Inteligencia artificial y simulación en urología / Artificial intelligence and simulation in urology

Introducción y objetivo: La inteligencia artificial (IA) está en pleno desarrollo, y su implementación en la medicina ha supuesto una mejora en la práctica clínica y quirúrgica. Una de sus múltiples aplicaciones es el entrenamiento quirúrgico, con la creación de programas que permiten evitar complicaciones y riesgos para el paciente. El objetivo de este artículo es analizar las ventajas de la IA aplicada al entrenamiento quirúrgico en urología.Material y métodosSe realiza una revisión de la literatura de los artículos publicados en inglés sobre la IA aplicada a la medicina, especialmente a la cirugía y a la adquisición de habilidades quirúrgicas.ResultadosEl entrenamiento quirúrgico ha evolucionado con el tiempo gracias a la IA. Se ha creado un modelo de aprendizaje quirúrgico en el que las habilidades se adquieren de forma gradual, evitando complicaciones al paciente. El uso de simuladores permite un aprendizaje progresivo en el que la cantidad y la complejidad de los procedimientos aumentan progresivamente. Adicionalmente, la IA se utiliza en pruebas de imagen para planificar cirugías o tratamientos.ConclusiónActualmente el uso de la IA en la práctica clínica diaria supone un avance en la medicina, y en particular en la formación quirúrgica. (AU)

Introduction and objective: Artificial intelligence (AI) is in full development and its implementation in medicine has led to an improvement in clinical and surgical practice. One of its multiple applications is surgical training, with the creation of programs that allow avoiding complications and risks for the patient. The aim of this article is to analyze the advantages of AI applied to surgical training in urology.Material and methodsA literary research is carried out to identify articles published in English regarding AI applied to medicine, especially in surgery and the acquisition of surgical skills.ResultsSurgical training has evolved over time thanks to AI. A model for surgical learning where skills are acquired in a progressive way while avoiding complications to the patient, has been created. The use of simulators allows a progressive learning, providing trainees with procedures that increase in number and complexity. On the other hand, AI is used in imaging tests for surgical or treatment planning.ConclusionCurrently, the use of AI in daily clinical practice has led to progress in medicine, specifically in surgical training. (AU)

Artificial intelligence and simulation in urology.

INTRODUCTION AND OBJECTIVE: Artificial intelligence (AI) is in full development and its implementation in medicine has led to an improvement in clinical and surgical practice. One of its multiple applications is surgical training, with the creation of programs that allow avoiding complications and risks for the patient. The aim of this article is to analyze the advantages of AI applied to surgical training in urology. MATERIAL AND METHODS: A literary research is carried out to identify articles published in English regarding AI applied to medicine, especially in surgery and the acquisition of surgical skills. RESULTS: Surgical training has evolved over time thanks to AI. A model for surgical learning where skills are acquired in a progressive way while avoiding complications to the patient, has been created. The use of simulators allows a progressive learning, providing trainees with procedures that increase in number and complexity. On the other hand, AI is used in imaging tests for surgical or treatment planning. CONCLUSION: Currently, the use of AI in daily clinical practice has led to progress in medicine, specifically in surgical training.

Artificial intelligence and simulation in urology. / Inteligencia artificial y simulación en urología.

INTRODUCTION AND OBJECTIVE: Artificial intelligence (AI) is in full development and its implementation in medicine has led to an improvement in clinical and surgical practice. One of its multiple applications is surgical training, with the creation of programs that allow avoiding complications and risks for the patient. The aim of this article is to analyze the advantages of AI applied to surgical training in urology. MATERIAL AND METHODS: A literary research is carried out to identify articles published in English regarding AI applied to medicine, especially in surgery and the acquisition of surgical skills. RESULTS: Surgical training has evolved over time thanks to AI. A model for surgical learning where skills are acquired in a progressive way while avoiding complications to the patient, has been created. The use of simulators allows a progressive learning, providing trainees with procedures that increase in number and complexity. On the other hand, AI is used in imaging tests for surgical or treatment planning. CONCLUSION: Currently, the use of AI in daily clinical practice has led to progress in medicine, specifically in surgical training.

