWHAZ) and regulator of G protein signaling 2 24-kDa (RGS2), observed in microarray. Furthermore, the in vivo effect of exogenous PGF(2alpha) (n = 3 cows per stage) on selected genes that were found to be differentially expressed during this developmental transition was examined. PGF(2alpha) increased the expression of a guanine nucleotide-binding protein (G protein) beta polypeptide 1 (GNB1) in D-4 CL and calcium/calmodulin-dependent kinase kinase 2 beta (CAMKK2) in D-10 CL. Therefore, GNB1, CAMKK2, YWHAZ, and RGS2 are candidate genes that may have a significant role in acquisition of luteal sensitivity to PGF(2alpha). Additional evidence supporting the significance of the microarray data was obtained from the observation that the amount of CAMKK2 paralleled the differential mRNA expression observed for this gene when examined by microarray analysis and by real-time RT-PCR. Furthermore, the two types of luteal steroidogenic cells known to be targets for PGF(2alpha) actions were demonstrated to be a cellular source for CAMKK2.

Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2alpha-induced luteolysis of the sheep corpus luteum.

Three experiments were designed to examine the mechanisms that govern prostaglandin (PGF2alpha)-induced regression of the sheep corpus luteum. Evidence is presented supporting the involvement of endothelin 1 (EDN1) in PGF2alpha-induced luteolysis. Experiment 1 measured effects of PGF2alpha when actions of EDN1 were blocked by sustained administration of a type-A endothelin (EDNRA) or type-B endothelin (EDNRB) antagonist in vivo. Experiment 2 examined antisteroidogenic actions of PGF2alpha and EDN1 in the presence of an EDNRA or EDNRB antagonist in Day-8 luteal minces. In experiment 3, luteal cellular expression of EDNRA and EDNRB was determined immunohistochemically. Relative gene expression of EDNRA and EDNRB receptors was examined by real-time RT-PCR in Day-8 sheep corpora lutea. EDNRA, but not EDNRB, participated in antisteroidogenic actions of EDN1. During the first 12 h after PGF2alpha-induced luteolysis, EDNRA antagonist did not prevent a decline in serum progesterone concentrations. Early actions of PGF2alpha are either direct or mediated by something other than EDN1. However, beyond 12 h after PGF2alpha, serum progesterone concentrations increased in EDNRA antagonist-treated animals until they were the same as saline-treated controls, whereas an EDNRB antagonist had no effect in vivo or in vitro. The EDNRA antagonist negated the antisteroidogenic actions of EDN1 but only partially abolished the actions of PGF2alpha in vitro. In contrast, the EDNRB antagonist was ineffective in abolishing antisteroidogenic actions of EDN1 and PGF2alpha. Whereas real-time RT-PCR demonstrated high expression of EDNRA and low expression of EDNRB, immunohistochemically, only EDNRA was located in small steroidogenic, endothelial, and smooth muscle cells. In summary, studies in ovine corpora lutea provided strong evidence that: 1) EDNRA, but not EDNRB, mediates antisteroidogenic actions of EDN1, 2) actions of PGF2alpha are both independent of and dependent upon mediation by EDN1, and 3) small steroidogenic cells are targets for antisteroidogenic effects of EDN1. Furthermore, the results from experiment 1 suggest that the intermediary role of EDN1 may be more important in later stages of luteal regression.

PKC epsilon and an increase in intracellular calcium concentration are necessary for PGF2 alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells.

The hypotheses that PKC epsilon is necessary for: 1) PGF2 alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKC epsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKC epsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2 alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2 alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.PKC epsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKC epsilon expression was ablated by 75%, the inhibitory effect of PGF2 alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKC epsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKC epsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2 alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 mumol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2 alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKC epsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2 alpha.

Mechanisms of reduced luteal sensitivity to prostaglandin F2alpha during maternal recognition of pregnancy in ewes.

