bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity of the prolactin receptor in the rat mammary gland and liver during various physiological states
Authors: Guillaumot P , Cohen H
Published: December 2008

This work was undertaken to determine variations in the 125I-labelled ovine prolactin (oPRL) binding in rat liver and mammary gland membranes, and to study the molecular forms of prolactin receptor in different physiological situations. Prolactin binding was determined using 125I-labelled oPRL in the 100 000 g pellet. 125I-Labelled oPRL was cross-linked to receptors in membranes from rat liver and mammary gland and subjected to SDS-PAGE under reducing conditions, followed by autoradiography of dried gels.

In the mammary gland, the specific binding of oPRL to membranes, expressed as mean ± s.e.m. fmol/mg protein increased from 1·36 ±0·24 on the day of dioestrus to 3·26 ±0·60 on the day of oestrus. It remained very low during pregnancy but increased during lactation to reach 4·78 ±0·99. In the liver, the specific binding of oPRL to membranes was higher than in the mammary gland on the days of dioestrus 1, dioestrus 2 and oestrus, but not on the day of pro-oestrus. It increased until day 14 of pregnancy when the specific binding of 125I-labelled oPRL was 17·01 ±0·30.

Cross-linking revealed different molecular forms in the mammary glands and the liver. In the mammary gland we observed four prolactin-binding forms of 80, 50, 40 and 16 kDa, all of which were specific for prolactin. The 80, 50 and 40 kDa forms were also able to bind to a Concanavalin A–Sepharose column, indicating that these binding forms were glycosylated while the smaller one (16 kDa) appeared to be unglycosylated. The 40 kDa prolactin receptor was seen at all stages studied: the oestrous cycle (dioestrus, pro-oestrus and oestrus), pregnancy (days 8, 14 and 22) and lactation. The 50 kDa form was observed in the mammary gland during the day of pro-oestrus and gestation. It was also observed in ovariectomized rats treated with oestradiol (OE2), suggesting that OE2 could be one of the factors involved in the induction of this receptor form. The 16 kDa form of the receptor was found in the mammary gland only during lactation. This form, while unglycosylated, bound specifically to prolactin, suggesting that it might play a specific role during lactation.

In the liver, two forms were shown, a major one of 40 kDa and a minor one of 80 kDa. No variation in receptor molecular weight was found in the liver during the physiological stages studied.

Journal of Endocrinology (1994) 141, 271–278
Putative aromatase inhibitor induces male sex determination in a female unisexual lizard and in a turtle with temperature-dependent sex determination
Authors: Wibbels T , Crews D
Published: December 2008

Treatment of developing embryos of two diverse species of reptiles with fadrozole (a potent and specific nonsteroidal inhibitor of aromatase activity in mammals) resulted in the induction of male sex determination. In the first experiment, males were produced in an all-female parthenogenic species of lizard (Cnemidophorus uniparens). In the second experiment, male sex determination was induced in a turtle (Trachemys scripta) with temperature-dependent sex determination. The results support the hypothesis that the endogenous production of oestrogen may represent a pivotal step in the sex determination cascade of reptiles. Further, the production of male C uniparens indicates that the genes required for male sexual differentiation have not been lost in this parthenogenic lizard.

Journal of Endocrinology (1994) 141, 295–299
Net uptakes of oestradiol-17β and progesterone across the portal-drained viscera and the liver of ewes
Authors: Freetly H C , Ferrell C L
Published: December 2008

The objective of this study was to determine whether circulating concentrations or prior exposure to oestradiol-17β (OE2) and progesterone affected their uptake by splanchnic tissues. Catheters were surgically placed in the portal vein, a branch of the hepatic vein, a mesenteric vein and the abdominal aorta of three multiparous ovariectomized Dorset ewes. Blood and plasma flow across the portal-drained viscera (PDV) and the liver, and net uptake of OE2 and O2 consumption in these same tissues were determined in ovariectomized ewes (control), during OE2 infusion into the jugular vein, 7 days after an OE2 implant had been given, and during OE2 infusion into the jugular vein 7 days after an OE2 implant. The above treatments were repeated for progesterone. Plasma flows across visceral organs were determined by marker dilution (para-aminohippuric acid), and OE2 and progesterone concentrations were determined by radioimmunoassay. During the infusion with OE2, OE2 arterial concentration (mean ± s.d.) was 346 ± 199 pg/ml, PDV net uptake was 9·7±5·6 μg OE2/h and hepatic net uptake was 15·5 ± 9·5 μg OE2/h. Hepatic uptake was 82% of the jugular OE2 infusion rate. Blood flow and oxygen consumption by hepatic tissue increased when ewes were exposed to an OE2 implant for 7 days. During the infusion with progesterone, progesterone arterial concentration (mean ± s.d.) was 8·8 ±3·4 ng/ml, PDV net uptake was 220 ± 118 μg progesterone/h and hepatic net uptake was 238 ± 52 μg progesterone/h. Hepatic net uptake was 23% of the progesterone jugular infusion rate.

