Carolyn A. Bondy

Mater , a maternal effect gene required for early embryonic development in mice

Maternal effect genes produce mRNA or proteins that accumulate in the egg during oogenesis. We show here that Mater, a mouse oocyte protein dependent on the maternal genome, is essential for embryonic development beyond the two-cell stage. Females lacking the maternal effect gene Mater are sterile. Null males are fertile.

Cellular pattern of type-I insulin-like growth factor receptor gene expression during maturation of the rat brain : comparison with insulin-like growth factors I and II

Insulin-like growth factors have a number of potent trophic effects on cultured neural tissue and most if not all of these effects appear to be mediated by the type-I insulin-like growth factor receptor. In order to establish the identity of cell types expressing this receptor in the rat central nervous system during development and maturity, we have used in situ hybridization to map sites of type-I insulin-like growth factor receptor mRNA synthesis in the developing and adult rat brain. In order to identify possible local sources of peptide ligands for this receptor, we have also mapped the sites of insulin-like growth factors I and II mRNA synthesis in parallel brain sections. From early development onward, there is a uniform and stable pattern of type-I insulin-like growth factor receptor gene expression in all neuroepithelial cell lineages, in which regional variations reflect primarily differences in cell density. In addition to this generalized pattern, during late postnatal development, high levels of type-I insulin-like growth factor receptor gene expression are found in specific sets of sensory and cerebellar projection neurons in conjunction with abundant insulin-like growth factor-I gene expression in these same neurons. While insulin-like growth factor-I expression is confined to the principal neurons in each system, receptor mRNA is also found in local interneurons. In the cerebral cortex and hippocampal formation, type-I insulin-like growth factor receptor mRNA and insulin-like growth factor-I are concentrated in different cell populations: receptor mRNA is abundant in pyramidal cells in Ammons horn, in granule cells in the dentate gyrus, and in pyramidal cells in lamina VI of the cerebral cortex. Insulin-like growth factor-I mRNA is found in isolated medium- to large-sized cells which are rather irregularly distributed throughout the hippocampus and isocortex. In the hypothalamus, receptor mRNA is concentrated in the suprachiasmatic nucleus but is in low abundance elsewhere, including the median eminence, while insulin-like growth factor-I mRNA is not detected in this region at all. Type-I insulin-like growth factor receptor and insulin-like growth factor-II mRNAs are both abundant in choroid plexus, meninges and vascular sheaths from early development to maturity, but insulin-like growth factor-II mRNA is not detected in cells of neuroepithelial origin at any stage of development. This study provides evidence for two fundamentally different patterns of gene expression for the brain type-I insulin-like growth factor receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Aortic Dilatation and Dissection in Turner Syndrome

Background— The risk for aortic dissection is increased among relatively young women with Turner syndrome (TS). It is unknown whether aortic dilatation precedes acute aortic dissection in TS and, if so, what specific diameter predicts impending deterioration. Methods and Results— Study subjects included 166 adult volunteers with TS (average age, 36.2 years) who were not selected for cardiovascular disease and 26 healthy female control subjects. Ascending and descending aortic diameters were measured by magnetic resonance imaging at the right pulmonary artery. TS women were on average 20 cm shorter, yet average aortic diameters were identical in the 2 groups. Ascending aortic diameters normalized to body surface area (aortic size index) were significantly greater in TS, and ≈32% of TS women had values greater than the 95th percentile of 2.0 cm/m2. Ascending diameter/descending diameter ratios also were significantly greater in the TS group. During ≈3 years of follow-up, aortic dissections occurred in 3 women with TS, for an annualized rate of 618 cases/100 000 woman-years. These 3 subjects had ascending aortic diameters of 3.7 to 4.8 cm and aortic size indices >2.5 cm/m2. Conclusions— The risk for aortic dissection is greatly increased in young women with TS. Because of their small stature, ascending aortic diameters of <5 cm may represent significant dilatation; thus, the use of aortic size index is preferred. Individuals with a dilated ascending aorta defined as aortic size index >2.0 cm/m2 require close cardiovascular surveillance. Those with aortic size index ≥2.5 cm/m2 are at highest risk for aortic dissection.

