bluuebunny/crossref_metadata_2025 huggingface dataset:
Age-related discrepancies between serum and pituitary gonadotrophin, and pituitary gonadotrophin subunit mRNA responses to castration and testosterone replacement in male rats
Authors: Pakarinen P. , Huhtaniemi I.
Published: December 2008

The responses of gonadotrophin gene expression, pituitary content and serum levels to castration alone and castration plus testosterone replacement (silicone elastomer implants) were compared in male rats at 10, 30, 60 and 90 days of age. Sham-operated animals served as controls. In addition, 30-day-old castrated rats were treated with dihydrotestosterone (DHT) and diethylstilboestrol (DES). When killed 7 days after castration, the increases in serum LH (six- to eightfold; P < 0·01) and FSH (two- to fourfold; P < 0·01) were similar at all ages studied. Likewise, testosterone reversed the effects of castration in a largely similar fashion at all ages. In contrast, great age-related differences were observed in the responses of gonadotrophin subunit mRNAs to the treatments. Castration increased the common α subunit mRNA two- to fourfold on days 10 and 30 (P < 0·01), sixfold on day 60 (P < 0·01), but not at all on day 90. Testosterone reversed the increases at all ages, but the levels were below those of controls only at 90 days (P < 0·01). The highest increases (sixfold; P < 0·01) of LH-β mRNA were seen on days 10 and 60, the others being two- to threefold higher (P < 0·05–0·01). Testosterone reversed this effect at 60 days and suppressed LH-β mRNA to below the control levels at other ages (P < 0·01). Castration had no effect on FSH-β subunit mRNA at 30 and 90 days but a four-to fivefold increase was seen on days 10 and 60 (P < 0·01). Testosterone suppressed these mRNAs at all ages, and they decreased to below the levels in controls at 30 and 60 days. Testosterone, DHT and DES had, at 30 days, practically the same effects on the LH parameters, whereas DHT was clearly less effective than testosterone and DES in suppressing those of FSH. In conclusion, although there was, in general, good agreement between gonadotrophin mRNA and serum levels in response to castration and testosterone replacement, there were specific ages when the post-castration increases in FSH and/or LH occurred with no detectable change in the respective mRNA levels. These findings indicate that altered transcription (or mRNA stability) is not solely responsible for the responses of the gonadotrophins to altered gonadal feedback, but that changes in translation efficiency and/or serum gonadotrophin stability are involved at specific ages of development.

Journal of Endocrinology (1992) 135, 507–515
Pituitary-dependent hyperadrenocorticism in a family of Dandie Dinmont terriers
Authors: Scholten-Sloof B. E. , Knol B. W. , Rijnberk A. , Mol J. A. , Middleton D. J. , Ubbink G. J.
Published: December 2008

Adrenocortical function studies were performed in seven Dandie Dinmont terriers with pituitary-dependent hyperadrenocorticism. The ability of dexamethasone at a dose rate of 0·1 mg/kg body weight to suppress cortisol secretion was only moderate in four out of the six dogs tested. Concentrations of α-melanocyte-stimulating hormone in plasma were highly increased. Responses to stimulation with corticotrophin-releasing hormone and the dopamineantagonist haloperidol, examined in three animals, were moderate or absent. These results indicate that adrenocortical stimulation, i.e. hyperadrenocorticotrophism, was caused by pituitary lesions which were functioning autonomously.

In six of the seven animals there was a very close familial relationship and the coefficients of relationship and the coefficients of inbreeding were significantly higher than in a representative control population.

It was concluded that these seven related terriers with hyperadrenocorticotrophism had the biochemical characteristics of de-novo neoplasms of proopiomelanocortin-producing cells, and there was evidence for a genetic involvement in tumorigenesis.

Journal of Endocrinology (1992) 135, 535–542
Distribution and properties of endo-oligopeptidases A and B in the human neuroendocrine system
Authors: Medeiros M. S. , Iazigi N. , Camargo A. C. M. , Oliveira E. B.
Published: December 2008

The thiol activated endo-oligopeptidases A and B were studied in the soluble fraction of human hypothalamus and various endocrine glands. For the identification, characterization and purification of the enzymes, Z-Gly-Pro-NH-Np and bradykinin were used as substrates. Endo-oligopeptidase B showed a molecular mass ranging from 55·5 to 65·5 kDa and isoelectric point from 4·7 to 4·95. Its activity in tissues was highest in the testis, with intermediate levels in the thyroid, neurohypophysis, adenohypophysis and hypothalamus and the lowest activity in the pineal gland. Endo-oligopeptidase A, 467-fold purified by immunoaffinity chromatography, exhibited a molecular mass of 65·5 kDa in the adenohypophysis but 58·5 kDa in other tissues. The isoelectric point ranged from 5·22 to 5·50. High endo-oligopeptidase A activity was observed in the adenohypophysis, testis and hypothalamus with lesser activity in the neurohypophysis and thyroid and the lowest in the pineal gland. Endo-oligopeptidase A cleaved the bonds Phe-Ser of bradykinin, Met-Arg of BAM-12P and Arg-Arg of neurotensin as described for rabbit brain and heart and bovine brain enzymes. This work shows that endo-oligopeptidase A also hydrolysed the bonds Tyr-Gly of LH-releasing hormone, Pro-Phe of angiotensin I and Tyr-Ile of angiotensin II.

