Henryk F. Urbanski

Morphologic evidence that neurokinin B modulates gonadotropin-releasing hormone secretion via neurokinin 3 receptors in the rat median eminence.

Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual‐label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin‐releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB‐immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH‐ir somata exhibited NK3R staining. ProNKB and NK3R‐ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB‐ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis. J. Comp. Neurol. 489:372–386, 2005.

Neuroendocrine Changes in the Aging Reproductive Axis of Female Rhesus Macaques (Macaca mulatta)

Abstract Female rhesus macaques show monthly menstrual cycles and eventually enter menopause at approximately 25 yr of age. To help identify early biomarkers of menopause in this nonhuman primate, we monitored reproductive hormones longitudinally from aged female macaques during the transitions from premenopause to perimenopause and postmenopause and found that, indeed, elevated plasma FSH was a better predictive factor of menopause onset than age. In a second experiment, we compared reproductive hormone profiles of young adult macaques (8–10 yr old) with those of regularly cycling old macaques (approximately 24 yr old). Indwelling vascular catheters were used for remote blood collection for at least 100 consecutive days, thereby covering three complete menstrual cycles in each macaque. Plasma levels of estradiol, progesterone, LH, FSH, follicular phase inhibin B, and anti-müllerian hormone (AMH) were determined during each menstrual cycle and were averaged for each animal; group mean differences were analyzed using one-way ANOVA. Old premenopausal macaques showed regular menstrual cycles that were qualitatively indistinguishable from those of young macaques; peak plasma levels of estradiol, progesterone, and LH were not significantly different. In marked contrast, peak plasma FSH concentrations were significantly higher, while inhibin B and AMH levels were generally lower, in the old premenopausal macaques compared with those in the young macaques. These data provide further evidence that rhesus macaques serve as an excellent model to study underlying mechanisms of human menopause. Furthermore, the data suggest that an age-related change in FSH, inhibin B, and AMH secretion may be the first endocrine manifestation of the transition into perimenopause, potentially having value in predicting the onset of the perimenopausal transition.

Two molecular forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) are expressed by two separate populations of cells in the rhesus macaque hypothalamus

Gonadotropin-releasing hormone represents the primary neuroendocrine link between the brain and the reproductive axis, and at least two distinct molecular forms of this decapeptide (GnRH-I and GnRH-II) are known to be expressed in the forebrain of rhesus macaques (Macaca mulatta). Although the distribution pattern of the two corresponding mRNAs is largely dissimilar, their expression appears to show some overlap in specific regions of the hypothalamus; this raises the possibility that some cells express both molecular forms of GnRH. To resolve this issue, double-label histochemistry was performed on hypothalamic sections from six male rhesus macaques, using a monoclonal antibody to GnRH-I and a riboprobe to monkey GnRH-II mRNA. In total, more than 2000 GnRH neurons were examined but in no instance were GnRH-I peptide and GnRH-II mRNA found to be coexpressed. This finding emphasizes that GnRH-I and GnRH-II are synthesized by two distinct populations of hypothalamic neurons, and suggests that they may be regulated by different neuroendocrine pathways.

Orexin neuronal changes in the locus coeruleus of the aging rhesus macaque

Orexin neuropeptides regulate arousal state and excite the noradrenergic locus coeruleus (LC), so it is plausible that an age-related loss of orexin neurons and projections to the LC contributes to poor sleep quality in elderly humans and nonhuman primates. To test this hypothesis we examined orexin B-immunoreactivity in the lateral hypothalamic area (LHA) and the LC of male rhesus macaques (Macaca mulatta) throughout the life span. Orexin perikarya, localized predominantly in the LHA, showed identical distribution patterns irrespective of age. Similarly, orexin neuron number and serum orexin B concentrations did not differ with age. In contrast, orexin B-immunoreactive axon density in the LC of old animals was significantly lower than that observed in the young or adult animals. Furthermore, the age-related decline was associated with a significant decrease in tyrosine hydroxylase (TH) mRNA in the LC, despite no change in TH-immunoreactive neuron number. Taken together, these data suggest that age-related decreases in excitatory orexin innervation to the noradrenergic LC may contribute to the etiology of poor sleep quality in the elderly.

GnRH systems of Cichlasoma dimerus (Perciformes, Cichlidae) revisited: a localization study with antibodies and riboprobes to GnRH-associated peptides

The distribution of cells that express three prepro-gonadotropin-releasing hormones (GnRH), corresponding to salmon GnRH, sea bream GnRH (sbGnRH), and chicken II GnRH, was studied in the brain and pituitary of the South American cichlid fish, Cichlasoma dimerus. Although the ontogeny and distribution of GnRH neuronal systems have previously been examined immunohistochemically with antibodies and antisera against the various GnRH decapeptides, we have used antisera against various perciform GnRH-associated peptides (GAPs) and riboprobes to various perciform GnRH+GAPs. The results demonstrate that: (1) the GnRH neuronal populations in the forebrain (salmon and sea bream GAPs; sGAP and sbGAP, respectively) show an overlapping pattern along the olfactory bulbs, nucleus olfacto-retinalis, ventral telencephalon, and preoptic area; (2) projections with sGAP are mainly located in the forebrain and contribute to the pituitary innervation, with projections containing chicken GAP II being mainly distributed along the mid and hindbrain and not contributing to pituitary innervation, whereas sbGAP projections are restricted to the ventral forebrain, being the most important molecular form in relation to pituitary innervation; (3) sbGnRH (GnRH I) neurons have an olfactory origin; (4) GAP antibodies and GAP riboprobes are valuable tools for the study of various GnRH systems, by avoiding the cross-reactivity problems that occur when using GnRH antibodies and GnRH riboprobes alone.

