P.S. MohanKumar

Developmental Programming: Impact of Excess Prenatal Testosterone on Intrauterine Fetal Endocrine Milieu and Growth in Sheep

Prenatal testosterone excess in sheep leads to reproductive and metabolic disruptions that mimic those seen in women with polycystic ovary syndrome. Comparison of prenatal testosterone-treated sheep with prenatal dihydrotestosterone-treated sheep suggests facilitation of defects by androgenic as well as androgen-independent effects of testosterone. We hypothesized that the disruptive impact of prenatal testosterone on adult pathology may partially depend on its conversion to estrogen and consequent changes in maternal and fetal endocrine environments. Pregnant Suffolk sheep were administered either cottonseed oil (control) or testosterone propionate in cottonseed oil (100 mg, i.m. twice weekly), from Day 30 to Day 90 of gestation (term is ∼147 d). Maternal (uterine) and fetal (umbilical) arterial samples were collected at Days 64–66, 87–90, and 139–140 (range; referred to as D65, D90, and D140, respectively) of gestation. Concentrations of gonadal and metabolic hormones, as well as differentiation factors, were measured using liquid chromatography/mass spectrometer, radioimmunoassay, or ELISA. Findings indicate that testosterone treatment produced maternal and fetal testosterone levels comparable to adult males and D65 control male fetuses, respectively. Testosterone treatment increased fetal estradiol and estrone levels during the treatment period in both sexes, supportive of placental aromatization of testosterone. These steroidal changes were followed by a reduction in maternal estradiol levels at term, a reduction in activin A availability, and induction of intrauterine growth restriction in D140 female fetuses. Overall, our findings provide the first direct evidence in support of the potential for both androgenic as well as estrogenic contribution in the development of adult reproductive and metabolic pathology in prenatal testosterone-treated sheep.

Diabetes-induced neuroendocrine changes in rats: role of brain monoamines, insulin and leptin

Diabetes is characterized by hyperphagia, polydypsia and activation of the HPA axis. However, the mechanisms by which diabetes produces these effects are not clear. This study was conducted to examine the effects of diabetes on the neuroendocrine system and to see if treatment with insulin and/or leptin is capable of reversing these effects. Streptozotocin-induced diabetic adult male rats were subjected to the following treatments: vehicle, insulin (2 U/day, s.c.), leptin (100 microg/kg BW) or leptin+insulin every day for 2 weeks. Food intake, water intake, and body weight were monitored daily. We measured changes in monoamine concentrations in discrete nuclei of the hypothalamus at the end of treatment. Diabetes produced a marked increase in food intake and water intake and this effect was completely reversed by insulin treatment and partially reversed by leptin treatment (P<0.05). Diabetes caused an increase in norepinephrine (NE) concentrations in the paraventricular nucleus with a concurrent increase in serum corticosterone. Treatment with insulin and leptin completely reversed these effects. Induction of diabetes also increased the concentrations of NE, dopamine and serotonin in the arcuate nucleus and NE concentrations in the lateral hypothalamus, ventromedial hypothalamus (VMH) and suprachiasmatic nucleus (P<0.05). Although insulin treatment was capable of reversing all these changes, leptin treatment was unable to decrease diabetes-induced increase in NE concentrations in the VMH. These data provide evidence that hypothalamic monoamines could mediate the neuroendocrine effects of diabetes and that insulin and leptin act as important signals in this process.

High fat diet affects reproductive functions in female diet-induced obese and dietary resistant rats

The incidence of ovulatory disorders is common in obese animal models. The mechanism behind this effect is unclear. We hypothesised that a high‐fat (HF) diet induces alterations in neuroendocrine mechanisms resulting in anovulation in diet‐induced obese (DIO) animals. Adult female DIO and diet‐resistant (DR) rats were fed either chow or a HF diet (45% calories from fat) for 6 weeks. Oestrous cyclicity and body weight were monitored regularly. At the end of treatment, rats were implanted with a jugular catheter to monitor luteinising hormone (LH) levels on the day of pro‐oestrous. Rats were sacrificed on the next pro‐oestrous, and their brains and ovaries were collected. Plasma from trunk blood was analysed for oestradiol and leptin concentrations. Ovaries were fixed and sectioned for histological analysis. Brains were removed, frozen and sectioned, and norepinephrine (NE) concentrations in discrete hypothalamic areas were measured using high‐performance liquid chromatography with electrochemical detection. A HF diet exposure affected oestrous cyclicity in both DIO and DR rats, with the effect being more pronounced in DIO animals. HF diet exposure increased leptin levels in both DIO and DR rats. Oestradiol levels were low in the DIO‐HF group. NE levels in the hypothalamus were unaffected by HF diet or genotype. A normal LH surge was observed in DR‐Chow rats and LH levels were low in the remaining groups. These results lead to the conclusion that DIO rats have an inherently reduced reproductive capacity and exposure to a HF diet decreases it further. A reduction in oestradiol and LH surge levels could contribute to this effect; however, the underlying mechanisms need to be investigated further.