Journal of Endocrinology (1992) 135, 579–588
Further studies on the mode of action of calcitonin on isolated rat osteoclasts: pharmacological evidence for a second site mediating intracellular Ca2+ mobilization and cell retraction
Authors: Alam A. S. M. T. , Bax C. M. R. , Shankar V. S. , Bax B. E. , Bevis P. J. R. , Huang C. L.-H. , Moonga B. S. , Pazianas M. , Zaidi M.
Published: December 2008

Calcitonin is a circulating polypeptide that inhibits bone resorption by inducing both quiescence (Q effect) and retraction (R effect) in osteoclasts. Two structurally related members of the calcitonin gene peptide family, calcitonin gene-related peptide (CGRP) and amylin, inhibit osteoclastic bone resorption selectively via the Q effect. In the present study, we have made measurements of cell spread area in response to the application of amylin, CGRP and a peptide fragment of CGRP, CGRP-(Val8Phe37). We found that, over a wide concentration range (50 pmol/l to 2·5 μmol/l), the selective Q effect agonists did not produce an R effect. Furthermore, the peptides, when used at a 50-fold higher molar concentration than calcitonin, did not antagonize calcitonin-induced cell retraction. Additionally, experiments designed to measure changes in the intracellular free calcium concentration ([Ca2 + ]i) in single osteoclasts revealed that, unlike calcitonin, the non-calcitonin Q effect agonists did not produce a rise in [Ca2+]i. The peptides were also unable to attenuate the peak rise in [Ca2+]i induced by calcitonin. The results support our hypothesis that the inhibitory activity of calcitonin on osteoclastic bone resorption is mediated by two sites which may or may not be part of the same receptor complex. One of these is the classical Q effect site coupled to adenylate cyclase via a cholera toxin-sensitive Gs. This site can be activated by nanomolar concentrations of calcitonin, amylin, CGRP or CGRP-(Val8Phe37). A novel R effect site, possibly coupled via a pertussis toxin-sensitive G protein to a [Ca2+]i elevating mechanism is predicted from this study. The site is highly specific for calcitonin and is not activated, even at micromolar concentrations, by amylin, CGRP or CGRP-(Val8Phe37). Whether or not the two sites are part of the same receptor complex or are two receptor subtypes remains to be determined.

Journal of Endocrinology (1993) 136, 7–15
Evidence that inhibin is an important factor in the regulation of FSH secretion during the mid-luteal phase in cows
Authors: Kaneko H. , Nakanishi Y. , Taya K. , Kishi H. , Watanabe G. , Sasamoto S. , Hasegawa Y.
Published: December 2008

To investigate the physiological importance of inhibin in the regulation of FSH secretion in cows, seven cyclic cows were treated with an inhibin antiserum raised against bovine 32 kDa inhibin in a castrated goat. The same animals treated with a castrated goat serum (control serum) served as controls. On day 12 of the oestrous cycle (day 0 = day of oestrus), four of seven cows were injected with 100 ml inhibin antiserum first, and the remaining three cows with 100 ml control serum first. Twelve days after the second oestrus following the first serum injection (42–46 days after the first serum injection), the former four cows were injected with control serum and the latter three with inhibin antiserum. Follicular development after the injections of control serum or inhibin antiserum was assessed by daily ultrasonographic examination.

Treatment with inhibin antiserum resulted in a marked increase (P < 0·01) in plasma concentrations of FSH and oestradiol-17β but not LH or progesterone, compared with those after treatment with control serum. Plasma concentrations of FSH increased significantly (P < 0·01) at 8 h after injection of antiinhibin serum when compared with the control value. Concentrations of FSH in the plasma remained high for 72 h, then declined to the control level by 84 h, concomitant with an abrupt decrease in the titre of free inhibin antibody in the plasma. High concentrations of oestradiol-17β were observed between 36 and 96 h after treatment. Treatment with inhibin antiserum markedly increased the number of small (≥ 4 < 7 mm in diameter), medium (≥ 7 < 10 mm) and large (≥ 10 mm) follicles by 48, 72 and 96 h after treatment when compared with the value before treatment. The number of large follicles returned to the pretreatment value at 168 h, whereas the number of small and medium follicles remained increased.

The present results provide strong evidence that inhibin is an important factor in the inhibitory regulation of FSH secretion during the mid-luteal phase of cows, and demonstrate that an increase in endogenous FSH secretion after immunoneutralization of circulating inhibin stimulates the rapid growth of a large number of follicles.

Journal of Endocrinology (1993) 136, 35–41
Effect of 3,3′-di-iodothyronine and 3,5-di-iodothyronine on rat liver mitochondria
Authors: Lanni A. , Moreno M. , Cioffi M. , Goglia F.
Published: December 2008

In the present study we report that 3,3′,5-tri-iodothyronine (T3) as well as two iodothyronines (3,5-diiodothyronine (3,5-T2) and 3,3′-di-iodothyronine (3,3′-T2)) significantly influence rat liver mitochondrial activity.

Liver oxidative capacity (measured as cytochrome oxidase activity/g wet tissue) in hypothyroid compared with normal rats was significantly reduced (21%, P > 0·01) and the administration of T3 and both iodothyronines restored normal values. At the mitochondrial level, treatment with T3 stimulated respiratory activity (state 4 and state 3) and did not influence cytochrome oxidase activity. On the other hand, both the mitochondrial respiratory rate and specific cytochrome oxidase activity significantly increased in hypothyroid animals after treatment with 3,3′-T2 or 3,5-T2 (about 50 and 40% respectively). The actions of both iodothyronines were rapid and evident by 1 h after the injection. The hepatic mitochondrial protein content which decreased in hypothyroid rats (9·6 mg/g liver compared with 14·1 in normal controls, P < 0·05) was restored by T3 injection, while neither T2 was able to restore it.

