bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization and distribution of angiotensin II in the rat epididymis
Authors: Zhao W , Leung P Y , Chew S B Cheng , Chan H C , Wong P Y D
Published: December 2004

The localization and distribution of angiotensin II (Ang II) in the rat epididymis was studied using immunohistochemical and RIA techniques. The immunohistochemical results showed that Ang II-like immunoreactivity progressively increased along the length of the rat epididymis (cauda>corpus>>caput) and was predominately localized in the basal region of the epididymal epithelium. Occasionally, immunostaining of lighter intensity was also found in the apical region. The concentration of Ang II in cultured rat cauda epididymal epithelial cells was further measured by RIA. In addition to that found in cultured epithelial cells, Ang II activity was also detected in the culture medium, suggesting a secretory role of the epithelium. These findings suggest that Ang II could be derived locally from epididymal epithelium and that it could play a role in local regulation of epithelial transport and, possibly, in the maintenance of sperm function as well, by exerting its paracrine and/or autocrine effect in various regions of the epididymis.

Journal of Endocrinology (1996) 149, 217–222
The expression of transforming growth factor-β by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3
Authors: Farquharson C , Law A S , Seawright E , Burt D W , Whitehead C C
Published: December 2004

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-β (TGF-β) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-β1 to -β3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-β mRNA and conditioned medium was assayed for TGF-β activity and isoform composition. Active TGF-β was only detected in 10−8m 1,25(OH)2D3-treated cultures (8·37 ng active TGF-β/mg protein). There was a significant decrease in total (latent+active) TGF-β activity in conditioned medium of 10−12 m (23·4%; P<0·05) and 10−10 m (20·7%; P<0·05) 1,25(OH)2D3-treated cultures but 10−8 m 1,25(OH)2D3 significantly increased (30·9%; P<0·01) TGF-β activity. The amounts of TGF-β1, -β2 and -β3 isoforms produced were similar in control, 10−10 or 10−12m 1,25(OH)2D3-treated cultures but the conditioned medium of 10−8 m 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-β mRNA demonstrated differential control of TGF-β gene expression with TGF-β1 and -β3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10−8, 10−10 and 10−12 m) whilst TGF-β2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-β secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth.

Journal of Endocrinology (1996) 149, 277–285
1,25-Dihydroxyvitamin D3 increases the sensitivity of human renal carcinoma cells to tumor necrosis factor α but not to interferon α or lymphokine-activated killer cells
Authors: Yacobi R , Koren R , Liberman U A , Rotem C , Wasserman L , Ravid A
Published: December 2004

Renal cell carcinoma is a chemotherapy-resistant tumor which is relatively responsive to immunotherapy. Immunotherapeutic regimes employ interferons or interleukin 2 with or without lymphokine-activated killer cells. Secondary cytokines, induced by interleukin 2 or interferon, may have an important impact on their anti-neoplastic activity. Notable among them is tumor necrosis factor (TNFα). We assessed the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the susceptibility of the human renal cell carcinoma cell line SK-RC-29 to the cytotoxic and cytostatic actions of TNFα, interferon a and lymphokine-activated killer cells. Using uptake of the vital dye neutral red as an indicator of viable cell number, we found that addition of 1,25(OH)2D3 (100 nm) to TNFα (30 ng/ml)-treated cultures resulted in a 2·6 ± 0·2-fold (mean ± s.e.) increase in the cytotoxic effect of the cytokine. The potentiating effect of 1,25(OH)2D3 was dose-dependent, and significant at concentrations equal to or higher than 10 nm. Another dihydroxylated vitamin D metabolite, 24,25(OH)2D3, had no effect on TNFα action. The cytotoxic effect of TNFα increased whereas the potentiation by 1,25(OH)2D3 decreased with cell density in culture. 1,25(OH)2D3, in contrast to its potentiating effect on TNFα action, did not modulate the cytostatic effect of interferon α or the susceptibility of SK-RC-29 to killing by lymphokine-activated killer cells. The findings reported here may explain some of the in vivo anti-tumor activity of 1,25(OH)2D3 and provide a rationale for the employment of active vitamin D analogs during immune anti-cancer therapy.

Journal of Endocrinology (1996) 149, 327–333
Regulation of cartilage glycosaminoglycan synthesis in the rainbow trout, Oncorhynchus mykiss, by 3,3′,5-tri-iodo-l-thyronine and IGF-I
Authors: Takagi Y , Th Björnsson B
Published: December 2004

The actions of 3,3′,5-tri-iodo-l-thyronine (T3) and recombinant human IGF-I (rhIGF-I) as well as their interaction on cartilage growth in rainbow trout (Oncorhynchus mykiss) were examined. Uptake of 3H-methyl thymidine and 35S-sulfate by isolated branchial cartilage was measured as a marker for chondrocyte proliferation and sulfated glycosaminoglycan synthesis respectively. When T3 (1·0 μg/g) was injected intraperitoneally, plasma T3 levels reached a transient peak after 1 day and decreased rapidly thereafter. Sulfate and thymidine uptake were not affected by T3 at 1 and 3 days post-injection, but at 6 days post-injection both were significantly higher in T3injected fish than those in controls. The stimulatory effects of a T3 injection on sulfate and thymidine uptake were dose-dependent over the range of 0·01, 0·1 and 1·0 μg/g. In vitro exposure of cartilage to T3 (0·075, 0·75, 7·5, 75 and 750 nm) for 6 days resulted in dose-dependent stimulation of sulfate uptake, with a maximum response at 7·5 nm and higher. T3 exposure (7·5 nm) for 2 or 3 days also increased sulfate uptake, but only slightly. Thymidine uptake was not clearly affected by T3. In vitro addition of rhIGF-I (0·075, 0·75 and 7·5 nm) increased sulfate uptake, but not thymidine uptake, dose-dependently. Compared with T3, rhIGF-I induced a greater maximum level of sulfate uptake: at 7·5 nm rhIGF-I increased the uptake 17-fold whereas T3 increased the uptake fourfold. When T3 (0·075, 0·75 or 7·5 nm) and rhIGF-I (0·1 or 1·0 nm) were added together, stimulative actions of T3 on sulfate uptake were largely additive to those of rhIGF-I. The results indicate that T3 as well as IGF-I are important modulators of sulfated glycosaminoglycan synthesis in rainbow trout cartilage.