Journal of Endocrinology (1994) 141, 353–358
Blockade of the neonatal rise in testosterone by a gonadotrophin-releasing hormone antagonist: effects on timing of puberty and sexual behaviour in the male marmoset monkey
Authors: Lunn S F , Recio R , Morris K , Fraser H M
Published: December 2008

In primates, plasma testosterone concentrations are elevated for some 3 months from birth. The function of this rise is uncertain, but studies in rats suggest that its prevention by castration or administration of gonadotrophin hormone-releasing hormone (GnRH) analogues has effects on development and expression of social and sexual behaviours, and adverse long-term effects on fertility. The consequences of suppression of this rise in testosterone by treatment with the GnRH antagonist antide have been investigated in the marmoset monkey. Eight sets of male:male twins were used, one of each set receiving s.c. injections of antide (10 mg/kg), on days 0, 3 and 7, then weekly from birth to 98 days of age, with the twin receiving vehicle only. Plasma samples were taken at weekly intervals for the determination of testosterone concentrations from birth until 2 years of age. Treatment with antide completely abolished the neonatal rise in testosterone seen in control animals. The timing of the onset of the pubertal testosterone rise was not significantly affected by treatment; however, the subsequent pattern of circulating testosterone showed a tendency to decreased plasma concentrations in the neonatally treated group from weeks 25 to 42, relative to controls, and this difference was significant between 43 and 70 weeks. This was associated with a similar depression in bioactive LH concentrations around this time. Thereafter, the testosterone concentrations were similar between treated and control groups. There was no effect of treatment on growth, based on sequential body weight data. At 20 months the animals underwent behaviour tests with ovariectomized females. During these encounters, males showed the full panoply of normal sexual behaviours, with response to female proceptivity, tongue flicking, sustainable penile erections, mounts with pelvic thrusting, intromissions and intravaginal ejaculations. No significant differences were observed for any of these parameters between the control and treated animals. No changes in aggressive behaviours between groups were evident. These results show that blockade of the postnatal rise in testosterone in the male marmoset monkey may result in attenuation of the pubertal rise in testosterone but is without major effect on adult basal testosterone concentrations or reproductive behaviour.

Journal of Endocrinology (1994) 141, 439–447
A monoclonal antibody to C-type natriuretic peptide – preparation and application to radioimmunoassay and neutralization experiment
Authors: Hama N , Itoh H , Suga S , Komatsu Y , Yoshimasa T , Nakao K
Published: December 2008

C-type natriuretic peptide (CNP), the third member of natriuretic peptides, has recently been discovered from the porcine brain. Using a polyclonal antiserum to CNP, we demonstrated that CNP-like immunoreactivity (CNP-LI) is present mainly in the central nervous system. Recently, however, we have discovered the production and secretion of CNP in vascular endothelial cells. These observations suggested that CNP may act not only as a neuropeptide but also as a local regulator of vascular tone or growth. In order to further clarify the pathophysiological significance of CNP, we aimed at the preparation of a monoclonal antibody to CNP.

A monoclonal antibody to CNP, KY-CNP-I, has been produced. This monoclonal antibody belongs to the immunogloblin G1 subclass and has high affinity for CNP. Using this monoclonal antibody, we established a specific radioimmunoassay (RIA) for CNP. The RIA detected CNP-LI in rat brain extracts and culture media conditioned with bovine endothelial cells. In addition, the pretreatment of cultured aortic smooth muscle cells with KY-CNP-I attenuated cyclic GMP production induced by CNP in vitro. The preadministration of KY-CNP-I to rats also attenuated plasma cyclic GMP increase after intravenous injection of CNP in vivo.

These results indicate that this monoclonal antibody is a useful tool to clarify the pathophysiological role of CNP as a neuropeptide and as a local vascular regulator.

Journal of Endocrinology (1994) 141, 473–479
Cellular mechanisms mediating the stimulation of ovine endometrial secretion of prostaglandin F2α in response to oxytocin: role of phospholipase A2
Authors: Lee J-S , Silvia W J
Published: December 2008

Four experiments were conducted to determine whether phospholipase (PL) A2 mediates the stimulatory effect of oxytocin on the release of prostaglandin (PG) F2α from ovine endometrial tissue. Caruncular endometrial tissue was collected from ovariectomized ewes on the day after a steroid replacement protocol had been completed. The replacement protocol consisted of progesterone for 10 days (12 mg/day) followed by oestradiol on days 10 and 11 (100 μg/day) and had been shown previously to provide endometrial tissue that would release PGF2α in response to oxytocin in vitro. In experiment 1, oxytocin (10−7 m) and melittin (1·76 × 10−6 m; a stimulator of PLA2) stimulated release of PGF2α from tissue explants (P<0·05). Aristolochic acid (10−4 m; an inhibitor of PLA2) decreased oxytocin-and melittin-induced release of PGF2α by 77% and 71% respectively (P<0·05). Experiment 2 was conducted to establish the minimum inhibitory dose of aristolochic acid. Basal release of PGF2α was inhibited at 10−5 m aristolochic acid, but 10−4 m was required to block the stimulatory effect of oxytocin. Experiment 3 was carried out to identify the precise intracellular locus at which aristolochic acid was exerting its effect. Oxytocin (10−7 m), AlF4− (5 × 10−2m NaF, 10−5m AlCl3), melittin (1·76 × 10−6 m) and arachidonic acid (AA; 20 μg/ml) stimulated release of PGF2α (P<0·05). Aristolochic acid (10−4 m) blocked the release of PGF2α stimulated by oxytocin, AlF4− or melittin by >80% (P<0·05). However, aristolochic acid reduced AA-induced release by only 22% (P=0·09). Thus, aristolochic acid blocked responsiveness to stimulatory agents that act on, or proximal to, PLA2 but not responsiveness to AA whose stimulatory effect is exerted distal to PLA2. Experiment 4 was intended to establish the specificity of the inhibitory effect of aristolochic acid. This was accomplished by examining the effect of aristolochic acid on the ability of oxytocin to stimulate activity of PLC. Aristolochic acid (10−4 m) increased basal PLC activity by 29% (P<0·05) but had no effect on the activity induced by oxytocin (P>0·1). Thus, aristolochic acid blocked the ability of oxytocin to stimulate PGF2α release (experiments 1–3) but not its ability to stimulate the activity of PLC (experiment 4). The effects of melittin and aristolochic acid are consistent with the hypothesis that the stimulatory effect of oxytocin on PGF2α release from ovine endometrial tissue is mediated through PLA2.