Our results suggest that T3 and both iodothyronines have different mechanisms of action. T3 acts on both mitochondrial mass and activity; the action on mitochondrial activity was not exerted at the cytochrome oxidase complex level. The action of the iodothyronines, on the other hand, is exerted directly on the cytochrome oxidase complex without any noticeable action on the mitochondrial mass.

Journal of Endocrinology (1993) 136, 59–64
Oestradiol negative feedback inhibition on LH secretion during lactation is prolonged in adolescent primiparous rhesus monkeys
Authors: Wilson M. E.
Published: December 2008

Lactational infertility in rhesus monkeys is significantly prolonged in adolescent primiparous compared with adult multiparous mothers. In order to determine if this longer period of infertility for young mothers is the result of a greater sensitivity to nursing-induced inhibition of LH release either by enhanced oestradiol negative feedback or a direct non-gonadally mediated suppression, the effects of periodic administration of oestradiol on serum LH concentrations in nursing ovariectomized adolescent primiparous (Prp; n = 5) and adult multiparous (Mlt; n = 7) mothers was assessed. Females were treated every 5–6 weeks with a 21-day time-release capsule of oestradiol which produced serum concentrations of approximately 250, 90 and 45 pmol/l by +6, +13 and +20 days after treatment. Thus, the design permitted assessment of LH and prolactin concentrations under a regime of 21 days of decreasing oestradiol levels followed by 2–3 weeks of no oestradiol treatment. Females were studied from week 3 to week 42 post partum and oestradiol treatment occurred during weeks 5, 11, 21, 26, 31, 36 and 41.

Behavioural observations indicated that the amount of time mothers nursed their offspring decreased in a similar fashion throughout the lactational period for both Prp and Mlt females. LH concentrations under the 'no oestradiol' conditions progressively increased throughout lactation reaching maximum levels by week 36 post partum in a similar manner for both Prp and Mlt mothers. These data suggest that differences in fertility between adolescent and adult nursing mothers observed previously cannot be attributed to a difference in a direct non-gonadally-mediated affect on LH.

With respect to oestradiol negative feedback inhibition of LH, oestradiol treatment effectively suppressed serum LH concentrations at all points during lactation up to week 31, at which time LH concentrations were maximally suppressed in both Prp and Mlt mothers at + 6 days after treatment but by day + 13 LH values were significantly higher in Mlt females. After oestradiol treatment during week 36, LH values were again maximally suppressed on day +6 in Prp females but not in Mlt females. However, the response of serum LH after oestradiol treatment during week 41 was similar between Prp and Mlt females at all sampling points. Thus, the effectiveness with which oestradiol suppressed LH concentrations diminished at an earlier point in lactation in Mlt compared with Prp mothers. Furthermore, this parity/age difference in oestradiol negative feedback inhibition of LH was not associated with differences in nursing behaviour. These data suggest that the prolonged period of lactational infertility in adolescent primiparous monkey mothers is the result of a hypersensitivity to oestradiol negative feedback inhibition of LH which is still functional at a time when the nursing stimulus has diminished.

Journal of Endocrinology (1993) 136, 127–136
Cell interactions in initiation of mammary epithelial proliferation by oestradiol and progesterone in prepubertal heifers
Authors: Woodward T. L. , Beal W. E. , Akers R. M.
Published: December 2008

The acute temporal effects of exogenous oestradiol (0·1 mg/kg per day), progesterone (0·25 mg/kg per day) or both together on the proliferative response of epithelial cells, fibroblasts, adipocytes and endothelial cells in the mammary tissue of prepubertal cross-bred heifers were determined. Mammary biopsies were taken immediately before, then 24, 48 and 96 h after the initiation of daily administration of hormones to three heifers per treatment group. Incorporation of [3H]thymidine into explants prepared from biopsies was evaluated after a 1-h incubation by measuring trichloroacetic acid (TCA)-insoluble radioactivity in explant homogenates as well as by quantitative histoautoradiography. Incorporation expressed as d.p.m./mg tissue or d.p.m./μg DNA was increased (P ≤ 0·05) approximately 11-fold by 96 h in oestradiol-treated heifers. Progesterone-treated animals were unresponsive and heifers treated with both hormones were intermediate in response compared with oestradiol-treated heifers. Autoradiographic data for ductal or terminal duct epithelial cells showed similar dramatic increases in labelling by 24 h with further increased (P < 0·01) labelling by 96 h (5·1% vs 0·1%) in heifers given oestradiol. As with incorporation, tissue from progesterone-treated heifers showed no time or treatment response compared with pretreatment biopsies and tissue from heifers given both showed intermediate responses, i.e. significantly increased labelling by 96 h compared with pretreatment (P ≤ 0·05) but less labelling (P < 0·05) than oestradiol-treated heifers. A proliferative response of epithelial cells in oestradiol-treated heifers occurred prior to the response of fibroblasts adjacent to epithelial cells (≤ 50 μm). Interestingly, labelling of endothelial cells was also markedly increased by 48 h in oestradiol-treated heifers. Non-adjacent fibroblasts (≥ 100 μm from ductal epithelial cells) and adipocytes showed no time or treatment response. These data demonstrate that exogenous oestradiol markedly and acutely stimulates proliferation of mammary ducts, while progesterone has no effect and may decrease the effectiveness of oestradiol. In contrast to mice, mammary epithelial response to oestradiol in prepubertal heifers is not first exhibited after a proliferative response of stromal cells. This suggests that the cell-type interactions involved in the control of mammogenesis are dissimilar in ruminants and rodents.