Journal of Endocrinology (1996) 149, 357–365
Characterization of serum-derived and recombinant rat IGF-I and their use for measuring true concentrations of IGF-I in rat plasma
Authors: Upton Z , Webb H , Tomas F M , Ballard F J , Francis G L
Published: December 2004

While numerous researchers have used rat models to investigate the in vivo actions of IGF-I, interpretation of the results in terms of true concentrations of rat IGF-I (rIGF-I) in plasma has been hampered by the absence of homologous reference standards. In order to overcome this we have produced recombinant rIGF-I (rrIGF-I) from Escherichia coli using procedures similar to those we have previously described for the production of other recombinant IGFs. The rrIGF-I is indistinguishable from serum-derived rIGF-I when characterized in a number of in vitro assays including ability to stimulate protein synthesis and inhibit protein degradation in cultured rat cells, as well as in interactions with the rat type-1 IGF receptor and with rat IGF-binding proteins. Moreover, both the serum-derived and the recombinant rat proteins are similar to recombinant human IGF-I (rhIGF-I) in these assays. However, differences between the human and rat IGFs are apparent when tested in immunoassays using some antibodies raised against rhIGF-I. Furthermore, the differences between rhIGF-I and rrIGF-I are even greater when rhIGF-I is used as the competing radiolabel in these assays, a situation that can lead to a two- to threefold underestimation of the actual concentration of IGF-I in rat plasma. These results indicate that, while immunoassays employing antibodies raised against rhIGF-I and rhIGF-I reference standards reliably indicate trends in IGF-I concentrations in rat plasma, the true amounts of rIGF-I present can only be assured in an assay using homologous tracer and reference peptides.

Journal of Endocrinology (1996) 149, 379–387
Functional characterisation of an ovine endometrial oxytocin receptor cDNA transiently expressed in Cos-7 cells
Authors: Riley P R , Abayasekara D R E , Stewart H J , Flint A P F
Published: December 2004

The entire coding region of an ovine endometrial oxytocin receptor (OTR) cDNA was generated by PCR, subcloned into the SV40 major late promoter expression vector pSVLJ and transiently expressed in Cos-7 cells. A specific OTR antagonist, 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH29]-vasotocin (OTA), was used to describe the binding kinetics of the expressed receptor which had a Kd of 4·5 nm and Bmax of 2·4 nm/mg protein (6·8 × 105 receptor molecules/transfected cell). The functional properties of the expressed OTR were determined by measuring oxytocin-induced phosphoinositide (PI) hydrolysis. Oxytocin increased PI turnover in OTR transfected cells fourfold in excess of residual endogenous activity, and stimulated phospholipase C (PLC) activity in a dose- and time-dependent manner, confirming that the expressed OTR cDNA was functional. Arginine vasopressin also stimulated PI turnover in a dose-dependent manner; thresholds of responses to oxytocin and arginine vasopressin were 10−9 m and 10−7 m respectively. OTA did not increase PI turnover and competitively inhibited the oxytocin-induced response. Direct activation of the pathway by aluminium fluoride and guanosine (3′-Othio)-triphosphate (GTPγS) confirmed that the OTR was G-protein linked. Co-incubation of GTPγS with oxytocin shifted the PI-response threshold from 10−7 m to 10−9 m and significantly increased the level of response, suggesting that maximum PI turnover was agonist-dependent. The G-protein involved in mediating the signal transduction pathway was pertussis toxin-insensitive and, therefore, probably a member of the Gq subfamily. The PLC inhibitor, U73122, had no effect on oxytocin-induced PI turnover, consistent with the response in endometrial tissue. These data suggest that the signalling pathway mediated by expressed OTR is similar to that attributed to OTR occupancy in ovine endometrium.

Journal of Endocrinology (1996) 149, 389–396
Differential release of interleukin-6 from human gestational tissues in association with labour and in vitro endotoxin treatment
Authors: Laham N , Brennecke S P , Bendtzen K , Rice G E
Published: December 2004

In this study, we quantified interleukin-6 (IL-6) concentrations in amniotic fluid at term and preterm labour, and determined the gestational tissue source of IL-6. In addition, aspects of the regulatory mechanisms involved in IL-6 release at the time of term labour and in response to bacterial endotoxin, lipopolysaccharide (LPS), have been established. IL-6 concentrations were 2-fold higher in amniotic fluid collected at term compared with preterm gestation, with an additional 2-fold increase in association with term labour. IL-6 was released from all choriodecidual and placental explants but was detected in only 33% of amniotic explant cultures of tissues obtained before labour onset. In contrast, IL-6 was detected in all amniotic, choriodecidual and placental cultures of tissues obtained after term labour onset and delivery, and the mean IL-6 release was significantly higher than that measured in explant cultures of both amniotic (80-fold increase, P<0·0001) and choriodecidual (3-fold increase, P<0·02) but not placental explants taken at the time of elective Caesarean section at term before labour onset. LPS significantly (P<0·05) increased the release of IL-6 from human choriodecidual and placental explants but not amniotic explants, in a time- and dose-dependent manner. IL-6 is a physiological constituent of amniotic fluid and its production by gestational tissues is differentially regulated by LPS and spontaneous labour onset and delivery.

