Vasilios T. Garyfallou

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.

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.

α-Adrenergic receptor antagonism and N-methyl-d-aspartate (NMDA) induced luteinizing hormone release in female rhesus macaques

The stimulatory influence of N-methyl-D-aspartate (NMDA), a glutamate receptor agonist, on LH secretion is well established in several mammalian species including the rhesus macaque. Although the mechanism of excitation appears to involve enhanced GnRH secretion, it is unclear whether the GnRH neurons respond directly to this excitation or whether stimulatory inter-neurons are involved. This study investigated the possibility that noradrenergic afferents play a major role in mediating the response of the primate hypothalamo-pituitary reproductive axis to NMDA. In situ hybridization histochemistry, using a cRNA probe coding for the NMDAR1 receptor subunit, revealed abundant mRNA in the locus coeruleus, a brain area rich in noradrenergic neurons. Furthermore, using double-label fluorescence immunocytochemistry, the tyrosine hydroxylase immunopositive neurons of the locus coeruleus showed immunoreactivity for the NMDAR1 receptor subunit protein. A second experiment examined whether prazosin, an alpha 1-adrenergic receptor antagonist, could attenuate NMDA-induced stimulation of LH release. Prazosin (either 1 or 5 mg/kg b.wt., i.v.) was administered to female rhesus macaques during the luteal phase of the menstrual cycle, 40 min before administration of NMDA (10 mg/kg b.wt., i.v.). Regardless of the prazosin pre-treatment, plasma LH concentrations showed a significant increase (P < 0.01) within 10 min of the administration of NMDA. Therefore, in spite of the evidence that at least some of the noradrenergic neurons of the primate hindbrain express the NMDAR1 receptor subunit, it is unlikely that noradrenergic inter-neuronal pathways alone play a major role in mediating the stimulatory action of NMDA on GnRH/LH secretion in primates. Indeed, because the GnRH neurons of the rhesus macaque are located diffusely in various regions of the hypothalamus and medial-septal/preoptic area, their net response to excitatory amino acids is likely to be more complicated, involving a combination of both stimulatory and inhibitory inter-neurons, and possibly also a direct interaction.

Influence of 17β-estradiol and progesterone on GABAergic gene expression in the arcuate nucleus, amygdala and hippocampus of the rhesus macaque

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, and the responsiveness of neurons to GABA can be modulated by sex steroids. To better understand how ovarian steroids influence the GABAergic system in the primate brain, we evaluated the expression of genes encoding GABA receptor subunits, glutamic acid decarboxylase (GAD) and a GABA transporter in the brains of female rhesus macaques. Ovariectomized adults were subjected to a hormone replacement paradigm involving either 17beta-estradiol (E), or E plus progesterone (E+P). Untreated animals served as controls. Using GeneChip microarray analysis and real-time RT-PCR (qPCR), we examined gene expression differences within and between the amygdala (AMD), hippocampus (HPC) and arcuate nuclei of the medial basal hypothalamus (MBH). The results from PCR corresponded with results from representative GeneChip probesets, and showed similar effects of sex steroids on GABA receptor subunit gene expression in the AMD and HPC, and a more pronounced expression than in the MBH. Exposure to E+P attenuated GAD1, GAD2 and SLC32A1 gene expression in the AMD and HPC, but not in the MBH. GABA receptor subunit gene expression was generally higher in the AMD and HPC than in the MBH, with the exception of receptor subunits epsilon and gamma 2. Taken together, the data demonstrate differential regulation of GABA receptor subunits and GABAergic system components in the MBH compared to the AMD and HPC of rhesus macaques. Elevated epsilon and reduced delta subunit expression in the MBH supports the hypothesis that the hypothalamic GABAergic system is resistant to the modulatory effects of sex steroids.

Photoperiodic modulation of GnRH mRNA in the male Syrian hamster

Male Syrian hamsters (Mesocricetus auratus) are seasonal breeders. They show marked testicular regression when exposed to short autumnal photoperiods, and then remain sexually quiescent for several months. By mid-winter, however, they show a loss in responsiveness to the inhibitory influence of short photoperiods and their testes begin to recrudesce. To shed light on the neuroendocrine mechanism responsible for mediating these reproductive changes, we examined the influence of photoperiod on the expression of GnRH mRNA in the hamster forebrain. Adult males were either exposed to short photoperiods (6L:18D) for 16 weeks or were maintained under long photoperiods (14L:10D); additional animals were exposed to short or long photoperiods for 22 weeks. As expected, exposure to short photoperiods for 12 weeks resulted in a marked decrease (P<0.01) in testicular mass and serum testosterone levels, but after 22 weeks these reproductive parameters were once again significantly elevated (P<0.01). In contrast, quantitative in situ hybridization histochemistry revealed no difference (P>0.05) between the GnRH mRNA levels of the short-photoperiod hamsters and their aged-matched long-photoperiod controls, although an age-related decrease (P<0.05) was evident in both photoperiod-treatment groups. These data emphasize that GnRH mRNA is highly expressed in hamsters even when their reproductive axis has been rendered sexually quiescent by exposure to short photoperiods, and that photoperiod-induced changes in GnRH secretion, rather than synthesis, are more likely to regulate the timing of the breeding season. On the other hand, the data indicate that GnRH mRNA levels show an aging-related decrease, regardless of photoperiod, suggesting that in the long term a decrease in GnRH gene expression may contribute to the reduced fertility of old hamsters.

