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Simultaneous multiple droplet deposition involves dispensing of multiple droplets at the same instant on the substrate. A newly defined parameter, arrangement angle is considered as one of the factor that influencing the coalescence of the multiple droplets. However, the effect of such factor on the coalescence is largely neglected in the past. The paper aims to investigate the effect of droplet centre-to-centre distance and arrangement angle on the spreading area of four droplet deposited simultaneously using simulation method. The project starts with single droplet deposition and following by simultaneous multiple droplet deposition. For single droplet deposition, the spreading area of 80% wt aqueous glycerin droplet on the glass substrate were obtained using simulation and experimental methods. A correlation function was introduced to match the simulation result with the experiment result. For simulations of multiple droplet deposition, three droplet centre-to-centre distances as 2.22 mm, 3 mm and 4 mm and three droplet arrangement angles as 90°, 110° and 120° were studied. The larger the droplet spacing, the larger the spreading area for arrangement angle of 90°. The spreading area are largest for spacing of 4 mm, followed by 2.22 mm and 3 mm for arrangement angle of 110° and 120°. The arrangement angle is proven as a factor that affect the spreading area.

We compared clinical outcomes between patients with healthcare-associated and community-acquired Klebsiella pneumoniae bacteraemia and identified predictors associated with mortality and high treatment cost in Hong Kong.This was a retrospective cohort study of patients with K. pneumoniae bacteraemia in a teaching hospital. Adult patients with K. pneumoniae in blood cultures were included. Demographics and clinical data were retrieved from medical records.The analysis included 208 patients. The mean age was 68.6 ± 16.8 years. The Pitt bacteraemia score was 2.2 ± 2.8. In all, 54.8% cases were healthcare-associated infections. The 30-day mortality rate was 32.7%. The mortality rate of patients with healthcare-associated bacteraemia was significantly higher than for community-acquired cases (p0.001). Extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae accounted for 15.4% of cases. Intra- abdominal infection was the most common infection (32.7%). Prior use of immunosuppressive agents and antimicrobial therapy were two major predisposing factors for infection. The treatment cost was USD12 282 ± 11 751 and the length of hospitalization was 9.0 ± 6.7 days. Multivariate analysis showed that liver disease (odds ratio (OR) = 3.06; 95% confidence interval (CI) = 1.38-6.78), malignancy (OR = 6.86; 95% CI = 3.25-14.48), pneumonia (OR = 5.25; 95% CI = 2.05-13.41) and Pitt score1 (OR = 2.50; 95% CI = 1.25-5.00) were associated with mortality. Malignancy (OR = 2.94; 95% CI = 1.33-6.49), Pitt score1 (OR = 4.15; 95% CI = 1.87-9.24) and age72 years (OR = 2.86; 95% CI = 1.35-5.88) were associated with high treatment cost.The 30-day mortality and treatment cost of patients with K. pneumoniae bacteraemia were high in Hong Kong. Based upon the risk factors identified, infection control and treatment algorithms for K. pneumoniae bacteraemia in patients with malignancy or liver disease are highly warranted.

Self-reported measurements of attentional bias are possibly influenced by social desirability, conscious awareness, or introspection. This study developed a visual dot-probe task to study the relationship between attentional bias and posttraumatic stress disorder among women with breast cancer. Fifty six women with breast cancer were presented with a series of face pairs, which were equally divided into positive-neutral and negative-neutral pairs. One face pair was shown for each trial, which consisted of the neutral and emotional versions of the same face displaying side-toside. Participants’ goal was to detect a small dot displayed on the screen after disappearance of the faces as quickly as possible in 80 trials. Negative/positive attentional bias was the mean latency to detect probes appearing on the side of neutral faces minus that of negative/positive faces. We investigated the relationships between the dot-probe task and the Chinese Impact of Event Scale (CIES-R) and demographic variables. Negative attentional bias as measured by the dot-probe task was positively correlated with the CIES-R total score ( r = 0.30, p< 0.05), the hyperarousal subscale (r = 0.32, p< 0.05), and the intrusions subscale ( r = 0.30, p< 0.05) but not the avoidance subscale ( r = 0.32, p = 0.14). This study has demonstrated that measuring attentional bias with a dot-probe task is possible. The dot-probe task may provide an alternative measurement to self-reported measurements and important information for psychotherapies. Future studies may examine the predictive values of the dot-probe task on treatment outcomes and the risk for developing symptoms of post-traumatic stress disorder.

