Biomedical subjects
Publications and source records attributed to D M Robertson.
The clinical and pathological features of a severely retarded 77-year-old man with recessive (true) microcephaly are reported. This case demonstrates that this major developmental error can be consistent with normal longevity.
1. The effect of inflammation induced by lipopolysaccharide (LPS) injection on plasma follistatin (FS) concentrations was investigated. 2. Plasma FS and tumour necrosis factor-alpha concentrations increase following LPS administration in ewes. 3. The rise in FS is similar, but more sustained, to that previously observed after surgery. 4. These results indicate a possible functional link between FS, inflammation and the acute-phase response.
Previous studies have shown that highly purified isoforms of human pituitary LH exhibited a 20-fold range of in vitro bioactivities. The aim of this study was to determine the corresponding plasma half-lives, metabolic clearance rates (MCR), and in vivo bioactivities of these human (h) LH isoforms. Cannulated adult male rats were administered hLH isoforms as a bolus i.v. injection. For the half-life studies, blood was then serially collected over a 6-h period, and serum was assayed for hLH using a specific immunofluorometric assay. All hLH (n = 19) isoforms exhibited biexponential disappearance profiles with an initial fast half-life (t 1/2) for component A of 12.8 +/- 3.7 min, followed by a slow component B with t 1/2 of 58.9 +/- 4.4 min. The prevalence of component B in relation to component A increased significantly (r = 0.81, P < 0.001) over a 3-fold range when correlated with the sialic acid content of the isoform. Similarly, the MCR showed a significant correlation (r = 0.77, P < 0.001) with sialic acid content. The basis for the two t 1/2 components was then investigated. In the first experiment, rat plasma containing primarily component B was collected 90 min after hLH isoform administration and injected into a second animal. Only component B was observed with no evidence of component A, which indicates that the two t 1/2 components are not the product of the redistribution of the hLH isoform between body compartments. In the second experiment, component B was found to be dependent on sialic acid content, as desialylated hLH isoforms showed a rapid disappearance (t 1/2 = 8.6 +/- 3.1) with the component B proportion decreasing to < 10% of that of the nondesialylated control. This data indicates that sialic acid protects component B from rapid clearance. In addition, the proportion of the two components is dependent on sialic acid content, suggesting that the molecular location of the sialic acid on the carbohydrate moieties of hLH has a critical role in the clearance process. To determine the in vivo bioactivity of the hLH isoforms, an acute in vivo bioassay was developed in male rats. The assay was based on the hLH dose-dependent increase in total testosterone release in the same rat model as used in the plasma disappearance studies. Using the second International Standard (IS) hLH (0.3 IU-2.6 IU/kg) as standard, a linear dose-response of 24-h integrated serum testosterone levels was observed, with an index of precision of 0.11. Using this in vivo assay, a 16-fold range in in vivo bioactivities (3,200 to 51,100 IU/mg) was observed for 14 hLH isoforms. These in vivo bioactivities correlated with sialic acid content (r = 0.78, P < 0.001), MCR (r = 0.56, P < 0.05) and LH in vitro bioactivity (r = 0.75, P < 0.001) as determined using mouse Leydig cells in culture. Desialylation lead to over a 100-fold decrease in in vivo bioactivity of hLH. It is concluded that hLH isoforms are cleared in vivo by a two-component clearance mechanism, the proportion of which varies between isoforms and is dependent on sialic acid content of the isoform. These findings suggest that the molecular location of sialic acid on the hLH isoform is critical in defining the plasma disappearance of component B, whereas the mechanism of elimination of component A may well involve the hepatic GalNAc-sulphate receptor. Using an in vivo bioassay, the 16-fold difference in bioactivity between isoforms is attributed primarily to differences in their in vitro activity at the cellular level with a minor influence (< 2-fold) due to differences in in vivo clearance.