Journal of Endocrinology (1994) 141, 491–496
IMMUNOFLUORESCENT EVIDENCE FOR THE ABUNDANCE OF FOCAL ADHESION KINASE IN THE HUMAN AND AVIAN OSTEOCLASTS AND ITS DOWN REGULATION BY CALCITONIN
Authors: Berry V , Rathod H. , Pulman L. B. , Datta H. K.
Published: December 2008

We show here that osteoclasts possess an abundant level of focal adhesion kinase, a novel cytosolic tyrosine kinase with unique structural features that may play an important role in the action of pp60c-src, cell surface integrins, and hormonal peptides. The presence of focal adhesion kinase in the bone cell osteoclast was determined using monoclonal antibodies to the kinase by employing immunofluorescent staining. The expression of focal adhesion kinase in the osteoclast was markedly suppressed following exposure to calcitonin. However, calcitonin-induced down regulation of the kinase was apparent only following a prolonged exposure. Our hypothesis that focal adhesion kinase is maximally expressed in the osteoclasts was confirmed when the transfection of avian osteoclasts and fibroblasts, with v-src containing plasmid pATV-8, induced increased expression of the kinase in the fibroblasts but did not alter the expression level of FAK in the osteoclasts.
Effects of close-arterial (external pudic) infusion of insulin-like growth factor-II on milk yield and mammary blood flow in lactating goats
Authors: Prosser C G , Davis S R , Farr V C , Moore L G , Gluckman P D
Published: December 2008

Five lactating goats were infused, via an external pudic arterial catheter, directly into the mammary gland with 0·9% (w/v) NaCl (20 ml/h), recombinant human insulin-like growth factor-I (IGF-I; 80 nmol/h), recombinant human IGF-II (133 nmol/h) or IGF-I and IGF-II combined. The infusion was for 6 h and milk yield was determined every 2 h. The ratio of milk yield in the infused relative to the non-infused gland was changed only slightly by saline (2%), but increased to 9% (P<0·05) in response to IGF-I and 8% (P<0·05) in response to IGF-II. When combined, both peptides increased this ratio by 6%. These effects were elicited within 2–4 h of the beginning of infusion. Mammary blood flow increased 50–80% (P<0·05) during all IGF infusions, but only 28% during saline treatment. Plasma insulin decreased 50% (P<0·01) during the infusion of IGF-I alone or in combination with IGF-II and 25% in response to IGF-II alone. Whereas plasma glucose increased by approximately 10% during infusion of IGF-I alone or with IGF-II, it was not altered by infusion of IGF-II only.

The rapidity and unilateral nature of the milk-yield response to IGF-I and IGF-II is consistent with their acting directly on mammary tissue itself. Thus, the present results demonstrate similar local and systemic actions induced by intramammary infusion of IGF-II and IGF-I, although the magnitude of the response to IGF-II tends to be less than that to IGF-I.

Journal of Endocrinology (1994) 142, 93–99
Effects of the opioid antagonist naloxone on release of luteinizing hormone in mares during the anovulatory season
Authors: Aurich C , Schlote S , Hoppen H-O , Klug E , Hoppe H , Aurich J E
Published: December 2008

To investigate an involvement of endogenous opioids in the regulation of circannual changes in reproductive activity, effects of the opioid antagonist naloxone on the concentration of immunoreactive and bioactive luteinizing hormone (LH) in plasma were measured in mares during the anovulatory season. Naloxone (0·5 mg/kg i.v.) caused a significant increase (P<0·05) in immunoreactive as well as bioactive LH concentration in plasma. The amplitude of the increase in LH concentrations measured with an in vitro bioassay was more pronounced than the amplitude of the increase in LH secretion determined by radioimmunoassay. This indicates that although in seasonal anovulatory mares the bioactivity of LH in plasma is low, highly bioactive LH is present in the anterior pituitary and can be released by naloxone. The LH response to naloxone did not depend on the degree of ovarian follicular activity. It can be concluded that a tonic opioid inhibition of LH release is present in mares during at least part of the anovulatory season and that endogenous opioids seem to be involved in the regulation of seasonal reproductive activity in the horse. In contrast to the situation during the breeding season, the opioid systems regulating LH release are activated independently of luteal progesterone.

Journal of Endocrinology (1994) 142, 139–144
Developmental changes in chicken and turkey insulin-like growth factor-I (IGF-I) studied with a homologous radioimmunoassay for chicken IGF-I
Authors: McMurtry J P , Francis G L , Upton F Z , Rosselot G , Brocht D M
Published: December 2008

The development of a homologous radioimmunoassay (RIA) for chicken insulin-like growth factor-I (cIGF-I) and its use to investigate the developmental changes in IGF-I in the chicken and turkey is described. A doubleantibody RIA has been developed using recombinantly derived cIGF-I as antigen, radiolabelled tracer and standard. The resulting immunoassay has a minimum detection limit of 0·035 ng and effective dose of 2·5 ng. Dose–response curves of chicken and turkey plasma and tissue extracts were parallel with cIGF-I standard. The antiserum is specific for IGF-I as no cross-reactivity with chicken IGF-II, insulin, glucagon, gastrin or avian pancreatic polypeptide was observed. We have also established that acid/ethanol extraction of chicken and turkey plasma reduced possible interference of IGF-binding proteins (IGFBPs) in the RIA. Comparison of IGF-I immunoactivity in unextracted and acid/ethanol-extracted samples following gel filtration under acidic and neutral conditions indicates that the cIGFBPs may be acid-labile. Analyses of samples from growing chickens and turkeys using the homologous avian reagents revealed higher IGF-I concentrations than if the IGF were quantified using heterologous mammalian-derived reagents. A similar pattern was observed when tissue extracts were assayed for IGF-I content. The application of the homologous RIA to monitor blood and tissue IGF-I levels during embryonic development and posthatch growth in avian species will provide more accurate comparisons of results from studies on the role of IGF-I in growth and metabolism of domestic birds.