Background We performed a pilot evaluation of a new formulation of levonorgestrel butanoate (LB) designed to be a long-acting injectable (6 months) contraceptive to determine pharmacodynamic end points in normal-body mass index (BMI) and obese women. Study design Obese (BMI ≥30 kg/m 2 ) and normal-BMI, otherwise healthy, women received a single intramuscular injection of LB after ovulation was confirmed in a baseline cycle. The primary outcome was return of ovulation in days. Results A total of 14 women enrolled and completed the study [normal BMI n =9, median BMI 22.7kg/m 2 (range 19.4–25.8); obese n =5, median BMI 35.7kg/m 2 (30.1–39.2)]. The first 6 subjects (normal BMI=4/9, obese BMI=2/5) received 40 mg of LB, and the remaining 8 received 20 mg. All women except one returned to ovulation prior to 6 months. Return to ovulation occurred earlier in the obese group; 3/5 obese and 0/9 normal BMI subjects returned to ovulation within 90 days (p=.03). No serious adverse events were reported during the study. Conclusion Return to ovulation was earlier than 6 months in both BMI groups but more so in the obese BMI group. Implications Since return of ovulation was earlier than expected for this LB injectable formulation, additional steps are needed to develop a preparation suitable as a longer-lasting product.

Hyperandrogenemia is associated with several clinical disorders in which both reproductive dysfunction and metabolic changes may coexist [i.e. polycystic ovary syndrome (PCOS), obesity and congenital adrenal hyperplasia]. Moreover, there is growing evidence that the elevated levels of circulating androgens in obese girls may lead to an increased neuroendocrine drive to the reproductive axis, similar to that associated with PCOS.To test whether androgen exposure in the childhood and adolescent period could lead to pubertal alterations in LH secretory patterns, female rhesus monkeys received subcutaneous testosterone implants prepubertally beginning at 1 year of age, maintaining a 3.7-fold increase (P = 0.001) in circulating testosterone levels over cholesterol-implant controls (n = 6/group) into the post-pubertal period. In early adulthood, pulsatile secretion of LH was measured over 12 h during the early follicular phase of a menstrual cycle, and responsiveness of the pituitary to gonadotrophin-releasing hormone was determined. In addition, ultrasounds were performed to assess ovarian morphology and glucose tolerance testing was performed to assess insulin sensitivity.The timing of menarche was similar between groups. Testosterone-treated animals had a significantly greater LH pulse frequency during the early follicular phase compared with controls (P = 0.039) when measured at 5 years of age. There was a larger LH response to GnRH when testosterone-treated animals were 4 years of age (P = 0.042), but not when the animals were 5 years old (P = 0.57). No differences were seen in insulin sensitivity or ovarian morphology, and the groups showed similar rates of ovulation in early adulthood.Exposure to increased levels of androgens over the course of pubertal development appears to trigger physiological changes in the neural drive to the reproductive axis that resemble those of obese hyperandrogenemic girls in early adulthood and are characteristic of PCOS.

Rabbit does nurse their litter once every 24 h during the night. We hypothesized that corticosterone, ghrelin, leptin, and metabolites such as glucose, liver glycogen, and free fatty acids could be affected in the pups by the time at which does nurse them. Therefore, we measured these parameters in pups nursed at 02:00 h (nighttime for the doe) to compare them with results from a previous study where does nursed at 10:00 h, during daytime. From postnatal day 7, pups were sacrificed either just before their scheduled time of nursing or at 4, 8, 12, 16, or 20 h after nursing ( n = 6 at each time point); additional pups were sacrificed at 4 h intervals between 48 and 72 h after nursing to study the persistence of oscillations during fasting. All pups developed locomotor anticipatory activity to nursing. Corticosterone, ghrelin, and free fatty acids exhibited a rhythm that persisted in fasted pups. Glucose concentrations were lower in fasted than in nursed pups, and glycogen was only detected in nursed subjects. Leptin values were stable and low in nursed subjects but increased significantly in fasted subjects up to 72 h after the expected nursing time. The rhythm of ghrelin persisted during fasting, contrary to our previous findings in pups nursed during daytime (i.e., outside the natural time of nursing for this species). Therefore, in 7-day-old rabbit pups, night nursing is a strong zeitgeber for corticosterone, ghrelin, free fatty acids, and energy metabolites but not for leptin.