Testosterone (T) is required for spermatogenesis, particularly in the conversion of round spermatids between stages VII and VIII of spermatogenesis. T is generally thought to be the major androgen involved in adult spermatogenesis due to the high local concentration within the testis, whereas its more potent 5alpha-reduced metabolite dihydrotestosterone (DHT) is thought to be the active androgen in peripheral tissues. The current study investigated whether 5alpha-reduction of T to DHT is involved in the restoration of spermiogenesis in vivo in a setting in which testicular T levels were markedly reduced. Adult male rats were given 3-cm T plus 0.4-cm estradiol implants for 9 weeks to suppress serum LH and testicular T levels and thereby inhibit spermatogenesis. Increasing doses of T (3-, 6-, 10-, and 24-cm implants) were then given for 4 days to restore spermatogenesis in the presence or absence of a 5alpha-reductase inhibitor (L685,273). The hourly production rates of round spermatids in stages I-III, IV-VI, VII, and VIII were assessed using stereological techniques, and the conversion of round spermatids between stages VII and VIII was then assessed as an index of androgen action on spermiogenesis. Testicular androgen levels were measured by HPLC and RIA. The 5alpha-reductase inhibitor significantly suppressed (P < 0.05) the hourly production rate of round spermatids at the 3- and 6-cm T doses, but not at the 10- and 24-cm doses. The conversion of round spermatids between stages VII and VIII was suppressed (P < 0.05) by the inhibitor only at the 3- and 6-cm doses. The 5alpha-reductase inhibitor had no effect on testicular T levels, but suppressed (P < 0.05) DHT levels at the 6-, 10-, and 24-cm doses. We conclude that the 5alpha-reduction of T is involved in the restoration of spermiogenesis at the lower administered doses of T and that these data are the first description of a role for 5alpha-reduced androgens in adult spermatogenesis.
The procession of round spermatids through stages VII and VIII of the rat spermatogenic cycle is critically dependent on testosterone (T). When intratesticular T levels are reduced, round spermatids appear to slough from the seminiferous epithelium, resulting in the disappearance of elongated spermatids. We hypothesized that T-dependent cell adhesion molecules are involved in Sertoli cell-round spermatid interactions. This study examined the hormonal regulation of one candidate cell adhesion molecule, N-cadherin, in vitro and its participation in Sertoli cell-round spermatid adhesion in coculture. Sertoli cells were isolated from 20-day-old Sprague-Dawley rats; treated with FSH and T, alone or in combination; and incubated for 48 h before determination of N-cadherin concentrations in Sertoli cell extracts by RIA. Both FSH and T significantly increased the cellular content of N-cadherin (3.7-fold), whereas FSH or T alone had no effect. Round spermatids were isolated from adult rats, and their adhesion to Sertoli cells was assessed in a 48-h coculture in the presence of FSH, T, or FSH plus T. Adherent round spermatids were quantitated by histological evaluation after staining with the periodic acid-Schiff reaction. A dose-dependent increase in round spermatid density (number of round spermatids bound per 10,000-microns2 Sertoli cell culture surface area) was observed with increasing T doses (7-28 ng/ml) in the presence of FSH (1 microgram/ml), whereas FSH and T alone at these doses produced no effect. T also increased the N-cadherin content of the cocultures in a dose-dependent manner in the presence of FSH. Addition of an N-cadherin antiserum to the Sertoli cell-round spermatid coculture in the presence of FSH and T significantly (P < 0.0001) reduced round spermatid density by 65%. It is concluded that both the production of N-cadherin by Sertoli cells and the binding of round spermatids to Sertoli cells are stimulated in a synergistic manner by T and FSH. Furthermore, the immunoneutralization data suggest the active involvement of N-cadherin in round spermatid-Sertoli cell adhesion in vitro. N-Cadherin may be one of the factors that subserve the androgen-dependent process of round to elongated spermatid maturation.
Dizygotic twinning is familial, suggesting that there may be an inherited abnormality of the control of ovulation that predisposes to double ovulation and, therefore, dizygotic twins. The present study examines 17 mothers of dizygotic twins (MODZT) and 8 control mothers of singletons by daily blood sampling throughout an entire menstrual cycle. Blood samples were assayed for LH, FSH, estradiol, progesterone, and inhibin. The process of follicular development was followed by transvaginal ultrasound. The pituitary LH response to iv GnRH was also assessed. Three of the 16 MODZT double ovulated during the study compared to none of the 8 control mothers (P < 0.05). The number of small follicles (<6 mm) declined significantly in control women at midcycle, but not in MODZT. There was no significant difference in serum FSH, LH, estradiol, or inhibin levels between the 2 groups at any stage of the menstrual cycle. During the follicular phase, serum progesterone levels were significantly higher in MODZT. The response to GnRH stimulation was not different between MODZT and controls. In conclusion, this study demonstrates an increased tendency to double ovulate in MODZT that may be due to a reduced rate of atresia in advanced follicles. Furthermore, the elevated progesterone levels in MODZT during the follicular phase suggest altered intrafollicular steroidogenesis that is independent of gonadotropins.