Journal of Endocrinology (1994) 142, 225–234
The regulation of plasma relaxin levels during human pregnancy
Authors: Johnson M R , Abbas A A , Allman A C J , Nicolaides K H , Lightman S L
Published: December 2008

The factors that determine the circulating levels of relaxin during pregnancy have been investigated by comparing the plasma levels of relaxin throughout pregnancy in women who became pregnant spontaneously (singleton, n=240) or following superovulation (singleton and multifetal pregnancies (two to ten conceptuses), n=83). Some of the women with multifetal pregnancies underwent selective fetal reduction to twin pregnancies. Relaxin levels were higher at 7–34 weeks of gestation in singleton pregnancies achieved following superovulation when compared with levels in spontaneously conceived singleton pregnancies (P<0·05–0·001). In samples obtained between 10 and 12 weeks of gestation (before fetal reduction for the multifetal pregnancies), plasma relaxin levels correlated with fetal number (r=0·526, P=0·0001). Reduction in fetal number to a twin pregnancy did not alter relaxin levels. These data suggest that the circulating levels of relaxin throughout pregnancy are determined during the cycle of conception by gonadotrophin stimulation, and within the first 10 weeks of pregnancy by the luteotrophic stimulus from the conceptus. Furthermore, once corpus luteum synthesis of relaxin is established, then reduction in the luteotrophic stimulus does not appear to affect it.

Journal of Endocrinology (1994) 142, 261–265
Biological effects of prostate specific antigen as an insulin-like growth factor binding protein-3 protease
Authors: Cohen P , Peehl D M , Graves H C B , Rosenfeld R G
Published: December 2008

Prostate specific antigen (PSA) is an insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) protease found in seminal plasma and produced by prostatic epithelial cells (PC-E) in vivo. We examined the effects of PSA-proteolysis of IGFBP-3 on the affinity of IGFBP-3 fragments for IGFs and on the mitogenic action of IGFs on PC-E. Recombinant human IGFBP-3 was cleaved by PSA, then incubated with 125I-IGF-I or -II in the presence of varying concentrations of unlabelled peptides, and then cross-linking electrophoresis and densitometric analysis were performed. While the affinity of IGF-II for the PSA-generated IGFBP-3 fragments fell slightly compared to intact IGFBP-3, the affinity of the PSA-generated IGFBP-3 fragments for IGF-I fell by ten fold. The addition of IGF-I or -II to PC-E in serum-free culture conditions resulted in a two-fold stimulation of cell number compared to control. The presence of IGFBP-3 in the media blocked the IGF-induced stimulation, but had no independent effect in the absence of IGFs. When PSA was added to PC-E cultures to which both IGF-I or -II and IGFBP-3 were added, the inhibitory effects of IGFBP-3 on IGF mitogenesis were reversed. We conclude that PSA decreases the affinity of IGFBP-3 for IGF and can potentiate IGF action in the presence of inhibitory IGFBP-3. This phenomenon may contribute to normal and malignant prostate growth.

Journal of Endocrinology (1994) 142, 407–415
Uncoupling between proliferation and differentiation of ovine granulosa cells in vitro
Authors: Monniaux D , Pisselet C , Fontaine J
Published: December 2008

Granulosa cells of ovarian follicles both proliferate and undergo differentiation. In vivo, an inverse relationship between proliferation and steroidogenesis is observed. However, both processes can be enhanced by insulin-like growth factor-I (IGF-I) in vitro. Studies were undertaken in the ewe to understand the mechanisms controlling the balance between proliferation and differentiation in cultured granulosa cells from antral follicles better. For this purpose, granulosa cells from ovine small follicles (1–3 mm in diameter) and large follicles (5–7 mm in diameter) were compared for progesterone secretion, cytochrome P450 side-chain cleavage (P450scc) expression and their proportions of non-proliferating (G0) cells, in response to IGF-I and FSH stimulation in vitro.

IGF-I mainly enhanced the proliferation of granulosa cells from small follicles but it strongly increased progesterone secretion and P450scc expression in granulosa cells from large follicles, in synergy with FSH. Blocking granulosa cell proliferation by the administration of colcemid or aphidicolin had no effect or a weak stimulating effect on progesterone secretion. At the beginning of the culture period, the proportion of non-proliferating cells, estimated by continuous [3H]thymidine labelling experiments, was clearly higher in large than in small follicles (91% vs 30%, P<0·001). For both cell types, treatment with IGF-I in vitro reduced the proportion of non-proliferating cells at 72 h of culture (40% vs 70% respectively in IGF-I-stimulated and unstimulated cells from large follicles, P<0·001, and 17% vs 30% respectively in IGF-I-stimulated and unstimulated cells from small follicles, P<0·001). Treatment with FSH had no effect on the proportion of non-proliferating cells. As revealed by immunohistochemistry experiments, IGF-I, in synergy with FSH, clearly increased the percentage of cells expressing P450scc enzyme and the intensity of staining in granulosa cells from large follicles. Unexpectedly, heavily stained cells in mitosis were observed in IGF-I-stimulated cells from large follicles after 96 h of culture, suggesting that dividing cells might also produce progesterone.