Major Vascular Anomalies in Turner Syndrome Prevalence and Magnetic Resonance Angiographic Features

Background—Turner syndrome (TS) is associated with aortic coarctation and dissection; hence, echocardiographic evaluation of all patients is currently recommended. X-ray angiography in clinically symptomatic patients has suggested a range of other vascular anomalies, but the true prevalence of such lesions in TS is unknown. To better understand the prevalence and pathogenesis of cardiovascular defects in TS, we prospectively evaluated a group of asymptomatic adult volunteers with TS using magnetic resonance (MR) angiography. Methods and Results—A total of 85 adults with TS and 27 normal female adult volunteers underwent gadolinium-enhanced 3D MR angiography. A high prevalence of aortic anomalies was seen in women with TS, including elongation of the transverse arch (49%), aortic coarctation (12%), and aberrant right subclavian artery (8%). Venous anomalies were also prominent, including persistent left superior vena cava (13%) and partial anomalous pulmonary venous return (13%). None of these anomalies were found in healthy female controls. The constellation of elongation of the transverse arch, aortic coarctation, and persistent left superior vena cava was significantly associated with women with TS. Neck webbing and increased thoracic anterior-to-posterior dimension diameters were strong predictors for arterial and venous anomalies. Conclusions—Thoracic vascular anomalies are common in TS, occurring in ≈50% of a group not preselected for cardiovascular disease. The highly significant association between neck webbing, increased chest diameter, and these vascular anomalies suggests that in utero, centrally localized lymphatic obstruction may contribute to these cardiovascular deformities in TS. Improved recognition of these often-undetected vascular lesions may be important for identification of patients in need of closer cardiovascular monitoring.

Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy

Longitudinal bone growth, and hence stature, are functions of growth plate chondrocyte proliferation and hypertrophy. Insulin‐like growth factor 1 (Igf1) is reputed to augment longitudinal bone growth by stimulating growth plate chondrocyte proliferation. In this study, however, we demonstrate that chondrocyte numbers and proliferation are normal in Igf1 null mice despite a 35% reduction in the rate of long bone growth. Igf1 null hypertrophic chondrocytes differentiate normally in terms of expressing specialized proteins such as collagen X and alkaline phosphatase, but are smaller than wild‐type at all levels of the hypertrophic zone. The terminal hypertrophic chondrocytes, which form the scaffold on which long bone growth extends, are reduced in linear dimension by 30% in Igf1 null mice, accounting for most of their decreased longitudinal growth. The expression of the insulin‐sensitive glucose transporter, GLUT4, is significantly decreased and the insulin‐regulated enzyme glycogen synthase kinase 3β (GSK3) is hypo‐phosphorylated in Igf1 null chondrocytes. Glycogen levels were significantly decreased and ribosomal RNA levels were reduced by almost 75% in Igf1 null chondrocytes. These data suggest that Igf1 promotes longitudinal bone growth by ‘insulin‐like’ anabolic actions which augment chondrocyte hypertrophy.— Wang, J., Zhou, J., Bondy, C. A. Igf1 promotes longitudinal bone growth by insulin‐like actions augmenting chondrocyte hypertrophy. FASEB J. 13, 1985–1990 (1999)

Testosterone inhibits estrogen-induced mammary epithelial proliferation and suppresses estrogen receptor expression