Activin, a dimer of the beta-subunits of inhibin, is a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors and has a widespread range of actions in a variety of tissues. The investigation of the physiology of activin action has been facilitated in recent years by the availability of immunoassays in addition to bioassays. Follistatin has been shown to bind to activin with a high affinity and therefore interferes in both radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). In this study we examined the effect of various surfactants and 1.4-dioxane on the measurement of activin in the presence of follistatin by radioimmunoassay. The addition of a combination of sodium deoxycholate. Tween 20 and sodium dodecyl sulphate removed the interference of follistatin in the radioimmunoassay. The measured content of activin in male rat serum, human male serum, human female serum and bovine follicular fluid rose from 3.29 to 4.15, < 0.48 to 2.87, 2.42 to 4.17 and 30.9 to 85.6 ng/ml, respectively, when assayed in the presence of the dissociating reagents. It was unclear whether the altered potencies were due to a dissociation of the follistatin/activin complex rather than the exposure of the epitope on activin recognized by the antiserum. Serum concentrations of activin were lower than those found in testicular cytosols, and after castration no change in serum activin levels was observed, suggesting that the testis does not contribute significantly to circulating activin levels. The use of the dissociating reagents in the radioimmunoassay will enable studies to be carried out that more accurately measure the activin content of various biological fluids, and thus lead to a greater understanding of the physiology of this growth factor.
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The aims of this study were to determine the plasma concentrations of follistatin in rams and to assess if the testis contributes to circulating follistatin and if there is uptake or production of follistatin by the head in rams. Catheters were inserted in the carotid artery, jugular vein and spermatic vein of intact rams during the non-breeding season (experiment 1; n = 5) and breeding season (experiment 2; n = 4). In experiment 1, blood samples were collected from 5 rams every 10 min for 4 h, commencing 20-60 min after surgery. After 2 h of sampling 1 microgram gonadotrophin-releasing hormone (GnRH) was injected intravenously. In experiment 2, blood samples were collected from 4 of the rams used in experiment 1 by venipuncture 30 and 15 min before surgery and every 15 min throughout surgery. Commencing 1 h after surgery, matched samples were taken from each of the vessels every 10 min for 4 h (1-4 h after surgery), then every hour for 20 h (4-24 h after surgery) and then every 10 min for 4 h (24-28 h after surgery). In both experiments, follistatin secretion was non-pulsatile and there were no significant differences between the concentrations of follistatin in any of the vessels. There was a significant (P < 0.05) increase in the concentrations of follistatin in each of the vessels throughout the 4 h of 10-min sampling in both experiments. In experiment 2 plasma concentrations of follistatin in the jugular vein were significantly (P < 0.05) lower before surgery than at other stages of the experiment. During the non-breeding season (experiment 1) the concentrations of follistatin in all vessels were about 2-fold higher (P < 0.001) than during the breeding season (experiment 2). Concentrations of follistatin were measured in the testicular tissue of the ram, bull, monkey and rat and were found to be 13.6, 2.1, 2.5, 0.8 ng/g testis respectively. In experiment 3, blood samples were collected every 15 min for 4 h from castrated rams (n = 6) in the absence of treatment with testosterone propionate (TP) and after 7 days of treatment with a physiological dose of TP during the breeding and non-breeding seasons. There was no effect of stage of breeding season or TP on the plasma concentrations of follistatin and these concentrations in the castrated rams were similar to the concentrations in the intact rams in experiment 2. In experiment 4, the function of Leydig cells was stimulated by administration of human chorionic gonadotrophin but this had no effect on plasma concentrations of follistatin. These experiments show that the concentrations of follistatin in the plasma of rams are measurable, that the testis is not the major contributor to circulating follistatin and that there is no significant uptake or production of follistatin by the head in rams. It appears that the contribution of the testis to circulating follistatin may vary with the stage of the breeding season, being greater during the non-breeding season than the breeding season. The gonadotrophins and testosterone do not appear to have a direct effect on the secretion of follistatin in rams. The increase in concentrations of circulating follistatin during surgery and more frequent blood sampling suggest a stress-related effect on the production of follistatin.
