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The pituitary intermediate lobe (IL) contains a single population of cells and has recently been shown to express endothelin (ET)-like peptides. The IL thus provides an excellent in vivo model to study regulation, function and processing of ET in an endocrine cell. The primary aims of the present study were to locate and characterize the precise molecular forms of ET in the ovine IL and determine if levels and/or processing of ET is under dopaminergic or other influences. We have developed a radioimmunoassay (RIA) that detects each form of ET and, when combined with reverse phase-HPLC (RP- HPLC), shows the ovine IL to contain predominantly the ET-1 isoform. In addition, using a specific anti-endothelin antiserum for immunohistochemistry (IHC), we localized ET-1 with alpha-melanocyte stimulating hormone (alpha-MSH) within the melanotroph. The effects of dopamine agonists, antagonists and hypothalamo-pituitary disconnection (HPD) on both tissue levels and processing of ET in the ovine IL were also examined. Normal sheep were treated chronically with haloperidol or bromocriptine to investigate the possibility of dopaminergic regulation of ET in the IL. In the haloperidol- treated group, plasma prolactin levels did not vary significantly from day 0 to day 8, but the bromocriptine treatment reduced prolactin levels (t = 9.4 P <0.01). Neither bromocriptine nor haloperidol, however, affected tissue ET peptide levels or forms. After HPD, the HPLC profile of pooled IL showed that ET-1 levels in the IL are slightly increased with no change in molecular forms.

In the present studies, we characterized the degradation of gonadotropin-releasing hormone (GnRH) by tissues of the ovine hypothalamo-pituitary axis. Membrane and soluble fractions of the medial basal hypothalamus, the pre-optic area, the median eminence and the anterior pituitary demonstrated greater GnRH-degrading activity than either hypophysial-portal or jugular plasma. The primary stable product of the membrane fractions was GnRH1-3, while the major product of the soluble fractions was GnRH1-5; both fragments were generated by plasma. Of all tissue fractions, the highest specific activity was observed in the soluble median eminence. Partial purification and characterization of soluble hypothalamic peptidase activity suggested that GnRH degradation by this tissue occurs via a two-step mechanism involving both post-proline cleaving enzyme and the metalloendopeptidase 3.4.24.15.

The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70-2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosi

The Fort Lawton tunnel project is a 2.67 km long bored tunnel with a finished 3.93 m diameter. It is designed to convey the combined sanitary and storm water run-off from the greater Seattle area. The project commences at the West Treatment plant and extends upgrade, beneath Discovery Park until reaching an existing brick lined sewer, 14.76 m beneath West Commodore Street, adjacent to the Seattle ship canal. The interconnection and bifurcation structure at this location added a unique complexity to the project., The Fort Lawton tunnel project is a 2.67 km long bored tunnel with a finished 3.93 m diameter. It is designed to convey the combined sanitary and storm water run-off from the greater Seattle area. The project commences at the West Treatment plant and extends upgrade, beneath Discovery Park until reaching an existing brick lined sewer, 14.76 m beneath West Commodore Street, adjacent to the Seattle ship canal. The interconnection and bifurcation structure at this location added a unique complexity to the project.

The Syar tunnel reaches from Strawberry Reservoir to Rays Valley to the west to portal at an elevation of 7 375 ft. It is a pressure tunnel with an elevation drop of over 110 ft in its 32 000 ft length. It lies somewhat parallel to the earlier Strawberry tunnel and considerable efforts were made to optimise the planning of the new tunnel to avoid the high water flows and adverse geological conditions that were encountered in the original Strawberry tunnel. The sedimentary rocks consist of shales, limestones, siltstones and claystones., The Syar tunnel reaches from Strawberry Reservoir to Rays Valley to the west to portal at an elevation of 7 375 ft. It is a pressure tunnel with an elevation drop of over 110 ft in its 32 000 ft length. It lies somewhat parallel to the earlier Strawberry tunnel and considerable efforts were made to optimise the planning of the new tunnel to avoid the high water flows and adverse geological conditions that were encountered in the original Strawberry tunnel. The sedimentary rocks consist of shales, limestones, siltstones and claystones.