The isoflavone daidzein directly affects porcine ovarian cell functions and modifies the effect of follicle-stimulating hormone.

The key biological active molecule of soya is the isoflavone daidzein, which possesses phytoestrogenic activity. The direct effect of soya and daidzein on ovarian cell functions is not known. This study examined the effect of daidzein on basic porcine ovarian granulosa cell functions and the response to follicle-stimulating hormone (FSH). We studied the effects of daidzein (0, 1, 10 and 100 µm), FSH (0, 0.01, 0.1, 1 IU/ml) and combinations of FSH (0, 0.01, 0.1, 1 IU/ml) + daidzein (50 µm) on proliferation, apoptosis and hormone release from cultured porcine ovarian granulosa cells and ovarian follicles. The expression of a proliferation-related peptide (PCNA) and an apoptosis-related peptide (Bax) was analysed using immunocytochemistry. The release of progesterone (P4) and testosterone (T) was detected using EIA. Leptin output was analysed using RIA. Daidzein administration increased granulosa cell proliferation, apoptosis and T and leptin release but inhibited P4 output. Daidzein also increased T release and decreased P4 release from cultured ovarian follicles. Follicle-stimulating hormone stimulated granulosa cell proliferation, apoptosis and P4, T and leptin release. The addition of daidzein promoted FSH-stimulated apoptosis (but not proliferation) but suppressed FSH-stimulated P4, T and leptin release. Our observations of FSH action confirm previous data on the stimulatory effect of FSH on ovarian cell proliferation, apoptosis and steroidogenesis and demonstrate for the first time the involvement of FSH in the upregulation of ovarian leptin release. Our observations of daidzein effects demonstrated for the first time that this soya isoflavone affected basic ovarian cell functions (proliferation, apoptosis and hormones release) and modified the effects of FSH. Daidzein promoted FSH action on ovarian cell proliferation and apoptosis and suppressed, and even inverted, FSH action on hormone release. The direct action of daidzein on basic ovarian cell functions and the ability of these cells to respond to FSH indicate the potential influence of soya-containing diets on female reproductive processes via direct action on the ovary.

Metabolic status and ghrelin regulate plasma levels and release of ovarian hormones in layer chicks.

The aim of the present study was to examine the role of nutritional status, the metabolic hormone ghrelin and their interrelationships in the control of chicken hormones involved in the regulation of reproduction. For this purpose, we identified the effect of food deprivation, administration of ghrelin 1-18 and their combination on plasma levels of testosterone (T), estradiol (E), arginine-vasotocin (AVT) and growth hormone (GH) as well as the release of these hormones by isolated and cultured ovarian fragments. It was observed that food deprivation reduces plasma T and E and increases plasma AVT and GH levels. Food restriction also reduced the amount of E produced by isolated ovaries, but it did not affect the ovarian secretion of T and AVT. No ovarian GH secretion was detected. Ghrelin administered to ad libitum fed chickens did not affect plasma T and E levels, but it did increase plasma GH and AVT concentrations. Moreover, it partially prevented the effect of food deprivation on plasma E and AVT levels, but not on T or GH levels. Ghrelin administration to control birds promoted ovarian T, but not E or AVT release and reduced T and no other hormonal outputs in birds subjected to food restriction. Our results (1) confirmed the ovarian origin of the main plasma T and E and the extra-ovarian origin of the main blood AVT and GH; (2) showed that food deprivation-induced suppression of reproduction may be caused by suppression of T and E and the promotion of AVT and GH release; (3) suggest the involvement of ghrelin in control chicken E, AVT and GH output; and (4) indicates that ghrelin can either mimic or modify the effect of the intake of low calories on chicken plasma and ovarian hormones, i.e. it can mediate the effect of metabolic state on hormones involved in the control of reproduction.

Obestatin directly controls chicken ovarian cell functions.