Prenatal exposure to excess testosterone modifies the developmental trajectory of the insulin‐like growth factor system in female sheep

Experimental elevation of maternal testosterone (T) from 30 to 90 days of gestation leads to intrauterine growth retardation (IUGR) and increased prepubertal growth rate in female lambs. This study tested the hypothesis that prenatal T treatment during mid‐gestation alters the trajectory of the fetal insulin‐like growth factor (IGF)–insulin‐like growth factor binding protein (IGFBP) system to promote IUGR and subsequent postnatal catch‐up growth in female lambs. Plasma IGF‐I and IGFBPs were measured by radioimmunoassay and Western ligand blot, respectively, on 65, 90 and 140 days (d) of gestation, at birth, ∼5 months (prepubertal, the catch‐up growth period), and ∼9.5 months (postpubertal). Northern blot analysis was used to measure hepatic mRNA content of IGF system components during fetal stages. At fetal 65 d, plasma protein and hepatic mRNA content of IGFBP‐1, an inhibitor of IGF bioactivity, was elevated in prenatal T‐treated fetuses although body weight did not differ. There was a transient increase in plasma IGF‐I and IGFBP‐3 concentrations at fetal 90 d in prenatal T‐treated fetuses. Hepatic IGF‐I mRNA and plasma IGFBP‐3 content were reduced by 140 d when body weight was reduced in prenatal T‐treated fetuses. Plasma IGFBP‐2 content was significantly reduced in prenatal T‐treated newborns, but by 4 months these females had significantly higher circulating IGF‐I and IGFBP‐3 concentrations and faster growth rates than control females. After puberty, plasma IGF‐I remained elevated in prenatal T‐treated females. These findings provide evidence that prenatal T excess programmes the developmental trajectory of the IGF/IGFBP system in female sheep to reduce IGF bioavailability during IUGR and increase IGF bioavailability during prepubertal catch‐up growth.

The rabies virus glycoprotein determines the distribution of different rabies virus strains in the brain

The contribution of rabies virus (RV) glycoprotein (G) in viral distribution in the brain was examined by immunohistochemistry following stereotaxic inoculation into the rat hippocampus. Viruses used in this study include the highly neuroinvasive challenge virus standard strains (CVS-N2C and CVS-B2C) and the nonneuroinvasive attenuated SN-10 strain, as well as SN-10-derived recombinant viruses expressing the G gene from CVS-N2C (RN2C) or CVS-B2C (RB2C). The distribution of recombinant viruses in the brain was similar to those of the parental viruses from which the G was derived. For example, while CVS-B2C- and RB2C-infected neurons were seen preferentially in the hippocampus, cortex, and hypothalamus, CVS-N2C- and RN2C-infected neurons were preferentially found in the hippocampus, cortex, and thalamus. SN-10 infected efficiently almost all the brain regions. To further study the role of the RV G in virus spreading, we examined the distribution of RV antigen in brains infected with a recombinant RV in which the SN-10 G was replaced with vesicular stomatitis virus (VSV) G (SN-10-VG) was examined. The spreading of SN-10-VG to the cortex and the thalamus was drastically reduced, but the number of infected neurons in hippocampus and hypothalamus, particularly the paraventricular nucleus, was similar to the SN-10 virus. This pattern of spreading resembles that of VSV. Together, our data demonstrate that it is the G protein that determines the distribution pattern of RV in the brain.

