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Biomedical subjects

D M Robertson

Publications and source records attributed to D M Robertson.

At least 199 records · Page 11Linked to original sources
PubMed1988

Serum inhibin concentrations rise throughout normal male and female puberty.

Using a newly developed, sensitive, and specific RIA, we measured the serum concentrations of inhibin, together with those of FSH, LH, and sex steroids, throughout puberty in 99 boys and 102 girls attending a suburban Melbourne school. Serum inhibin levels rose from a geometric mean level of 161 U/L (range, 87-310; 67% confidence interval) at stage I puberty in boys to 442 U/L (range, 300-626) at stage V, while corresponding values in girls were 97 U/L (range, 46-204) and 231 U/L (range, 187-372), respectively. Serum inhibin concentrations were strongly correlated with age and serum FSH, LH, testosterone, and estradiol; all hormones increased in parallel in both boys and girls. After adjustment for age, the partial correlation coefficients remained significant only for testosterone in the boys. We hypothesize that gonadal inhibin production is stimulated by rising gonadotropin levels during pubertal development.

Adolescent↗
PubMed1988

Ovine follicular fluid inhibits thymidine incorporation by 3T3 fibroblasts in vitro.

The ability of antral follicular fluid obtained from sheep follicles to inhibit 3T3 fibroblasts maintained for 48 h in concentrations of fetal calf serum optimal for cell growth was examined. Addition of pooled follicular fluid to cultures resulted in a dose-dependent and reversible inhibition of [3H]thymidine incorporation. Serum from ovariectomized ewes, fetal calf serum, bovine inhibin, oestradiol-17 beta, testosterone, cortisol or progesterone were without effect over a range of doses. Treatment of pooled follicular fluid with charcoal-dextran did not reduce inhibitory activity which was only partly removed by heating at 85 degrees C. Fluid obtained from large follicles (greater than 5 mm) was more potent as an inhibitor than was fluid obtained from smaller (less than 5 mm) follicles. Gel chromatography of pooled fluid resolved two peaks of inhibitory activity associated with material of Mr approximately 180,000 and less than 10,000 respectively. No inhibitory activity was evident in fractions of serum from ovariectomized ewes chromatographed in an identical manner. These results indicate that ovine follicular fluid contains two components able to inhibit reversibly mitosis of 3T3 fibroblasts in vitro.

Animals↗
PubMed1988

Effects of 31 kDa bovine inhibin on FSH and LH in rat pituitary cells in vitro: antagonism of gonadotrophin-releasing hormone agonists.

The effects of 31 kDa bovine inhibin on the release of FSH and LH stimulated by gonadotrophin-releasing hormone (GnRH) or its agonist analogue buserelin have been studied using 5-day-old cultures of pituitary cells prepared from adult male Sprague-Dawley rats. Exposure of cultures to increasing concentrations of inhibin for 3 days before and during a 4-h stimulation with GnRH resulted in the progressive suppression of both basal and stimulated gonadotrophin release. At the highest inhibin concentrations FSH release was abolished (inhibin median inhibitory concentration (IC50) = 0.15 U/ml) whereas LH release was suppressed by 75% (IC50 = 0.93 U/ml). To correct for the reduced size of the FSH pool resulting from inhibin pretreatment, the amount of FSH or LH released by an agonist was expressed as a proportion of the total hormone available for release in each case. Following this adjustment, concentrations of inhibin producing maximal effects increased the GnRH median effective concentration for FSH release 4.1-fold and that for LH release 2.2-fold, with inhibin IC50 values of 0.45 and 0.32 U/ml respectively. Inhibin also suppressed the maximum proportion of both FSH and LH that excess GnRH released in 4 h by 36%, with IC50 values of 0.53 and 0.76 U/ml respectively. These effects were not changed by reduction of the inhibin pretreatment period from 3 days to 1 day or by exclusion of inhibin during the stimulation period. After a 3-day pretreatment, inhibin inhibited gonadotrophin release by buserelin less effectively than that by GnRH, but the pattern of antagonism was the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The isolation of polypeptides with FSH suppressing activity from bovine follicular fluid which are structurally different to inhibin.

Three proteins (31, 35 and 39 kDa) with inhibin-like activity have been isolated from bovine follicular fluid with identical NH2-terminal amino acid sequences. These polypeptides are distinct from inhibin, based on their different NH2-amino acid sequence, molecular masses, absence of a subunit structure, absence of inhibin immunoactivity and the failure of inhibin antiserum to neutralize their bioactivity in vitro. Their inhibin-like biological activities based on their ability to suppress FSH cell content by pituitary cells in culture are 5-10% of bovine 31 kDa inhibin.