The aim of the present in-vitro study was to examine the role of obestatin in the direct control of basic avian ovarian granulosa cell functions – proliferation, apoptosis and secretory activity. In addition, the effects of obestatin on hormone release by cultured ovarian granulosa cells and follicular fragments (containing both granulosa and theca cells) were examined. We identified the effect of obestatin addition (0.1, 10 or 100 ng/ml medium) on the accumulation of markers of proliferation (PCNA, cyclin B1, MAPK/ERK1,2) and nuclear (TdT) and cytoplasmic (bax, caspase 3) apoptosis, as well as the release of progesterone (P), testosterone (T) and estradiol (E) by cultured chicken granulosa cells. Furthermore, the action of obestatin addition (0.1, 10 or 100 ng/ml medium) on the release of P, T, E and argininevasotocin (AVT) by cultured fragments of chicken ovarian follicles was examined. The accumulation of proliferation and apoptosis markers was assessed by immunocytochemistry and SDS PAGE-Western immunoblotting. The release of hormones was determined by an EIA. It was observed that obestatin addition could inhibit the accumulation of proliferation markers (PCNA and cyclin B1, but not of MAPK/ERK1,2), promote the expression of nuclear (TdT) and cytoplasmic (bax, caspase 3) apoptosis markers and suppress P, T, and E release by cultured granulosa cells. In cultured ovarian follicular fragments, obestatin promoted P, T, and E, but not AVT, release. These observations represent the first demonstration that (i) obestatin can directly control avian ovarian cell proliferation, apoptosis and hormone release and (ii) the interrelationship between theca and granulosa cells can determine the characteristics of obestatin action on ovarian secretory activity.

Ricinus communis L. stem bark extracts regulate ovarian cell functions and secretory activity and their response to Luteinising hormone.

Ricinus communis L. has ethnopharmacological contraceptive reputation but its stem bark has unexplored mechanisms of action in female reproductive system. In the present study, the effect of methanolic and aqueous extracts from the stem bark of the plant was examined on basic porcine ovarian granulosa cell functions and its response to Luteinising hormone (LH)-the upstream hormonal regulator. Systemic treatment of methanolic and aqueous extracts stimulated cell proliferation (proliferating cell nuclear antigen, PCNA) and also promoted cell apoptosis (caspase-3). Aqueous extract has inverted the stimulatory effect of LH on PCNA but not on caspase-3. Methanolic extract stimulated as well as inhibited progesterone release and stimulated testosterone secretion. Whereas aqueous extract inhibited both steroid releases and suppressed the stimulatory effect of LH on progesterone release and promoted the inhibitory effect of LH on testosterone release. In conclusion, the present study unveils the mechanism of action of R. communis stem bark in in vitro condition. These suggest its possible contraceptive efficacy by exerting its regulatory role over LH and on basic ovarian cell functions and secretion activity.

Comparative proteomic analysis of the breast muscle response to chronic corticosterone administration in broiler chickens showing long or short tonic immobility.

Broilers of the same genetic origin were classified as short or long tonic immobility duration (STI and LTI, respectively) phenotypes and treated chronically with vehicle (control) or corticosterone (CORT) dissolved in drinking water between 27 and 42 d of age. Differential expression of proteins and mRNA was examined using 2-dimensional gel electrophoresis and real-time PCR to elucidate the mechanism behind the severe retardation of broiler breast muscle growth caused by LTI and CORT. The majority of the 13 proteins found to be differentially expressed in breast muscle of STI and LTI broilers are involved in either glycolysis (5 proteins) or myofilament formation (5 proteins). Of the 16 proteins differentially expressed in breast muscle following CORT treatment, 6 are structural proteins, 5 are categorized as cellular defense and stress proteins, and 3 (pyruvate kinase, l-lactate dehydrogenase, and creatine kinase) are involved in responses to stress and muscle damage. Real-time PCR results indicated that expression of these proteins is transcriptionally and posttranscriptionally regulated. Protein synthesis capacity, estimated by the RNA-to-protein ratio, was significantly lower in the breast muscle of CORT-treated broilers than in untreated control broilers. The level of Leu, Gly, and Ser in serum was significantly higher in CORT-treated broilers than in the control birds. Therefore, we conclude that CORT treatment retards the growth of skeletal muscle by suppressing protein synthesis and augmenting protein catabolism, paralleling the response to severe stress and muscle damage, and the negative effect of LTI on muscle growth is likely mediated through glucose metabolism. No interaction was observed between CORT and tonic immobility affecting growth performance or any parameter examined in the current study.