Journal of Endocrinology (1993) 136, 149–157
Reproducibility of the cortisol response to stimulation with a low dose of ACTH(1–24): the effect of basal cortisol levels and comparison of low-dose with high-dose secretory dynamics
Authors: Crowley S. , Hindmarsh P. C. , Honour J. W. , Brook C. G. D.
Published: December 2008

We compared the reproducibility and repeatability of the acute adrenal response to low doses (90 and 500 ng/1·73 m2) of Synacthen (ACTH(1–24)) with that of the standard dose (250 μg/1·73 m2). We also examined the effect of basal cortisol levels on peak values achieved after stimulation with a low dose.

ACTH(1–24) was given to six male volunteers: 90 ng/1·73 m2 twice at 90-min intervals on day 1, and 90 and 500 ng/1·73 m2 once on day 2 and 250 μg/1·73 m2 once on day 3. The rise in serum cortisol concentration with repeated low doses of ACTH was not attenuated (161 ± 49 (s.d.) nmol/l on initial vs 150 ± 41 nmol/l on repeat stimulation; P = 0·5) and this was reproducible (161 ± 49 nmol/l on day 1 vs 148 ± 15 nmol/l on day 2; P = 0·6). A dose of 500 ng ACTH(1–24)/1·73 m2 produced a maximal adrenal response in that the rise in serum cortisol concentration at 20 min was identical with that produced at the same time by the standard dose of 250 μg/1·73 m2. There was a strong positive correlation between the basal cortisol level and peak cortisol concentration after low-dose ACTH stimulation (r = 0·93, P < 0·001) but not between the basal cortisol level and the incremental rise (r= −0·1, P = 0·69).

These results suggest that the cortisol response to low-dose ACTH stimulation is reproducible and not attenuated by repeat stimulation at 90-min intervals. The incremental rise in serum cortisol concentration after ACTH stimulation appears constant in these situations and is not influenced by the basal cortisol level. When there is concern that the standard dose may be excessive and mask subtle but important changes in adrenal function, the low dose (500 ng) of ACTH should be used.

Journal of Endocrinology (1993) 136, 167–172
Comparison of the effects of insulin-like growth factors-I and -II on the human osteosarcoma cell line OHS-4
Authors: Fournier B. , Ferralli J. M. , Price P. A. , Schlaeppi J. M.
Published: December 2008

The effects of insulin-like growth factor-I (IGF-I) and IGF-II on the human osteoblast cell-line OHS-4 were investigated. Both IGF-I and IGF-II stimulated cell proliferation at nanomolar concentrations and alkaline phosphatase activity was decreased in a dose-dependent manner with either IGF-I or IGF-II. The production of the bone-specific protein osteocalcin was not influenced by either IGF-I or IGF-II. However, they acted synergistically with 1,25-dihydroxyvitamin D3 at concentrations ranging from 10 to 100 nmol/l. Neither IGF-I nor IGF-II had an effect on either the basal or the parathyroid hormone-stimulated level of adenylate cyclase activity, and likewise they had no effect on phosphodiesterase activity. Binding and cross-linking experiments confirmed the presence of both type-I and type-II IGF receptors on the OHS-4 cells. The present study shows that IGF-I and IGF-II have similar effects on the parameters studied in these osteoblastic cells. They influenced both proliferation and differentiation markers.

Journal of Endocrinology (1993) 136, 173–180
Corticotrophin-releasing factor as a mediator of the acute-phase response in rats, mice and rabbits
Authors: Hagan P. M. , Poole S. , Bristow A. F.
Published: December 2008

The acute-phase response involves a number of separate physiological components, including induction of acute-phase protein synthesis by the liver. This response can be induced in vivo by administration of the endogenous leucocytic mediator interleukin-1β. A number of in-vivo effects of interleukin-1β have been reported to be mediated by corticotrophin-releasing factor (CRF), including activation of the hypothalamo-pituitary-adrenal axis and induction of fever, and in this report we have examined a possible involvement of CRF in mediating interleukin-1β-induced acute-phase protein synthesis.

Interleukin-1β stimulated the elevation of species-specific plasma acute-phase proteins in rats, mice and rabbits. Co-injection of interleukin-1β with the specific CRF receptor antagonist α-helical-CRF9–41 NH2 abolished or attenuated acute-phase protein synthesis induced by interleukin-1β in all three species for up to 12 h after injection. The inhibitory effect of α-helical-CRF9–41NH2 was reduced or absent 24 h after injection. Neutralizing anti-CRF antisera had no effect on acute-phase protein synthesis in the mouse and, paradoxically, potentiated acute-phase protein synthesis induced by interleukin-1β in the rat. These results indicate a possible mediatory role for CRF in regulation of acute-phase protein synthesis, and suggest that CRF may mediate induction of acute-phase protein synthesis by a different mechanism from that involved in regulation of corticotrophin secretion.

Journal of Endocrinology (1993) 136, 207–216
Arginine vasopressin-induced natriuresis in the anaesthetized rat: involvement of V1 and V2 receptors
Authors: Smith C. P. , Balment R. J.
Published: December 2008

The present study was undertaken to determine the involvement of the two established vasopressin receptor subtypes (V1 and V2) in arginine vasopressin (AVP)-induced natriuresis and also to determine whether changes in mean arterial pressure (MAP) and/or the renally active hormones atrial natriuretic peptide (ANP), angiotensin II (AII) and aldosterone are a prerequisite for the expression of AVP-induced natriuresis.

