Peripubertal GnRH and testosterone co-treatment leads to increased familiarity preferences in male sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) treatment is effective for the medical suppression of the hypothalamic-pituitary-gonadal axis in situations like central precocious puberty and gender dysphoria. However, its administration during the peripubertal period could influence normal brain development and function because GnRH receptors are expressed in brain regions that regulate emotions, cognition, motivation and memory. This study used an ovine model to determine whether chronic peripubertal GnRHa-treatment affected the developmental shift from preference of familiarity to novelty. Experimental groups included Controls and GnRHa-treated rams. To differentiate between effects of altered GnRH signaling and those associated with the loss of sex steroids, a group was also included that received testosterone replacement as well as GnRHa (GnRHa + T). Preference for a novel versus familiar object was assessed during 5-min social isolation at 8, 28 and 46 weeks of age. Approach behavior was measured as interactions with and time spent near the objects, whereas avoidance behavior was measured by time spent in the entrance zone and attempts to escape the arena via the entry point. Emotional reactivity was measured by the number of vocalizations, escape attempts and urinations. As Control and GnRHa-treated rams aged, their approach behaviors showed a shift from preference for familiarity (8 weeks) to novelty (46 weeks). In contrast, relative to the Controls the GnRHa + T rams exhibited more approach behaviors towards both objects, at 28 and 46 weeks of age and preferred familiarity at 46 weeks of age. Vocalisation rate was increased in GnRHa treated rams in late puberty (28 weeks) compared to both Control and GnRHa + T rams but this effect was not seen in young adulthood (46 weeks). These results suggest that the specific suppression of testosterone during a developmental window in late puberty may reduce emotional reactivity and hamper learning a flexible adjustment to environmental change. The results also suggest that disruption of either endogenous testosterone signalling or a synergistic action between GnRH and testosterone signalling, may delay maturation of cognitive processes (e.g. information processing) that affects the motivation of rams to approach and avoid objects.

Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes.

This study was designed to identify genes that regulate the transition from FSH- to LH-dependent development in the bovine dominant follicle (DF). Serial analysis of gene expression (SAGE) was used to compare the transcriptome of granulosa cells isolated from the most oestrogenic growing cohort follicle (COH), the newly selected DF and its largest subordinate follicle (SF) which is destined for atresia. Follicle diameter, follicular fluid oestradiol (E) and E:progesterone ratio confirmed follicle identity. Results show that there are 93 transcript species differentially expressed in DF granulosa cells, but only 8 of these encode proteins known to be involved in DF development. Most characterised transcripts upregulated in the DF are from tissue development genes that regulate cell differentiation, proliferation, apoptosis, signalling and tissue remodelling. Semiquantitative real-time PCR analysis confirmed seven genes with upregulated (P< or =0.05) mRNA expression in DF compared with both COH and SF granulosa cells. Thus, the new genes identified by SAGE and real-time PCR, which show enhanced mRNA expression in the DF, may regulate proliferation (cyclin D2; CCND2), prevention of apoptosis or DNA damage (growth arrest and DNA damage-inducible, beta; GADD45B), RNA synthesis (splicing factor, arginine/serine rich 9; SFRS9) and unknown processes associated with enhanced steroidogenesis (ovary-specific acidic protein; DQ004742) in granulosa cells of DF at the onset of LH-dependent development. Further studies are required to show whether the expression of identified genes is dysregulated when abnormalities occur during DF selection or subsequent development.

Modulation of the phosphorylation state of tau in situ: the roles of calcium and cyclic AMP.