During maternal recognition of pregnancy, the conceptus stimulates endometrial secretion of PGF2alpha and PGE2. However, PGF2alpha is less effective in causing luteal regression in pregnant than in non-pregnant ewes. Experiments were conducted to elucidate mechanisms for reduced luteal sensitivity to PGF2alpha during maternal recognition of pregnancy. Corpora lutea (CL) were collected from pregnant and non-pregnant ewes 0, 4, or 12h following treatment with PGF2alpha on day 12 after estrus. Luteal PTGHS2 mRNA did not differ due to PGF2alpha or pregnancy status. Luteal PTGES mRNA was reduced in both pregnant and non-pregnant ewes after PGF2alpha treatment; while, luteal PTGFS mRNA was reduced 4h after PGF2alpha in pregnant, but not non-pregnant ewes. The result was a greater ratio of PTGES to PTGFS mRNA in pregnant ewes. Luteal mRNA for HPGD did not differ between pregnant and non-pregnant ewes on day 12. Luteal END1 mRNA was reduced in pregnant as compared to non-pregnant ewes prior to PGF2alpha challenge. Luteal END1 mRNA was increased after PGF2alpha in pregnant and non-pregnant ewes; however, ECE1 mRNA was reduced 4h after PGF2alpha in pregnant, but not non-pregnant ewes. The in vitro conversion of PGF2alpha to PGFM was greater in CL of pregnant than non-pregnant ewes at day 14. Luteal conversion of PGF2alpha to PGFM appears to be regulated post-transcriptionally. During maternal recognition of pregnancy, mechanisms of reduced luteal sensitivity to PGF2alpha may include a shift in prostaglandin production to the luteotropin PGE2, a reduction of ECE1 mRNA, and increased catabolism of PGF2alpha.

Participation of specific PKC isozymes in the inhibitory effect of ET-1 on progesterone accumulation in cells isolated from early- and mid-phase corpora lutea.

Expression of PKC alpha, beta I, beta II, epsilon and micro has been demonstrated in the whole bovine CL with PKC epsilon being differentially expressed as a function of development. In experiment 1 we have investigated the amount of mRNA encoding PKC epsilon at different stages of luteal development (days 1, 4, 10 and 17). In experiment 2, the cellular source of luteal PKC isozymes was determined. Enriched steroidogenic (SC) and endothelial (EC) cells from day-10 CL were used to examine this question by Western blot analysis and immuno-histochemistry. In experiment 3, Western blot analysis was used to examine the ability of ET-1 to activate luteal PKC isozymes in day-10 CL. In experiment 4, the role of luteal PKC isozymes in the ET-1 mediated inhibition of P(4) accumulation in steroidogenic cell cultures from day-4 and day-10 CL was examined. Abundance of PKC epsilon mRNA gradually increased from day-1 to -10 with no further increase on day-17. In experiment 2, PKC epsilon was exclusively detected in SC (LLC and SLC). In contrast, PKC alpha, beta I and beta II were detected in both SC and EC, with EC expressing higher amounts of PKC isozymes. In day-10 CL, ET-1 induced cellular redistribution of PKC alpha, beta I, epsilon but not beta II. Inhibitors specific for conventional PKC isozymes as well as PKC epsilon were able to negate the inhibitory effects of ET-1 on P4 accumulation in the day 10 CL. In the day-4 CL, the inhibitory effect of ET-1 might be mediated via conventional PKC. Thus, an exclusive presence of PKC epsilon in luteal steroidogenic cells, its higher expression along with the ability of ET-1 to stimulate its activation in day-10 CL strongly suggests that this PKC isoform may play an important regulatory role in decreasing P(4) during luteal regression. Inhibition of P(4) by ET-1 in the early CL may be mediated via conventional PKC isozymes.

Developmental sensitivity of the bovine corpus luteum to prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1): is ET-1 a mediator of the luteolytic actions of PGF2alpha or a tonic inhibitor of progesterone secretion?

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.

Effects of selective protein kinase c isozymes in prostaglandin2alpha-induced Ca2+ signaling and luteinizing hormone-induced progesterone accumulation in the mid-phase bovine corpus luteum.

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.

Prostaglandin F2alpha (PGF2alpha) independent and dependent regulation of the bovine luteal endothelin system.