Most biomedical facilities that use rhesus macaques (Macaca mulatta) limit the amount of blood that may be collected for experimental purposes. These limits typically are expressed as a percentage of blood volume (BV), estimated by using a fixed ratio of blood (mL) per body weight (kg). BV estimation ratios vary widely among facilities and typically do not factor in variables known to influence BV in humans: sex, age, and body condition. We used indicator dilution methodology to determine the BV of 20 adult rhesus macaques (10 male, 10 female) that varied widely in body condition. We measured body composition by using dual-energy X-ray absorptiometry, weight, crown-to-rump length, and body condition score. Two indicators, FITC-labeled hydroxyethyl starch (FITC-HES) and radioiodinated rhesus serum albumin ((125)I-RhSA), were injected simultaneously, followed by serial blood collection. Plasma volume at time 0 was determined by linear regression. BV was calculated from the plasma volume and Hct. We found that BV calculated by using FITC-HES was consistently lower than BV calculated by using (125)I-RhSA. Sex and age did not significantly affect BV. Percentage body fat was significantly associated with BV. Subjects categorized as having 'optimal' body condition score had 18% body fat and 62.1 mL/kg BV (by FITC-HES; 74.5 mL/kg by (125)I-RhSA). Each 1% increase in body fat corresponded to approximately 1 mL/kg decrease in BV. Body condition score correlated with the body fat percentage (R(2) = 0.7469). We provide an equation for calculating BV from weight and body condition score.

Anxiety is a normal aspect of human personality, which can manifest in a variety of disorders and other negative traits. The primary treatment for anxiety is the class of drugs known as the selective serotonin reuptake inhibitors (SSRIs), which bind to the serotonin reuptake transporter. The upstream region of the gene that codes for this transporter contains a polymorphism that is an insertion/deletion event that in turn, produces long (l) and short (s) alleles in the population. This particular polymorphism in the serotonin transporter, the 5HTTLPR (serotonin transporter linked polymorphic region), is thought to be involved in the genesis of anxious traits and disorders. Most studies with human subjects have examined adult behavior, which may derive from diverse experiential and environmental backgrounds, as well as genetic differences. To better isolate the effect of genetics, we genotyped 128 infant and juvenile monkeys for the 5HTTLPR and tested for behavioral response in four testing paradigms designed to elicit fearful-anxious behaviors: a free play, remote-controlled car, human intruder, and novel fruit test. The s/s monkeys were found to be behaviorally inhibited in the free play test, engaged in more fear behaviors in the remote-controlled car test, and threatened more in the stare portion of the human intruder test, even though a small number of monkeys were assessed. There was no difference between genotypes of either sex in the prolactin response to fenfluramine. These data indicate greater anxiety in the s/s monkeys for distinct facets of anxious behavior, which are independent of a global neurohormonal challenge test. These neurobehavioral data support recent neuroimaging findings in humans indicating the importance of the 5HTTLPR for amygdala-dependent anxious behavior.

Many patients with hyperandrogenemia are overweight or obese, which exacerbates morbidities associated with polycystic ovary syndrome (PCOS). To examine the ability of testosterone (T) to generate PCOS-like symptoms, monkeys received T or cholesterol (control) implants ( n = 6/group) beginning prepubertally. As previously reported, T-treated animals had increased neuroendocrine drive to the reproductive axis [increased luteinizing hormone (LH) pulse frequency] at 5 yr, without remarkable changes in ovarian or metabolic features. To examine the combined effects of T and obesity, at 5.5 yr (human equivalent age: 17 yr), monkeys were placed on a high-calorie, high-fat diet typical of Western cultures [Western style diet (WSD)], which increased body fat from

Specific alterations in the pulsatility of luteinizing hormone (LH) are linked to obesity-related subfertility in ovulatory women. Vervet monkeys (Chlorocebus aethiops sabaeus) are an Old World nonhuman primate that develops obesity and has a menstrual cycle similar to humans. We evaluated follicular-phase LH pulses in 12 adult normal-weight female vervets. Serum was collected every 10 min for 4 h by using a tether device in conscious, freely moving monkeys on menstrual cycle days 2 through 5. Serum estradiol was collected daily during the follicular phase to identify the luteal–follicular transition. For comparison, we used data from 12 ovulatory normal-weight women who had undergone frequent blood sampling of early-follicular LH. LH pulse frequency was similar, with 2.8 ± 0.7 LH pulses during 4 h in vervets compared with 2.3 ± 0.7 LH pulses during 4 h in women. The LH pulse mass (percentage change in the pulse peak over the preceding nadir) was 123.2% ± 27.4% in vervets and 60.9% ± 14.9% in humans. The first day of low serum estradiol after the follicular-phase peak was denoted as the day of the luteal–follicular transition. Luteectomy was performed on luteal days 7 through 9, and corpora lutea were confirmed by histology. We demonstrate that follicular LH patterns in vervets are similar to those in humans and that the luteal phase is easily identified by monitoring daily serum estradiol. These findings demonstrate that vervet monkeys are a suitable animal model for evaluating LH pulse dynamics longitudinally in studies of diet-induced obesity.