The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hLH) are glycoproteins which are microheterogeneous in terms of their charge and molecular size, as well as their in vitro and in vivo bioactivities. The aim of this study was to determine the contribution of variations in sialic acid (N-acetyl neuraminic acid) content to the structural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was released by partial acid hydrolysis (0.1 M TFA, 80 degrees C, 1 h) and derivatised with the fluorescent label DMB (1,2-diamino-4,5-methylenedioxybenzene) in conjunction with an internal standard (N-glycoyl-neuraminic acid). The derivatives were then separated by reversed-phase HPLC. This method allowed quantitation of the sialic acid content over a range of 5-100 pmol with between assay variation of < 6% for sialic acid released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparison of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromatography with pulsed amperometric detection yielded similar results, confirming the reliability of the fluorescence detection method. The sialic acid contents of 9 hFSH isoforms varied between 1.5-13.7 mol Neu5AC/mol FSH, whilst a range of 1.1-9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. The sialic acid content of the hFSH isoforms was also observed to be related to the hormonal specific activity in a radioreceptor assay, confirming that alterations in the carbohydrate structure can influence the FSH-receptor interaction. In contrast, the sialic acid content of the hLH isoforms was found to be not related to specific activity at the receptor level.
PURPOSE: To obtain long-term photographic follow-up of retinal astrocytic hamartomas in patients with tuberous sclerosis to learn about their stability or possible growth patterns. METHODS: Sixteen patients with a confirmed diagnosis of tuberous sclerosis and in whom retinal astrocytic hamartomas were photographed before 1986 at the Mayo Clinic underwent a complete ophthalmic examination, and fundus photographs were taken. The new photographs were compared with previous photographs, and changes in size, character, or number of retinal hamartomas were determined. The minimum follow-up period was five years. RESULTS: A total of 37 astrocytic hamartomas were found. Follow-up ranged from almost six years to more than 34 years, with an average of 16 years. Hamartomas in three patients showed progressive or new calcification. In a fourth patient a retinal hamartoma appeared to originate from a site that had been previously photographically documented to be normal. The remaining hamartomas appeared unchanged over the follow-up period. CONCLUSIONS: Although most retinal lesions in tuberous sclerosis remain stable, some become calcified over time. Additionally, new lesions may develop from previously normal-appearing retina.
PURPOSE/METHODS: We observed a clinicopathologic correlation of a case of adenocarcinoma of the retinal pigment epithelium. RESULTS/CONCLUSIONS: The ultrasonographic findings of this tumor demonstrated a collar button profile with high internal reflectivity. The ultrasonographic characteristics in this case reflected the variable vascularity and cystic spaces within the tumor.
PURPOSE: To report unusual and heretofore unreported visually damaging manifestations of severe central serous chorioretinopathy. METHODS: Case studies. RESULTS: Each of six male patients (average age, 40 years) had a form of severe central serous chorioretinopathy with at least one eye containing fibrin in the subretinal space that then developed into a subretinal fibrotic scar. Scar formation was followed by a tenting up of the macula, vascularization of the fibrosis (subretinal neovascularization), or a retinal pigment epithelial rip. Four of the seven eyes with subretinal fibrosis had severe visual loss (20/400 or worse). CONCLUSION: Subretinal fibrin and other extracellular matrix molecules appear to stimulate the retinal pigment epithelium to undergo fibrous metaplasia, which results in subretinal fibrotic scar formation and other sequelae, all of which can lead to severe visual loss.