We have examined the genes of the endothelin system that are targets for regulation by prostaglandin F2alpha (PGF2alpha). The effects of a luteolytic dose of PGF2alpha ) on the mRNA encoding endothelin converting enzyme-1 (ECE-1), pre-pro endothelin-1 (pp ET-1) and the ET receptors ETA, ETB, in bovine corpus luteum (CL) during the early (days 1 and 4), mid (day 10) or late (day 17) luteal phases were examined. The effect of the PGF(2alpha) treatment on ECE-1 protein, Big ET-1 and the biologically active mature ET-1 peptide were also examined. Most importantly, the direct ECE-1 activity was determined. Before day 10 of the cycle, in a PGF2alpha-independent manner, the amounts of mRNA encoding ET-1, ECE-1, ETA, and ETB were increased steadily from day 1. After day 10 of the cycle, expression of mRNA encoding pp ET-1 and ETA acquired responsiveness to exogenous PGF2alpha and both genes were up-regulated by the PGF2alpha treatment. This effect of PGF2alpha was also detected for the proteins corresponding to the mature ET-1. The enzymatic activity of ECE-1 remained unchanged throughout the lifespan of the CL in spite of the detected changes in mRNA and protein. The results suggest that the luteal endothelin system is regulated in a PGF2alpha-independent and -dependent manner. Importantly, an alteration in luteal ET-1 availability is most likely achieved by modulating the expression of mRNA encoding pp ET-1 and not by the amount or activity of ECE-1. This interpretation is supported by the observation that the activity of ECE-1 remained unchanged throughout the ovarian cycle. The combined effects of greater ET-1 availability and gene expression encoding the ETA receptor in the late luteal phase could render the CL, at this developmental stage, more sensitive or responsive to ET-1. If the luteal tissue is responsive to the available ET-1 during the early phase of the ovarian cycle, an additional role for ET-1 should be considered beyond mediating the luteolytic actions of PGF2alpha. Agents blocking the actions of ET-1 might be the best approach to interfere with the luteal ET system and test its physiological role(s) in vivo.

Expression and activation of protein kinase C isozymes by prostaglandin F(2alpha) in the early- and mid-luteal phase bovine corpus luteum.

Western blotting was used to identify the array of protein kinase C (PKC) isozymes expressed in the early (Day 4) and midcycle (Day 10) bovine corpus luteum (CL). PCKalpha, betaI, betaII, epsilon, and micro isozymes were detected in total protein samples prepared from both Day-4 and Day-10 corpora lutea. In contrast, specific antibodies for PKCgamma, eta, lambda, and theta isozymes failed to detect protein bands in the luteal samples. PKCbetaII and epsilon isozymes were expressed differentially at these two developmental stages of the bovine CL. In the Day-4 luteal samples, PKCepsilon was barely detectable; in contrast, in the Day-10 samples, the actin-corrected ratio for PKCepsilon was 1.16 +/- 0.13. This ratio was higher than the detected ratio for PKCbetaI and micro at this developmental phase of the CL (P < 0.01), but it was comparable with the ratio detected for the PCKalpha and betaII. The amount of PKCbetaII was, although not as dramatic, also greater in the Day-10 CL (actin-corrected ratio was 0.85 +/- 0.2) than in the Day-4 CL (0.35 +/- 0.09 [P < 0.01]). The actin-corrected ratios for all other PKC isozymes, alpha (Day 4 = 0.93 +/- 0.16, Day 10 = 0.97 +/- 0.09), betaI (Day 4 = 0.54 +/- 0.073, Day 10 = 0.48 +/- 0.74), and micro (Day 4 = 0.21 +/- 0.042, Day 10 = 0.21 +/- 0.38) were not different at these 2 days of the cycle. An experiment was designed to test whether activation of specific isozymes differed between CL that do or do not regress in response to PGF(2alpha). Bovine CL from Day 4 and Day 10 of the estrous cycle were collected and 1 mm CL fragments were treated in vitro for 0, 2.5, 5, 10 or 20 min with PGF(2alpha) (0.1, 1.0, and 10 nM) or minimal essential medium-Hepes vehicle. Translocation of PKC from cytoplasm to membrane fraction was used as indication of PKC activation by PGF(2alpha). Evidence for PKC activation was observed in both Day-4 and Day-10 luteal samples treated with 10 nM PGF(2alpha). Therefore, if PKC, an intracellular mediator associated with the luteal PGF(2alpha) receptor, contributes to the lesser sensitivity of the Day-4 CL, it is likely due to the differential expression of the epsilon and betaII isozymes of PKC at this stage and not due to an inability of the PGF(2alpha) receptor to activate the isozymes expressed in the early CL.