Overall, these results support the hypothesis that the growth-promoting and the cytodifferentiative effects of IGF-I are clearly distinct. Moreover, they suggest that uncoupling between proliferation and steroidogenesis may occur in cultured ovine granulosa cells. The loss of proliferative activity accompanying terminal follicular growth in vivo could be reversed in vitro. During terminal follicular growth in vivo, the existence of an active mechanism inhibiting granulosa cell proliferation, and unrelated to terminal differentiation, is therefore strongly suspected.

Journal of Endocrinology (1994) 142, 497–510
Lactogenic actions of different growth hormone preparations in pregnant and lactating rats
Authors: Carón R W , Jahn G A , Deis R P
Published: December 2008

We studied the capacity of different GH preparations, natural human (h)GH, recombinant hGH (rhGH), rat (r)GH, ovine (o)GH, bovine (b)GH and porcine (p)GH, and ovine prolactin (oPRL), to stimulate lactogenesis in ovario-hysterectomized pregnant rats or intact lactating rats treated with bromocriptine (BC). Ovariohysterectomy (OVX-HYS) performed at 0800 h on day 19 of pregnancy induced lactogenesis, i.e. increases in mammary casein and lactose and positive response to the oxytocin test, 28 h later. Lactogenesis was prevented by treatment with BC (1·5 mg/kg) immediately after surgery (OVX-HYS-BC). The hormones were given at doses of 0·25 or 0·5 mg/rat (except rhGH given only at 0·5 mg/rat) at 1200 and 2000 h on day 19. Casein was increased by both doses of oPRL and hGH, rhGH and 0·25 mg oGH, and lactose by both doses of oPRL, rhGH and 0·25 mg rGH. The other GH preparations had no effect. The oxytocin test demonstrated the presence of milk in the mammary tissues of the OVX-HYS rats and in the OVX-HYS-BC plus oPRL (0·25 and 0·5 mg) or rhGH-treated groups.

Injection of BC to pregnant rats at 2000 h on day 20 and at 0800 h on day 21 decreased litter growth on the first 4 days postpartum. Two-thirds of the litters resumed growth after day 4, indicating the recuperation of milk production, while the rest never recuperated. Serum prolactin in BC-treated rats was reduced until day 4 postpartum. On day 6 the rats which had recuperated had normal values, while those which had still not recuperated had lower values. BC-treated rats were injected s.c. with 0·25 mg each of oPRL, hGH, rGH, oGH, bGH or pGH, or 0·25 or 0·5 mg rhGH/rat, immediately postpartum and 12, 24 and 36 h later. hGH and 0·5 mg rhGH induced levels of milk production similar to controls except on day 3. oPRL and rhGH (0·25 mg), induced a partial reversion of the effect of BC. rGH and oGH had a slight effect on days 1 and 2 and all the litters resumed growth on day 7. In contrast, pGH and bGH were inactive.

The affinity of hGH for the prolactin receptor, measured as displacement of 125I-labelled oPRL binding to crude liver membranes, was comparable with that of oPRL. While rhGH was ten times less active than oPRL, rPRL was 100 times lower and all the other GH preparations had at least 104 times lower capacity to displace 125I-labelled oPRL.

These results indicate that both natural and recombinant hGHs are potent inductors of milk synthesis in pregnant or lactating rats, most probably due to their actions at the level of the prolactin receptor. rGH and oGH have a partial action, while pGH and bGH seem to be inactive. The actions of non-human GHs may be explained by their somatogenic properties exclusively, and indicate that GH may play a role in the optimization of milk production during lactation and an accessory role in the induction of lactogenesis in pregnant rats.

Journal of Endocrinology (1994) 142, 535–545
Dexamethasone potentiates the stimulatory effect of insulin-like growth factor-I on collagen production in cultured human fibroblasts
Authors: Bird J L E , Tyler J A
Published: December 2008

We examined the effects of insulin-like growth factor-I (IGF-I) and dexamethasone on the production of collagen by cultures of human infant foreskin fibroblasts, and the interaction between these two factors. IGF-I at 500 ng/ml maximally increased collagen accumulation fourfold. Collagen was increased twofold relative to total protein production. Dexamethasone at a concentration of 1 μmol/l reduced collagen production by between 25% and 40% in unstimulated cells and those cultured with up to 100 ng IGF-I/ml. However, dexamethasone did potentiate collagen production in cells stimulated with 250 ng IGF-I/ml. This potentiation was independent of any effects of IGF-I or dexamethasone on prostaglandin (PG)E2 production. Transforming growth factor-β (TGF-β) is also a potent stimulator of collagen formation. However, no potentiation of TGF-β-stimulated collagen production by dexamethasone was apparent. The mechanism by which dexamethasone potentiates IGF-I-stimulated collagen production was investigated. Dexamethasone treatment increased IGF-I binding to the type 1 IGF receptor without altering the binding affinity. Dexamethasone also attenuated the secretion of IGF-binding proteins by IGF-I-maintained cells.

Journal of Endocrinology (1994) 142, 571–579
Tumour necrosis factor-α differentially alters progesterone and prostaglandin F2α production by porcine luteal cells
Authors: Richards R G , Almond G W
Published: December 2008

