Vascular and parenchymal lesions along with enhanced neurogenesis characterize the brain of asymptomatic stroke-prone spontaneous hypertensive rats.

BACKGROUND AND OBJECTIVES: Spontaneously hypertensive stroke-prone rats (SHRSPs) develop hypertension, cerebrovascular abnormalities and a stroke phenotype in association with higher levels of proteinuria. Here, we focus on cerebral abnormalities preceding lesions detectable by MRI. METHODS: Longitudinal assessment of brain histology was performed in salt-loaded male SHRSPs (n = 26) and Wistar-Kyoto (WKY) normotensive control animals (n = 27). Groups of rats were sacrificed at different time points: Time 0, before the salt diet administration; Time 1, when proteinuria achieved 40 mg/day; Time 2, when proteinuria exceeded 100 mg/day. RESULTS: At Time 0, no brain lesions were observed. At Time 1, changes of the cortical penetrating arteries, vasogenic oedema, lacunae and focal cell loss appeared in SHRSPs and worsened at Time 2, although no lesions were yet detected by MRI. Staining for proliferation markers revealed a significant boost of cellular mitosis in the subventricular zone (SVZ) of SHRSPs. Moreover, we observed higher immunopositivity for nestin, glial fibrillary acidic protein and doublecortin (markers for neural stem cells, astrocytes and immature neurons, respectively). At Time 2, apoptotic caspase-3 as well as 4-hydroxynonenal-positive neurons were associated to decreased nestin and doublecortin staining. High expression levels of glial fibrillary acidic protein were maintained in the SVZ. No comparative alterations and SVZ activation were recorded in WKYs. CONCLUSION: Appearance of vascular changes in SHRSPs, before any MRI-detectable brain lesion, is coupled to active neural proliferation in the SVZ. With disease progression, only newborn astrocytes can survive, likely because of the neurotoxicity triggered by brain oedema and oxidative stress.

Organizational effects of DDE on brain vasotocin system in male Japanese quail.

p,p'-DDE, or ethylene, 1,1-dichloro-2,2-bis(p-chlorophenyl), is the main metabolite of the pesticide DDT, or 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane. It is an androgen receptor antagonist and testosterone hydroxylase modulator that is also more persistent than its parent compound. In a previous study we demonstrated that embryonic exposure to different doses of p,p'-DDE accelerated onset of puberty in females and reduced male reproductive behavior. In the present study we investigated the long-term effects of the exposure to p,p'-DDE on the differentiation of male Japanese quail (Coturnix japonica) limbic circuits related to male copulatory behavior: the parvocellular vasotocin (VT) system. We observed a decrease in the density of VT-immunoreactive fibers within the medial preoptic nucleus, bed nucleus of the stria terminalis, and lateral septum in p,p'-DDE-treated birds, while no differences could be detected in the magnocellular neurons of the supraoptic nucleus. In particular the lowest dose of p,p'-DDE causes the highest decrease of VT immunoreactivity. This study provides further evidence for VT system sensitivity towards endocrine disrupting chemicals and demonstrates that the VT system may be an appropriate and sensitive biomarker for early p,p'-DDE exposure in birds.

Selective activation of estrogen receptor alpha in Japanese quail embryos affects reproductive organ differentiation but not the male sexual behavior or the parvocellular vasotocin system.

Estradiol is crucial for normal female differentiation in birds. Developmental effects of estrogen are believed to be mediated by slow genomic actions through the nuclear estrogen receptors alpha (ERalpha) and/or beta (ERbeta). Consequently, exogenous compounds that interfere with the ERs may disrupt sexual differentiation of the reproductive organs and of the brain areas controlling sexual behaviors. The present study was conducted to elucidate the role of ERalpha in xenoestrogen-induced disruption of sexual differentiation in the Japanese quail (Coturnix japonica). Embryonic treatment with the synthetic estrogen, ethinylestradiol (EE(2)), and with the ERalpha-selective agonist, propyl pyrazole triol (PPT), induced oviductal malformations in females and retention of oviducts in males. Both EE(2) and PPT caused weight asymmetry between left and right testes and reduced the cloacal gland area in males. EE(2) significantly reduced the copulatory behavior in males whereas PPT had no effect on this behavior. The sexually dimorphic parvocellular vasotocin-immunoreactive (VT-ir) system in the medial preoptic nucleus (POM), the lateral septum (SL) and the medial part of the nucleus of the stria terminalis (BSTm), was not affected by EE(2) or PPT. Our results suggest that xenoestrogen-induced effects on reproductive organ differentiation are mediated by ERalpha, whereas demasculinization of male copulatory behavior and the VT-ir system appears not to be induced by activation of ERalpha alone.

Photoperiod-independent hypothalamic regulation of luteinizing hormone secretion in a free-living Sonoran desert bird, the Rufous-winged Sparrow (Aimophila carpalis).

