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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Novel expression and functional role of ghrelin in chicken ovary.

Ghrelin has recently emerged as pleiotropic regulator of a wide array of endocrine and non-endocrine functions. The former likely includes the control of gonadal function, as expression of ghrelin and its putative receptor, the GH secretagogue receptor type 1a (GHS-R1a), has been described in mammalian gonads, and direct effects of ghrelin in the control of testicular secretion and cell proliferation have been reported. Yet, the expression and/or functional role of ghrelin in gonads from non-mammalian species remain to be analyzed. The present study aimed to evaluate the expression of ghrelin and GHS-R genes in the chicken ovary, and to assess the potential involvement of ghrelin in the direct control of chick ovarian function. To this end, RT-PCR assays for ghrelin and GHS-R1a mRNAs were performed in ovarian tissue, and cultures of chicken ovarian cells were conducted in the presence of increasing doses (1, 10 or 100 ng/ml) of the ghrelin analog, ghrelin 1-18. Our results demonstrate that both ghrelin and GHS-R1a mRNAs are expressed in chick ovarian tissue. Moreover, challenge of ovarian granulosa cells with ghrelin 1-18 was able to induce markers of proliferation (i.e. expression of both PCNA and cyclin), and to modulate markers of apoptosis (i.e. decreased expression of caspase-3, bax, bcl-2 and TUNEL-positive cells). Moreover, ghrelin 1-18 increased the expression of PCNA, cyclin, bax and p53 in cultures of ovarian follicular fragments, where it also stimulated the release of progesterone, estradiol, arginine-vasotocin (AVT) and IGF-I, but not of testosterone. In conclusion, our study provides novel evidence for the gonadal expression of the genes encoding ghrelin and its cognate receptor in a non-mammalian species, i.e. the chicken ovary, and unravels the potential involvement of this newly discovered molecule in the control of key gonadal functions in the chick, such as proliferation, apoptosis, and hormone release.

The role of protein kinase A and cyclin-dependent (CDC2) kinase in the control of basal and IGF-II-induced proliferation and secretory activity of chicken ovarian cells.

The aim of these experiments was to study the role of protein kinase A (PKA), cyclin-dependent kinase 2 (CDC2) and insulin-like growth factor II (IGF-II) in the control of ovarian function in domestic fowl, as well as the role of PKA and CDC2 in mediating the effects of IGF-II on the ovary. For this purpose, we studied the influence of an inhibitor of PKA (KT5720; 50 ng/ml), a CDC2 blocker (olomoucine; 1 microg/ml), IGF-II (0, 1, 10 or 100 ng/ml) and their combinations on cultured fragments of chicken ovarian follicular wall. Accumulation of PKA and CDC2 and secretion of progesterone (P4), testosterone (T), estradiol (E2) and arginine-vasotocin (AVT) were evaluated by using SDS-PAGE-Western blotting and RIA/EIA. IGF-II addition to culture medium stimulated T, E2 and AVT secretion and inhibited P4 secretion. These changes were associated with an increase in PKA and a decrease in CDC2 accumulation. The PKA blocker KT5720, when given alone, increased accumulation of PKA and secretion of T and E2, but not AVT and inhibited P4 secretion. The PKA blocker also prevented and even reversed the effects of IGF-II on PKA and steroid hormones secretion, but enhanced the action of IGF-II on AVT. The inhibitor of CDC2, olomoucine, when given alone, suppressed the expression of CDC2 and the secretion of P4 and AVT (but not T and E2). When given together with IGF-II, it augmented IGF-II-induced suppression of CDC2 and reversed the effects of IGF-II on P4 (but not on T, E2 or AVT). These observations demonstrate the involvement of PKA, CDC2 and IGF-II in regulating the secretory activity of avian ovarian cells. Our data also suggest the involvement of PKA in the mediation of IGF-II effects on P4, T, E2 and AVT secretion. CDC2 can mediate the effects of IGF-II on ovarian P4 secretion but not on other hormones.

Effects of autonomic neuropathy on counterregulation and awareness of hypoglycemia in type 1 diabetic patients.