The annual killifish, Austrofundulus limnaeus, typically enters embryonic diapause at two distinct points of development, termed diapause II and III. This study explores the role of maternal and embryonic steroid hormones, including 17-β-estradiol (E2), androstenedione (A4) and testosterone (T), in regulating the developmental decision to enter or escape diapause II. Steroid hormone levels were measured in tissues isolated from adult female killifish during the normal lifespan of this species and in individuals of the same age that were producing either high or low proportions of escape embryos. Levels of steroid hormones were also measured during early development and in fertilized eggs that were predicted to be on either an escape or diapausing developmental trajectory. Decreases in maternal E2 levels associated with age are correlated with decreasing escape embryo production. Maternal production of escape embryos is correlated with increased ratios of E2 to T in adult ovary tissue. Interestingly, neither hormone is significantly different in fish producing embryos on different developmental pathways when examined independently. Levels of steroid hormones in fertilized eggs are not correlated with entry or escape from diapause II, though levels of A4 tend to be higher in escape embryos. Escape embryos exhibit faster hormone metabolism and earlier hormone synthesis than embryos that will enter diapause II. Incubation of embryos in exogenous E2 is associated with a 7-fold increase in escape embryo production, and significantly elevated A4 levels. These data suggest that steroid hormones may be critical factors involved in determining developmental pathways in embryos of A. limnaeus.

Objective To use a nonhuman primate model and determine whether individuals sensitive to stress-induced reproductive dysfunction have lower activity of central serotonergic neurons under nonstressed conditions. Design The activity of the central serotonergic system was assessed by measuring responsiveness to a fenfluramine challenge (5 mg/kg, IV) in sedated monkeys previously categorized as highly stress resistant (HSR; n=4; normal menstrual cyclicity through two stress cycles), medium stress resistant (MSR; n=5; ovulatory in the first stress cycle but anovulatory in the second stress cycle), or low stress resistant (i.e., stress sensitive, SS; n=4; anovulatory as soon as stress is initiated). To control for differences in pituitary stores of prolactin or ACTH, the animals were subsequently administered a bolus of thyrotropin-releasing hormone (3 μg/kg) plus corticotropin releasing factor (CRF), (3 μg/kg). Setting Oregon National Primate Research Center, Animal Services Building. Patient(s) Female cynomolgus macaques exhibiting normal menstrual cycles. Intervention(s) Administration of fenfluramine, a serotonin-releasing drug. Main outcomes measure(s) Serum concentrations of prolactin (PRL) and cortisol (F). Result(s) Prolactin release in response to fenfluramine was significantly greater in the HSR group compared with the MSR or SS groups. In contrast, cortisol was higher in the SS group compared with the other two groups. Similar responses were not evident after thyrotropin-releasing hormone + CRF stimulation. Conclusion(s) The lower PRL response to fenfluramine in the stress-sensitive animals suggests that stress-sensitive individuals have decreased activity in central serotonergic neurons. However, the F data suggest that the hypothalamic-pituitary-adrenal axis in stress-sensitive individuals is highly responsive to even small increases in serotonin.

In the female rabbit, coitus induces a massive release of hypothalamic gonadotropin-releasing hormone (GnRH) within 20 min. The GnRH surge is preceded by an increase in hypothalamic norepinephrine (NE) release. Presumably, coitus stimulates NE, hence GnRH, release by increasing the activity of tyrosine hydroxylase (TH, the rate-limiting enzyme for NE synthesis) and/or decreasing the activity of norepinephrine transporter (NET, the key protein for NE re-uptake). Since NE cell bodies are located primarily in the brainstem, we hypothesize that coital signals are relayed to hypothalamic GnRH-secreting neurons via brainstem NE-containing perikarya. In support of this hypothesis, we found that both c-fos and TH mRNA expressions in brainstem noradrenergic areas, particularly in the A1 and A2 cell groups, increased within 30 min and returned to precoital levels within 60 min after coitus. Here we analyzed coitally induced changes in NET mRNA expression at 0, 15, 30 and 60 min postcoitus in the brainstem by in situ hybridization, using 35S-labeled rabbit NET RNA probes. In comparison with nonmated females (i.e., at 0 min), the expression of NET mRNA significantly increased (P0.05) within 15 min postcoitus in the A1, but not the A2 area. By 30 min postcoitus, NET gene expression increased in the caudal portion of the A1 and in the caudal and central portion of the A2. By 60 min postcoitus, NET mRNA expression in the caudal and rostral portion of the A1 and the caudal and central portion of the A2 was still higher than NET mRNA expression in nonmated rabbits (P0.05). No change in NET mRNA expression was observed in the A6. The results suggest that coitus increases NET mRNA expression in A1 and A2 noradrenergic areas within 15-30 min, and this enhanced NET mRNA expression was maintained for at least 60 min, particularly in the A2. These findings, in combination with our previous observation on increased TH gene expression within 30 min, but not 60 min, after coitus, further suggest that the coitus-induced NET transcriptional events within brainstem NE neurons may play an important role in the maintenance, and particularly in the termination, of hypothalamic NE release, hence regulating the size and duration of the coitus-induced GnRH surge.

