Testicular development of Zebu bulls after chronic treatment with a gonadotropin-releasing hormone agonist

The objective was to compare testis characteristics of Zebu bulls treated with the GnRH agonist, deslorelin, at different times and for different durations during their development. An additional objective was to determine the usefulness of a stain for the transcription factor GATA-binding protein 4 (GATA-4) as a specific marker for Sertoli cell nuclei in cattle. Bulls (54) were allocated to nine groups (n = 6) and received s.c. deslorelin implants as follows: G1 = from birth to 3 mo of age; G2 = from 3 to 6 mo; G3 = from 6 to 9 mo; G4 = from 9 to 12 mo; G5 = from birth to 15 mo; G6 = from 3 to 15 mo; G7 = from 6 to 15 mo; G8 = from 12 to 15 mo; and G9 (control) = no implant. Bulls were castrated at 19 mo of age. Paraffin sections (10 mu m) were subjected to quantitative morphometry and GATA-4 immunohistochemistry. At castration, all bulls in the control group (6/6) had attained puberty (scrotal circumference ! 28 cm), whereas a smaller proportion (P < 0.05) had reached puberty in G2 (2/5) and G6 (1/ 6). Bulls in G2 and G6 also had a lesser (P < 0.05) testis weight compared with the control group. Total volume of seminiferous epithelium and total daily sperm production in G2 and G6 were only half that observed in the control group. Spermatids were observed in less than 50% of seminiferous tubules in G2, G6, and G7 compared with 82% in the control group (P < 0.05). Staining for GATA-4 was specific for and abundant in the Sertoli cell nucleus in both pre- and postpubertal bulls, and no other cell nucleus inside the seminiferous tubule was positive for GATA-4. Total number of Sertoli cells was not affected by treatment (P = 0.45), but nuclear volume was smaller in G2 and G6 (P < 0.05) compared with the control group. In conclusion, treatment of Zebu bulls with deslorelin had no apparent beneficial effect on testis development and delayed puberty when treatment was initiated at 3 mo of age. Staining for GATA-4 was a useful method for identifying and quantifying Sertoli cell nuclei in both pre- and postpubertal bulls.

Effect of naloxone-precipitated morphine withdrawal on c-fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing, hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 It for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis. however these were in CRH negative neurons. (C) 2004 Published by Elsevier Ireland Ltd.

Testicular germ cell tumors exhibit evidence of hormone dependence

The aim of this investigation was to test the hypothesis that testicular germ cell tumors (TGCTs) are hormone-dependent cancers. Human TGCT cells were implanted in the left testis of male severe combined immunodeficient mice receiving either no treatment or hormone manipulation treatment [blockade of gonadotropin-releasing hormone secretion and/or signaling using leuprolide or leuprolide plus exogenous testosterone]. Real-time RT-PCR analysis was used to determine the expression profiles of hormone pathway-associated genes. Tumor burden was significantly smaller in mice receiving both leuprolide and testosterone. Real-time RTPCR analysis of follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor and P450 aromatase revealed changes in expression in normal testis tissue related to presence of xenograft tumors and manipulation of hormone levels but a complete absence of expression of these genes in tumor cells themselves. This was confirmed in human specimens of TGCT. Reduced TGCT growth in vivo was associated with significant downregulation of LH receptor and P450 aromatase expression in normal testes. In conclusion, manipulation of hormone levels influenced the growth of TGCT in vivo, while the presence of xenografted tumors influenced the expression of hormone-related genes in otherwise untreated animals. Human TGCTs, both in the animal model and in clinical specimens, appear not to express receptors for FSH or LH. Similarly, expression of the P450 aromatase gene is absent in TGCTs. Impaired estrogen synthesis and/or signaling may be at least partly responsible for inhibition of TGCT growth in the animal model. (c) 2005 Wiley-Liss, Inc.

The stability of lipidic analogues of GnRH in plasma and kidney preparations: the stereoselective release of the parent peptide

The conjugation of a lipoamino acid to the N-terminus of Gonadotropin releasing hormone (GnRH) produces a lipophilic peptide from which the parent GnRH peptide is released into solution on treatment with plasma and kidney enzyme preparation. Our findings show that one stereoisomer of the Laa is cleaved very rapidly, providing a bolus dose of the peptide while the opposite stereoisomer is cleaved much more slowly, providing prolonged elevation of peptide concentration. The Laa-Glu linkage appears to act as a two phase prodrug system. © 2005 Elsevier Ltd. All rights reserved.

Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus)

The cane toad (Bufo marinus) was used as a model to study male anuran reproductive endocrinology and to develop a protocol for non-invasive sperm recovery. Circulating testosterone concentrations in 6-hourly samples did not vary significantly (P < 0.05) over a 24 h period although there was a tendency (P = 0.06) for testosterone to be elevated at 19:00 h relative to other times of the day, which may be related to the nocturnal activity pattern of this species. Testosterone secretion after intraperitoneal (IP) injection of either a GnRH agonist (5 mu g IP) or hCG (1000 IU) was also examined. While the GnRH agonist did not produce a significant increase above basal plasma testosterone (0.29, 95% C.I. of 0.05-1.10 ng/ml), injection of hCG resulted in an increase (P < 0.01) of plasma testosterone with peak concentrations at approximately 120 min (4.17, 95% C.I. of 2.69-7.44 ng/ml) after injection. Non-invasive pharmaceutical sperm recovery was attempted following IP injection of graded doses of GnRH agonist, hCG or FSH. Urine was collected at 3, 6 and 12 h after treatment to assess sperm quality and quantity. The optimal protocol for sperm recovery in cane toads was injection of either 1000 or 2000 IU hCG; there was no significant difference in the quality of the spermic urine samples obtained using either dose of hCG or with respect to collection time. The findings indicated that hCG can be used to assess testicular steroidogenic status and also to induce sperm recovery in the cane toad. The hCG protocols developed in this study will have application in studies on the reproductive biology of rare and endangered male anurans. (c) 2005 Elsevier B.V. All rights reserved.

The Randomized Nitric Oxide Tocolysis Trial (RNOTT) for the treatment of preterm labor

Objective: This study was undertaken to assess the effectiveness of glyceryl trinitrate (GTN) patches in comparison with beta2 sympathornimetics (beta2) for the treatment of preterm labor. Study design: A multicenter, multinational, randomized controlled trial was conducted in tertiary referral teaching hospitals. Women in threatened preterm labor with positive fetal fibronectin or ruptured membranes between 24 and 35 weeks' gestation were recruited and randomly assigned to either beta2 or GTN with rescue beta2 tocolysis if moderate-to-strong contractions persisted at 2 hours. Obstetric and neonatal outcomes were assessed. Results: Two hundred thity-eight women were recruited and randomly assigned, 117 to beta2 and 121 to GTN. On a strict intention-to-treat basis, there was no significant difference in the time to delivery using Kaplan-Meier curves (P = .451). At 2 hours, 27% of women receiving beta2 had moderate or stronger contractions compared with 53% in the GTN group (P < .001). This led to 35% of women in the GTN group receiving rescue treatment. If delivery or requirement for beta2 rescue are regarded as treatment failure, then a significant difference was observed between the 2 arms (P = .0032). There were no significant differences in neonatal outcomes. Conclusion: GTN is a less efficacious tocolytic compared with beta2 sympathomimetics. (C) 2004 Elsevier Inc. All rights reserved.

Dissection of peripheral and central endogenous opioid modulation of systemic interleukin-1 beta responses using c-fos expression in the rat brain

In opiate addicts or patients receiving morphine treatment, it has been reported that the immune system is often compromised. The mechanisms responsible for the adverse effects of opioids on responses to infection are not clear but it is possible that central and/or peripheral opioid receptors may be important. We have utilised an experimental immune challenge model in rats, the systemic administration of the human pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) to study the effects of selectively blocking peripheral opioid receptors only (using naloxone methiodide) or after blocking both central and peripheral opioid receptors (using naloxone). Pre-treatment with naloxone methiodide decreased (15%) IL-1 beta-induced Fos-immunoreactivity (Fos-IR) in medial parvocellular paraventricular nucleus (mPVN) corticotropin-releasing hormone (CRH) neurons but increased responses in the ventrolateral medulla (VLM) C1 (65%) and nucleus tractus solitarius (NTS) A2 (110%) catecholamine cell groups and area postrema (136%). However no effect of blocking peripheral opioid receptors was detected in the central nucleus of the amygdala (CeA) or dorsal bed nucleus of the stria terminalis (BNST). We next determined the effect of blocking both central and peripheral opioid receptors with naloxone and, when compared to the naloxone methiodide pre-treated group, a further 60% decrease in Fos-IR mPVN CRH neurons induced by IL-1 beta was detected, which was attributed to block of central opioid receptors. Similar comparisons also detected decreases in Fos-IR neurons induced by IL-1 beta in the VLM A1, VLM C1 and NTS A2 catecholamine cell groups, area postrema, and parabrachial nucleus. In contrast, pre-treatment with naloxone increased Fos-IR neurons in CeA (98%) and dorsal BNST (72%). These results provide novel evidence that endogenous opioids can influence central neural responses to systemic IL-1 beta and also suggest that the differential patterns of activation may arise because of actions at central and/or peripheral opioid receptors that might be important in regulating behavioural, hypothalamic-pituitary-adrenal axis and sympathetic nervous system responses during an immune challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Topological side-chain classification of beta-turns: Ideal motifs for peptidomimetic development