Journal of Endocrinology (1996) 149, 431–439
GnRH-induced calcium mobilisation and inositol phosphate production in immature and mature rat ovarian granulosa cells
Authors: Anderson L , Hillier S G , Eidne K A , Miro F
Published: December 2004

In rat ovarian granulosa cells the effects of GnRH are determined by the state of granulosa cell development with mainly inhibitory actions in immature cells and stimulatory actions in differentiated mature cells. These developmentally related effects of GnRH may arise from changes in either one or more of the signal transduction pathways activated by GnRH. The present study therefore measured downstream signalling events associated with the activation of the phospholipase C (PLC) signal transduction pathway in both mature and immature rat ovarian granulosa cells. Results showed that GnRH produced similar total inositol phosphate and intracellular calcium ([Ca2+ ]i) responses in both immature and mature granulosa cells. In contrast to the biphasic GnRH-induced [Ca2+]i response in pituitary gonadotropes, stimulation of the endogenously expressed GnRH receptor in both immature and mature granulosa cells produced a prompt monophasic rise in [Ca2+]i. This calcium transient was abolished by pretreating either cell type with a potent GnRH receptor antagonist or the PLC inhibitor U73122, demonstrating a GnRH receptor-specific activation of PLC. Similarly, pretreatment of cells with the [Ca2+]i antagonists thapsigargin or cyclopiazonic acid abolished the GnRH-induced calcium transient, whereas EGTA and nifedipine, a voltage-operated calcium channel (VOCC) antagonist, had no effect. These results suggest that in either immature or mature granulosa cells GnRH mobilises calcium from thapsigargin/cyclopiazonic acid-sensitive [Ca2+]i stores but does not involve the influx of extracellular calcium through VOCCs. We conclude that GnRH-induced stimulation of the PLC signal transduction pathway is independent of the stage of granulosa cell maturity and that alternative mechanisms account for the opposite effects of GnRH on gonadotrophin-induced steroidogenesis in mature and immature rat granulosa cells in vitro.

Journal of Endocrinology (1996) 149, 449–456
Temperature and non-aromatizable androgens: a common pathway in male sex determination in a turtle with temperature-dependent sex determination?
Authors: Crews D , Cantú A R , Bergeron J M
Published: December 2004

This study addressed the hypothesis that, in the red-eared slider turtle, Trachemys scripta, non-aromatizable androgens are the physiological equivalent of temperature in determining male development. In the first experiment, eggs were treated in the middle of the temperature-sensitive period with 1·0 or 10·0 μg androsterone, 5α-dihydrotestosterone, 3α-androstanediol, or 3β-androstanediol, while at an all-male, male-biased, or one of two female-biased incubation temperatures. In the second experiment, eggs were treated with the same dosages of dihydrotestosterone at different stages of embryonic development while at a male-biased, threshold, or a female-biased incubation temperature. Results of experiment one indicated that hormone-induced masculinization is specific to non-aromatizable androgens. Results of experiment two indicated that the sensitivity to dihydrotestosterone corresponds to the temperature-sensitive window during development. Further, there is a dose–response relationship but no apparent synergism between exogenous dihydrotestosterone and incubation temperature. When considered with other research, it is suggested that non-aromatizable androgens and their products are involved in the initiation of male sex determination whereas oestrogens and their aromatizable androgen precursors are involved in the initiation of female sex determination.

Journal of Endocrinology (1996) 149, 457–463
Acquired and naturally occurring resistance of thyroid follicular cells to the growth inhibitory action of transforming growth factor-β1 (TGF-β1)
Authors: Asmis L M , Kaempf J , Von Gruenigen C , Kimura E T , Wagner H E , Studer H
Published: December 2004

While the multifunctional proteins of the transforming growth factor-β (TGF-β) family have a potent antiproliferative effect on thyroid follicular cell growth, increased expression of TGF-β in proliferating thyroid cells and in thyroid tumours has recently been described, suggesting a secondary counter-regulatory role of these proteins. We have studied further this apparent paradox in vitro using FRTL-5 cells, 5 continuous cell strains from feline multinodular goitres (MNG) and 29 primary cultures prepared from human MNG.

While dose dependent inhibition of FRTL-5 cell growth was confirmed, a variable fraction of these cells was naturally resistant towards TGF-β1, thus explaining the large interassay variability of growth inhibition (36 to 98% within 2 days, n=19). After 40 days of continuous exposure, FRTL-5 cells became fully refractory towards TGF-β1 inhibition, due to the selective growth of naturally resistant subclones, as demonstrated for example by microscopic observation of three-dimensionally growing collagen-embedded cell clusters. The refractoriness could still be demonstrated even after several cell passages. In addition, 2 out of 5 feline thyroid cell strains obtained from feline MNG and 18 out of 29 primary cultures from human MNG showed a high degree of refractoriness towards TGF-β.

We conclude that constitutively TGF-β resistant cells may occur in thyroid glands and that persistent TGF-β refractoriness may secondarily be acquired. Resistant cells may escape regular growth control mechanisms and hence may contribute to the notorious heterogeneity of thyroid growth and to nodular transformation.

