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SUMMARY

[4-¹⁴C]Cholesterol was cleaved to pregnenolone and progesterone by a washed particulate fraction isolated from involved lymph nodes obtained from a radical mastectomy specimen. Difficulties were experienced in isolating subcellular fractions from primary breast tumours and normal breast tissue. However, an acetone powder fraction, obtained from normal tissue derived from a radical mastectomy specimen, yielded pregnenolone from [4-¹⁴C]cholesterol.

[7α-³H]17α-Hydroxyprogesterone was converted to androstenedione and to 17α,20α-dihydroxypregn-4-en-3-one and its 17α,20β-epimer by homogenates of secondary breast carcinoma tissue and normal breast tissue obtained from radical mastectomy specimens. These results, in conjunction with those obtained previously (Adams & Wong, 1968a), show that human breast carcinoma tissue contains the enzymes necessary to convert cholesterol to androgens and oestrogens.

ABSTRACT

This study aimed to determine the effect of streptozotocin (STZ)-induced diabetes on pulsatile LH secretion in the mature male rat. LH pulse frequency was reduced by 56% and pulse amplitude by 54%, with a consequential decrease of 72% in mean LH levels 8 days after i.v. administration of STZ (55 mg/kg) to castrated Wistar rats compared with castrated non-diabetic controls. Twice daily insulin treatment completely reversed all parameters of pulsatile LH secretion to control values. Food-restricted non-diabetic controls, studied to distinguish the metabolic effect of diabetes from that of concurrent weight loss, demonstrated a 34% reduction in LH pulse frequency but no significant changes in LH pulse amplitude or mean LH levels compared with non-diabetic controls given free access to food. To distinguish whether the decreased LH pulse amplitude in diabetes was due to a reduction in either the quantity of hypothalamic gonadotrophin-releasing hormone (GnRH) released per secretory episode or to decreased pituitary responsiveness to GnRH, the responsiveness of the pituitary to exogenous GnRH (1–1000 ng/kg body weight) was tested in diabetic rats after castration, using a full Latin square experimental design. The net LH response (total area under response curve over 40 min following GnRH) was decreased by 33% (P=0·001) in diabetic compared with control rats. The decreased LH pulse frequency in STZ-induced diabetes therefore suggests that the metabolic effect of diabetes is to decelerate directly the firing rate of the hypothalamic GnRH pulse generator independent of testicular feed-back. These effects were fully reversed by insulin treatment and were only partly due to the associated weight loss. The impaired pituitary responsiveness to GnRH is at least partly involved in the reduction of LH pulse amplitude.

Journal of Endocrinology (1991) 131, 49–55

Abstract

Parturition in the sheep is preceded by an increase in the synthesis of prostaglandins by intrauterine tissues. Prostaglandin G/H synthase (PGHS) is the central enzyme involved in prostanoid production. Its expression is enhanced during late gestation in the ewe. Recent studies have identified two PGHS isozymes, termed PGHS-1 and PGHS-2. The labour-associated expression of the two isozymes of PGHS in the sheep has not been characterized.

This study investigated the changes in expression of immunoreactive PGHS-1 and PGHS-2 in ovine amnion and placenta following glucocorticoid-induced labour. Ewes underwent surgery to implant fetal and maternal vascular cannulae and uterine electromyogram electrodes between 118 and 125 days of gestation. Fetal sheep were administered either the glucocorticoid betamethasone (n=5) or saline (control n=6) by direct transabdominal intrafetal injection. Ewes from the betamethasone-injected group were killed in the first stage of labour as indicated by uterine electromyographic activity. Ewes from the saline-injected group were killed at the same time to obtain age-matched control tissue. The time taken to euthanasia following induced-labour onset in the glucocorticoid-injected animals was 56·6 ± 0·8 h post-injection.

Plasma endocrine profiles in the maternal and fetal circulation following glucocorticoid injection were comparable to those observed following normal spontaneous delivery. At post-mortem, amnion and cotyledons were collected in liquid N₂ and stored at −70 °C. Solubilized tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PGHS-1 and PGHS-2 isozymes. Fetal amnion contained PGHS-1 isozyme at day 133 of gestation, as demonstrated in the saline-injected animals. Slightly higher PGHS-1 immunoreactivity was observed following induced-labour onset, although this did not reach statistical significance (P>0·05). PGHS-2 enzyme was not detectable in amnion. PGHS-2 expression was also not induced following labour onset.

In contrast, PGHS-2 demonstrated enhanced expression following glucocorticoid-induced labour in ovine cotyledon. This tissue contained PGHS-1 enzyme, but immunoreactive levels were minimal and demonstrated limited regulation at labour.

These data suggest that the previously reported rise in placental PG production at term in the sheep is predominantly due to increased expression of the PGHS-2 isozyme. This suggests that PGHS-2 contributes to PG production at term labour in sheep or is induced by the mechanisms controlling ovine parturition. PGHS-1 isozyme is produced constitutively in ovine amnion and may contribute to the gestational increase in PG formation by intrauterine tissues.