In Sprague–Dawley rats which were anaesthetized with Inactin (5-ethyl-5-(1′-methylpropyl)-2-thiobarbiturate) and infused with 0·077 mol NaCl/l, infusion of 63 fmol AVP/min was found to be natriuretic whereas an approximately equipotent dose of the specific V2 agonist [deamino-cis1, d-Arg8]-vasopressin (dDAVP) did not induce natriuresis. The specific V1 antagonist [β-mercapto-β,β-cyclopenta-methylene-propionyl1, O-Me-Tyr2, Arg8]-vasopressin when administered prior to infusion of 63 fmol AVP/min did not inhibit AVP-induced natriuresis. AVP-induced natriuresis was not accompanied by changes in MAP or in the plasma concentrations of the renally active hormones ANP, AII or aldosterone.

These results suggest that neither the V1 nor the V2 receptor subtypes are involved in AVP-induced natriuresis. In addition, it was found that changes in MAP, plasma ANP, All or aldosterone concentrations were not a prerequisite for AVP-induced natriuresis.

Journal of Endocrinology (1993) 136, 283–288
Receptor binding of insulin-like growth factor-I to mammary microsomes from non-pregnant, pregnant and lactating sheep
Authors: Winder S. J. , Wheatley S. D. , Forsyth I. A.
Published: December 2008

Sucrose density centrifugation was used to prepare a partially purified membrane fraction from the mammary glands of non-pregnant, pregnant and lactating sheep. The binding of125 I-labelled insulin-like growth factor-I (IGF-I) was dependent on membrane protein concentration, pH, time and temperature. The binding showed the characteristics of a type-1 IGF receptor, being displaced by IGF-I (median effective dose (ED50) 0·55 nmol/l), less effectively by IGF-II (ED50 8·8 nmol/l) and least effectively by insulin. Glucagon, ovine prolactin and ovine placental lactogen could not displace binding. A molecular weight of 135 000 was determined by affinity cross-linking using disuccinimidyl suberate; this was consistent with the reported size of the type-1 receptor α-subunit. Scatchard analysis was used to determine binding affinity and numbers of IGF-I-binding sites. A single class of high-affinity binding sites was found in all physiological states. In non-pregnant sheep and sheep at days 40, 75 and 110–120 of pregnancy and at term, the binding affinity was similar (apparent dissociation constant (Kd) 2·73 ±0·31 nmol/l, n = 22). In lactating sheep (weeks 1, 4 and 10), the binding affinity was significantly (P = 0·02) higher (Kd 0·77± 0·06 nmol/l n = 9). Binding capacity was similar in non-pregnant and pregnant sheep (1005 ± 113 fmol/mg, n = 19), but fell by parturition and remained low in lactation (570±52 fmol/mg membrane protein, n = 12). The results suggest that the mammary growth of pregnancy is not regulated at the level of the type-1 IGF receptor.

Journal of Endocrinology (1993) 136, 297–304
Variations in oxytocin secretion during the 4-day oestrous cycle of the rat
Authors: Windle R. J. , Forsling M. L.
Published: December 2008

Oxytocin concentrations in the plasma, pituitary and hypothalamus of female rats were determined in the morning and evening over the 4-day oestrous cycle. Vasopressin concentrations were also determined to allow calculation of the ratios of the two hormones. The results were compared with those from male rats. Plasma oxytocin concentrations were significantly higher in the evening than in the morning on the day of oestrus. Although the evening concentration achieved was similar on each day of the cycle, morning plasma oxytocin concentrations showed a progressive rise from oestrus to pro-oestrus so that no significant diurnal increases were observed on the other days of the cycle. Vasopressin concentrations in the plasma were also seen to increase over the days of oestrus, dioestrus day 1 and dioestrus day 2. On pro-oestrus the plasma concentrations of vasopressin remained unchanged. The ratio of oxytocin:vasopressin fell during the light hours of the cycle. The hypothalamic content of both hormones showed a rise during the hours of daylight parallel to that seen in the plasma, whereas the pituitary content fell over the same period. The diurnal pattern of hormone release observed in male rats was similar to that in females at oestrus. However, the plasma oxytocin concentrations were significantly higher in the male.

The plasma clearance rate of vasopressin did not vary significantly during the oestrous cycle. However, the plasma clearance rate for oxytocin did show significant variation, being highest on dioestrus day 1 and lowest on dioestrus day 2.

Journal of Endocrinology (1993) 136, 305–311
Specificity studies of particulate binding sites for steroid hormones in subcellular fractions of the porcine corpus luteum
Authors: Bramley T. A. , Menzies G. S.
Published: December 2008

We have studied the binding of a number of radiolabelled steroids and lipophilic substances to porcine corpus luteum (CL) particulate fractions. Following preincubation of CL homogenates with radiolabelled progesterone or pregnenolone prior to fractionation on continuous sucrose density gradients, a broad peak of binding was observed associated with a particulate fraction of buoyant density 1·05–1·10 g/cm3. Progesterone content also peaked at a similar buoyant density (1·06–1·12 g/cm3). Pretreatment of luteal homogenates with digitonin perturbed the buoyant density of the progesterone-binding particulate fraction to 1·10–1·14 g/cm3 and sharpened the binding peak. Progesterone content was also perturbed to a similar extent by digitonin pretreatment, without release of the steroid. Oestrogens were also sequestered by this fraction, but steroid precursors (cholesterol, cholesterol ester), corticosteroids (cortisol, corticosterone), sterol conjugates (oestrone sulphate, pregnanediol glucuronide) and other lipophilic substances (arachidonic acid, phospholipid, prostaglandins E1, E2 and F2α) were not bound. Androgens were bound weakly by fractions from control gradients but, in the presence of digitonin, significant binding could be demonstrated. Radiolabelled steroids were shown to interact directly with luteal membrane fractions, rather than interacting first with cytosolic steroid receptors which then bound to membranes. Furthermore, [3H]progesterone was not bound by porcine granulosa cell particulate fractions. These observations suggest that this fraction may be involved in sequestration or packaging of progesterone for secretion by the luteal cell.