Effects of N-methyl-D-aspartate (NMDA) on seasonal cycles of reproduction, body weight and pelage colour in the male Siberian hamster.

Siberian hamsters (Phodopus sungorus) transferred from stimulatory photoperiods (long days: LD) to inhibitory photoperiods (short days: SD) undergo testicular regression within 8 weeks. This reproductive response to photoperiod was blocked by systemic daily treatment with the glutamatergic agonist N‐methyl‐D‐aspartate (NMDA: 20 mg/kg BW, sc). This powerful effect of NMDA demonstrates the potential for endogenous glutamate to regulate reproductive function.

Effect of age and caloric restriction on circadian adrenal steroid rhythms in rhesus macaques

Dietary caloric restriction (CR) slows aging, extends lifespan, and reduces the occurrence of age-related diseases in short-lived species. However, it is unclear whether CR can exert similar beneficial effects in long-lived species, like primates. Our objective was to determine if CR could attenuate purported age-related changes in the 24-h release of adrenal steroids. To this end, we examined 24-h plasma profiles of cortisol, and dehydroepiandrosterone sulfate (DHEAS) in young and old, male and female rhesus macaques (Macaca mulatta) subjected to either ad libitum (AL)-feeding or CR (70% of AL) for 2-4 years. Hormone profiles from young monkeys showed pronounced 24-h rhythms. Cortisol concentrations were higher in old males but not females, whereas DHEAS rhythms were dampened with age in both sexes. The cortisol rhythms of old CR males resembled those of young control males. However, CR failed to prevent age-related declines in DHEAS and further dampened DHEAS rhythms in both sexes. Apart from the partial attenuation of the age-related cortisol elevation in the old males, 24-h adrenal steroid rhythms did not benefit from late-onset CR.

Effect of Caloric Restriction on the 24‐Hour Plasma DHEAS and Cortisol Profiles of Young and Old Male Rhesus Macaques

Abstract: Although dietary caloric restriction (CR) can retard aging in laboratory rats and mice, it is unclear whether CR can exert similar effects in long‐lived species, such as primates. Therefore, we tested the effect of CR on plasma levels of dehydroepiandrosterone sulfate (DHEAS), a reliable endocrine marker of aging. The study included six young (∼10 years) and ten old (∼25 years) male rhesus macaques, approximately half of the animals in each age group having undergone >4 years of 30% CR. Hourly blood samples were collected remotely for 24 hours, through a vascular catheter, and assayed for DHEAS and cortisol. Both of these adrenal steroids showed a pronounced diurnal plasma pattern, with peaks occurring in late morning, but only DHEAS showed an aging‐related decline. More importantly, there was no significant difference in plasma DHEAS concentrations between the CR animals and age‐matched controls. These data fail to support the hypothesis that CR can attenuate the aging‐related decline in plasma DHEAS concentrations, at least not when initiated after puberty.

Dehydroepiandrosterone and age-related cognitive decline

In humans the circulating concentrations of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) decrease markedly during aging, and have been implicated in age-associated cognitive decline. This has led to the hypothesis that DHEA supplementation during aging may improve memory. In rodents, a cognitive anti-aging effect of DHEA and DHEAS has been observed but it is unclear whether this effect is mediated indirectly through conversion of these steroids to estradiol. Moreover, despite the demonstration of correlations between endogenous DHEA concentrations and cognitive ability in certain human patient populations, such correlations have yet to be convincingly demonstrated during normal human aging. This review highlights important differences between rodents and primates in terms of their circulating DHEA and DHEAS concentrations, and suggests that age-related changes within the human DHEA metabolic pathway may contribute to the relative inefficacy of DHEA replacement therapies in humans. The review also highlights the value of using nonhuman primates as a pragmatic animal model for testing the therapeutic potential of DHEA for age-associate cognitive decline in humans.

Neuronal elements in the testis of the rhesus monkey: Ontogeny, characterization and relationship to testicular cells

Intrinsic neuron-like cells expressing the catecholamine-biosynthetic enzyme tyrosine hydroxylase (TH) were recently identified in the testis of the prepubertal rhesus monkey. In this study, we characterized the neuron-like nature of these cells and examined distribution and frequency of neuronal elements in the testes of monkeys during postnatal development, puberty and adulthood. Using immunohistochemical methods, we detected both nerve fibers and cell bodies, immunoreactive for the neuronal markers neurofilament 200 (NF-200) and synaptosomal associated protein of 25 kDa (SNAP-25), TH and neuropeptide Y (NPY) in perivascular locations, intermingled with interstitial cells and close to the wall of seminiferous tubules. Marked age-related differences in the numbers of these neuronal elements became apparent, when we quantified NF-200-immunoreactive neuronal elements. Thus, intrinsic neuron-like cell bodies were found only in the testes from immature animals (i.e., until about 3 years of age). Conversely, nerve fibers, presumably representing mainly the extrinsic innervation, were observed at all ages although they became more prominent after the pubertal increase in LH and testosterone levels. Interestingly, another testicular cell type known to contain potent regulatory substances, mast cells, was found to be in close anatomical proximity to nerve fibers. The number of these cells, positively identified with an antibody to tryptase, increased significantly after puberty following the same pattern as nerve fibers. These results confirm that the testicular nervous system of the monkey is composed of two components, intrinsic nerve cells and extrinsic fibers, both of which are catecholaminergic and peptidergic in nature. Furthermore, both components show a marked degree of plasticity during development, especially around the time of puberty. The intratesticular locations of neuron-like cells and fibers suggest that catecholamines and neuropeptides are likely to have multiple sites of actions, and may affect Leydig cells, cells of the tubular wall and vascular cells directly and/or indirectly via intermediation of mast cells.