Amino Acid Sequence↗
PubMed1987

Effects of bovine inhibin, transforming growth factor-beta and bovine Activin-A on granulosa cell differentiation.

To determine whether inhibin and its related peptides might act locally to control granulosa cell function and differentiation, the dose- and time-dependent effects of bovine inhibin, the homo-dimer of the beta-chain of bovine inhibin (Activin-A) and porcine TGF beta on rat granulosa cell aromatase activity and progesterone synthesis were investigated in vitro. TGF beta enhanced FSH-induced aromatase activity and progesterone synthesis, and accelerated the peak response for progesterone synthesis. Activin-A on the other hand, augmented FSH-induced aromatase activity while arresting progesterone synthesis, and anti-luteinization effect. By contrast, exogenous inhibin had no detectable effect on the steroidogenic potential of these cells. Thus TGF beta and Activin, unlike their similar effects on the release of FSH by the pituitary, appear to affect ovarian granulosa cell function in different fashion, under conditions where inhibin itself has no effect.

Activins↗
PubMed1987

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXV. Separation of isoforms of the glycoprotein hormones from human pituitary extracts.

Procedures for the high-resolution separation of microheterogeneous forms of the glycoprotein hormones, lutropin, follitropin and thyrotropin, present in human pituitary extracts are described. The sequential use of preparative isoelectric focusing and ion-exchange high-performance liquid chromatography (HPLC) allows the further discrimination of the isoforms of these glycoprotein hormones, previously considered homogeneous on the basis of their electrophoretic mobility. The ability of ion-exchange HPLC stationary phases to probe for slight differences in charge asymmetry expressed on the surface of these proteins raises the possibility of their rapid purification via the tandem on-line use of the same ion-exchange support with several different elution systems. Retention maps, illustrating the basis of this approach, are described.

Amino Acids↗
PubMed1987

Effect of mass loadability, protein concentration and n-alkyl chain length on the reversed-phase high-performance liquid chromatographic behaviour of bovine serum albumin and bovine follicular fluid inhibin.

The chromatographic behaviour of bovine serum albumin and partially purified preparations of the 58 kilodalton form of bovine follicular fluid inhibin has been investigated using two different n-alkylsilica stationary phases. In particular, the effects of mass loadability, protein concentration and loading conditions on the relative retention, peak width and recovery of these proteins have been studied over a dynamic range up to 100 mg protein per g packing material per injection. The influence of variable amounts of more abundant contaminating proteins such as bovine serum albumin in crude inhibin preparations on the chromatographic purification of trace quantities of the 58 kilodalton inhibin protein has also been examined. Based on these observations, recommendations are offered for the selection of protein loading conditions with n-alkylsilica stationary phases, particularly for the trace recovery and purification of hydrophobic proteins similar to inhibin where self-aggregation, adsorption to glass or plastic surfaces, and aberrant chromatographic behaviour on size exclusion or adsorptive chromatographic supports may occur.

Animals↗
PubMed1987

A technique for accurate placement of episcleral iodine-125 plaques.

Confirmation of iodine-125 plaque position was determined with a modified fiberoptic light pipe that directed light at right angles to the long axis of the fiberoptic pipe. While the examiner observed the interior of the eye with indirect ophthalmoscopy, the point source of light from the fiberoptic light pipe was moved along the margins of the episcleral plaque. The position of the plaque relative to the location of the underlying melanoma could then be verified by transillumination.

Brachytherapy↗
PubMed1987

Choroidal melanoma treatment with iodine 125 brachytherapy.

Twenty-six patients were treated for malignant melanoma of the choroid with iodine 125 episcleral plaques. Patients were followed up for at least two years, with a mean follow-up of 45 months. Tumor shrinkage was documented by A-scan ultrasonography in all but one patient. The average reduction in tumor thickness was 46%. Visual acuity remained within two Snellen lines of preoperative levels in 14 (54%) of the 26 patients. Of the 12 patients who lost more than two lines of central visual acuity, eight developed radiation changes affecting either the optic nerve or retina, and three developed cataracts; enucleation accounted for the loss of vision in the final patient. Enucleation was necessary in one other patient in this series because of tumor growth. The mean duration between treatment with iodine 125 and the development of radiation changes affecting the optic nerve or retina was 32 months.

Adult↗
PubMed1987

Circulating immunoactive inhibin in the luteal phase and early gestation of women undergoing ovulation induction.