Journal of Endocrinology (1996) 149, 485–496
Periovulatory endocrinology in high ovulating Meishan sows
Authors: Hunter M G , Picton H M , Biggs C , Mann G E , McNeilly A S , Foxcroft G R
Published: December 2004

The present study was designed to investigate the hormone profiles (oestradiol, LH, FSH, inhibin, progesterone) in high ovulating Meishan sows (MS; n = 9) and in contemporary Large-White hybrid control sows (LW; n = 9) during the follicular phase, the periovulatory period and the early luteal phase. Ovulation rate was higher in MS than LW animals (23·7 and 16·6 respectively; P<0·001) and overall was correlated with the area of the oestradiol peak (P<0·05) and inhibin concentrations (P<0·05). Both the duration of and the area of the oestradiol peak were greater in MS than LW (P<0·01; P<0·02), as were inhibin concentrations both before and after the LH surge (P<0·05). Neither basal nor peak concentrations of LH or FSH differed between the breeds (P>0·05), although FSH concentrations were correlated with the area under the oestradiol peak (P<0·05). Finally, the time-interval from the onset of the LH surge until the rise in plasma progesterone was shorter in MS than LW (54·5 and 74·3 h respectively; P<0·01). In conclusion, these results show for the first time that the higher ovulation rate in MS is associated with enhanced oestradiol and inhibin secretion with no significant difference in LH or FSH concentrations. The more rapid luteinization response to the LH surge by MS in terms of plasma progesterone concentrations may be important in ensuring the high level of embryo survival in this breed.

Journal of Endocrinology (1996) 150, 141–147
Fasting alters somatostatin binding to liver membranes of rainbow trout (Oncorhynchus mykiss)
Authors: Pesek M J , Sheridan M A
Published: December 2004

Somatostatins are a diverse family of peptides that influence various aspects of animal growth, development, and metabolism. Recent work in our laboratory has shown that somatostatins stimulate hepatic lipolysis in rainbow trout. In this study we characterized somatostatin-binding sites in trout hepatic membrane preparations. We also examined changes in binding characteristics brought about by food deprivation. Binding of [Tyr11]-somatostatin-14 (SS-14) was saturable, reversible, and time- and temperature-dependent. Under optimal conditions, [Tyr11]-SS-14 specific binding averaged 5·7 ± 0·3%. While SS-14 and SS-28 (an N-terminally extended form of SS-14 and derived from the same gene as SS-14) displaced [Tyr11]-SS-14 specific binding (ED50 values of approximately 50 nm and 100 nm respectively), salmon SS-25 (containing [Tyr7,Gly10]-SS-14 at its C terminus and presumably derived from a gene different from that giving rise to SS-14/SS-28), except at pharmacological concentrations, did not. Significant specific binding was also detected in brain, esophagus, stomach, upper and lower intestine, pancreas, and adipose tissue. Scatchard analysis suggested the existence of two classes of hepatic somatostatin-binding sites: a high-affinity site with a Kd of 23 nm and Bmax of 1·4 pmol/mg protein and a low-affinity site with a Kd of 379 nm and Bmax of 4·9 pmol/mg protein. Fasting resulted in reduced growth and elevated plasma levels of SS-14 compared with fed animals. SS-14 binding capacity of the high-affinity class in liver membranes isolated from fasted fish increased by 120% over that from fed counter-parts. No difference in Kd for the high-affinity binding class or in either Kd or Bmax of the low-affinity class was noted between fasted and fed animals. These data support the role of the liver as a target of somatostatin and suggest that fasting enhances hepatic sensitivity to SS-14 binding.

Journal of Endocrinology (1996) 150, 179–186
Dopaminergic regulation of pituitary function in the late-gestation fetal sheep
Authors: Matthews S G , Fraser M , Challis J R G
Published: December 2004

Development of the fetal ovine pituitary is essential for normal maturation and initiation of the parturition process, as well as for orchestrating endocrine responses to stress in utero. Increases in the biosynthesis of ACTH and prolactin (PRL) occur in the late-gestation fetal sheep pituitary. In the anterior lobe (AL) of the pituitary, pro-opiomelanocortin (POMC) biosynthesis and processing are primarily regulated by corticotrophin-releasing hormone and vasopressin. However, POMC in the intermediate lobe (IL) and PRL in the AL are known to be primarily regulated by dopamine, via the D2 receptor, in adult sheep. Because of the importance of ACTH and PRL during gestation we have investigated a potential role of dopamine in the control of both IL melanotrophs and AL lactotrophs and corticotrophs, in late gestation.

Catheters were implanted into a maternal femoral artery and vein, fetal carotid artery and jugular vein as well as into the amniotic cavity. At day 130 of gestation, fetuses were infused intravenously with either the specific D2 receptor agonist bromocriptine (n = 5) or vehicle (n=5), for 5 days. Blood samples were taken throughout the experiment and pituitaries were removed at the end of the treatment period. Bromocriptine caused a significant decrease (>50%) in POMC mRNA levels in the IL. In contrast, bromocriptine had no significant effect on POMC mRNA levels or distribution in the AL. Fetal arterial ACTH and cortisol concentrations were unaffected by the bromocriptine infusion, compared with vehicle-infused controls. There was a dramatic decrease (>80%) in plasma PRL concentrations, compared with the control fetuses. However, PRL mRNA levels in the AL were not significantly affected by bromocriptine. In conclusion, we have found that bromocriptine inhibits aspects of both melanotroph and lactotroph function in late-gestation fetal sheep. The data indicate that the fetal pituitary possesses functional D2 receptors in late gestation.