Chronic Exposure to Low Levels of Oestradiol‐17β Affects Oestrous Cyclicity, Hypothalamic Norepinephrine and Serum Luteinising Hormone in Young Intact Rats

Chronic exposure to oestrogens is known to inhibit the secretion of luteinising hormone (LH) in rats, leading to anovulation. Hypothalamic catecholamines, norepinephrine and dopamine play an important role in LH regulation. However, the effects of chronic exposure to low levels of oestradiol on hypothalamic catecholamines have not been investigated thoroughly. In the present study, adult female Sprague–Dawley rats were either sham implanted or implanted with 17β‐oestradiol (E2) pellets (20 ng/day) for 30 (E‐30), 60 (E‐60) or 90 (E‐90) days. E2 exposure affected oestrous cyclicity and ovarian morphology in a duration‐dependent manner. There was no change in oestrous cyclicity in E‐30 rats; however, 75% of E‐60 and 95% of E‐90 rats were acyclic (P < 0.05). Cycling rats from E‐30 or the control group were killed at different time points on the afternoon of pro‐oestrous. E‐30 rats in oestrous, constant oestrous rats in the E‐60 and E‐90 groups and a group of old constant oestrous (OCE) rats were killed at 12.00 h. LH was measured in the serum by radioimmunoassay. Individual hypothalamic nuclei that are involved in LH regulation were microdissected and analysed for norepinephrine and dopamine levels using high‐performance liquid chromatography/electrochemical detection. Norepinephrine levels in the hypothalamic nuclei increased significantly in control and E‐30 groups during the afternoon of pro‐oestrous, which was accompanied by a rise in LH levels (P < 0.05). On the day of oestrous, norepinephrine concentrations in hypothalamic nuclei and serum LH were significantly lower in E‐60, E‐90 and OCE rats compared to E‐30 and control rats. On the other hand, dopamine levels declined significantly in one hypothalamic nucleus. These results indicate that chronic E2 exposure affects hypothalamic catecholamine and serum LH levels in a duration‐dependent manner. This coincides well with the loss of cyclicity observed in these animals. These results suggest that repeated exposure to endogenous oestrogens could play a role in reproductive senescence.

Aging alters norepinephrine release in the medial preoptic area in response to steroid priming in ovariectomized rats

Changes in luteinizing hormone (LH) secretion that are observed in aging animals have been attributed to a reduction in hypothalamic norepinephrine (NE). The reason for the reduction in NE levels with aging is unclear. We hypothesized that the responsiveness of noradrenergic neurons to ovarian steroids is altered during aging. To test this, regularly cycling female Sprague-Dawley rats (young: 4-5 months old and middle age: 8-11 months old) were implanted with a push-pull cannula in the medial preoptic area (MPA) and ovariectomized bilaterally. On the 8th day after ovariectomy, they were injected with estrogen (30 microg/100 microl corn oil, s.c.) at 1000 h and on the 9th day they were implanted with a jugular catheter. On the 10th day they were injected with progesterone (2 mg/100 microl corn oil, s.c.) at 1000 h and subjected to push-pull perfusion. Perfusate samples from the MPA were collected at the rate of 10 microl/min every 30 min from 1300 to 1800 h and blood samples (0.3 ml) were collected hourly. The perfusate samples were analyzed for NE and dopamine (DA) concentrations using high performance liquid chromatography with electrochemical detection and serum LH levels were determined by RIA. In young animals, NE release (mean+/-S.E., pg/min) was 4.0+/-1.1 pg/min at 1300 h and increased significantly (p<0.05) to 10.4+/-4.3 pg/min at 1500 h and remained elevated until 1600 h and then declined to 6.8+/-2.5 at 1730 h. In contrast, the increase in NE release occurred briefly in middle-aged animals and was delayed by an hour. LH patterns in both age groups followed the pattern in NE release. There was no change in the release of DA in both young and middle-aged animals. It is concluded that the altered responsiveness of noradrenergic neurons to steroid priming in middle-aged rats probably plays a critical role in the alterations seen in LH secretion in older animals.