OBJECTIVE: The recent EURODIAB Study has identified autonomic neuropathy as an independent risk factor for severe hypoglycemia in patients with type 1 diabetes. We tested the hypothesis that counterregulatory catecholamine responses and awareness of hypoglycemia are impaired to a greater extent in type 1 diabetic patients with autonomic neuropathy (AN+) than in those without autonomic neuropathy (AN-). RESEARCH DESIGN AND METHODS: We studied 22 type 1 diabetic patients (8 AN+, 14 AN-) matched for age, duration of diabetes, glycemic control, and history of hypoglycemic episodes. We also studied 33 nondiabetic control subjects using the stepped hypoglycemic clamp technique and determined glycemic thresholds and magnitudes of counterregulatory hormone responses and of hypoglycemia symptoms. RESULTS: Both groups of diabetic patients had reduced awareness of hypoglycemia as evidenced by an elevated glycemic threshold for autonomic symptoms > or =2 SD above normal but neither the magnitude nor thresholds for symptoms differed in AN+ patients and AN-patients. Both groups also had impaired glucagon, epinephrine, norepinephrine, growth hormone and cortisol responses to hypoglycemia. However, in AN+ patients compared with AN-patients, magnitudes of epinephrine and norepinephrine responses (194+/-49 vs. 784+/-206 pmol/l, P < 0.007, and 316+/-56 vs. 610+/-87 pmol/l, P < 0.02, respectively) and epinephrine and norepinephrine glycemic thresholds (2.33+/- 0.10 vs. 2.82+/-0.10 mmol/l, P < 0.009 and 2.34+/-0.06 vs. 2.79+/-0.10 mmol/l, P < 0.008, respectively) were impaired to a greater extent. This was associated with a 50% greater requirement of exogenous glucose to prevent more severe hypoglycemia during the 2.3 mmol/l glycemic plateau (P < 0.002). No differences were observed between other counterregulatory hormone responses in AN+ and AN- patients. CONCLUSIONS: We conclude that in patients with type 1 diabetes, autonomic neuropathy further reduces counterregulatory catecholamine responses. Since this should increase the risk for severe hypoglycemia, one might consider safer therapeutic goals in these patients.

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.

Development of sexually dimorphic vasotocinergic system in the bed nucleus of stria terminalis in chickens.

The bed nucleus of stria terminalis (BnST) of the domestic fowl contains two groups of parvicellular vasotocinergic neurons that are sexually dimorphic. In adult cockerels, arginine vasotocin (AVT) synthesis is well expressed in the dorsolateral and ventromedial portions of the BnST, whereas in corresponding brain areas of hens, AVT synthesis is completely lacking. In the present study, in situ hybridization and immunocytochemical methods were used to compare the ontogeny of sexually dimorphic AVT gene expression in the BnST of male and female chickens from day 12 of embryonic development (E12) until the onset of sexual maturation. By E12, both parvicellular groups of AVT-immunoreactive (AVT-ir) perikarya in the developing BnST can be distinguished in some males, whereas in females their presence is questionable. A quantitative analysis, beginning at E14, showed that the parvicellular dorsolateral portion of the BnST of male embryos had more AVT perikarya compared with females. In contrast, no evident sex difference in distribution pattern and number of AVT mRNA containing neurons in this BnST portion was observable by in situ hybridization at E15. At E18, as well as on the first and second days posthatch (D1 and D2), no differences in the number of AVT synthesizing cells and intensity of immunoreactive staining in male versus female chickens were found. Between D2 and D7, the number of AVT-ir cells in the BnST declined rapidly in both sexes until it disappeared completely in females before D35. In males, another increase in sexually dimorphic AVT-ir cells and innervation of the lateral septum was associated with the onset of puberty and fully matched a pattern observed in adult fowls. These results demonstrate that the sexually dimorphic part of the AVT system undergoes sexual differentiation during early stages of ontogeny.

Anatomical and neurochemical definition of the nucleus of the stria terminalis in Japanese quail (Coturnix japonica).

This study in birds provides anatomical, immunohistochemical, and hodological data on a prosencephalic region in which the nomenclature is still a matter of discussion. In quail, this region is located just dorsal to the anterior commissure and extends from the level of the medial part of the preoptic area at its most rostral end to the caudal aspects of the nucleus preopticus medialis. At this caudal level, it reaches its maximal elongation and extends from the ventral tip of the lateral ventricles to the dorsolateral aspects of the paraventricular nucleus. This area contains aromatase-immunoreactive cells and a sexually dimorphic population of small, vasotocinergic neurons. The Nissl staining of adjacent sections revealed the presence of a cluster of intensely stained cells outlining the same region delineated by the vasotocin-immunoreactive structures. Cytoarchitectonic, immunohistochemical, and in situ hybridization data support the notion that this area is similar and is probably homologous to the medial part of the nucleus of the stria terminalis of the mammalian brain. The present data provide a clear definition of this nucleus in quail: They show for the first time the presence of sexually dimorphic vasotocinergic neurons in this region of the quail brain and provide the first detailed description of this region in an avian species.

Improved glucose counterregulation and autonomic symptoms after intraportal islet transplants alone in patients with long-standing type I diabetes mellitus.

BACKGROUND: Defective glucose counterregulation and hypoglycemia unawareness are both well-recognized risk factors for recurrent episodes of severe hypoglycemia in patients with type I diabetes. At present, no conventional therapy is available to routinely overcome these acquired impairments in long-standing diabetes. METHODS: To test the hypothesis that successful intraportal islet transplantation could improve this syndrome, hormonal counterregulatory responses and symptoms were studied during stepped hypoglycemic clamp tests before and after intraportal islet transplantation in three patients with type I diabetes who were prone to severe hypoglycemia. RESULTS: As compared with matched nondiabetic control subjects, before islet transplantation, glucagon responses were absent while epinephrine and cortisol responses were either markedly decreased or absent in all diabetic subjects. One patient also had decreased norepinephrine and growth hormone responses. Autonomic warning symptoms were absent in all patients during hypoglycemia. One month after successful islet transplantation, there was no improvement in the glucagon response. However, glycemic thresholds and/or peak incremental responses of epinephrine, norepinephrine, and cortisol improved in all patients. Moreover, all patients had developed autonomic warning symptoms so that glycemic thresholds were detectable within the examined range. CONCLUSION: We conclude that intraportal islet transplantation does not restore hypoglycemia-induced glucagon secretion, but it improves the responses of most counterregulatory hormones and hypoglycemic warning symptoms even in long-standing type I diabetes.