In ovo leptin administration accelerates post-hatch muscle growth and changes myofibre characteristics, gene expression and enzymes activity in broiler chickens.

To evaluate the effect of maternal leptin on muscle growth, we injected 0 µg (control, CON), 0.5 µg (low leptin dose, LL) or 5.0 µg (high leptin dose, HL) of recombinant murine leptin dissolved in 100 µl of PBS into the albumen of broiler eggs prior to incubation. The newly hatched chicks were all raised under the same conditions until 21 days of age (D21), when body weight was measured and samples of gastrocnemius muscle were collected and weighed. Myosin ATPase staining was applied to identify myofibre types and measure the cross-sectional area (CSA) of myofibres. Real-time PCR was performed to quantify leptin receptor (LEPR), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor (GHR) and myostatin (MSTN) mRNA expression in the gastrocnemius muscle. The activity of calpains (CAPNs) in the gastrocnemius muscle was measured using a quantitative fluorescence detection kit. Male chickens treated with both high and low doses of leptin had significantly higher (p < 0.05) body weight on D21. The high leptin significantly increased the CSA (p < 0.05) of gastrocnemius muscle in male chickens, which coincided with a 93% increase (p < 0.05) in IGF-1 mRNA expression. Likewise, the LL dose increased the weight of gastrocnemius muscle in male chickens (p < 0.05), which was accompanied by a 41% down-regulation (p < 0.05) of MSTN mRNA expression and a decreased activity of CAPNs. However, all these changes were not observed in female chickens. The proportion of myofibre types did not altered. No significant change was detected for LEPR and GHR mRNA expression. These results indicate that in ovo leptin treatment affects skeletal muscle growth in chickens in a dose-dependent and sex-specific manner. The altered expression of IGF-1, MSTN mRNA and activity of CAPNs in skeletal muscle may be responsible for such effects.

The effect of equol injection in ovo on lipid metabolism and hepatic lipogenic gene expression in broilers.

This study investigated the effects of in ovo administration of equol (Eq) on post-hatch growth and hepatic lipid metabolism in broiler chickens. Fertilized eggs (146 eggs/group) were injected with 0 µg (control, Con), 20 µg (low dose, L) and 100 µg (high dose, H) Eq in the albumen on the 7th day of incubation. Except a trend increase in the weight of total fat (P = 0.09), Eq had no effect on growth or liver weight in broilers at 49 days of age. Males presented higher liver and BWs and lower total fat and relative liver weights than females (P < 0.01). However, there were no significant effects of Eq or Eq-gender interactions on growth performance or tissues weight (P > 0.05). With respect to lipid parameters in the serum, the results showed that female broilers presented higher triacyglycerol (TG) and low-density lipoprotein cholesterol concentrations than males, whereas there was no gender difference in serum total cholesterol (TC) or high-density lipoprotein cholesterol (HDLC) concentration (P > 0.05). Eq administration significantly decreased serum TG and TC but increased HDLC concentrations in serum of broilers at 49 days of age (P < 0.05), whereas there were no interactions between gender and Eq (P > 0.05). To elucidate the mechanism behind the significant changes of serum TG and TC levels, the expression of genes involved in lipid metabolism in the liver was investigated in female chickens using reverse transcription-PCR. Carnitine palmitoyl transferase I (CPTI) messenger RNA (mRNA) was significantly upregulated by 20 and 100 µg Eq (P < 0.05). High-dose Eq significantly decreased fatty acid synthase (FAS) and enhanced cholesterol-7alpha-hydroxylase (CYP7A1) mRNA levels in the liver (P < 0.05). Eq had no significant effects on acetyl-CoA carboxylase, sterol regulatory element binding protein-1c, malic enzyme, low-density lipoprotein receptor or 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in the liver (P > 0.05). These results in female broilers suggest that Eq decreased blood TG by upregulating CPTI and downregulating FAS mRNA expression in the liver, and that high serum cholesterol levels stimulated CYP7A1 gene transcription in the liver.