This study examined the effects of tumour necrosis factor-α (TNFα) on basal and stimulated progesterone secretion, as well as prostaglandin F2α production, by small, large and mixed porcine luteal cells and assessed the action of TNFα in the presence and absence of indomethacin. Corpora lutea were isolated from gilts on days 8–9 of the oestrous cycle and enzymatically dissociated. Luteal cells were either subjected to elutriation to isolate small and large cells or were separated from erythrocytes by a polysucrose gradient to serve as the mixed luteal cell group. Then 24-well culture plates were seeded with 150 000 small, 30 000 large and mixed (30 000 large+100 000-250 000 small) luteal cells suspended in 1 ml medium 199 media supplemented with 5 μg insulin/ml, 40 ng cortisol/ml and 50 μg low-density lipoproteins/ml. Cells were cultured for up to 24 h in a humidified incubator at 37 °C with 5% CO2 in air. TNFα time- and dose-dependently suppressed (P<0·05) LH-induced, but not basal, progesterone secretion by small luteal cells. Moreover, TNFα inhibited (P<0·05) forskolin-mediated, but not cyclic AMP-mediated, progesterone secretion by small luteal cells. The LH-stimulated progesterone secretion by small luteal cells was not affected by TNFα in the presence of indomethacin. Progesterone secretion by large and mixed luteal cells was not affected by TNFα. Prostaglandin F2α production by small and mixed, but not large, luteal cells was enhanced (P<0·05) by TNFα. These data demonstrated that TNFα acts primarily on a target cell(s) in the small cell fraction, and the cytokine-induced inhibition of LH-stimulated progesterone secretion by small cells was mediated by prostaglandin F2α and involved a site(s) proximal, but not distal, to cyclic AMP generation.

Journal of Endocrinology (1994) 143, 75–83
Consequences of thyroid hormone deficiency induced by the specific ablation of thyroid follicle cells in adult transgenic mice
Authors: Wallace H , McLaren K , Al-Shawi R , Bishop J O
Published: December 2008

The herpes simplex type 1 virus thymidine kinase (HSV1-TK) reporter gene was coupled to a bovine thyroglobulin promoter (TG-tk construct). Within the thyroid glands of transgenic mice expression was confined to thyroid follicle cells. Infusion of Ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) to 8 to 12 week transgenic females led to the complete loss of thyroid HSV1-TK activity (at 3 to 4 days) and thyroid follicles (between 7 and 14 days). During the first 5 days of treatment a single reciprocal oscillation in circulating thyroxine (T4) and TSH levels occurred. By 14 days the circulating triiodothyronine (T3) and T4 levels of all treated animals were below the detection limits of the assays, while TSH levels were elevated ten-fold and continued to increase thereafter. During 14 days of treatment the thyroids regressed, protein content fell by 80–90% and the C cells, normally dispersed within the central region of each gland, came together in aggregates. Pituitary GH levels in females rose and fell back to normal within 14 days and between 14 and 28 days fell to a level comparable with that of GH-deficient lit/lit mice. The levels of hepatic GH receptor mRNA and the predominant 6·6 kb T3 receptor mRNA were unaffected by thyrocyte ablation. Thyrocyte ablation had no effect on the level of prolactin (Prl) receptor mRNA in females, but increased Prl receptor mRNA levels in males and eliminated group 1 major urinary protein (MUP) mRNA in females. T4 replacement reversed the effects of thyrocyte ablation on MUP mRNA in females and on Prl receptor mRNA in males. Despite the many physiological changes induced by thyrocyte ablation, ablated mice have been maintained for up to 1 year without thyroid hormone supplementation. T4-deficient females were normally fertile and carried pups to term. Although transgenic males expressed HSV1-TK ectopically in spermatids and spermatozoa at levels similar to thyrocyte levels, a rate of Ganciclovir infusion which successfully ablated the thyrocytes did not affect the testis. As an alternative to infusion by minipump, thyrocyte ablation could be achieved by 6 twice-daily injections of Ganciclovir, at a level of 112 μg Ganciclovir/g body weight per day, and fetuses in utero could be thyrocyte ablated by administering 50 or 15 μg/g body weight per day to pregnant females between days 14 and 18 of gestation. These data demonstrate the potential value of transgenic thyrocyte ablation in the study of the effects of thyroid hormone deprivation.

Journal of Endocrinology (1994) 143, 107–120
Expression of inhibin α,βA and βB messenger ribonucleic acids in the normal human ovary and in polycystic ovarian syndrome
Authors: Jaatinen T-A , Penttilä T-L , Kaipia A , Ekfors T , Parvinen M , Toppari J
Published: December 2008

We studied the cellular distribution of inhibin α, βA and βB mRNAs in the normal human ovary and in polycystic ovarian syndrome (PCOS) by in situ hybridization. Our results show that human granulosa cells express inhibin α, βA and βB subunit mRNAs, and theca cells express inhibin α and βA subunit mRNAs. The co-localization of α and βA mRNAs in theca cells supports the hypothesis that inhibin also has an autocrine function in these cells. We did not detect any inhibin subunit mRNA in the granulosa cells of atretic follicles, while theca cells also expressed α subunit mRNA in those follicles. The present findings suggest that the expression of inhibin subunits is regulated differently in human follicular granulosa and theca cells. It has been speculated that inhibin may be involved in the development of PCOS. Our results show that the cellular localization of inhibin subunit mRNAs is not disturbed in PCOS ovaries.

Journal of Endocrinology (1994) 143, 127–137
Expression of 17β-hydroxysteroid dehydrogenase in human granulosa cells: correlation with follicular size, cytochrome P450 aromatase activity and oestradiol production
Authors: Ghersevich S A , Poutanen M H , Martikainen H K , Vihko R K
Published: December 2008