Journal of Endocrinology (1996) 150, 187–194
A type I collagen substrate increases PTH/PTHrP receptor mRNA expression and suppresses PTHrP mRNA expression in UMR106–06 osteoblast-like cells
Authors: Celic S , Chilco P J , Zajac J D , Martin T J , Findlay D M
Published: December 2004

We have previously shown that the response of osteoblasts to parathyroid hormone (PTH) can be influenced at the receptor level by growth on the physiological substrate, type I collagen, or by treatment with retinoic acid. We have also shown differential expression of genes when cells of the osteoblast lineage are grown on type I collagen. The aim of this study was therefore to examine the effect of retinoic acid and growth on type I collagen on PTH/PTH-related protein (PTHrP) receptor mRNA expression in the osteosarcoma osteoblast-like cell line UMR106–06.

PTH/PTHrP receptor mRNA levels, as assessed by Northern blot, of cells grown on collagen were increased up to 2-fold compared with cells on plastic and in a concentration-dependent manner with respect to collagen. An increase was seen as early as 6 h and was maintained over a 24 h period. This was not due to increased mRNA stability. Retinoic acid decreased the level of receptor mRNA on both plastic and collagen at each time but did not alter mRNA stability. For all treatments PTH/PTHrP receptor mRNA abundance, relative to glyceraldehyde-3-phosphate dehydrogenase, increased steadily over 24 h after subculture of cells. In contrast, PTHrP mRNA levels were reduced in cells on collagen, compared with plastic. PTH-stimulated cAMP levels of cells grown on collagen were increased compared with plastic at 24 h, but not earlier. Consistent with the mRNA data, retinoic acid decreased the amplitude of cAMP responses in cells on plastic and collagen. There was no evidence for changes in adenylate cyclase per se, since forskolin-induced cAMP levels did not change with either treatment. This study shows that known modulators of osteoblast maturation also affect signal transduction in these cells by regulating gene expression of the PTH/PTHrP receptor as well as the PTHrP ligand.

Journal of Endocrinology (1996) 150, 299–308
Inter-relationships between nonapeptide hormones and cyclic nucleotides within cultured porcine granulosa cells
Authors: Sirotkin A V
Published: December 2004

The reciprocal control of nonapeptide hormone (oxytocin, vasopressin) and cyclic nucleotide (cAMP, cGMP) release by porcine granulosa cells was studied. In particular, the influence of vasopressin and oxytocin treatment (10–10 000 ng/ml) on basal and LH-induced cAMP and cGMP output, as well as the effects of dibutyryl cAMP (dbcAMP; cAMP analogue) and forskolin (a stimulator of cAMP formation; 0·1–1000 ng/ml) on vasopressin and oxytocin secretion by cultured porcine granulosa cells were examined.

It was observed that the addition of arginine-8-vasopressin or oxytocin stimulated both cAMP and cGMP output from granulosa cells. Moreover, both vasopressin and oxytocin also increased LH-stimulated cAMP and cGMP release. On the other hand, both dbcAMP and forskolin decreased vasopressin secretion. Oxytocin release was stimulated under the influence of dbcAMP. The same stimulating effect occurred with forskolin given at a low dose (1 ng/ml), whilst higher doses of forskolin (10 or 1000 ng/ml) were inhibitory.

The present observations demonstrate the reciprocal influence of nonapeptide hormones and cyclic nucleotides in porcine ovarian cells. Oxytocin and vasopressin, like LH, exert their action on the ovary via the activation of cAMP- and cGMP-dependent intracellular mechanisms. cAMP in turn inhibits vasopressin release through a negative feedback mechanism. On the other hand, a reciprocal stimulation of oxytocin and cAMP output in granulosa cells is suggested. Thus, cyclic nucleotides can be both regulators of nonapeptide hormone secretion and mediators of their action within porcine ovaries.

Journal of Endocrinology (1996) 150, 343–348
Disrupted co-ordination of Pax-8 and thyroid transcription factor-1 gene expression in a dedifferentiated rat thyroid tumour cell line derived from FRTL-5
Authors: van der Kallen C J H , Spierings D C J , Thijssen J H H , Blankenstein M A , de Bruin T W A
Published: December 2004

The mutant rat thyroid cell line FRTL-5/TA, isolated from a non-functional tumour which originated spontaneously from wild-type FRTL-5 cells, shows autonomous TSH-independent growth and loss of the thyroid-specific phenotype, lacking thyroid-specific expression of thyroglobulin (Tg) and thyroid peroxidase (TPO) genes. To investigate the role of the transcription factors Pax-8 and thyroid transcription factor-1 (TTF-1) in rat thyroid tumorigenesis, RNA expression of these two thyroid-specific nuclear factors was measured in FRTL-5/TA tumour cells and compared with the expression in wild-type FRTL-5 cells. TTF-1 gene expression was similar to that in wild-type FRTL-5, and showed a similar down-regulation after stimulation with TSH. The finding suggested normal TTF-1 mRNA and protein expression in both cell lines. By contrast, Pax-8 mRNA transcript signal was markedly reduced in FRTL-5/TA cells, reaching levels as low as 8% of the normal, basal level in FRTL-5 cells. These data indicated that the loss of thyroid-specific expression of Tg and TPO genes in FRTL-5/TA cells was not related to changes in TTF-1 gene expression but rather to reduced Pax-8 gene expression. It was concluded that a disruption of the co-ordinated expression of TTF-1 and Pax-8 is implicated in the loss of thyroid phenotype of FRTL-5/TA cells in terms of reduced Tg and TPO expression.