The impact of dalteparin (Fragmin) on thrombin generation in pregnant women with venous thromboembolism: significance of the factor V Leiden mutation.

Hypercoagulability is observed in patients with inherited thrombophilia, e.g. factor V Leiden (FVL) mutation. Pregnancy represents a hypercoagulable state as well. This study addresses the effects of the FVL mutation on haemostatic activation during pregnancy as indicated by prothrombin fragments (F1+2). 233 pregnant women with no history of venous thromboembolism were studied. Additionally, two patient groups (25 pregnant FVL carriers and 36 pregnant women without thrombophilic diathesis) in whom low molecular weight heparin (dalteparin) was used prophylactically against rethrombosis were investigated. None of the women developed clinical signs of venous thromboembolism during pregnancy or after delivery. Untreated women exhibited substantial hypercoagulability. F1+2 levels were similar in FVL carriers and non-carriers (difference n. s.). After sufficient adjustment for anti-factor Xa activity (> or =0.15; < or =0.4 U/mL), heparinized women without any thrombophilic diathesis had significantly lower levels of F1+2 than untreated pregnant women. This was evident only in the first and second trimenon (p <0.001). F1+2 levels in heparinized FVL carriers were quite similar to the levels observed in untreated pregnant women, however. In conclusion, our data support the thesis that in comparison to asymptomatic patients, thrombin generation is exaggerated in symptomatic FVL carriers. Coagulation activation during pregnancy can be reduced by dalteparin.

Selective screening for the Factor V Leiden mutation: is it advisable prior to the prescription of oral contraceptives?

The cumulative thrombotic risk of Factor V (FV) Leiden and oral contraceptives (OC) recommends screening for the mutation. Assuming that a family history of thrombosis increases the patient's likelihood of bearing FV Leiden, a selective rather than universal screening would be performed. We studied the utility of a family history of thrombosis for screening of FV Leiden before prescription of OC and, furthermore, the utility of screening even if oral contraception is favoured. 101 patients who had their first and single thromboembolic event while using OC were interviewed. 609 women without any history of thromboembolism recruited by gynecologists completed a standard questionnaire. 101 of these women, age-matched and currently using OC, were selected for a case-control study. Regarding patients with previous thromboembolism, a family history in a first-degree relative had a positive predictive value (PPV) of only 14% for FV Leiden. A PPV of 12% was calculated by investigating the 609 thrombosis-free women. Inherited FV Leiden (odds ratio = 4.9) and acquired risk factors (odds ratio = 10.1) were both found to be the most prominent, but independent additional risks. Nevertheless, FV Leiden carriers, both heterozygotes and homozygotes, did not suffer earlier from thromboembolism than patients without the mutation. In conclusion, family history is an unreliable criterion to detect FV Leiden carriers. Screening for factor V Leiden can be worthwhile even if the advantages of oral contraception are higher assessed than the thrombotic risk. Affected women knowing about their additional risk could contribute to the prevention of thrombosis in risk situations.

PIP: The cumulative thrombotic risk of Factor V Leiden (FVL) and oral contraceptive (OC) use raises the possibility of either selective or universal screening for this mutation before OCs are prescribed. Family history of venous thromboembolism as a criterion to detect FVL carriers was evaluated in a case-control study of 101 women from Bavaria, Germany, who had their first and single thromboembolic event while using OCs and 101 healthy age-matched OC users. A questionnaire was administered to a broader group of 609 OC users without a history of thromboembolism. Analysis of the 609 women revealed a 7.4% prevalence of FVL, but no association between this mutation and a family history of thromboembolism. Among women with a previous thromboembolism, a family history in a first-degree relative had a positive predictive value of only 14% for FVL. The sensitivity of family history was under 50%. 35% of cases compared with 8% of controls carried the FVL mutation. The most significant independent risk factors of thromboembolism were inherited FVL (odds ratio, 4.9) and acquired risk factors--i.e., surgery, leg fractures, distortions, confinement to bed for more than 1 week, or a restricted sitting position more than 6 hours in the 4 weeks before the index date (odds ratio, 10.1). Both heterozygote and homozygote FVL carriers did not suffer earlier from thromboembolism than patients without the mutation. These findings indicate that family history is not an effective predictor of FVL. However, even if the advantages of OC use are greater than the thrombotic risk, screening for FVL may be indicated to permit high-risk women to take preventive action.