The aim of this study was to examine the expression and regulation of type 1 17β-hydroxysteroid dehydrogenase (type 1 17-HSD) enzyme protein and mRNA, and 17-HSD activity in human granulosa cells. The cells were obtained from patients taking part in an in vitro fertilization programme. The cells from each patient were divided into two groups: cells obtained from preovulatory follicles (LGC=granulosa cells from large follicles ≥ 18 mm in diameter), and cells from other visible follicles (SGC=granulosa cells from small follicles, less than 15 mm in diameter). The identity of 17-HSD enzyme protein expressed in human granulosa cells with placental cytosolic 17-HSD (type 1 17-HSD) was assessed by immunoblot analysis using polyclonal antibodies, and the enzyme was immunolocalized in the cytoplasm of granulosa cells. Type 1 17-HSD protein concentration, 17-HSD and cytochrome P450 aromatase (P450arom) activities and oestradiol (OE2) production in cells from LGC were significantly lower than the corresponding values obtained in SGC in the same patient (paired t-test). The type 1 17-HSD protein concentration, 17-HSD activity and P450arom activity were 140±16% (mean±s.e.m.), 121±22% and 113±26% higher in cells from SGC, which was also reflected in a 70±12% higher OE2 production in these cells. In freshly isolated cells from LGC or SGC, a high correlation between 17-HSD and P450arom activities was observed (r=0·93, P<0·001). In long-term cultured cells, type 1 17-HSD was stably expressed at least until day 9, while P450arom expression decreased. In addition, treatments with gonadotrophins did not affect type 1 17-HSD protein concentration and 17-HSD activity. In contrast to this, both P450arom activity and OE2 production were significantly increased (P<0·05). The data, therefore, suggest that type 1 17-HSD and P450arom are expressed in parallel during the latest stages of follicular maturation but, in cultured granulosa-luteal cells, the enzymes are regulated by distinct mechanisms.

Journal of Endocrinology (1994) 143, 139–150
Liposomal delivery of antioxidant enzymes protects against hydrogen peroxide- but not interleukin-1β-induced inhibition of glucose metabolism in rat pancreatic islets
Authors: Welsh N , Margulis B , Bendtzen K , Sandler S
Published: December 2008

The aim of the present investigation was to evaluate the putative involvement of oxygen free radicals in interleukin-1β (IL-1β)-induced suppression of islet glucose oxidation. Isolated adult rat pancreatic islets were exposed for 1 h to liposomally encapsulated superoxide dismutase (SOD; 10 mg/ml), catalase (CAT; 10 mg/ml) and glutathione peroxidase (GPX; 5 mg/ml), after which IL-1β (25 U/ml) or hydrogen peroxide (H2O2; 0·1 mm) was added, and the incubation was continued overnight. The following day, samples were taken from the incubation media for nitrite determinations, and islet glucose oxidation rates were measured. The CAT activity increased fourfold after addition of CAT-containing liposomes. It was found that IL-1β induced a marked increase in islet nitrite production, as an index of nitric oxide formation, and that this was paralleled by a decrease in islet glucose oxidation rates. H2O2-treated islets exhibited a modest decrease in glucose oxidation rates and a minor increase in the release of nitrite to the media. Treatment of islets with liposomes containing the antioxidant enzymes SOD, CAT and GPX, either alone or in combination, did not decrease the effect of IL-1β. However, the H2O2-induced decrease in glucose oxidation rates was counteracted by the combination of the antioxidants. It was concluded that, provided the intracellular delivery of the antioxidant enzymes to the islet cells was effective, oxygen free radicals probably do not play a decisive role in IL-1β suppression of islet glucose metabolism.

Journal of Endocrinology (1994) 143, 151–156
Androgen and oestrogen responsiveness of stromal cells derived from the human hyperplastic prostate: oestrogen regulation of the androgen receptor
Authors: Collins A T , Zhiming B , Gilmore K , Neal D E
Published: December 2008

Stromal cells derived from collagenase-digested benign hyperplastic adult prostates were isolated and grown in culture. Androgen and oestrogen receptor status were determined and growth in response to mibolerone (a synthetic androgen) and oestradiol-17β was measured. In addition, the ability of oestrogens to regulate the androgen receptor in stromal cells was investigated.

[3H] Thymidine incorporation into DNA was stimulated by mibolerone in primary and secondary cultures, but sensitivity was lost with subsequent passages. Androgen stimulation of [3H] thymidine incorporation was consistently inhibited by the anti-androgen cyproterone acetate. Oestradiol-17β also stimulated [3H]thymidine incorporation into DNA, and this effect was inhibited by the anti-oestrogen tamoxifen. Sensitivity to oestradiol was lost with subsequent passages. A combination of mibolerone and oestradiol was not synergistic in increasing [3H] thymidine incorporation into DNA, but maximal stimulation occurred at 100-fold lower concentrations of mibolerone and oestradiol when the two hormones were applied in combination. Specific high-affinity [3H] mibolerone- and [3H]oestradiol-binding sites were demonstrated by radioligand binding in intact cells. The affinity for oestradiol binding to its receptor exceeded that quantified for mibolerone binding to the androgen receptor, whilst the number of oestradiol-binding sites was approximately tenfold less than that quantified for mibolerone. Treatment with oestradiol down-regulated the number of [3H] mibolerone binding sites 1·7-fold (P<0·005) as early as day 2 after oestradiol treatment.

In conclusion, we successfully cultured stromal cells derived from hyperplastic prostates which retained sensitivity to androgen and oestrogen. These results suggest that mibolerone and oestradiol exert their biological effects independently of each other, but there is a close relationship in which one steroid increases the sensitivity of the other in the stromal cells of the hyperplastic prostate.