We investigated the regulation of luteinizing hormone (LH) in the male Rufous-winged Sparrow,Aimophila carpalis, a resident of the Sonoran desert that breeds after irregular summer rains. Although the testes develop in March due to increasing photoperiod and regress in September due to decreasing photoperiod, LH does not consistently increase in the spring as in other photoperiodic birds. However, throughout the year increased plasma LH is correlated with rainfall. To investigate this rainfall-associated regulation of LH secretion, we quantified immunocytochemical labeling for gonadotropin-releasing hormone I (GnRH-I), proGnRH (the GnRH precursor), and gonadotropin-inhibitory hormone (GnIH) in the hypothalamus of free-living adult males caught before (low LH), and during (high LH) the monsoon rainy season. Compared to pre-monsoon birds, birds caught during the monsoon season had larger immunoreactive GnRH-I (GnRH-I-ir) and proGnRH-ir cell bodies, as well as fewer, less densely labeled proGnRH-ir cell bodies. Birds caught during the monsoon had fewer, less densely labeled GnIH-ir cell bodies than birds caught before the monsoon. Further, there was no GnIH-ir labeling in the median eminence on either capture dates, suggesting that GnIH is not released to the pituitary gland via the portal vein at this time of year, but there were fewer GnIH-ir fibers in the preoptic area of birds caught during the monsoon season. Our data support the hypothesis that environmental factors associated with increased rainfall during the monsoon season stimulate GnRH synthesis and release to increase LH secretion. These data also suggest that GnIH could inhibit GnRH neuronal activity prior to the monsoon season.

Effects of early embryonic exposure to genistein on male copulatory behavior and vasotocin system of Japanese quail.

Genistein is a phytoestrogen, particularly abundant in soybeans that can bind estrogen receptors and sex hormone binding proteins, exerting both estrogenic and antiestrogenic activity. In this study we used the Japanese quail embryo as a test end-point to investigate the effects of early embryonic exposure to genistein on male copulatory behavior and on vasotocin parvocellular system. Both differentiate by the organizational effects of estradiol during development and may therefore represent an optimal model to study the effects of xenoestrogens. We injected two doses of genistein (100 and 1000 microg) into the yolk of 3-day-old Japanese quail eggs. Other eggs were treated with either 25 microg of estradiol benzoate or sesame oil as positive and negative controls. At the age of 6 weeks, behavioral tests revealed a significant decrease of all aspects of copulatory behavior (in comparison to the control group) in estradiol-treated birds. In contrast, genistein-treated animals demonstrated various degrees of decrease in the mean frequencies of some aspects of the sexual behavior. The computerized analysis of vasotocin innervation in medial preoptic, stria terminalis and lateral septum nuclei revealed a statistically significant decreased immunoreactivity in treated animals compared to control ones. These results demonstrate that genistein, similarly to estradiol, has an organizational effect on quail parvocellular vasotocin system and on copulatory behavior. In conclusion, present results confirm, in this avian model, that embryonic exposure to phytoestrogens may have life-long effects on sexual differentiation of brain structures and behaviors.

Early embryonic administration of xenoestrogens alters vasotocin system and male sexual behavior of the Japanese quail.

The copulatory behavior and the parvocellular vasotocin (VT) system of the nucleus of the stria terminalis (BST) are sexually dimorphic in the Japanese quail. Embryonic administration of estradiol benzoate (EB) induces an organizational effect determining the disappearance of such a dimorphism (male shows behavior and cerebral phenotype of the female). The VT parvocellular system can therefore be considered an accurate marker of the sexual differentiation of brain circuits and a very sensitive indicator of the activity of estrogen-like substances on neural circuits. To test this hypothesis we administered diethylstilbestrol (DES), a powerful synthetic xenoestrogen, genistein (GEN), a phytoestrogen produced by soy, and bisphenol A (BPA). After 3 days of incubation, quail eggs were injected with vehicle, EB, DES, GEN or BPA. Administration of BPA caused an early blockage of development and no further analyses were done on the BPA groups. At puberty, the copulatory behavior of EB- or DES-treated male quail was totally abolished, whereas only the highest doses of GEN determined a significant decrease of the behavior. After the tests, the animals were sacrificed and perfused. The fractional area (FA) covered by VT immunoreactivity was analyzed in BST, medial preoptic nucleus, and lateral septum by computerized image analysis. The FA was significantly reduced after treatment with EB, DES and GEN at high doses. These results confirm that the sexually dimorphic VT system of the Japanese quail is a sensible indicator of the effects of xenoestrogens at the level of the central nervous system.

Organizational effects of diethylstilbestrol on brain vasotocin and sexual behavior in male quail.

In Japanese quail, we previously described a sexual dimorphism of the parvocellular vasotocin system of the limbic region that, as the reproductive behavior, is steroid-sensitive and is organized during embryonic life by the exposure to estradiol. We verified in this study whether diethylstilbestrol, a chemical xenoestrogen, has analogous organizational effects on the vasotocin system of limbic regions and on copulatory behavior of male Japanese quail. We injected in the yolk sac of 3 day-old quail embryos diethylstilbestrol or estradiol benzoate (a treatment which suppresses male copulatory behavior in adulthood and reduces vasotocin innervation), or sesame oil (control). No further hormonal manipulations were performed after hatching. Sexual behavior was recorded in males at the age of 6 weeks. Estradiol- and diethylstilbestrol-treated males exhibited a total suppression of copulatory behavior. After behavioral tests, all males were sacrificed and brain sections processed for vasotocin immunocytochemistry. Significant decrease in the density of vasotocin immunoreactivity was detected in the medial preoptic nucleus, in the bed nucleus of stria terminalis, and in the lateral septum of diethylstilbestrol-treated males. The magnocellular vasotocin neurons were, in contrast, not affected. In conclusion, the present data demonstrate that embryonic treatment with diethylstilbestrol induces a full sex reversal of behavioral phenotype as well as a significant decrease of vasotocin expression in the preoptic-limbic region in male Japanese quail. Therefore, the parvocellular vasotocin system could represent an optimal model to investigate the effects of pollutants on neural circuits controlling reproductive functions.