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized-oestrogen-supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol-17beta (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge-like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2-infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER-alpha and ER-beta mRNAs were expressed in the locus coeruleus, although the expression was greater for ER-alpha than for ER-beta. We conclude that i.v. infusion of E2, which induces a preovulatory surge-like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER-mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2-stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.

Leptin acts as a metabolic activator of the neuroendocrine reproductive axis in several rodent species, but whether leptin plays a similar role in primates is unknown. To explore this question, we examined the effects of leptin on gonadotropin and testosterone secretion in male rhesus monkeys that were fasted for 2 days. Mean plasma levels of LH and FSH, LH pulse frequency, and LH pulse amplitude were significantly higher in leptin-treated animals compared with saline-treated controls during the second day of the fast. To identify targets for leptin's action, we used in situ hybridization and computerized imaging to map leptin receptor (Ob-R) messenger RNA (mRNA) distribution. Ob-R mRNA was observed in the anterior pituitary and several areas of the brain, including the arcuate and ventromedial nuclei of the hypothalamus. Ob-R mRNA was coexpressed in both POMC and neuropeptide Y neurons in the arcuate nucleus, whereas little or no coexpression of Ob-R mRNA was evident in GnRH neurons. These results suggest that leptin is a metabolic signal to the reproductive axis in primates and imply that both POMC and neuropeptide Y neurons are involved in mediating leptin's effects in the brain.

Gonadotropin-releasing hormone (GnRH) is a key hypothalamic peptide that controls the secretion of pituitary gonadotropins, particularly luteinizing hormone (LH), and hence gonadal function. Hypothalamic GnRH is released in a pulsatile manner. In the female, the pattern of GnRH pulses, i.e., pulse frequency and amplitude, varies during different reproductive stages and among different species. Several central and peripheral signals modulate GnRH neuronal activities. Some of these signals are stimulatory to GnRH release, e.g., norepinephrine (NE) and neuropeptide Y (NPY); some are inhibitory, e.g., beta-endorphin and interleukin-1; others are both stimulatory and inhibitory, e.g., estradiol-17 beta (E2). The neuronal structures and chemical interactions that result in pulsatile GnRH release remain unresolved. However, the core of the so-called 'GnRH pulse-generator' likely involves NE and NE transporter (NET, the protein for pre-synaptic re-uptake of NE). Both secretion and re-uptake of NE may determine hypothalamic NE availability. Many of the GnRH-stimulating and GnRH-inhibiting signals may influence the 'pulse-generator' by acting on GnRH neurons as second level signals. Hypothalamic GnRH is also released in a "surge" manner that is triggered either by increasing levels of circulating steroids (E2 and progesterone) during the preovulatory period in spontaneous-ovulating species, or by coitus in induced-ovulating animals. The sequential steps and mechanisms by which the GnRH surge occurs after E2 or coitus are not clear. However, it is unlikely that the E2 or coital stimuli act directly on GnRH neurons; E2 receptors have not been found in GnRH cells whereas coital signals must stop in the brainstem before they reach the hypothalamus. The brainstem may be an extra-hypothalamic site where both E2 and coital stimuli are transformed into GnRH-stimulating signals. One such signal may be NE whose brainstem cell bodies send terminals into the hypothalamus. Evidence from our laboratory suggests that a hypothalamic NE surge occurs at the time of the preovulatory GnRH surge in both the monkey and rabbit. Moreover, gene expression of both tyrosine hydroxylase (the rate-limiting enzyme for NE synthesis) and NET (the rate-limiting factor for synaptic NE transmission) in the brainstem increases after E2 in the monkey and after coitus in the rabbit. Other hypothalamic and/or brainstem signals, i.e., NPY, galanin, beta-endorphin, nitrous oxide and gamma aminobutyric acid, are likely involved in generating, maintaining and/or modulating the GnRH surge process. A better understanding of the up-stream GnRH-regulating signals will help improve treatments for many reproductive disorders associated with stress, obesity, infection and aging.

