Intact osmoregulatory centers in the preterm ovine fetus: Fos induction after an osmotic challenge.

We previously demonstrated a functional systemic dipsogenic response in the near-term fetal sheep (128-130 days; 145 days = full-term) with swallowing activity stimulated in response to central and systemic hypertonic saline. Preterm fetal sheep (110-115 days) do not consistently demonstrate swallowing in response to hypertonic stimuli, and it is unclear whether this is due to immaturity of osmoreceptor mechanisms or neuronal pathways activating swallowing motor neurons. To determine whether osmoreceptive regions in the preterm fetus are activated by changes in plasma tonicity, we examined Fos expression with immunostaining in these neurons in response to an osmotic challenge. Nine preterm fetal sheep [five hypertonic saline-treated fetuses (Hyp) and four isotonic saline-treated fetuses (Iso)] were prepared with vascular and intraperitoneal catheters. Seventy-five minutes before tissue collection, hypertonic (1.5 M) or isotonic saline was infused (12 ml/kg) via an intraperitoneal catheter to fetuses. Brains were examined for patterns of neuronal activation (demonstrated by Fos protein expression). Hyp demonstrated increases in plasma osmolality (~10 mosmol/kg H(2)O) and Na concentrations (5 meq/l). Increased Fos expression was detected in Hyp in the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), median preoptic nucleus (MnPO), supraoptic (SON), and paraventricular nuclei (PVN) compared with Iso animals. Neuronal activation within the OVLT, SFO, and MnPO indicates intact osmoregulatory mechanisms, whereas activation of the SON and PVN suggests intact fetal neural pathways to arginine vasopressin neurons. These results suggest that preterm fetal swallowing insensitivity to osmotic stimuli may be due to immaturity of integrated motor neuron pathways.

Evidence for a causal role of low energy availability in the induction of menstrual cycle disturbances during strenuous exercise training.

Cross-sectional and short-term prospective studies in humans support the concept that low energy availability, and not other factors associated with exercise, causes the development of exercise-induced reproductive dysfunction. To rigorously test this hypothesis, we performed a longitudinal study, examining the role of low energy availability on both the development and the reversal of exercise-induced amenorrhea, using a monkey model (Macaca fascicularis). Eight adult female monkeys developed amenorrhea (defined as absence of menses for at least 100 d, with low and unchanging concentrations of LH, FSH, E2, and P4) after gradually increasing their daily exercise to 12.3 +/- 0.9 km/d of running over a 7- to 24-month period. Food intake remained constant during exercise training. To test whether amenorrhea is caused by low energy availability, four of the eight amenorrheic monkeys were provided with supplemental calories (138-181% of calorie intake during amenorrhea) while they maintained their daily training. All four monkeys exhibited increased reproductive hormone levels and reestablished ovulatory cycles, with recovery times for circulating gonadotropin levels ranging from 12-57 d from the initiation of supplemental feeding. The rapidity of recovery within the reproductive axis in a given monkey was directly related to the amount of energy that was consumed during the period of supplemental feeding (r = -0.97; P < 0.05). Repeated measurements of plasma T3 concentrations, a marker of cellular energy availability, revealed a tight correlation between the changes in reproductive function and T3 levels, such that T3 significantly decreased (27%) with the induction and significantly increased (18%) with the reversal of amenorrhea (P < 0.05). These data provide strong evidence that low energy availability plays a causal role in the development of exercise-induced amenorrhea.

Longitudinal changes in reproductive hormones and menstrual cyclicity in cynomolgus monkeys during strenuous exercise training: abrupt transition to exercise-induced amenorrhea.

Cross-sectional studies of exercise-induced reproductive dysfunction have documented a high proportion of menstrual cycle disturbances in women involved in strenuous exercise training. However, longitudinal studies have been needed to examine individual susceptibility to exercise-induced reproductive dysfunction and to elucidate the progression of changes in reproductive function that occur with strenuous exercise training. Using the female cynomolgus monkey (Macaca fascicularis), we documented changes in menstrual cyclicity and patterns of LH, FSH, estradiol, and progesterone secretion as the animals developed exercise-induced amenorrhea. As monkeys gradually increased running to 12.3 +/- 0.9 km/day, body weight did not change significantly although food intake remained constant. The time spent training until amenorrhea developed varied widely among animals (7-24 months; mean = 14.3 +/- 2.2 months) and was not correlated with initial body weight, training distance, or food intake. Consistent changes in function of the reproductive axis occurred abruptly, one to two menstrual cycles before the development of amenorrhea. These included significant declines in plasma reproductive hormone concentrations, an increase in follicular phase length, and a decrease in luteal phase progesterone secretion. These data document a high level of interindividual variability in the development of exercise-induced reproductive dysfunction, delineate the progression of changes in reproductive hormone secretion that occur with exercise training, and illustrate an abrupt transition from normal cyclicity to an amenorrheic state in exercising individuals, that is not necessarily associated with weight loss.