Journal of Endocrinology (1996) 150, 377–382
Potent inhibitory effects of steroids in an in vitro model of angiogenesis
Authors: Jaggers D C , Collins W P , Milligan S R
Published: December 2004

The regulation of angiogenesis in the ovarian follicle and corpus luteum is unclear. Steroids are produced at very high concentrations in these tissues and we therefore examined the effect of steroids on angiogenesis in vitro. Explants of rat aorta were embedded in collagen gel and cultured in serum-free medium. Capillary-like microvessels were produced from the explants and microvessel number and length were measured in the presence and absence of steroids. At a concentration of 10 μg/ml, cortisol, progesterone, 17α-hydroxyprogesterone and medroxyprogesterone acetate produced degeneration of microvessels after 7 days of steroid treatment (P<0·01). Androstenedione and tetrahydro-S-(11-deoxytetrahydro-cortisol) (tetrahydro S) produced degeneration at a slower rate: androstenedione inhibited microvessel growth after 11 days (P<0·01) and tetrahydro S after 14 days (P<0·05). Oestriol had no effect on microvessels; oestrone had a slow degenerative effect with significant inhibition seen after 14 days (P<0·01). Oestradiol-17β at a concentration of 10 μg/ml completely inhibited microvessel growth from the explant cultures (P<0·01) while at 1 μg/ml it caused degenerative effects on growing microvessels. The effects of oestradiol and cortisol were reversible on removal of steroid-containing medium and replacement with 10% serum. We conclude that oestradiol may modulate angiogenesis in tissues in which the steroid concentration is high.

Journal of Endocrinology (1996) 150, 457–464
Localization of prostaglandin H synthase type 2 protein and mRNA in term human fetal membranes and decidua
Authors: Gibb W , Sun M
Published: December 2004

Prostaglandin (PG) production by human fetal membranes (amnion and chorion laeve) may be important in the onset and progression of labour, cervical ripening and membrane rupture. Prostaglandin H synthase (PGHS) is a key enzyme in PG formation and has two isoforms, a constitutive form (PGHS-1) and an inducible form (PGHS-2). The present study examined the cellular distribution of the PGHS-2 enzyme and PGHS-2 mRNA in term human fetal membranes and decidua prior to and following labour, using immunohistochemistry and in situ hybridization with an 35S-labelled oligonucleotide probe. The PGHS-2 protein was found to be localized in amnion epithelial cells and chorion laeve trophoblast, but was absent or at low levels in the decidual stroma in most tissues, although cells surrounding some of the blood vessels in the decidua did express PGHS-2. In situ hybridization demonstrated that PGHS-2 mRNA had a similar distribution and was localized to amnion epithelial cells, cells in the amnion-chorion mesenchyme, chorion laeve trophoblast and, occasionally, to cells surrounding blood vessels in the decidua. Of particular note was the high mRNA expression in some cells and low expression in other cells, particularly in the chorion, and the low level of PGHS-2 mRNA in decidua. There was no observable difference in the cellular localization of PGHS-2 protein or PGHS-2 mRNA in tissues obtained prior to and following labour. The studies indicate that, at term, the inducible form of PGHS, PGHS-2, is expressed at a high level in fetal tissues in a number of different cell types rather than in the maternal decidua.

Journal of Endocrinology (1996) 150, 497–503
Follistatin concentrations in male sheep increase following sham castration/castration or injection of interleukin-1β
Authors: Phillips D J , Hedger M P , McFarlane J R , Klein R , Clarke I J , Tilbrook A J , Nash A D , de Kretser D M
Published: December 2004

Plasma follistatin (FS) concentrations were determined after castration (n=5) or sham castration (n=4) of mature rams. Both treatments resulted in a prolonged increase in FS between 7 and 19 h after surgery, which returned to pretreatment concentrations by 24 h. Tumour necrosis factor-α (TNF-α), a sensitive marker of an acute-phase response, was undetectable in plasma, indicating that the FS response was not induced by trauma due to surgery. In a second experiment, injection of castrated rams (n=4) with ovine recombinant interleukin-1β, an acute-phase mediator, resulted in a sustained rise in FS concentrations within 4 h of injection. Plasma TNF-α concentrations increased transiently within 1 h of interleukin-1β injection, indicating that an acute-phase response had been initiated. Plasma follicle-stimulating hormone (FSH) concentrations were significantly decreased at 8 and 24 h after interleukin-1β injection, strongly suggestive of an inhibitory effect of increased FS concentrations on the secretion of FSH. Injection of castrated rams (n=2) with a control preparation of recombinant interleukin-2 did not induce an acute-phase response, and plasma FS and FSH concentrations were unaffected. These data show that the testis is not a major source of circulating FS, that the increase in circulating FS following sham castration/castration is not due to an acute-phase response, but that conversely FS concentrations are modulated by the acute-phase mediator, interleukin-1β.

Journal of Endocrinology (1996) 151, 119–124
Expression of mRNAs for insulin-like growth factor binding proteins-2, -3 and -4 in the ovine ovary throughout the oestrous cycle
Authors: Perks C M , Wathes D C
Published: December 2004

Ovarian localization of the IGFs and their binding proteins is species-specific. In the ewe we have demonstrated previously that IGF-II mRNA is found at high concentrations in the theca, whereas IGF-I mRNA is undetectable in ovine follicles at any stage of development, although both are present in the corpus luteum. In this study we have investigated the localization of mRNA for IGF-binding proteins (IGFBPs)-2, -3 and -4 using in situ hybridization to further elucidate the roles of the IGFs and their possible modulation during follicular development. Results were quantified by optical density (OD) measurements of autoradiographs using image analysis. There was intense follicular expression of mRNAs for both IGFBP-2 and -4 although IGFBP-3 mRNA was undetectable. Concentrations of IGFBP-2 mRNA varied significantly (P<0·01) with respect to size, being highest in small (<2 mm) follicles but with no variation attributable to health, whereas expression of IGFBP-4 mRNA was significantly (P<0·01) influenced by health and not size, being higher in healthy than atretic follicles. IGFBP-2, -3 and -4 mRNAs were all present at low concentrations in the corpus luteum (OD 0·04 ± 0·005, n=6; 0·05 ± 0·02, n=5; 0·1 ± 0·02, n=8 respectively), whereas only IGFBP-2 and -4 mRNAs were found in ovarian stroma (OD 0·04 ± 0·006, n=4; 0·04 ± 0·007, n=9 respectively). These data suggest that changes in follicular distribution of IGFBPs-2 and -4 may modulate the actions of the IGFs during follicular growth thus contributing to the process of follicle selection.