Pulsatile secretion of hypothalamic gonadotropin-releasing hormone (GnRH) is suppressed by alpha-adrenergic antagonists in ovariectomized (OVX) rabbits, thus suggesting that initiation of GnRH pulses requires the presence of norepinephrine (NE) stimulation. Terminals of NE neurons are located in proximity with GnRH cells in the hypothalamus, including the arcuate nucleus-median eminence (AME) region. Synaptic NE molecules may be catabolized or transported back to NE terminals (i.e. reuptake) via specific NE transporter proteins (NET). Thus, the amount of synaptic NE acting on GnRH cells is a function of the rate of NE release, metabolism and reuptake. Hypothetically, the rise and fall of a GnRH pulse may be associated with the similar fluctuations of synaptic NE release and/or NET activity. To test this hypothesis, we examined the effects of AME administration of desipramine (DMI, a specific NET blocking drug) on GnRH release. First, we delivered 0.2-10 mM doses of DMI continuously for 1 h via an AME microdialysis (microD) system into intact male rabbits. We found that each AME-DMI infusion, between dosages of 1 mM and 10 mM, stimulated a GnRH pulse, and that the size of these GnRH pulses were proportional to the dosage of DMI. To confirm the specificity of DMI on NET, we measured catecholamine content in microD samples by HPLC. The temporal (60 min) DMI induced a pattern of NE release that included a rising limb within the first 20-30 min; although NE returned to baseline values within the period of DMI treatment. Neither epinephrine nor dopamine levels were changed by DMI. Second, a median dose of DMI (5 mM) was given by microD for 60 min in four separate rabbit models: gonadal intact females (F-INT), intact males (M-INT), gonadectomized females (F-GDX) and castrated males (M-GDX). Individual microD samples were measured for NE and GnRH. Regardless of gender or gonadal status, 5 mM of DMI concomitantly induced a pulse-like release of NE and GnRH. Furthermore, the response of GnRH to DMI was greater in GDX rabbits than in INT animals of both genders. Third, we administered DMI (5 mM) for 30 min via a push-pull perfusion (PPP) system during four repeated 90 min intervals, in either F-INT or ovariectomized (F-GDX) females, and measured GnRH in PPP samples. In both F-INT and F-GDX, each DMI challenge induced a GnRH pulse. In F-INT, all sequential DMI-induced GnRH pulses were nearly equal in size. In contrast, in F-GDX, the first DMI-induced GnRH pulse was greater than subsequent ones. Collectively, these observations are consistent with the concept of noradrenergic regulation of pulsatile GnRH release, and we conclude that the temporal activity of NET may be an integral part of the mechanism by which GnRH pulses operate.

Certain brain peptides and catecholamines function in activating the hypothalamohypophysial-ovarian axis in both rabbits and rhesus monkeys. The natural stimulus for a surge release of GnRH is coitus in rabbits, whereas the initial excitatory signal is ovarian steroids in monkeys. Despite this contrast in initial signals, specific neurochemicals may serve as common stimuli for GnRH secretion in both species. Evidence is presented that one such substance is norepinephrine (NE), which is released from the mediobasal hypothalamus before, or simultaneously with, GnRH, although the latency in time between stimulus (coitus/estrogen) and response (GnRH/LH release) is very different. Moreover, both stimuli activate NE gene expression in cells located in the brainstem. We suggest that the brainstem is an extra-hypothalamic site where preovulatory signals for GnRH surges are developed in both rabbits and primates.