Serum inhibin was measured by radioimmunoassay in 19 women undergoing ovulation induction within an in vitro fertilization program. In 16 nonpregnant subjects, inhibin rose during the luteal phase to reach a peak (2.5 U/ml) 6 days after oocyte retrieval; it then fell to undetectable levels by day 14 (less than 0.37 U/ml). Serum inhibin was significantly correlated (P less than 0.001) with both serum progesterone and estradiol concentrations and inversely correlated with serum follicle-stimulating hormone (FSH) (P less than 0.001). In the pregnant group (n = 3), inhibin levels reached an initial peak (3.7 U/ml) on day 6, then declined to a nadir (1.5 U/ml) on day 8 after oocyte retrieval and subsequently rose to 3.4 U/ml on day 14. These data suggest (1) an inverse relationship between inhibin and FSH, which is in support of the inhibin hypothesis; (2) that the human corpus luteum secretes inhibin; and (3) a trophoblastic/placental origin of inhibin during gestation.

Estradiol↗
PubMed1987

Inhibin--a non-steroidal regulator of pituitary follicle stimulating hormone.

Inhibin is a gonadal glycoprotein produced by the granulosa and Sertoli cell under the influence of FSH and acts to specifically suppress pituitary FSH secretion. Recently, ovarian inhibin has been purified from several species and its amino acid sequence deduced using cloning techniques. Inhibin consists of two disulphide-linked heterologous subunits of which the smaller may exist in two different forms accounting for two different forms of inhibin in humans and pigs. Heterogeneity of inhibin also exists as a result of proteolytic processing of the molecule during its passage into the circulation. Significant homology exists between the subunits of inhibin and the dimeric peptides TGF-beta and Mullerian inhibitory substance (MIS), suggesting they are all derived from a common ancestral gene. Furthermore, dimers of the smaller subunit of inhibin (FSH-releasing protein (FRP) or activin) have now been found in follicular fluid (FF) and, along with TGF-beta, shown to be potent and specific stimulators of FSH secretion. These proteins may be involved in controlling FSH by another as yet unknown pathway and may prove to be the FSH-releasing factor, analogous to LHRH, which has been postulated to exist for some years. Inhibin can no longer be simply considered as an isolated FSH-suppressing protein. The physiological significance and relationship between inhibin and its related proteins represent one of the most challenging and interesting areas in reproductive endocrinology. Further studies, particularly with the development and use of sensitive assays for both the FSH releasing hormone and inhibin will clarify their role in reproduction and their usefulness in monitoring or treating fertility.

Animals↗
PubMed1987

Changes in testicular inhibin after a single episode of heating of rat testes.

Brief heating (43 C for 15 min) of the scrota of adult rats was used to induce reversible spermatogenic damage to the testes. In these animals the changes in testicular inhibin content and an index of inhibin production rate, measured after efferent duct ligation, were examined and correlated with serum gonadotropin levels. The effect of heating was not evident until after 1 week when testis weight, inhibin content, and inhibin production rate were significantly reduced and both serum FSH and LH were elevated. By 2 weeks, the maximal effects were observed, and, thereafter, all parameters gradually returned to control values (FSH: by 6 weeks; testis and epididymal weight, inhibin content, inhibin production rate, and seminiferous tubule fluid production: by 17 weeks). Throughout the study, serum testosterone levels showed no significant changes. Significant inverse correlations were found between serum FSH levels and inhibin content (r = -0.502, P less than 0.001) or inhibin production rate (r = -0.533, P less than 0.001), and these were taken as supportive evidence for the hypothesis that inhibin is involved in the feedback control of pituitary FSH secretion. Although serum LH levels were also negatively correlated to the corresponding inhibin content (r = -0.669, P less than 0.001) or inhibin production rate (r = -0.420, P less than 0.001), recent findings of Leydig cell dysfunction in these animals led us to relate the transient rise in LH to the altered state of Leydig cell function.

Animals↗
PubMed1987

Circulating immunoreactive inhibin levels during the normal human menstrual cycle.