Journal of Endocrinology (1996) 151, 241–249
The steroidogenic effects of β-endorphin and joining peptide: a potential role in the modulation of adrenal androgen production
Authors: Clarke D , Fearon U , Cunningham S K , McKenna T J
Published: December 2004

This study examines the androgen-stimulating properties of pro-opiomelanocortin-derived peptides, ACTH, β-endorphin (β-End) and joining peptide (JP). Ten different cell suspensions were prepared from ten human adrenal glands. ACTH and JP stimulated cortisol, androstenedione (Δ4) and dehydroepiandrosterone (DHEA) production (P<0·05); β-End stimulated only Δ4 and DHEA production. β-End brought about significant increases in the Δ4 or DHEA to cortisol ratios. The addition of β-End (10−10 m) suppressed ACTH-stimulated cortisol production from 7573 ± 2960 to 5994 ± 2654 pmol/106 cells (means ± s.e.m.; P<0·05). The addition of β-End did not affect ACTH-stimulated Δ4 production (210 ± 88 and 236 ± 105 pmol/106 cells). JP (10−10 m) inhibited ACTH-stimulated cortisol production so that the mean values fell to 5186 ± 2588 and also inhibited DHEA production, from 240 ± 48 to 180 ± 33 pmol/106 cells. These results suggest that the relative production of androgen to cortisol is greater in response to β-End and JP than in response to ACTH. If blood levels of these peptides rise to herald adrenarche as reported for β-End, suppression of cortisol production may result in an increase in ACTH to correct cortisol levels resulting in an increase in Δ4 and DHEA levels. This may explain the occurrence of increasing androgen levels at adrenarche.

Journal of Endocrinology (1996) 151, 301–307
A comparison of cytokine and hormone production by decidual cells and tissue explants
Authors: Lonsdale L B , Elder M G , Sullivan M H F
Published: December 2004

Previous work has shown that enzymatic digestion of human placental tissue can induce the production of the cytokine interleukin-1β. Most studies of the feto-maternal interface of human pregnancy have used decidual cells prepared in a similar way, but the effects of tissue dissociation on the production of growth factors, cytokines, prostaglandins or hormones have not been investigated. Our studies show human decidual explants produce substantially lower levels of a range of factors than do human decidual cells cultured under the same conditions, indicating that induction may be a general process during the dissociation of tissues in vitro as the production of interleukins-1β, -6 and -8, granulocyte-macrophage colony-stimulating factor, transforming growth factor-β2, tissue necrosis factor-α, prostaglandins E2 and F2α, and prolactin were all affected. The induction of cytokine production (expressed per mg tissue protein) ranged from 10- to 300-fold, indicating that isolated cells cultured in vitro may not reflect accurately the in vivo situation.

Journal of Endocrinology (1996) 151, 309–313
Benign prostatic stromal cells are regulated by basic fibroblast growth factor and transforming growth factor-β1
Authors: Collins A T , Robinson E J , Neal D E
Published: December 2004

The current study was undertaken, using cultures of prostatic epithelial and stromal cells, to determine the functional interactions between androgens, basic fibroblast growth factor (FGF2) and transforming growth factor-β1 (TGFβ1) and their importance in maintaining stromal homeostasis. Treatment of stromal cells with TGFβ1 significantly increased intracellular FGF2 and FGF2 sequestered to the extracellular matrix. FGF2 was also detected in stromal conditioned medium (SCM), but at levels 70-fold less than found in cell lysates. TGFβ1 (0·1 ng/ml) treatment caused an initial increase of 86% in secreted FGF2 levels, but high concentrations of TGFβ1 (5 ng/ml) decreased FGF2 levels by 38%, relative to the untreated control. Further studies showed that epithelial conditioned medium (ECM), androgen-treated, stromal conditioned medium (ASCM), but not SCM were mitogenic for stromal cells. Both ECM and ASCM caused a threefold increase in DNA synthesis. FGF2 may be the mediator of these interactions, since the mitogenic effect of both ECM and ASCM was significantly reduced by the addition of anti-FGF2 neutralising antibody. We hypothesise that the lack of response of stromal cells to SCM is due to TGFβ1 blocking the mitogenic effect of FGF2. Thus down-regulation of TGFβ1 synthesis, by androgens, results in stromal proliferation by ASCM.