The mammalian testis is innervated by extrinsic catecholaminergic nerves and responds to catecholamines with steroid secretion. Although the primate testis has also been shown to be innervated, potential differences in the density of this innervation between immature and sexually developed individuals have not been described. A recent study demonstrated that the primate ovary contains a network of neuron-like cells and that some of these cells are catecholaminergic. It is thus possible that the male gonad is also endowed with a similar intragonadal source of catecholamines. The present study addresses these two issues. Catecholaminergic nerves were identified as such by their content of immunoreactive tyrosine hydroxylase (TH; the rate-limiting step in catecholamine biosynthesis), and in some cases by glyoxylic acid histochemistry. Fibers containing TH were abundant in testes from juvenile animals (1-2 yr of postnatal life), but the density of this innervation was not maintained in adult animals, whose testis showed only a few TH-positive fibers scattered in the interstitial tissue. Testicular norepinephrine (NE) concentration was much lower in adult than in juvenile animals, suggesting that the marked increase in testicular weight that occurs with the attainment of sexual maturity is not accompanied by corresponding changes in NE content. At the ultrastructural level, testicular nerve fibers contained pleiomorphic, dense-core and clear vesicles, suggesting the presence of catecholamines and other neurotransmitters. In addition to this extrinsic catecholaminergic innervation, prepubertal testes, but not adult gonads, contained an intrinsic population of TH-immunopositive neuron-like elements, identified as cells by confocal scanning laser microscopy. To determine whether the prepubertal monkey testis indeed expresses the TH gene, testicular RNA was subjected to reverse transcriptase polymerase chain reaction to amplify the 5' end of TH mRNA, which encodes the regulatory domain of the enzyme. The cDNA that was obtained predicts an amino acid sequence similar, but not identical, to that encoded by the alternatively spliced type 1 TH mRNA form present in the adrenal gland. These results indicate 1) that the primate testis receives a dual catecholaminergic input, one provided by the extrinsic innervation and the other by neuron-like cells located within the gonad itself, and 2) that the influence exerted by both sources on testicular function may be more prominent during the prepubertal period than in adulthood. The presence in the testis of a TH mRNA variant encoding amino acid substitutions in its 5' end suggests that regulation of testicular TH enzyme activity may include a gonad-specific component.

Concomitant fluctuations in median eminence perfusate GnRH and plasma LH occur in rhesus macaques during the periovulatory period and after ovariectomy. The association between GnRH and LH pulses during the follicular and luteal phases of the monkey menstrual cycle is less clearly defined. However, observed LH patterns suggest higher amplitude and slower pulses of GnRH in the luteal than in the follicular phase of the menstrual cycle. The present studies were planned to compare the GnRH/LH patterns in individual monkeys by simultaneous push-pull perfusion (PPP) and blood sampling during different ovarian steroid milieus. In the initial trial, placement of two push-pull cannulae (PPCs) in the median eminence and a jugular vein catheter caused immediate loss of regular menstrual cycles in 3 monkeys, although cycles resumed over 3-6 mo postoperatively. After the return of normal reproductive cycles, PPP was performed for 12 h on either Day 7, 8, or 9 of the luteal phase. The results showed an unexpected and profound decline in LH and progesterone (P4) concentrations during the initial 4 h. No pulses of LH or P4 were observed in the remaining 8 h. All 3 monkeys exhibited menstrual bleeding 2-3 days after PPP. In subsequent trials, we continuously infused the opioid receptor antagonist nalmefene (Nmf, 1 mg/h, i.v.), starting the fourth day after PPC implantation into 11 monkeys. Menstrual cycles with accompanying fluctuations of circulating estradiol-17 beta (E2) and P4 returned in less than 40 days in these macaques and continued without further Nmf treatment. Trials of 12-h PPP/blood sampling were performed during the follicular phase with (n = 4) or without (n = 4) Nmf, and during the luteal phase with (n = 6) or without (n = 3) Nmf. Endocrine data from the 3 animals without Nmf during the luteal phase were combined with the hormonal values that were obtained in the initial trial because all 6 animals exhibited similar GnRH, LH, and P4 profiles, i.e., low levels and infrequent or absent pulses. Treatment with Nmf during luteal sampling enhanced hypothalamic GnRH secretion (10-fold increase in mean GnRH levels over those without Nmf) and reinitiated distinctive serum LH and P4 pulses. In contrast, patterns of hypothalamic GnRH and serum LH during the follicular phase were similar with or without Nmf treatment. These GnRH/LH profiles consisted of low-amplitude hourly pulses. Collectively, the observations suggest that stress-induced activation of opiatergic neurons can inhibit the GnRH pulse generator and that these neuronal systems are more sensitive to such inhibition in the presence of elevated levels of circulating P4. However, our observation that Nmf accelerated the reinstatement of ovarian cycles after surgery, when circulating E2 and P4 were very low, suggests that GnRH secretions are influenced by activation of different opioid receptor subtypes in response to different stresses. Some of these GnRH/opioid interactions are independent of P4.