beta-turns are important topological motifs for biological recognition of proteins and peptides. Organic molecules that sample the side chain positions of beta-turns have shown broad binding capacity to multiple different receptors, for example benzodiazepines. beta-turns have traditionally been classified into various types based on the backbone dihedral angles (phi 2, psi 2, phi 3 and psi 3). Indeed, 57-68% of beta-turns are currently classified into 8 different backbone families (Type I, Type II, Type I', Type II', Type VIII, Type VIa1, Type VIa2 and Type VIb and Type IV which represents unclassified beta-turns). Although this classification of beta-turns has been useful, the resulting beta-turn types are not ideal for the design of beta-turn mimetics as they do not reflect topological features of the recognition elements, the side chains. To overcome this, we have extracted beta-turns from a data set of non-homologous and high-resolution protein crystal structures. The side chain positions, as defined by C-alpha-C-beta vectors, of these turns have been clustered using the kth nearest neighbor clustering and filtered nearest centroid sorting algorithms. Nine clusters were obtained that cluster 90% of the data, and the average intra-cluster RMSD of the four C-alpha-C-beta vectors is 0.36. The nine clusters therefore represent the topology of the side chain scaffold architecture of the vast majority of beta-turns. The mean structures of the nine clusters are useful for the development of beta-turn mimetics and as biological descriptors for focusing combinatorial chemistry towards biologically relevant topological space.

Quantitative analysis of MC1R gene expression in human skin cell cultures

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells. Antibody mediated detection of MC1R protein in membrane extracts demonstrated exogenous receptor in MC1R transfected cell lines, as well as endogenous MC1R in melanoma cells. However, radioligand binding procedures were required to detect MC1R protein of normal human melanocytes and no surface expression of MC1R was detected in any of the non-melanocytic cells examined. This was consistent with their low level of mRNA, and suggests that, if present, the levels of surface receptor are significantly lower than that in melanocytes. The capacity of such limited levels of MC1R protein to influence non-melanocytic skin cell biology would likely be severely compromised. Indeed, the MC1R agonist [NIe(4), D-Phe(7)] alpha-melanocyte stimulating hormone (NDP-MSH) was unable to elevate intracellular cyclic adenosine monophosphate (cAMP) levels in the keratinocyte and fibroblast cells examined, whereas a robust increase was elicited in melanocytes. Although there are a variety of cell types with detectable MC1R mRNA, the expression of physiologically significant levels of the receptor may be more restricted than the current literature indicates, and within epidermal tissue may be limited to the melanocyte

Evidence that the Bed Nucleus of the Stria Terminalis Contributes to the Modulation of Hypophysiotropic Corticotropin-Releasing Factor Cell Responses to Systemic Interleukin-1β

Systemic infection activates the hypothalamic-pituitary-adrenal (HPA) axis, and brainstem catecholamine cells have been shown to contribute to this response. However, recent work also suggests an important role for the central amygdala (CeA). Because direct connections between the CeA and the hypothalamic apex of the HPA axis are minimal, the present study investigated whether the bed nucleus of the stria terminalis (BNST) might act as a relay between them. This was done by using an animal model of acute systemic infection involving intravascular delivery of the proinflammatory cytokine interleukin-1 (IL-1, 1 g/kg). Unilateral ibotenic acid lesions encompassing the ventral BNST significantly reduced both IL-1-induced increases in Fos immunoreactivity in corticotropin-releasing factor (CRF) cells of the hypothalamic paraventricular nucleus (PVN) and corresponding increases in adrenocorticotropic hormone (ACTH) secretion. Similar lesions had no effect on CRF cell responses to physical restraint, suggesting that the effects of BNST lesions were not due to a nonspecific effect on stress responses. In further studies, we examined the functional connections between PVN, BNST, and CeA by combining retrograde tracing with mapping of IL-1-induced increases in Fos in BNST and CeA cells. In the case of the BNST, these studies showed that systemic IL-1 administration recruits ventral BNST cells that project directly to the PVN. In the case of the CeA, the results obtained were consistent with an arrangement whereby lateral CeA cells recruited by systemic IL-1 could regulate the activity of medial CeA cells projecting directly to the BNST. In conclusion, the present findings are consistent with the hypothesis that the BNST acts as a relay between the CeA and PVN, thereby contributing to CeA modulation of hypophysiotropic CRF cell responses to systemic administration of IL-1.