Journal of Endocrinology (1996) 151, 125–135

ABSTRACT

Confluent monolayers cultured from the rat cauda epididymidis have been shown to respond to angiotensin I (AI) and angiotensin II (AII) when studied under short-circuit conditions and bathed on both sides with Krebs–Henseleit solution. Both the decapeptide AI and the octapeptide AII elicited transient increases in short-circuit current (SCC) when added to the basolateral as well as to the apical surfaces, with the effect of basolateral application greater than that of apical application. The maximal responses produced by AI and AII were similar with median effective concentrations of 20 to 80 nmol/l. The increase in SCC by AII was dependent upon extracellular Cl⁻ and was inhibited by addition of a Cl⁻ channel blocker, diphenylamine 2-carboxylate, to the apical surface. These patterns of activity suggest that the SCC responses to angiotensins result from electrogenic chloride secretion. Pretreating the monolayers with captopril (100 nmol/l), an angiotensin-converting enzyme (ACE) inhibitor, reduced the response to basolateral application of AI, but completely abolished the response to AI added apically. These results suggest that the response to apical addition of AI was due to conversion of AI to AII which interacts with apical angiotensin receptors. This conversion was mediated by ACE which has been detected in epididymal monolayers. Of the endogenous ACE activity, 86% was found to be inhibited by captopril (100 nmol/l).

Responses of the epididymal monolayers to angiotensins were mediated by specific angiotensin receptors. [Sar¹,Ile⁸]-AII, a specific antagonist of the AII receptor, completely inhibited the responses to AI and All but had no effect on the responses to bradykinin and endothelin. The effects of All were mediated by eicosanoid formation since piroxicam, a cyclooxygenase inhibitor, inhibited the AII-induced increase in SCC. This is the first study to demonstrate an effect of angiotensin on epididymal functions. We propose that angiotensin formed locally in the epididymis may play a role in the regulation of electrolyte and fluid transport.

Journal of Endocrinology (1990) 125, 449–456

ABSTRACT

Some, though not all, previous studies have suggested that the inositol lipid which is hydrolysed during transmembrane signalling in response to receptor activation might be drawn from a metabolically discrete and relatively small hormone-sensitive lipid pool that turns over more rapidly than the bulk of membrane inositol lipid. In order to seek evidence for the existence of this putative hormone-sensitive lipid pool, we have double-labelled cells by growing them for 3 days in a medium containing [¹⁴C]inositol and then supplying them with [³H]inositol for the final 2 h before stimulation. We anticipated that stimulation of these doubly labelled cells might provoke the formation, from the postulated hormone-sensitive pool, of small quantities of relatively ³H-enriched inositol phosphates, and that these could be harvested from cells (provided that the cytosolic inositol monophosphatase and inositol 1,4-bisphosphate/inositol 1,3,4-trisphosphate 1-phosphatase activities are first inhibited by Li⁺). Experiments of this type, using both vasopressin-stimulated WRK1 rat mammary tumour cells and 3T3 mouse fibroblasts stimulated by prostaglandin F_2α, have largely failed to demonstrate the formation of relatively ³H-enriched inositol phosphates. There was a tendency for phosphatidyl-inositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to have slightly higher ³H: ¹⁴C ratios than phosphatidylinositol, but the ³H: ¹⁴C ratios of the inositol phosphates formed in stimulated cells were not substantially greater than the ³H: ¹⁴C ratios of the inositol lipids. We therefore conclude, at least for the two cell lines that we studied, that hormone-stimulated inositol lipid hydrolysis can call, either directly or indirectly, upon the majority of the inositol lipid complement of the stimulated cell.

Journal of Endocrinology (1989) 122, 379–389

Abstract

Sex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79·0, 68·1 and 63·2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and hot-nested PCR analyses indicated that rabbit SHBG is produced from a 1·6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX-1λT for expression of a glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5α-dihydrotestosterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (K_d) for rabbit and human SHBGs produced in E. coli were 11·1 ± 1·1 nm and 2·1 ± 0·6 nm respectively, and rabbit SHBG formed a less stable protein-steroid complex (t_½=5 min) than human SHBG (t_½>60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5′-terminal half of SHBG from one species and 3′-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.

Journal of Endocrinology (1997) 153, 373–384

Seasonal effects were studied on basal levels of hormones in the serum of adult male Sprague–Dawley rats, which were born and raised under rigorously controlled laboratory conditions. Groups of 90-day-old rats were killed at monthly intervals by rapid decapitation. Significant fluctuations were observed throughout the observation period of 19 months in serum levels of TSH, prolactin, androgens, tri-iodothyronine and LH. Minor fluctuations were observed in serum levels of FSH, corticosterone, parathyroid hormone and thyroxine. The results indicate that male laboratory rats exhibit circannual and semi-annual fluctuations in serum levels of several hormones even though the animals were born, raised and maintained in constant laboratory conditions.