Impact of vitamin D supplementation on markers of inflammation in adults with cystic fibrosis hospitalized for a pulmonary exacerbation.

Patients with cystic fibrosis (CF) suffer from chronic lung infection and inflammation leading to respiratory failure. Vitamin D deficiency is common in patients with CF, and correction of vitamin D deficiency may improve innate immunity and reduce inflammation in patients with CF. We conducted a double-blinded, placebo-controlled, randomized clinical trial of high-dose vitamin D to assess the impact of vitamin D therapy on antimicrobial peptide concentrations and markers of inflammation. We randomized 30 adults with CF hospitalized with a pulmonary exacerbation to 250,000 IU of cholecalciferol or placebo, and evaluated changes in plasma concentrations of inflammatory markers and the antimicrobial peptide LL-37 at baseline and 12 weeks post intervention. In the vitamin D group, there was a 50.4% reduction in tumor necrosis factor-α (TNF-α) at 12 weeks (P<0.01), and there was a trend for a 64.5% reduction in interleukin-6 (IL-6) (P=0.09). There were no significant changes in IL-1ß, IL-8, IL-10, IL-18BP and NGAL (neutrophil gelatinase-associated lipocalin). We conclude that a large bolus dose of vitamin D is associated with reductions in two inflammatory cytokines, IL-6 and TNF-α. This study supports the concept that vitamin D may help regulate inflammation in CF, and that further research is needed to elucidate the potential mechanisms involved and the impact on clinical outcomes.

In ovo leptin administration inhibits chorioallantoic membrane angiogenesis in female chicken embryos through the STAT3-mediated vascular endothelial growth factor (VEGF) pathway.

Previous studies indicate that leptin regulates placental angiogenesis and fetal growth in mammals and that in ovo leptin administration affects embryonic development and hatch weight in the chicken. To test the hypothesis that leptin affects embryonic growth through modifying chorioallantoic membrane (CAM) angiogenesis, we injected 0.5 µg of recombinant murine leptin into the albumen of fertilized eggs before incubation. On embryonic day 12 (E12), the number and the total area of blood vessels on CAM were measured, and expression of genes involved in angiogenesis was quantitated to show the possible mechanisms. Leptin in ovo administration decreased (P < 0.05) both the total area of blood vessels and the number of small-sized capillaries on CAM of E12 female chicken embryos, which coincided with significantly decreased (P < 0.05) embryo weight on E12 and BW at hatching. Vascular endothelial growth factor (VEGF) and inducible and endothelial nitric oxide synthases (iNOS and eNOS) were all downregulated (P < 0.05) in CAM both at the mRNA and protein/activity levels with reduced (P < 0.05) nitric oxide (NO) concentration in chorioallantoic fluid of female embryos. Furthermore, signal transducer and activator of transcription-3 (STAT3) was found to be diminished (P < 0.05) both at the mRNA and protein levels and associated with decreased (P < 0.05) binding of STAT3 to VEGF promotor in the CAM of leptin-treated E12 female embryos. These data suggest that in ovo leptin administration affects CAM angiogenesis and embryo growth in female chicken embryos, probably through STAT3-mediated VEGF/NO pathways.

The effect of equol injection in ovo on posthatch growth, meat quality and antioxidation in broilers.