Distribution of NMDA and AMPA receptors in the cerebellar cortex of rhesus macaques

The distribution of glutamate receptors in the cerebellar cortex of the rhesus macaque was examined by light microscopic immunocytochemistry using an antibody specific to the N-methyl-D-aspartate (NMDA) R1 receptor subunit (i.e. NMDAR1) as well as antibodies specific to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits (i.e. GluR1, GluR2/3, and GluR4). NMDAR1 immunolabeling was most prevalent in the Purkinje cell perikarya and dendrities, but was also significant in the stellate and basket cells of the granular layer and Golgi cells of the molecular layer. On the other hand, GluRl and GluR4 immunolabeling was concentrated principally in the processes of the Bergmann glia located in the vicinity of the Purkinje cell perikarya. Although GluR2/3 immunolabeling also occurred in these Bergmann glia processes as well as in the Bergmann fibers, it was more pronounced in the Purkinje cell perikarya and dendrites; additionally, significant GluR2/3 labeling was evident in the stellate and basket cells of the molecular layer and medium-size soma of the granular layer (most likely Golgi cells). In situ hybridization histochemistry (ISHH), using cRNA probes to NMDAR1. GluR1.GluR2, and GluR3, showed glutamate receptor mRNA distribution patterns consistent with those disclosed in the immunocytochemical study. Furthermore, the ISHH findings suggest that the positive immunocytochemical labeling of Purkinje cells with the GluR2/3 antibody is most likely due to the gene expression of both GluR2 and GluR3 AMPA receptor subtypes. Taken together, the results are potentially important for the elucidation of mechanisms that control aspects of cerebellar function, such as long-term depression.

Regional distribution of glutamic acid decarboxylase (GAD65 and GAD67) mRNA in the hypothalamus of male rhesus macaques before and after puberty.

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme in the gamma-aminobutyric acid (GABA) biosynthetic pathway, and is coded for by two mRNAs, GAD65 and GAD67. Using in situ hybridization, we examine the distribution pattern of both GAD mRNAs in the hypothalamus and thalamus of prepubertal and adult male rhesus macaques. Qualitatively, GAD65 and GAD67 mRNAs showed a similar wide, but highly specific distribution pattern, supporting the view that GABAergic neurons play an important role in modulating neuroendocrine function. However, no quantitative difference in the intensity of hybridization signal was detected between prepubertal and adult animals in any of the hypothalamic or thalamic nuclei. Therefore, although GABAergic neurons are anatomically well-placed to control the secretion of gonadotropin-releasing hormone (GnRH) in primates, it is unlikely that the onset of puberty and the associated increase in GnRH secretion is triggered by a change in GAD gene transcription.

Lesions in the bed nucleus of the stria terminalis, but not in the lateral septum, inhibit short-photoperiod-induced testicular regression in Syrian hamsters

The transfer of adult male hamsters from long days (LD) to short days (SD) (i.e. < 12 h of light per day) typically results in marked testicular regression and a decline in plasma testosterone concentrations. To help disclose key brain regions responsible for mediating this photoperiodic response male hamsters received either chemical (i.e. N-methyl-D-aspartate; NMDA) or radiofrequency current lesions in the bed nucleus of the stria terminalis (BNST), and were then exposed to SD for 15 or 12 weeks, respectively. Although body weights were similar between sham-lesioned controls and the NMDA-lesioned hamsters, the latter showed a significant attenuation of testicular regression; additionally, their plasma testosterone concentrations remained at typical LD levels. When radiofrequency current-lesioned hamsters were transferred from LD to SD they also failed to show significant signs of testicular regression, nor a decline in plasma testosterone concentrations, nor a complete arrest of spermatogenesis. In contrast, sham-lesioned controls or hamsters that were lesioned dorsally to the BNST at a site primarily involving the lateral septum all showed the expected degree of testicular regression, a decline in plasma testosterone concentrations, and complete arrest of spermatogenesis; body weights were similar in all of the experimental group. Taken together, these findings suggest that the BNST, a brain area traditionally not associated with reproductive function, may play an important role in mediating photoperiodic information to the neural circuits that control the reproductive axis.

High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function

Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signalling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through pre-pubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of tumour necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signalling of the testis in health and disease.

Expression profiling of genes encoding glutamate and GABA receptor subunits in three immortalized gnrh cell lines

Gonadotropin-releasing hormone (GnRH) plays a central role in regulating development and function of the reproductive axis, and its secretion is known to be influenced by glutamate and GABA. In the present study, we used gene microarrays and RT-PCR to compare the expression profiles of glutamate and GABA receptor subunits in three immortalized GnRH cell lines: GT1-1, GT1-7, and Gn10. All of these cell lines expressed the AMPA glutamate receptor subunit genes GluR2 and GluR4, but only the GT1-1 and GT1-7 cells expressed the kainate glutamate receptor subunit gene KA2. Additionally, GluRdelta2, a subunit that can form heteromeric receptors with kainate and AMPA subunits, was present in GT1-1 and Gn10 cells but not in GT1-7 cells. Genes encoding the GABA(A) receptor alpha3, beta2, beta3, epsilon, and pi subunits, as well as the GABA(B) receptor 1 subunit, were evident in all three cell lines. However, the gene encoding the expression of GABA(A) receptor gamma subunit was noticeably absent. Taken together, these data demonstrate comprehensive screening of neurotransmitter receptor genes in a controlled neuronal culture system, and reveal novel features.