Journal of Endocrinology (1993) 136, 371–380
Paracrine effects via the epidermal growth factor receptor in the rodent testis may be mediated by non-Leydig interstitial cells
Authors: Moore A. , Morris I. D.
Published: December 2008

The epidermal growth factor (EGF) receptor is expressed in a wide variety of cell types and is known to be present in the testis of many species including man. In the present study, specific 125 I-labelled EGF binding was observed in isolated interstitial cell preparations from both the intact and Leydig cell-depleted rat testis. It was demonstrated that the population of cells to which 125I-labelled EGF binds has a different buoyant density from either of the two adult Leydig cell populations, and remains unchanged in the absence of Leydig cells following in-vivo treatment with ethane dimethane sulphonate (EDS). Cells of this density (1·064 g/ml) identified by electron microscopy were fusiform mesenchymal cells, identical to those suggested by others to be able to differentiate into Leydig cells in vitro, i.e. Leydig cell precursors. In a culture system using two interstitial cell preparations of different buoyant densities from immature rats, both EGF and transforming growth factor-α (TGF-α) caused increased [3H]thymidine incorporation in the less dense cell preparation. TGF-α was more potent than EGF. EGF increased testosterone production in both fractions in amounts which could be related to the amount of 3β-hydroxysteroid dehydrogenase (3β-HSD)-positive cells. This study demonstrated that rat Leydig cells (defined as those cells which bind 125I-labelled human chorionic gonadotrophin, have distinct buoyant densities, are 3β-HSD positive and are sensitive to EDS), do not bind 125I-labelled EGF. Rather, EGF binds to a mesenchymal cell without LH receptors which is resistant to EDS. Growth factors which act via the EGF receptor increased [3H]thymidine incorporation in a Leydig cell-depleted interstitial fraction which may reflect an action upon the progenitor of the mature Leydig cell.

Journal of Endocrinology (1993) 136, 439–446
Inhibitory effects of insulin-like growth factor (IGF)-binding proteins-1 and -3 on IGF-activated glucose consumption in mouse BALB/c 3T3 fibroblasts
Authors: Okajima T. , Iwashita M. , Takeda Y. , Sakamoto S. , Tanabe T. , Yasuda T. , Rosenfeld R. G.
Published: December 2008

We have examined the biological effects of insulinlike growth factor-binding proteins (IGFBPs) on insulin-like growth factor (IGF)-activated glucose consumption in a BALB/c 3T3 subline. The method employed was a colorimetric measurement of glucose consumption, allowing the detection of changes from the initial glucose concentration in conditioned medium, following the addition of IGFs and IGFBPs. Human IGFBP-1, purified from amniotic fluid, inhibited IGF-activated glucose consumption, although it had no effect on insulin-activated glucose consumption. The median effective dose (ED50) of IGFBP-1 to cause inhibitory effects on IGF-activated glucose consumption was 100–200 μg/l and was similar for both IGF-I and IGF-II at a concentration of 1·0 μg IGF/1. Therefore, at IGF concentrations of comparable activity, the inhibitory effects of IGFBP-1 were greater for IGF-I than for IGF-II, because of the higher activity of IGF-I in this assay. Recombinant human IGFBP-3 also inhibited IGF-activated glucose consumption, without affecting insulin-stimulated glucose consumption. The inhibitory effects of IGFBP-3 were greater for IGF-II than for IGF-I when IGFBP-3 was coincubated with either of the IGFs, at both IGF concentrations of comparable activity and equivalent molar concentrations. Thus, it became clear that the inhibitory effects of these IGFBPs on IGF biological action depended primarily upon their affinity for the specific IGF ligand and molar ratio of IGFBP/IGF peptide. Interestingly, when cells were pretreated with IGFBP-3, prior to the simultaneous addition of IGFs and IGFBP-3, the inhibitory effect was higher for IGF-I than for IGF-II. Either no effect or a minor inhibitory effect on IGFactivated glucose consumption was detected with IGFBP pretreatment alone. When the ED50 for inhibition of IGF action by IGFBPs in this in-vitro assay was compared with the physiological concentrations of IGFs and IGFBPs in normal human serum and in amniotic fluid, it was estimated that the IGFBP-1 concentration present in serum was not sufficient to modulate IGF action effectively while the concentration in amniotic fluid was enough for effective suppression. IGFBP-3 exhibited an ED50 low enough to suppress IGF-II and possibly IGF-I action when cells were pretreated with IGFBP-3. Thus, our data suggested that IGFBP-1 in amniotic fluid and IGFBP-3 in serum could be a potent inhibitor for IGF action. IGFBP-1 in serum, however, may not be able to function as a direct inhibitor under physiological conditions but, rather, may modulate IGF action together with other IGFBPs.

Journal of Endocrinology (1993) 136, 457–470
Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins
Authors: Phaneuf S. , Europe-Finner G. N. , Varney M. , MacKenzie I. Z. , Watson S. P. , López Bernal A.
Published: December 2008

Phosphoinositide hydrolysis is important in mediating the actions of oxytocin and prostaglandin (PG) F2α on uterine contractions during labour. We have measured the effect of oxytocin, PGF2α and other agents on the formation of inositol phosphates (IPs) in cultured human myometrial cells labelled with [3H]inositol and on changes in intracellular free Ca2+ concentration ([Ca2 + ]i) in cells loaded with Fura-2.