Journal of Endocrinology (1996) 151, 315–322
Mechanisms of amino acid-induced insulin secretion from the glucose-responsive BRIN-BD11 pancreatic B-cell line
Authors: McClenaghan N H , Barnett C R , O'Harte F P M , Flatt P R
Published: December 2004

The effects of different classes of amino acids known to be transported and utilized by pancreatic B-cells were examined using the novel glucose-responsive pancreatic B-cell line, BRIN-BD11. Amino acids tested included α-aminoisobutyric acid, l-alanine, l-arginine, l-glutamine, glycine, l-leucine, l-lysine, l-proline and l-serine. At non-stimulatory (1·1 mmol/l) glucose, acute incubations with either 1 or 10 mmol/l amino acid evoked 1·3- to 4·7-fold increases of insulin release. Raising glucose to 16·7 mmol/l enhanced the effects of all amino acids except l-glutamine, and increased insulin output at 10 mmol/l compared with 1 mmol/l amino acid. Glyceraldehyde (10 mmol/l) also served to promote 10 mmol/l amino acid-induced insulin secretion with the exceptions of l-arginine, glycine, l-lysine and l-proline. At 16·7 mmol/l glucose, diazoxide (300 μmol/l) significantly decreased the secretory response to all amino acids except l-glutamine. Likewise, verapamil (20 μmol/l) or depletion of extracellular Ca2+ reduced insulin output indicating the importance of Ca2+ influx in the actions of amino acids. These data indicate that BRIN-BD11 cells transport and utilize amino acids, acting in association with glycolysis, K+-ATP channels and/or voltage-dependent Ca2+ channels to promote Ca2+ influx and insulin secretion. The response of BRIN-BD11 cells to glucose and amino acids indicates that this is a useful cell line for future research on the mechanisms of nutrient regulation of insulin secretion.

Journal of Endocrinology (1996) 151, 349–357
Luminal peptide YY-releasing factors in the isolated vascularly perfused rat colon
Authors: Plaisancié P , Dumoulin V , Chayvialle J-A , Cuber J-C
Published: December 2004

Peptide YY (PYY) is produced in endocrine L cells primarily localized in the distal bowel. These open-type L cells make contact with the intestinal chyme which may thus affect their secretory activity. The aim of the present study was to examine a large variety of luminal compounds found in colonic contents for their potential as PYY-releasing factors, using the isolated vascularly perfused rat colon. The release of PYY into the portal effluent was measured by a specific RIA. Luminal administration of 5 mm glucose or 0·5% (w/v) starch for 30 min did not induce significant release of PYY. Oleic acid (10 and 100 mm) also did not significantly increase PYY secretion. A pharmacological concentration of glucose (250 mm) and a mixture of amino acids (total concentration 250 mm) both induced PYY secretion (200% of basal). Pectin, a polygalacturonic acid, evoked dose-dependent secretion of PYY-like immunoreactivity over the range 0·1–0·5% (w/v). The maximal response was observed after infusion of 0·5% pectin which induced a prompt and sustained release of PYY (300% of basal). Galacturonic acid itself (5%) produced marked PYY secretion. Gum arabic (0·5%) induced a gradual increase in portal PYY concentration (maximal response 250% of the basal value) whereas cellulose (0·5%) did not elicit PYY secretion. Luminal n-butyrate over the range 0·5–5 mm produced a dose-dependent release of PYY (maximal response 300% of the basal value with 5 mm n-butyrate). Increasing the concentration of n-butyrate to 100 mm provoked a gradual decrease in PYY secretion. Propionate was a less potent stimulant than n-butyrate, and acetate did not increase PYY secretion above the basal value. At a concentration of 2 or 20 mm, taurocholate, cholate and deoxycholate brought about PYY secretion while hyodeoxycholate was without effect.

In conclusion, glucose and amino acids may mediate PYY release but only when they are present at high supraphysiological concentrations in the colon while oleic acid does not produce any PYY secretion. Physiological concentrations of fibers (pectin, gum arabic), short-chain fatty acids (n-butyrate, propionate) and bile salts (taurocholate, cholate, deoxycholate) are all potent stimulants of PYY release. Whether the release of PYY by luminal factors is coupled to water and electrolyte transfer via a local/paracrine pathway remains an open question which requires additional work with the isolated vascularly perfused colon preparation.

Journal of Endocrinology (1996) 151, 421–429
Dopaminergic regulation of adrenocorticotrophic hormone, α-melanocyte-stimulating hormone and cortisol secretion in the ovine fetus
Authors: Hagan D M , Brooks A N
Published: December 2004

During ovine fetal development there is a progressive maturation of the hypothalamo–pituitary–adrenal axis which culminates in the onset of birth. The role and regulation of the intermediate lobe of the fetal pituitary gland in relation to this maturational process remains controversial. To test the hypothesis that the pars intermedia of the ovine fetal pituitary is under tonic inhibitory dopaminergic control we treated fetal sheep at day 131 of gestation with a 3-day intravenous infusion of one of the following: the dopamine antagonist sulpiride (0·3 mg/0·5 ml/h; n=12), the dopamine agonist bromocriptine (0·03 mg/0·5 ml/h; n=7) or vehicle (0·1 m tartaric acid in saline; n=8) alone. Fetal plasma concentrations of α-MSH were significantly (P<0·01) increased by treatment with sulpiride and decreased (P<0·05) by bromocriptine. The increase in α-MSH after sulpiride was characterised by an increase in the amplitude of α-MSH pulses whereas bromocriptine virtually abolished all pulses of α-MSH. Immunoreactive ACTH (IR-ACTH) concentrations were significantly (P<0·05) elevated after sulpiride but were unaffected by bromocriptine. There were no changes in the pulsatile characteristics of IR-ACTH secretion. Cortisol concentrations were unchanged by either treatment. We conclude that fetal α-MSH and IR-ACTH are secreted in a pulsatile fashion and are tonically inhibited by dopaminergic pathways. The lack of an effect of endogenously raised α-MSH on plasma cortisol concentrations provides evidence that this POMC-derived peptide is not responsible for the regulation of cortisol biosynthesis and/or secretion.

Journal of Endocrinology (1996) 151, 439–447