Central leptin stimulates ingestive behavior and urine flow in the near term ovine fetus.

Leptin inhibits ingestive behavior and induces diuresis and natriuresis. To examine whether leptin influences fetal physiologic functions, we investigated the effect of central leptin on ovine fetal swallowing activity and urine flow. Six pregnant ewes with singleton fetuses (130 +/- 2 d gestation) were prepared with maternal and fetal arterial and venous catheters, fetal lateral intra-ventricle cannula, fetal bladder and amniotic fluid catheters. Electromyogram wires were placed in the fetal thyrohyoid muscle and upper and lower nuchal esophagus and electrodes were implanted on the parietal dura. Five days after surgery, recombinant human leptin was infused into the lateral ventricle and the fetus monitored for 8 h. Central leptin increased fetal swallowing activity during low-voltage electrocortical activity from basal values (0.96 +/- 0.08 swallows/min) at 2 h (1.41 +/- 0.24 swallows/min), 4 h (2.81 +/- 0.57 swallows/min), 6 h (2.53 +/- 0.59 swallows/min) and 8 h (2.08 +/- 0.39 swallows/min, p < 0.05). In comparison to basal values, low voltage electrocortical activity decreased (57 +/- 5% to 42 +/- 4%) and high voltage electrocortical increased (43 +/- 5% to 61 +/- 4%). In response to leptin, fetal urine flow initially decreased from basal values at 2 h (0.12 +/- 0.03 to 0.08 +/- 0.02 ml/kg/min, p < 0.05) then subsequently increased at 4 h and 6 h (0.20 +/- 0.04; 0.21 +/- 0.04 ml/kg/min, respectively, p < 0.05). Central leptin significantly increases near term ovine fetal swallowing activity and urine output, suggesting that leptin contributes to in utero development of ingestive behavior.

Central neuropeptide Y stimulates ingestive behavior and increases urine output in the ovine fetus.

We hypothesized that central neuropeptide Y (NPY) increases swallowing activity and alters renal function in the near-term ovine fetus. Six ewes with singleton fetuses (130 +/- 2 days of gestation; 148 days = term) were chronically prepared with arterial and venous catheters, a fetal lateral cerebroventricular cannula, and fetal bladder and amniotic fluid catheters. For determination of fetal swallowing, electromyogram wires were placed in the fetal thyrohyoid muscle and the upper and lower nuchal esophagus. Electrodes were implanted on the parietal dura for determination of fetal electrocorticogram (ECoG). After 5 days of recovery, fetal swallowing, ECoG, blood pressure, and heart rate were monitored during a 3-h basal period. At t = 3 h, ovine NPY (0.05 mg/kg) was administered into the lateral ventricle, and fetuses were monitored for an additional 8 h. A control study of central administration of artificial cerebral spinal fluid was performed on an alternate day. Central NPY significantly increased swallowing activity during low-voltage ECoG from basal activity (1.26 +/- 0.15 swallows/min) at 4 h (1.93 +/- 0.37 swallows/min), 6 h (1.69 +/- 0.27 swallows/min), and 8 h (2.38 +/- 0.31 swallows/min). NPY significantly increased fetal urine flow (basal: 0.13 +/- 0.02; 4 h: 0.21 +/- 0.04; 6 h: 0. 19 +/- 0.03 ml.kg(-1).min(-1)). These results demonstrate that central NPY stimulates fetal swallowing activity and increases urine output, which may contribute to the in utero development of ingestive behavior.

Central Fos expression in fetal and adult sheep after intraperitoneal hypertonic saline.