PURPOSE: The objectives of this study are to determine the incidence of superficial tissue breakdown and/or exposure of the Allen implant, to identify potential risk factors for superficial tissue breakdown and/or exposure of the Allen implant, and to provide a comparison for future studies of other orbital implants. METHODS: The authors retrospectively studied 186 consecutive patients managed with enucleation and Allen implant placement from January 1967 through December 1991. Information was obtained from a review of the patients' records and from telephone and letter surveys. RESULTS: More than half of the patients had follow-up more than 5 years and approximately one third had follow-up of 10 or more years. The mean length of follow-up was 7.8 years. Two allen implants (1.1%) became exposed 5.3 and 11.5 years, respectively, after initial implant placement and were subsequently replaced by dermis fat grafts. Superficial tissue breakdown without implant exposure occurred over four additional Allen implants (2.2%); the patients satisfactorily were treated medically or surgically without the need for removal or replacement of the Allen implant. Most prostheses have horizontal and vertical excursions of 30 degrees or more. CONCLUSION: The satisfactory motility, the low incidence of superficial tissue breakdown, and the even lower incidence of exposed Allen implants observed in our long-term follow-up study provide evidence that the Allen implant is a satisfactory choice to replace the enucleated eye. The absence of implant exposure occurring before 5 years of follow-up underscores the need for long-term follow-up to properly evaluate newer orbital implants.
Inhibin was first isolated in 1985. Major progress has been made in defining various aspects of its structure and physiology, using a heterologous radioimmunoassay. Current research is aimed at characterizing the nature of the circulating forms of inhibin and is examining whether there are sex-specific roles for inhibins A and B. It has been recognized that various forms of epithelial and stromal ovarian cancer produce members of the inhibin peptide family but the precise nature of these products is not yet clear. The recognition that the inhibin subunits together with follistatin are expressed locally within the pituitary has lead to an investigation of their possible roles in intrapituitary regulation. It is clear that these peptides also have intragonadal roles. Of particular current interest is the nature of the signals that control the specificity of cellular peptide production and that determine whether a particular cell produces inhibin or activin. The inhibins are members of a complex family with many potential roles in physiology and pathophysiology. The role of the inhibins in feedback control of follicle stimulating hormone in the male, particularly, remains unclear. New applications for inhibin and related peptides are likely to be developed.
Rat pituitary cells express messenger RNA for the activin-binding protein, follistatin (FS), and rat and bovine pituitary cell cultures secrete FS into the medium. In the present study, a previously validated, heterologous RIA for ovine FS was employed to investigate FS synthesis, secretion, and regulation in cultures of ovine anterior pituitary cells. The validity of the RIA was confirmed by the finding that FS immunoreactivity in ovine pituitary cell culture-conditioned medium diluted in parallel with purified bovine FS, and fractionation of the conditioned medium resulted in the coelution of activin-binding activity with the FS immunoactivity. The concentration of endogenous ovine FS achieved in the culture medium (0.08-0.6 nM) was in the range over which bovine FS suppresses FSH secretion in these cultures (IC50 = 0.5 nM). To characterize the relationship between endogenous FS and FSH secretion, dispersed ovine pituitary cells were preincubated with 10% fetal bovine serum for 2 days, then cultured between days 2-5 in the presence of a chemically defined serum substitute. Under these conditions, FS was continuously secreted at a rate of 12.1 +/- 1.8 ng/10(6) cells.day (mean +/- SEM; n = 18), whereas FSH was secreted at 64 +/- 13 ng/10(6) cells.day (n = 7). The secretion of FS and FSH changed in a reciprocal way as culture conditions were altered either by maintaining exposure of the cells to fetal bovine serum or by plating the cells at a 6- to 10-fold higher seeding density. Under the latter circumstance, for instance, FS secretion during the 3-day test period decreased to 47 +/- 14% (n = 10) and FSH secretion increased to 137 +/- 6% (n = 6) of the respective values in cultures of dispersed