In order to investigate the long-term effects of equol (Eq) on growth and meat quality in broilers, 0 µg (control, Con), 20 µg (low dose, L) and 100 µg (high dose, H) Eq, respectively, were injected into fertile eggs (146 eggs per group) on 7 days of embryos. After hatch, chickens were fed under the same conditions and slaughtered at 49 days of age for sample collection and analysis. The results showed that body weight and composition were marginally affected by Eq administration (P > 0.05). Compared with their male counterparts, the meat quality of female broilers was affected greatly after Eq administration. The redness (a*) of meat color in the L and H groups of female broilers was significantly decreased by 24.10% and 21.50% (P < 0.01), respectively; cooking loss decreased by 12.11% and 16.82%, respectively, in the L and H groups (P < 0.01); 24 h and 48 h drip loss was significantly decreased by 60.27% and 45.72% (P < 0.05), respectively, in the H group. However, for male broilers, only cooking loss was significantly decreased by high dosage of Eq treatment (P < 0.05). The antioxidative status was analyzed for discovering further the mechanism behind the improvement of the water-holding capacity caused by Eq in female broilers. The activity of glutathione peroxidase (GSHPx) in plasma was greatly increased by 15.94% in the L group (P < 0.01), whereas the total superoxide dismutase activity (T-SOD) and the content of malondialdehyde in plasma were not changed (P > 0.05). The T-SOD activity in the breast muscle of the L and H groups were significantly improved by 23.14% and 18.82% (P < 0.05), respectively. GSHPx in the breast muscle of the H group showed a tendency to increase (P = 0.06 < 0.1). These results indicate that Eq injection in ovo does not affect the growth of broilers, but significantly improves the water-holding capacity of the muscle, especially in female broilers, which is related to the improvement of antioxidative status.

Clinical assessment of efficacy and safety of DDAVP.

UNLABELLED: The efficacy of DDAVP (1-deamino-8-D-arginine-vasopressin, desmopressin) in mild haemophilia A and von Willebrand disease (VWD) has been established and the use of this well tolerated drug has become clinical routine. In case of increased fluid intake and based on very rarely occurring hyponatraemia, the indication of administration of DDAVP intravenously (i. v.) has to be performed diligently in elderly patients and in children below the age of five years. Aim, patients: Due to clinical practice we were interested in finding prospective parameter potentially correlating with adverse reactions of DDAVP and initiated this study. From 2007 to 2008, we included 49 patients suspicious to suffer from mild haemophilia A (n = 1) or VWD (n = 48) and investigated efficacy and safety of DDAVP after intravenous administration (mean: 0.29±0.032 μg/kg body weight). They underwent clinical and laboratory investigation and were questioned with regard to potential adverse reactions immediately and three days after administration of DDAVP. RESULTS, CONCLUSION: Most adverse reactions were mild and no serious adverse drug reactions were either observed or reported by the subjects. We identified significant changes of heart rate, blood pressure and leucocytes after conduct of the DDAVP test. The value of these findings has to be investigated in later prospective randomized studies. Further research on identification of prospective parameter is currently ongoing.

The effect of obestatin on porcine ovarian granulosa cells.

The aim of our in vitro experiments was to investigate the role of obestatin, a newly discovered metabolic hormone produced in the stomach and other tissues, in the direct control of ovarian cell proliferation, apoptosis and secretion. Porcine granulosa cells were cultured in the presence of obestatin (0, 1, 10 and 100ng/ml medium). The expression of intracellular peptides associated with proliferation (PCNA, cyclin B1, MAP kinase), as well as markers of apoptosis (Bax, p53, Caspase 3), were detected using immunocytochemistry and Western immunoblotting. Secretion of progesterone (P4), testosterone (T) and estradiol (E2) was measured by EIA. Addition of obestatin (1-100ng/ml) to the culture medium significantly stimulated the expression of PCNA and resulted in an increase in expression of cyclin B1 and MAPK. It also significantly increased the percentage of cells containing the apoptotic and anti-proliferating peptides p53, Caspase 3 and Bax. At 10 and 100ng/ml, obestatin promoted the secretion of P4, but not T or E2. Our results are the first demonstration that obestatin directly controls porcine ovarian cell functions: it can stimulate proliferation (accumulation of rPCNA, cyclin B1 and MAPK), apoptosis (expression of p53, Caspase 3 and Bax) and the secretion of progesterone.

Effects of ghrelin and its analogues on chicken ovarian granulosa cells.