Alterations in situ in the phosphorylation state of the microtubule-associated protein tau were examined in response to increasing intracellular levels of Ca2+ through N-methyl-D-aspartate (NMDA)-receptor activation, or activating cyclic AMP (cAMP)-dependent protein kinase (cAMP-PK), in rat cerebral-cortical slices. Increasing intracellular concentrations of Ca2+ by treatment of the brain slices with the glutamate analogue NMDA in depolarizing conditions (55 mM KCl) resulted in dephosphorylation of tau. Addition of KCl+NMDA to the slices resulted in a 40% decrease in 32P incorporation into tau, whereas addition of KCl or NMDA alone had no effect on tau phosphorylation. The KCl+NMDA-induced dephosphorylation of tau was blocked by the non-competitive NMDA-receptor antagonist MK801. Determine the involvement of the Ca2+/calmodulin-dependent phosphatase, calcineurin, in the KCl+NMDA-induced dephosphorylation of tau, slices were pretreated with the calcineurin inhibitor Cyclosporin A. Pretreatment of the rat brain slices with Cyclosporin A completely abolished the dephosphorylation of tau induced by the addition of KCl+NMDA. The dephosphorylation of tau in situ was site-selective, as indicated by the loss of 32P label from only a few select peptides. Activation of cAMP-PK by stimulating adenylate cyclase in rat cerebral-cortical slices with forskolin resulted in a 73% increase over control levels in 32P incorporation into immunoprecipitated tau. Two-dimensional phosphopeptide mapping revealed that most of the sites on tau phosphorylated in brain slices in response to increased cAMP levels were the same as those phosphorylated on isolated tau by purified cAMP-PK. Although the state of tau phosphorylation is certainly regulated by many protein phosphatases and kinases in vivo, to our knowledge this study provides the first direct evidence of a specific protein phosphatase and kinase that modulate the phosphorylation state of tau in situ.

BW373U86: a nonpeptidic delta-opioid agonist with novel receptor-G protein-mediated actions in rat brain membranes and neuroblastoma cells.

BW373U86 is a potent and highly selective nonpeptidic agonist for delta-opioid receptors. To determine its ability to couple with G protein-linked second messenger systems, this study examined the effects of BW373U86 on the inhibition of adenylyl cyclase and the stimulation of low-Km GTPase activity. In rat striatal membranes, BW373U86 inhibited basal adenylyl cyclase activity in a GTP-dependent manner, with maximal inhibition levels similar to those of the prototypic delta agonist [D-Ser2,Thr6]Leu-enkephalin (DSLET). However, BW373U86 was approximately 100 times more potent than DSLET in inhibiting adenylyl cyclase. Analysis of the inhibitory activity across 10 brain regions revealed that both low and high concentrations of BW373U86 inhibited adenylyl cyclase activity in a manner similar to that of DSLET. Inhibition of adenylyl cyclase by BW373U86 was delta receptor selective, because the delta receptor-selective antagonist naltrindole was significantly more potent than naloxone and the mu receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 was ineffective in blocking BW373U86 inhibition. BW373U86 also inhibited adenylyl cyclase activity in membranes prepared from NG108-15 cells, with an IC50 value 5 times lower than that of DSLET. This increased potency was not observed in concentration-effect curves for agonist-stimulated low-Km GTPase in NG108-15 membranes. BW373U86 is a competitive inhibitor of [3H]diprenorphine at delta receptors of NG108-15 cell membranes. However, unlike DSLET, BW373U86 displacement of [3H]diprenorphine binding to NG108-15 cell membranes was not affected by sodium and guanine nucleotides. This lack of GTP effect on binding apparently produced slow dissociation rates for this agonist, because naltrindole was less potent in blocking BW373U86 inhibition of adenylyl cyclase when membranes were preincubated with this agonist. These results demonstrate the novel finding that the binding of a full agonist to a G protein-coupled receptor is not regulated by GTP, and they also show how the lack of regulation in receptor binding affects agonist potency.

Inhibition of protein phosphorylation by opioid-inhibited adenylyl cyclase in rat brain membranes.

Opioid inhibition of adenylyl cyclase is a major second messenger system associated with opioid receptors in brain. To identify membrane phosphoproteins whose phosphorylation state is modulated by opioid inhibition of adenylyl cyclase, rat striatal membranes were preincubated with opioid agonists in the presence of 500 microM 5'-adenylyl-imidodiphosphate (which acted as a substrate for adenylyl cyclase, but not for protein kinase) before addition of [gamma-32P]ATP. Under these conditions, adenylyl cyclase in the membranes formed cyclic AMP, which stimulated cyclic AMP-dependent protein kinase. This process was confirmed by observing forskolin-stimulated phosphorylation of two bands of MW 85 and 63 kDa, which were also stimulated directly by cyclic AMP. Forskolin-stimulated phosphorylation of these two bands was inhibited by 15 to 30% by opioid agonists such as D-Ala2-Met5-enkephalinamide. This inhibition of phosphorylation was mediated by opioid receptors, because it required both sodium and GTP, and was blocked by naloxone. These results suggest that these two proteins may be primary targets of opioid-inhibited adenylyl cyclase in striatal membranes.