Serum inhibin concentrations were measured daily by RIA in six normal women throughout one menstrual cycle. The RIA was specific for inhibin, and inhibin subunits and related proteins cross-reacted minimally in it. In the early to midfollicular phase, inhibin levels changed little, while in the late follicular phase, inhibin levels rose, in parallel with estradiol (r = 0.43; P less than 0.05; n = 22), to a peak level of 714 (407-1267) U/L (geometric mean +/- 67% confidence limits) coincident with the midcycle LH and FSH surges. An inverse relationship was found between serum inhibin and FSH during the mid- to late follicular phase (r = 0.42; P less than 0.01; n = 45). Inhibin levels rose further during the luteal phase to a peak level of 1490 (1086-2028) U/L 7-8 days after the LH surge, and they correlated positively with serum progesterone (r = 0.76; P less than 0.001; n = 49) and inversely with serum FSH (r = 0.43; P less than 0.01; n = 49) throughout the luteal phase. We conclude that 1) circulating inhibin is detectable throughout the normal menstrual cycle; 2) in the late follicular phase, inhibin levels rise in parallel with estradiol, consistent with the concept that both are products of the maturing follicle; 3) in the luteal phase, the profile of inhibin suggests that it is a secretory product of the corpus luteum; and 4) the inverse relationship between inhibin and FSH in the follicular phase is consistent with the inhibin hypothesis, while at midcycle there is loss of the inhibitory effect of inhibin on FSH secretion. The inverse relationship between FSH and inhibin during the luteal phase suggests a hitherto unsuspected role for inhibin in the feedback regulation of FSH secretion.

Adult↗
PubMed1987

Influence of dose and route of administration of bovine follicular fluid and the suppressive effect of purified bovine inhibin (Mr 31,000) on plasma FSH concentrations in ovariectomized ewes.

Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of Mr 31,000. Various doses (0.25, 0.5, 1 or 2 ml) of bovine follicular fluid, given either by the intravenous (i.v.) or intracarotid route (i.c.) resulted in significant linear dose-related suppression of plasma FSH and interval to maximum suppression. Control ewes (1.0 ml steer plasma) showed no significant change in FSH over the same period. Doses of 470 and 2590 U of pure inhibin given i.v. caused a significant suppression of FSH in plasma in all ewes. The in-vivo potency estimate of the high dose (2760 U, 1420-4690 fiducial limits) agreed well with the in-vitro assay of potency. There were no significant changes observed in mean plasma LH after treatment with the higher dose of pure inhibin. There were no rebound effects of treatment with bovine follicular fluid or pure inhibin on FSH concentrations above that of controls. It is concluded that the form of bovine inhibin of Mr 31,000, which is believed to be the predominant circulating form, is biologically active when administered in vivo.

Animals↗
PubMed1987

Isolation of inhibin from ovine follicular fluid.

Two forms of inhibin with molecular weights of 65,000 and 30,000 (65 and 30 kD) were isolated from ovine follicular fluid using a combination of gel permeation chromatography, reversed-phase high-performance liquid chromatography and preparative polyacrylamide gel electrophoresis. The 65 kD form was partially purified approximately 315-fold whilst the 30 kD form was isolated as two isoforms (29 and 30 kD) of similar biological activity and in greater than 95% purity (1210-fold purification and 4.2% recoveries). On reduction the 30 kD form resolved into four components of 36, 31, 20-21 and 16 kD of which the 20-21 and 16 kD components were similar to the corresponding inhibin subunits isolated from porcine and bovine follicular fluid. The 36 kD component was established as a non-reducible inhibin-like material, based on its binding to antiserum raised against bovine 58 kD inhibin. The nature of the remaining non-reducible 31 kD component is unknown. Two NH2-terminal amino acid sequences (first 13 amino acids) identified in purified 30 kD inhibin were identical to the corresponding subunit amino acid sequences of bovine 31 kD inhibin.

Animals↗
PubMed1987

Immunization against an inhibin subunit produced by recombinant DNA techniques results in increased ovulation rate in sheep.

Seven Merino-Border Leicester cross-bred ewes were immunized with a purified fusion protein, produced by recombinant DNA methods, of the alpha subunit of bovine inhibin. Four animals were immunized with the fusion protein alone and three with a conjugate made by coupling the fusion protein to keyhole limpet haemocyanin (KLH) using glutaraldehyde. Each animal received four injections of the fusion protein over 93 days. The animals were synchronized using progestagen sponges and subjected to laparoscopy for the determination of ovulation rates in two consecutive cycles (days 115 and 135). The immunized animals had overall mean ovulation rates for each cycle of 3.4 and 3.4 which was significantly (P less than 0.001) above the rates of 1.1 and 1.4 determined for the controls, which had either received no treatment (n = 5) or had been immunized with 300 micrograms KLH (n = 4). Analysis of antisera taken on day 115 showed significant fusion protein antibodies and iodinated inhibin-binding capacity in the test but not control groups. Furthermore, antisera to the fusion protein in four out of seven ewes neutralized the inhibin bioactivity of ovine follicular fluid in an in-vitro bioassay. These data demonstrate that neutralization of inhibin can be effected by immunization with bovine inhibin alpha subunit and that such immunization results in increased ovulation rates as predicted from the biological role of inhibin as a suppressor of FSH.

Animals↗