The aim of these in vitro experiments was (1) to examine the effects of ghrelin on the basic functions of ovarian cells (proliferation, apoptosis, secretory activity); (2) to determine the possible involvement of the GHS-R1a receptor and PKA- and MAPK-dependent post-receptor intracellular signalling cascades; (3) to identify the active part of the 28-amino acid molecule responsible for the effects of ghrelin on ovarian cells. We compared the effect of full-length ghrelin 1-28, a synthetic activator of GHS-R1a, GHRP6, and ghrelin molecular fragments 1-18 and 1-5 on cultured chicken ovarian cells. Indices of cell apoptosis (expression of the apoptotic peptide bax and the anti-apoptotic peptide bcl-2), proliferation (expression of proliferation-associated peptide PCNA), and expression of protein kinases (PKA and MAPK) within ovarian granulosa cells were analysed by immunocytochemistry. The secretion of progesterone (P(4)), testosterone (T), estradiol (E(2)) and arginine-vasotocin (AVT) by isolated ovarian follicular fragments was evaluated by RIA/EIA. It was observed that accumulation of bax was increased by ghrelin 1-28, GHRP6 and ghrelin 1-18, but not by ghrelin 1-5. Expression of bcl-2 was suppressed by addition of ghrelin 1-28, GHRP6 and ghrelin 1-5, but promoted by ghrelin 1-18. The occurrence of PCNA was reduced by ghrelin 1-28, GHRP6, ghrelin 1-18 and ghrelin 1-5. An increase in the expression of MAPK/ERK1, 2 was observed after addition of ghrelin 1-28, GHRP6 and ghrelin 1-18, but not ghrelin 1-5. The accumulation of PKA decreased after treatment with ghrelin 1-28 and increased after treatment with GHRP6 and ghrelin 1-18 but not ghrelin 1-5. Secretion of P(4) by ovarian follicular fragments was decreased after addition of ghrelin 1-28 or ghrelin 1-5 but stimulated by GHRP6 and ghrelin 1-18. Testosterone secretion was inhibited by ghrelins 1-28 and 1-18, but not by GHRP6 or ghrelin 1-5. Estradiol secretion was reduced after treatment with ghrelin 1-28 but stimulated by ghrelins 1-18 and 1-5; GHRP6 had no effect. AVT secretion was stimulated by ghrelin 1-28, GHRP6 and ghrelin 1-18, but inhibited by ghrelin 1-5. The comparison of the effects of the four ghrelin analogues on nine parameters of ovarian cells suggest (1) a direct effect of ghrelin on basic ovarian functions-apoptosis, proliferation, steroid and peptide hormone secretion; (2) that the majority of these effects can be mediated through GHS-R1a receptors; (3) an effect of ghrelin on MAPK- and PKA-dependent intracellular mechanisms, which can potentially mediate the action of ghrelin at the post-receptor level; (4) that ghrelin residues 5-18 may be responsible for the major effects of ghrelin on the avian ovary.

Leptin directly controls proliferation, apoptosis and secretory activity of cultured chicken ovarian cells.

The aim of our in-vitro experiments was to examine, whether leptin can directly control functions of avian ovarian cells and to outline potential intracellular mediators of its effects. Granulosa cells or fragments of ovarian follicular wall were cultured with leptin (0, 1, 10 or 100 ng/mL medium). The expression of peptides involved in apoptosis (TdT, bax, its binding protein, bcl-2, ASK-1 and p53), cell cycle-related peptides (PCNA and cyclin B1), release of hormones (progesterone, testosterone, estradiol, arginine-vasotocin), as well as the expression of protein kinases (PKA, MAPK/ERK1,2 and CDK/p34) in the ovarian cells were examined by using immunocytochemistry, TUNEL, SDS-PAGE-Western immunoblotting, EIA and RIA. It was found that leptin inhibited expression of all markers of cytoplasmic apoptosis (bax, ASK-1 and p53), stimulated expression of anti-apoptotic peptide bcl-2, but did not affect nuclear DNA fragmentation (TdT). Furthermore, leptin inhibited expression of PCNA (marker of S-phase of mitosis), but not of cyclin B1 (marker of G phase of cell cycle). Moreover, it promoted release of progesterone and estradiol, suppressed release of testosterone, but did not affect arginine-vasotocin. Finally, leptin inhibited expression of MAPK/ERK1,2 and CDK/p34 and stimulated expression of PKA. The present observations demonstrate that leptin can directly control basic chicken ovarian functions - inhibit cytoplasmic apoptosis and proliferation (S-phase, but not G-phases of mitosis), regulate secretory activity (release of steroids, but not nonapeptide hormone) and expression of MAPK, PKA and CDC2, which might be potential intracellular mediators of leptin action.