Substance P (SP) is present in large quantities in the brainstem and hypophysiotropic areas of the brain, but its roles in gonadotropin and prolactin secretion are controversial. The aim of this study was to measure luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) release from the pituitary after either intracerebroventricular (ICV) injection or infusion of SP or its C- and N-terminal fragments in intact (INT) and ovariectomized (OVX) conscious rabbits. A single injection of SP into the 3rd cerebral ventricle (3CVT) in INT and OVX rabbits augmented plasma LH concentrations, especially when SP was applied during the initial phase of an LH peak. Injection of SP during the declining phase of LH release was not effective. Injection of SP into the 3CVT was followed by increased plasma PRL concentrations in OVX but not in INT rabbits. Both SP 1-11 and SP 1-7 failed to alter LH, FSH, and PRL secretion when the peptides were slowly infused into the 3CVT, although ICV infusion of SP 6-11 did cause a delayed increase in LH release. The results support a stimulatory role of SP on LH and prolactin release. The results further indicate that although the stimulatory effect of SP on LH is ovarian steroid-independent, in the absence of ovarian steroids, SP is stimulatory only during the rising phase of an LH pulse. A dual role of SP-ergic transmission in modulating LH secretion is discussed.

Both neuropeptide Y (NPY) and norepinephrine stimulate gonadotropin-releasing hormone (GnRH) secretion in intact or ovariectomized (OVx) estradiol-treated rabbits. The mechanism by which NPY stimulates GnRH is currently unknown. We have tested the hypothesis that NPY increases GnRH release via an alpha-adrenergic pathway. Adult female rabbits were OVx and had Silastic capsules containing 17 beta-estradiol inserted subcutaneously that maintained plasma estradiol levels similar to those in ovarian intact rabbits. One week later, push-pull (PP) perfusion cannulae, with tips positioned in the mediobasal hypothalamus (MBH), and jugular vein catheters were placed in all does. Blood and PP perfusate samples were obtained every 20 min during 7 h perfusion of the MBH with Krebs-Ringer phosphate buffer (KRP). Intrahypothalamic treatment with NPY (n = 5), prazosin (alpha 1-adrenergic antagonist; n = 7), yohimbine (alpha 2-adrenergic antagonist; n = 7), NPY plus prazosin (n = 7) or NPY plus yohimbine (n = 6) dissolved in KRP occurred during hours 4 through 6. GnRH in hypothalamic perfusate and luteinizing hormone (LH) and prolactin (PRL) in peripheral plasma were measured by specific radioimmunoassays. As anticipated, NPY alone significantly increased MBH-GnRH secretion (0.93 +/- 0.24 vs. 2.46 +/- 0.37 pg/ml; p less than 0.05). In contrast, NPY infused concomitantly with prazosin did not increase MBH-GnRH release (1.26 +/- 0.50 vs. 0.78 +/- 0.19 pg/ml; p greater than 0.05) whereas NPY plus yohimbine did stimulate GnRH secretion (1.15 +/- 0.13 vs. 2.65 +/- 0.89 pg/ml; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty-four-hour pulsatile patterns of bioactive luteinizing hormone (LH) and immunoactive estradiol (E2) and progesterone (P4) were measured during the mid-follicular (FP, Days 5, 6, or 7) and mid-luteal (LP, Days 7, 8, 9, or 10) phases of the menstrual cycle in six intact and in four ovariectomized (OVX) rhesus monkeys. Blood samples were collected remotely at 7.5-min (FP) and 15-min (LP, OVX) intervals via catheters in freely moving, vested monkeys. Hormonal patterns were analyzed by the MUNRO computer program. A significant coupling of LH and E2 pulses was observed during the FP and between the LH and P4 pulses during the LP. No diurnal rhythm of LH pulse frequency was observed during the FP or in OVX monkeys. In contrast, day/night differences in frequency were seen during the luteal phases (0.35 +/- 0.03 vs. 0.24 +/- 0.05 pulses/h, day vs. night, p less than 0.05). Naloxone (NAL), an opiate antagonist, was continuously infused (2 mg/h.i.v.) for 12 h at night during the LP. NAL treatment increased nighttime LH pulse frequency to 0.33 +/- 0.07 pulses/h, which was not different from the daytime LH pulse frequency, thereby abolishing the normal diurnal pattern. Interestingly, NAL treatment did not achieve the approximately 1 pulse/h frequency seen in the FP or OVX condition. These results support the hypothesis that P4 modulates the activity of an opioid system that is responsible both for a slowing of LH pulse frequency during the LP and for an additional nightly reduction in hypothalamic LH pulse-generating activity.