Oxytocin induced the formation of [3H]IPs in a concentration-dependent manner with an EC50 (concentration of agonist producing 50% of the maximal response) of 1·4 ±0·5 nmol/l (mean ± s.e.m.). The maximal response was obtained with 1 μmol oxytocin/l and represented a stimulation of 670% over basal. PGF2α also stimulated the formation of [3H]IPs and the response at 1 μmol/l was a 204% stimulation over basal. The effects of PGF2α were independent of extracellular Ca2 + but the effect of oxytocin was reduced with low extracellular Ca2 +. Cyclic AMP formation, induced by forskolin or PGE2, had no effect on the stimulated levels of [3H]IPs. Pertussis toxin (PT) reduced the oxytocin-stimulated formation of [3H]IPs in a concentration-dependent manner. The maximal effect of PT resulted in an 80% reduction in the formation of [3H]IPs. However, PGF2α stimulation was not affected by PT treatment.

To analyse the action of PT further, we studied its effect on oxytocin-induced changes in [Ca2 + ]i. The basal [Ca2 +]i was 112 ±4 nmol/l (n=225 cells) and was not affected by PT treatment (109 ± 3 nmol/l; n= 200 cells). In the absence of PT, 1 μmol oxytocin/l increased [Ca2 + ]i to a peak of 522 ±26 nmol/l, and in PT-treated cells, the [Ca2 + ]i peak was reduced to 348 ± 16 nmol/l. Similar inhibitory effects of PT were obtained at oxytocin concentrations ranging from 1 to 100 nmol/l.

Our data suggest that in human myometrial cells, the oxytocin-induced production of [3H]IPs and increase in [Ca2 + ]i are mediated by a PT-sensitive G-protein. However, a significant fraction of the oxytocin response appears to be mediated by a PT-insensitive G-protein, possibly a member of the Gq family.

Journal of Endocrinology (1993) 136, 497–509
Involvement of inhibin in the regulation of FSH secretion in prepubertal bulls
Authors: Kaneko H. , Yoshida M. , Hara Y. , Taya K. , Araki K. , Watanabe G. , Sasamoto S. , Hasegawa Y.
Published: December 2008

To investigate the physiological importance of inhibin in the regulation of FSH secretion in prepubertal bulls, animals (6-month-old) were passively immunized against inhibin. Five animals were given an i.v. bolus injection of 50 ml inhibin antiserum raised against bovine 32 kDa inhibin in a castrated male goat, and four bulls were given the same amount of castrated male goat serum (control serum) as controls. Treatment with the inhibin antiserum resulted in a marked increase (P < 0·01) in plasma concentrations of FSH within 12 h compared with control animals, and FSH levels in immunized animals remained high until 168 h after the injection. Concentrations of plasma LH and testosterone in the immunized animals were not different from those in the control animals. The present findings provide strong evidence that inhibin plays an important role in the inhibitory regulation of FSH secretion in prepubertal bulls.

Journal of Endocrinology (1993) 137, 15–19
Opioids and testicular activity in the frog, Rana esculenta
Authors: Facchinetti F. , Genazzani A. R. , Vallarino M. , Pestarino M. , Polzonetti-Magni A. , Carnevali O. , Ciarcia G. , Fasano S. , D'Antonio M. , Pierantoni R.
Published: December 2008

The presence and activity of brain, pituitary and testicular β-endorphin (β-EP)-like material have been studied in the frog, Rana esculenta, using reverse-phase high-pressure liquid chromatography, coupled with radioimmunoassay and immunocytochemistry. In-vivo and in-vitro treatments with naltrexone were carried out to assess the putative physiological activity of opioid peptides.

β(1–31) and (1–27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, β-EP(1–31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. β-EP immunoreactivity was present in the brain within the nucleus preopticus and nucleus infundibularis ventralis while positive fibres in the retrochiasmatic regions projected to the median eminence. In the testis, interstitial cells, canaliculi of the efferent system, spermatogonia and spermatocytes showed positive immunostaining for β-EP. In intact animals, naltrexone treatment increased plasma and testicular androgen levels and this effect was confirmed in in-vitro incubations of minced testes. Naltrexone also induced a significant increase in germ cell degeneration.

Our results indicated that an opioid system modulates the hypothalamus-pituitary-gonadal axis in the frog, Rana esculenta and, for the first time, we have shown that the testicular activity of a non-mammalian species may be regulated by opiates locally.

Journal of Endocrinology (1993) 137, 49–57
Pro-oestrous-specific role for polyamines in mediating the secretion of prolactin induced by thyrotrophin-releasing hormone in the rat
Authors: Wynne-Jones G. A. , Gurney A. M.
Published: December 2008

The activity of ornithine decarboxylase (ODC) in the rat anterior pituitary gland varies during the oestrous cycle, with a rise in activity seen at pro-oestrus. This enzyme, which is rate-limiting for the synthesis of the polyamines, can be specifically and irreversibly blocked by α-difluoromethylornithine (DFMO). A previous study showed that when this drug was administered to rats in vivo on the afternoon of pro-oestrus, it suppressed the normal surge in plasma prolactin levels that occurred later that day. The effect of DFMO was associated with reduced levels of putrescine in the anterior pituitary gland, suggesting that ODC activity in the lactotroph might be involved in the prolactin surge. We have examined the effects of DFMO on the secretion of prolactin from anterior pituitary cells, isolated either from male rats or from females at different stages of the oestrous cycle. The drug was found to reduce prolactin secretion stimulated by thyrotrophin-releasing hormone (TRH), but only in cells isolated from pro-oestrous animals and only for 2 days after cell isolation. Basal secretion was unaffected by DFMO. The results imply that ODC is important for TRH-stimulated prolactin secretion at pro-oestrus, and it is specific for pro-oestrus. The prolactin surge could therefore be influenced by this ODC-dependent effect of TRH. The pro-oestrous-specific response to TRH may be a consequence of the increased ODC activity seen at this time. Alternatively, the increased ODC activity could be a consequence of coupling to TRH receptors, which are known to increase in number at pro-oestrus.