Journal of Endocrinology (1994) 143, 269–277
Isolation and characterization of distinct bioactive forms of LH from male buffalo pituitaries: differences localized to their α subunits
Authors: Sairam M R , Zaky A A H , Hassan A A
Published: December 2008

The isolation of highly purified forms of pituitary LH from Egyptian male (Nile) buffaloes is described. The total LH content (receptor binding activity) which was approximately 30 to 50 fold higher than FSH in the pituitary could be divided into three pools based upon fractionation patterns on a cation exchanger. The acidic fraction which also contained FSH was not purified to homogeneity. A basic fraction (bu-LH-2; 300 mg/kg anterior pituitary) and a very basic fraction (bu-LH-3; 80 mg/kg) were both highly purified and free of FSH activity as tested by specific FSH receptor and immunoassays. The basic buffalo LH fraction, bu-LH-2, was as active as highly purified ovine LH (oLH). The most basic form of buffalo LH, bu-LH-3, was, however, about twice as active as highly purified oLH in the in vitro bioassay using mouse Leydig tumour (MA-10) cells. In a receptor binding assay employing 125I-labelled buffalo LH (bu-LH-3) and porcine testicular membranes, the affinity of bu-LH-3 was about five times higher than purified oLH. The Mr of both forms of purified buffalo LH and subunits was similar to that of oLH. Amino acid composition of buffalo LH was also very similar to oLH except for small differences. Fractionation by fast protein liquid chromatography on Mono-Q columns revealed further evidence of microheterogeneity in each of the pools of buffalo LH with bu-LH-3 exhibiting a predominant single component. By reverse-phase high-pressure liquid chromotography analysis we have localized differences in the two purified isoforms of male buffalo LH to the α subunit. It is suggested that differences in biological potencies could be due to variations in terminal glycosylation and/or differences in branching of this subunit which is known to be important for signal transduction.

Journal of Endocrinology (1994) 143, 313–323
Vitamin D metabolism in the Damara mole-rat is altered by exposure to sunlight yet mineral metabolism is unaffected
Authors: Pitcher T , Sergeev I N , Buffenstein R
Published: December 2008

Vitamin D may be endogenously synthezised in the skin in the presence of sunlight or, alternatively, acquired from dietary sources. Cryptomys damarensis appear to have a naturally impoverished vitamin D status with low plasma concentrations of both 25-hydroxyvitamin D (25(OH)D; <5 ng/ml) and 1,25-dihydroxyvitamin D (1,25(OH)2D; <20 pg/ml). We attribute this to their underground habitat and herbivorous habits. We questioned whether these subterranean mammals could utilize sunlight-mediated pathways and therefore compared vitamin D metabolism and function when animals were (a) housed naturally (control), (b) given an oral vitamin D3 (D3) supplement (1 IU/g dry matter food eaten per day) and (c) exposed to 10 h of sunlight. Control animals exhibited a highly efficient apparent fractional absorption of both calcium (Ca) and inorganic phosphorus (Pi) (>90%), passive mode of intestinal mineral uptake, yet tightly regulated serum ionized calcium (Ca2+). The ratio of 25(OH)D-1α-hydroxylase (1-OHase) to 25(OH)D-24R-hydroxylase (24-OHase) activity in the kidney, corresponded with a state of vitamin D deficiency. Cryptomys damarensis responded to both oral D3 supplementation and sun exposure by an increase in plasma concentration of 1,25(OH)2D with a commensurate decline (P<0·05) in 1-OHase activity, and a resulting decrease (P<0·05) in the ratio of 1-OHase:24-OHase activity. Despite these changes, the intestinal mode of Ca uptake and plasma total Ca, Ca2+ and Pi remained unchanged with either treatment. Responses to sunlight were less pronounced than that of oral D3 supplementation. These data confirm that naturally vitamin D-deficient mole-rats can convert vitamin D to the active hormone 1,25(OH)2D, and indicate that mole-rats function optimally at the low concentrations of vitamin D metabolites found naturally. Furthermore, these animals exhibit a highly efficient vitamin D-independent mode of intestinal Ca absorption.

Journal of Endocrinology (1994) 143, 367–374
Growth hormone and prolactin affect oxytocin, vasopressin, progesterone and cyclic nucleotide secretion by bovine granulosa cells in vitro
Authors: Sirotkin A V , Nitray J
Published: December 2008

The effects of growth hormone (GH) and prolactin (PRL) (1, 10, 100, 1000 or 10 000 ng/ml medium) on oxytocin, vasopressin, progesterone, cAMP and cGMP release by cultured bovine granulosa cells were studied. It was found that GH significantly stimulated oxytocin, vasopressin and cAMP but suppressed progesterone secretion. PRL tended to have the same pattern of action on nonapeptide, cAMP and steroid release, but its effect was not as great, with only a high supraphysiological dose (10 000 ng/ml) producing a statistically significant effect. No significant influence of GH on cGMP output was observed. Physiological doses of PRL (1, 10, 100 or 1000 ng/ml) significantly inhibited cGMP production whilst a high dose (10 000 ng/ml) resulted in stimulation.

These observations suggested that GH may regulate ovarian oxytocin, vasopressin, progesterone and cAMP secretion. The effects of PRL on the release of these substances appeared to be non-specific, possibly resulting from its structural similarity to GH.

Journal of Endocrinology (1994) 143, 417–422
Angiotensin II (All)-binding sites in nuclei from rat liver: partial characterization of the mechanism of All accumulation in nuclei
Authors: Jiménez E , Vinson G P , Montiel M
Published: December 2008

Isoelectric focusing analysis showed a single angiotensin II (All)-receptor complex migrating to pI 6·8 in nuclear preparations, while in plasma membranes a charge heterogeneity of the All receptor subtype AT1 was observed. 125I-Labelled All binding sites were found in intact nuclei and were not detected in nuclear extracts. Neither disruption of cytoskeletal elements by colchicine nor prevention of endosome acidification by chloroquine had any effect on nuclear accumulation of AIL Nevertheless, the monovalent ionophore monensin inhibited nuclear accumulation of 125I-Labelled All. Our findings are consistent with the hypothesis that processing through the Golgi apparatus could be involved in the nuclear accumulation of AIL

Journal of Endocrinology (1994) 143, 449–453