Developmental programming of cardiovascular disorders: Focus on hypertension

Increasing evidence suggests that adult cardiovascular disorders, e.g. hypertension, can be “programmed” in utero. The mechanisms that affect the developing fetus and lead to future cardiovascular disease are not fully established. This review addresses the possible involvement of maternal nutrition, sex steroids and other endocrine factors in the programming of hypertension in adulthood. Some possible mechanisms of subsequent development of hypertension in adulthood, such as elevated sympathetic and renin–angiotensin system activity, and failure of nephron development, also are discussed. Previous studies suggest that maternal undernutrition could be a major factor in fetal programming, but in light of the increased worldwide prevalence of obesity, maternal overnutrition is now receiving increased attention. Special emphasis is given here to this phenomenon. Obesity is associated with increased serum and tissue levels of proinflammatory cytokines, and loss of sensitivity to the adipokine leptin. It is postulated that this causes dysregulation of the hypothalamo–pituitary–adrenal axis, resulting in increased levels of circulating glucocorticoids. These factors could play a major role in programming, during the in utero period, of future hypertension in the offspring of obese mothers.

Leptin inhibits norepinephrine efflux from the hypothalamus in vitro: role of gamma aminobutyric acid

Leptin, a hormone secreted by adipocytes, produces a number of central and neuroendocrine effects, the mechanisms behind which are not completely understood. Hypothalamic norepinephrine (NE) is involved in many of the neuroendocrine effects that are associated with leptin. Therefore, we hypothesized that leptin could affect hypothalamic NE activity to bring about its central and neuroendocrine effects. Because gamma aminobutyric acid (GABA) is known to affect the release of NE, we also tested the possibility that leptin-induced changes in NE could be mediated through GABA. The mediobasal hypothalami from adult male rats were incubated in an in vitro incubation system for four consecutive incubation periods of 60 min each at 37 degrees C in Krebs Ringers Henseleit (KRH) solution in an atmosphere of 95% O(2) and 5% CO(2.) After determining the basal release, the hypothalami were challenged with 0, 0.1, 1 or 10 nm of leptin, bicuculline (a GABA-A receptor antagonist; 10 microM) and bicuculline (10 microM) +10 nM of leptin during the second incubation period. Residual effects of leptin were measured in the third incubation where tissues were incubated with KRH alone, and the viability of tissues was determined in the fourth incubation when tissues were exposed to high K(+) KRH. NE levels in the incubation medium were measured using high-performance liquid chromatography with electrochemical detection (HPLC-EC). Leptin inhibited NE efflux from the hypothalamus in a dose-dependent manner. Moreover, incubation of hypothalami with 10 nM of leptin and bicuculline, a completely blocked the leptin-induced decrease in NE efflux. These results demonstrate for the first time that leptin could act directly on the hypothalamus to inhibit NE efflux through GABA. It was concluded that leptin could probably produce its central and neuroendocrine effects by modulating NE and GABA levels in the hypothalamus.

Differential effects of inhalation exposure to PM2.5 on hypothalamic monoamines and corticotrophin releasing hormone in lean and obese rats

Acute exposure to airborne pollutants, especially particulate matter (PM2.5) is known to increase hospital admissions for cardiovascular conditions, increase cardiovascular related mortality and predispose the elderly and obese individuals to cardiovascular conditions. The mechanisms by which PM2.5 exposure affects the cardiovascular system is not clear. Since the autonomic system plays an important role in cardiovascular regulation, we hypothesized that PM2.5 exposure most likely activates the paraventricular nucleus (PVN) of the hypothalamus to cause an increase in sympathetic nervous system and/or stress axis activity. We also hypothesized that these changes may be sustained in obese rats predisposing them to higher cardiovascular risk. To test this, adult male Brown Norway (BN) rats were subjected to one day or three days of inhalation exposures to filtered air (FA) or concentrated air particulate (CAP) derived from ambient PM2.5. Corpulent JCR-LA rats were exposed to FA or CAP for four days. Animals were sacrificed 24h after the last inhalation exposure. Their brains were removed, frozen and sectioned. The PVN and median eminence (ME) were microdissected. PVN was analyzed for norepinephrine (NE), dopamine (DA) and 5-hydroxy-indole acetic acid (5-HIAA) levels using HPLC-EC. ME was analyzed for corticotrophin releasing hormone (CRH) levels by ELISA. One day exposure to CAP increased NE levels in the PVN and CRH levels in the ME of BN rats. Repeated exposures to CAP did not affect NE levels in the PVN of BN rats, but increased NE levels in JCR/LA rats. A similar pattern was observed with 5-HIAA levels. DA levels on the other hand, were unaffected in both BN and JCR/LA strains. These data suggest that repeated exposures to PM2.5 continue to stimulate the PVN in obese animals but not lean rats.