We hypothesized that neural structures, involved in sensing extracellular body fluid composition in adult animals during an osmotic challenge, would show similar patterns of activation in fetal sheep. Eight adult sheep [4 hypertonic saline-treated adults (HYP-A), 4 isotonic saline-treated adults] and six near-term fetal sheep [3 hypertonic saline-treated fetuses (HYP-F), 3 isotonic saline-treated fetuses; 130 days gestation] were prepared with vascular and intraperitoneal catheters. Seventy-five minutes before tissue collection, hypertonic (1.5 M) or isotonic saline was infused via an intraperitoneal catheter to adult (18 ml/kg) or fetal sheep (6 ml/kg). Brains were examined for patterns of neuronal activation (demonstrated by Fos protein expression). HYP-A and HYP-F demonstrated similar acute increases in plasma osmolality ( approximately 10 mosmol/kgH2O) and comparable patterns of Fos expression within the organum vasculosum of the lamina terminalis (HYP-A, 67 +/- 2 vs. HYP-F, 63 +/- 6; means +/- SE) and hypothalamic supraoptic (SON; HYP-A, 107 +/- 8 vs. HYP-F, 102 +/- 7) and paraventricular nuclei (PVN; HYP-A, 71 +/- 18 vs. HYP-F, 124 +/- 19). Fewer activated neurons were detected in HYP-A vs. HYP-F within the subfornical organ (HYP-A, 33 +/- 8 vs. HYP-F, 91 +/- 17) and median preoptic nucleus (HYP-A, 33 +/- 5 vs. HYP-F, 70 +/- 6). In adults and fetuses, counterstaining for arginine vasopressin revealed that neurons within the SON and PVN respond to osmotic challenge. These findings demonstrate that central osmoregulatory centers in adult and near-term fetal sheep are similarly activated by osmotic challenge.

Fos response of fetal sheep anterior circumventricular organs to osmotic challenge in late gestation.

We hypothesized that the anterior circumventricular organs (ACVO) and the supraoptic (SON) and hypothalamic paraventricular nuclei (PVN), among other structures that play a role in sensing extracellular body fluid volume and composition in postnatal animals (as demonstrated by Fos protein production by the immediate-early gene c-fos), would show similar activation in fetal sheep during an osmotic challenge. The brains of 10 fetal sheep [6 treated, 4 controls; 129-131 days of gestational age (dGA) = 0.87 gestation] were immunostained for Fos. Seventy-five minutes before tissue collection the dams were given intravenous 20% mannitol (1 ml . min-1 . kg-1 for 10 min). Subsequently, the ACVO, SON, and PVN were scored for the amount of neuronal Fos immunostaining. The subfornical organ (SFO; 24.5 +/- 9.0 vs. 1.7 +/- 1.2), the organum vasculosum of the lamina terminalis (OVLT; 26.8 +/- 5.6 vs. 7.0 +/- 2.0), the SON (39.8 +/- 3.0 vs. 0.15 +/- 0.1), and the PVN (59.8 +/- 7.9 vs. 0.7 +/- 0.7) had increases (P < 0.05) in the average number of Fos-positive cells per field compared with controls, whereas the median preoptic nucleus did not. Double immunostaining for Fos and arginine vasopressin (AVP) or oxytocin (OT) indicated that AVP- but not OT-immunopositive neurons in SON and PVN respond to osmotic challenge. These results demonstrate that the SFO, OVLT, SON, and PVN are activated by osmotic challenge in fetal sheep at 130 dGA.

Immunocytochemical localization of Fos in perfused nonhuman primate brain tissue: fixation and antisera selection.

Immunocytochemical localization of immediate early gene proteins, such as Fos, provides a powerful tool with which to demonstrate activated neuronal populations in response to specific stimuli. In contrast to studies using rat brain tissue that consistently show good Fos detection with a variety of antisera, studies using brain tissue from other species yield variable Fos detection. This may be partly due to differences in Fos protein sequences among species or to perfusion and fixation methods. To determine the ability of various Fos antisera to detect neuronal activation in nonhuman primate tissue, we tested nine Fos antisera and compared these antibodies under conditions of intense or physiological stimulation. Monkey brain tissue was either perfused and postfixed with 4% paraformaldehyde or perfused with 4% paraformaldehyde and postfixed with 2.5% acrolein in 4% paraformaldehyde. In rat tissue, stained for comparison, several antisera resulted in good to excellent Fos detection. However, few antisera tested in monkey tissue resulted in excellent Fos staining. We demonstrate that detection of Fos in monkey brain tissue perfused with 4% paraformaldehyde can be improved by postfixation in a dilute acrolein solution. Our findings emphasize the importance of choosing appropriate antisera and perfusion-fixation procedures to optimize Fos detection in nonhuman primate tissue.