The role of ghrelin and some intracellular mechanisms in controlling the secretory activity of chicken ovarian cells.

The general aim of these in-vitro experiments was to determine whether ghrelin controls the secretory activity of chicken ovarian cells and whether its action is mediated by TK-, MAPK-, CDK- or PKA-dependent intracellular mechanisms. We postulated that particular protein kinases could be considered as mediators of ghrelin action (a) if they are controlled by ghrelin, and (b) if blockers of these kinases modify the action of ghrelin. In our in-vitro experiments we investigated whether ghrelin altered the accumulation of TK, MAPK, CDK and PKA in chicken ovarian cells and whether ghrelin, with or without blockers of MAPK, CDK and PKA, affected the secretion of progesterone (P4), testosterone (T), estradiol (E2) or arginine-vasotocin (AVT). In the first series of experiments, the influence of a ghrelin 1-18 analogue (1, 10 or 100 ng/mL) was studied on the expression of TK, MAPK and PKA in cultured chicken ovarian granulosa cells. The percentage of cells containing TK/phosphotyrosine MAPK/ERK1, 2 and PKA was determined using immunocytochemistry. Ghrelin increased the expression of both TK and MAPK. The low concentration of ghrelin (1 ng/mL) increased the accumulation of PKA in ovarian cells whilst the high concentration (100 ng/mL) decreased it. The 10 ng/mL concentration had no effect. In the second series of experiments, the effects of the ghrelin analogue combined with an MAPK blocker (PD98059; 100 ng/mL), a CDK blocker (olomoucine; 1 microg/mL), or a PKA blocker (KT5720; 100 ng/mL), were tested for their effects on the secretion of hormones by cultured fragments of chicken ovarian follicular wall. P4, T, E2 and AVT secretions were measured using RIA and EIA. Ghrelin increased T and decreased E2, but did not affect P4 or AVT secretion. The PKA blocker promoted P4 secretion and suppressed E2 and AVT, but did not affect T secretion. It prevented or even reversed the effect of ghrelin on T and E2, but did not modify its effect on AVT secretion. The MAPK blocker enhanced P4 and T and reduced AVT, but did not affect E2 secretion. It was able to prevent or reverse the effect of ghrelin on T and E, and it induced a stimulatory effect of ghrelin on AVT secretion. The CDK blocker reduced the secretion of AVT, but had no effect on steroid hormone secretion. It induced the stimulatory influence of ghrelin on the secretion of P4 and AVT, but did not modify the effect of ghrelin on other hormones. These observations clearly demonstrate that ghrelin is a potent regulator of the secretory activity of ovarian cells and of TK, MAPK and PKA. Furthermore, they suggest that MAPK-, CDK- and PKA-dependent intracellular mechanisms are involved in the control of ovarian secretion and that they mediate the effects of ghrelin on these processes.

Development and performances of a Japanese quail line homozygous for the diabetes insipidus (di) mutation.

A strain of Japanese quail with the polyuria disorder (excessive urination) was developed from founders that regurgitated water spontaneously. A back-cross with a nonpolyuric quail line showed that the polyuric strain was fixed for an autosomal recessive mutation that also induced polydipsia (excessive drinking). Plasma levels and brain mRNA contents for avian Arg vasotocin were little affected by the mutation, but plasma avian Arg vasotocin was 13-fold higher and brain mRNA contents were significantly increased in both normal and mutant quail following a 24-h water deprivation. Affected and normal birds had similar performance traits (egg production and quality, feed intake, and gross carcass traits), but residual feed consumption was higher in polydipsic males. These results are consistent with the hypothesis that this strain was fixed for a mutation similar to the di gene described in the chicken and which induces nephrogenic diabetes insipidus. This new strain of Japanese quail might constitute a convenient model for the analysis of the underlying mechanisms of the disorder in birds and for comparative study with mammals.