Journal of Endocrinology (1993) 137, 133–139
Effects of melanocyte-stimulating hormone on tyrosinase expression and melanin synthesis in hair follicular melanocytes of the mouse
Authors: Burchill S. A. , Ito S. , Thody A. J.
Published: December 2008

Tyrosinase mRNA, synthesis and activity were measured in the skin during the first 2 weeks of life of C3H-HeAvy mice. Tyrosinase mRNA levels were found to peak on days 3–4 and were followed by increases in tyrosinase synthesis and activity which peaked on days 6–7 and 7–8 respectively. These changes in tyrosinase expression were presumably associated with the growth of the first coat of hair that in neonatal C3H-HeAvy mice is yellow in colour as a result of the increased proportion of phaeomelanin. By the time hair growth had ceased there was no expression of tyrosinase at both mRNA and protein levels. Daily administration of α-melanocyte stimulating hormone (α-MSH) enhanced the expression of tyrosinase mRNA transcripts, tyrosinase synthesis and activity. The increase in tyrosinase activity paralleled the change in the amount of tyrosinase, suggesting that the primary action of α-MSH is to stimulate new synthesis of the enzyme. This induction of tyrosinase was associated with the growth of hair that was darker in colour than that of the controls and contained an increased proportion of eumelanin. This increase in eumelanin reflected a decrease in phaeomelanin content. It was concluded that, through its actions on the enzyme tyrosinase, α-MSH is able to switch the synthesis of phaeomelanin to that of eumelanin in hair follicular melanocytes.

Journal of Endocrinology (1993) 137, 189–195
The effects of cortisol on hepatic and renal gluconeogenic enzyme activities in the sheep fetus during late gestation
Authors: Fowden A. L. , Mijovic J. , Silver M.
Published: December 2008

The effects of cortisol on hepatic and renal gluconeogenic enzyme activities were investigated in sheep fetuses during late gestation and after experimental manipulation of plasma cortisol levels by fetal adrenalectomy and exogenous infusion of cortisol. Hepatic and renal gluconeogenic enzyme activities increased with increasing gestational age in parallel with the normal rise in fetal cortisol levels towards term (146± 2 days). For the majority of enzymes this increase in activity towards term was prevented when the prepartum cortisol surge was abolished by fetal adrenalectomy and stimulated prematurely in fetuses younger than 130 days by exogenous infusion of cortisol. When the data from all the fetuses were combined irrespective of treatment or gestational age, there were significant positive correlations between the log plasma cortisol concentration in utero and the activities of glucose-6-phosphatase, fructose diphosphatase, phosphoenolpyruvate carboxykinase and aspartate transaminase in the fetal liver and kidney, and pyruvate carboxylase in the fetal liver but not in the kidney. No correlation was observed between log plasma cortisol and alanine aminotransferase activity in either fetal liver or kidney. These findings show that cortisol is a physiological regulator of most of the fetal gluconeogenic enzymes and enhances the glucogenic capacity of the sheep fetus during late gestation.

Journal of Endocrinology (1993) 137, 213–222
Angiotensin II (AII) and adrenal gland AII receptors in rats with congenital hypothyroidism
Authors: Matilla M. J. , Montiel M. , Jiménez E.
Published: December 2008

Plasma angiotensin II (AII) concentration, plasma renin concentration (PRC), lung angiotensin-converting enzyme (ACE) activity and adrenal gland AII receptor isoforms have been evaluated in the postnatal development of the rat, in order to determine the alterations of the renin-angiotensin system (RAS) which occur in congenital hypothyroidism. The developmental AII profiles observed in control and hypothyroid rats were quite similar. Thus, AII was elevated at birth and declined at week 5 of life to reach adult values. However, AII levels were lower in hypothyroid than in euthyroid animals before their stabilization. On the other hand, PRC in newborn hypothyroid and euthyroid animals was higher than in adulthood, being significantly increased immediately after birth in hypothyroid rats. Pulmonary ACE activity in both experimental groups was low at birth and increased with age. This increase was greater in euthyroid rats than in congenitally hypothyroid animals from day 28 of life. These results confirm that plasma AII levels in rats are predominantly controlled by plasma renin activity, although other factors, such as renin substrate availability, may be responsible for the reduced plasma AII concentration in congenital hypothyroidism during the first weeks of life.

The developmental profile of the adrenal gland AII receptor showed four isoforms, with pI values of 6·8, 6·7, 6·5 and 6·3. AII receptor-complex expression increased with age but, in congenitally hypothyroid rats, a higher induction of AII receptor isoforms migrating to pI 6·8, 6·5 and 6·3 was observed.

These findings show that thyroid hormone deficiency in early life can have an important role in the postnatal induction of the RAS components, which may be essential for the regulation of blood pressure, and for fluid and electrolyte balance in developing rats.

Journal of Endocrinology (1993) 137, 231–238