cells. FS secretion was increased nearly 3-fold (P < 0.05) in a dose-dependent manner by continuous exposure of ovine pituitary cells between days 2-5 to recombinant human activin A (1-10 nM), which concomitantly increased FSH secretion. Recombinant human inhibin A (0.003-10 nM); the synthetic glucocorticoids, RU28362 and dexamethasone (each 1-100 nM); the sex steroids, testosterone (1-100 nM), 17 beta-estradiol (0.001-5 nM), and progesterone (4-2500 nM); and the vitamin A derivative, retinoic acid (0.3-32 microM), each inhibited FSH secretion from these cultures, but only the last agent significantly (P < 0.05) increased FS secretion. Inhibin prevented the stimulation of FSH secretion by activin A without affecting its stimulation of FS secretion.(ABSTRACT TRUNCATED AT 400 WORDS)
The role of FSH in spermatogenesis is unclear as testosterone alone has been reported to be sufficient in the gonadotropin-deficient rat. This study examined the effects of recombinant FSH on the restoration of spermatogenesis after gonadotropin withdrawal by GnRH immunization. Adult Sprague-Dawley rats received GnRH immunogen (100 micrograms, sc, every 4 weeks) to induce gonadotropin deficiency, with severe spermatogenic regression occurring by 12 weeks. Recombinant human FSH was then given (10 or 50 IU/kg, sc, daily) for 7, 14, and 21 days, with data from both dosages combined in the analyses. Testes were perfusion fixed, and germ cell numbers were quantified by the optical disector technique. After 7 days of FSH, testis weight significantly increased by 43% (P < 0.01), with no further increases at 14 and 21 days. GnRH immunization severely reduced germ cell numbers, which were then significantly (P < 0.05) restored in all cell types, except elongated spermatids, by 7 days of FSH; type A spermatogonia (45%-->61% of control), type B spermatogonia/preleptotene spermatocytes (46%-->65%), leptotene/zygotene spermatocytes (39%-->55%), pachytene spermatocytes in stages I-VIII (11%-->30% control) and IX-XIV (4.3%-->22% control), and round spermatids in stages I-VIII (1.4%-->4.4% control). Prolonged FSH treatment did not further increase type A spermatogonial or pachytene spermatocyte number, whereas round spermatids increased to a peak of 12.8% of the control value. At no stage did FSH increase elongated spermatid numbers above 1% of the control level. The incorporation of bromode-oxyuridine into spermatogonial and early spermatocyte nuclei did not change after GnRH immunization or FSH treatment. Sertoli cell number was not altered by any treatment; however, Sertoli cell nuclear volume was significantly decreased from the control value by GnRH immunization (142 +/- 9 vs. 455 +/- 22 microns 3; P < 0.01) and increased after 7 and 14 days of FSH treatment to 212 +/- 10 and 259 +/- 24 microns 3, respectively. FSH treatment restored serum inhibin levels to normal, but did not increase serum or testicular androgen levels. We conclude that recombinant FSH partially restores spermatogenesis in the gonadotropin-deficient rat by increasing the number of spermatogonia and promoting subsequent maturational steps up to the round spermatid stage. Spermatid elongation was not restored by FSH, indicating the need for an additional factor(s), most likely testosterone.
A unique kindred manifesting medullary thyroid carcinoma and corneal nerve thickening without other aspects of the multiple endocrine neoplasia syndrome (MEN) was analyzed by linkage analysis using four highly polymorphic (CA)n repeat markers (sTCL-1, D10S141, ZNF22, and sJRH-1). Additionally, the RET protooncogene was examined for specific mutations by DNA sequence analyses in all affected family members. Screening of 11 family members spanning 4 generations revealed 7 subjects with corneal nerve thickening; of these subjects, 3 had abnormal pentagastrin-stimulated calcitonin studies, and these 3 subjects were each found to have C-cell hyperplasia or medullary thyroid carcinoma at surgery. Linkage analysis showed cosegregation of alleles (as defined by the above markers), with the presence of both corneal nerve thickening and medullary thyroid carcinoma/C-cell hyperplasia (maximum LOD score, 2.69; consistent with, but not proving linkage). DNA sequence analysis showed that none of the affected individuals had mutations in either exon 10 or 11, or in exon 16 of the RET protooncogene, regions where mutations have been described for MEN type 2A (MEN-2A) and MEN-2B families, respectively. Thus, compared to the defined syndromes of MEN-2A and MEN-2B, this kindred appears to represent a true clinical overlap syndrome whose genetic basis may be distinct from these two syndromes.