This study investigated the effect of sex steroids and tamoxifen on primate mammary epithelial proliferation and steroid receptor gene expression. Ovariectomized rhesus monkeys were treated with placebo, 17β estradiol (E2) alone or in combination with progesterone (E2/P) or testosterone (E2/T), or tamoxifen for 3 days. E2 alone increased mammary epithelial proliferation by ~sixfold (P<0.0001) and increased mammary epithelial estrogen receptor (ERα) mRNA expression by ~50% (P<0.0001; ERβ mRNA was not detected in the primate mammary gland). Progesterone did not alter E2s proliferative effects, but testosterone reduced E2‐induced proliferation by —40% (P< 0.002) and entirely abolished E2‐induced augmentation of ERα expression. Tamoxifen had a significant agonist effect in the ovariectomized monkey, producing a ~threefold increase in mammary epithelial proliferation (P<0.01), but tamoxifen also reduced ERα expression below placebo level. Androgen receptor (AR) mRNA was detected in mammary epithelium by in situ hybridization. AR mRNA levels were not altered by E2 alone but were significantly reduced by E2/T and tamoxifen treatment. Because combined E2/T and tamoxifen had similar effects on mammary epithelium, we investigated the regulation of known sex steroid‐responsive mRNAs in the primate mammary epithelium. E2 alone had no effect on apolipoprotein D (ApoD) or IGF binding protein 5 (IGFBP5) expression, but E2/T and tamoxifen treatment groups both demonstrated identical alterations in these mRNAs (ApoD was decreased and IGFBP5 was increased). These observations showing androgen‐induced down‐regulation of mammary epithelial proliferation and ER expression suggest that combined estrogen/androgen hormone replacement therapy might reduce the risk of breast cancer associated with estrogen replacement. In addition, these novel findings on tamoxifens androgen‐like effects on primate mammary epithelial sex steroid receptor expression suggest that tamoxifens protective action on mammary gland may involve androgenic effects.— Zhou, J., Ng, S., Adesanya‐Famuiya, O., Anderson, K., Bondy, C. A. Testosterone inhibits estrogen‐induced mammary epithelial proliferation and suppresses estrogen receptor expression. FASEB J. 14, 1725–1730 (2000)

Distinctive anatomical patterns of gene expression for cGMP-inhibited cyclic nucleotide phosphodiesterases.

Type III cGMP-inhibited phosphodiesterases (PDE3s) play important roles in hormonal regulation of lipolysis, platelet aggregation, myocardial contractility, and smooth muscle relaxation. We have recently characterized two PDE3 subtypes (PDE3A and PDE3B) as products of distinct but related genes. To elucidate their biological roles, in this study we compare cellular patterns of gene expression for these two enzymes during rat embryonic and postnatal development using in situ hybridization. PDE3B [corrected] mRNA is abundant in adipose tissue and is also expressed in hepatocytes throughout development. This mRNA is also highly abundant in embryonic neuroepithelium including the neural retina, but expression is greatly reduced in the mature nervous system. Finally, PDE3B [corrected] mRNA is localized in spermatocytes and renal collecting duct epithelium in adult rats. PDE3B mRNA is highly expressed in the cardiovascular system, including myocardium and arterial and venous smooth muscle, throughout development. It is also abundant in bronchial, genitourinary and gastrointestinal smooth muscle and epithelium, megakaryocytes, and oocytes. PDE3A [corrected] mRNA demonstrates a complex, developmentally regulated pattern of gene expression in the central nervous system. In summary, the two different PDE3s show distinctive tissue-specific patterns of gene expression suggesting that PDE3B [corrected] is involved in hormonal regulation of lipolysis and glycogenolysis, while regulation of myocardial and smooth muscle contractility appears to be a function of PDE3A [corrected]. In addition, the present findings suggest previously unsuspected roles for these enzymes in gametogenesis and neural development.

A physiologic role for testosterone in limiting estrogenic stimulation of the breast.

ObjectiveThe normal ovary produces abundant testosterone in addition to estradiol (E2) and progesterone, but usually only the latter two hormones are “replaced” in the treatment of ovarian failure and menopause. Some clinical and genetic evidence suggests, however, that endogenous androgens normally inhibit estrogen-induced mammary epithelial proliferation (MEP) and thereby may protect against breast cancer. DesignTo investigate the role of endogenous androgen in regulating mammary epithelial proliferation, normal-cycling rhesus monkeys were treated with flutamide, an androgen receptor antagonist. To evaluate the effect of physiological testosterone (T) supplementation of estrogen replacement therapy, ovariectomized monkeys were treated with E2, E2 plus progesterone, E2 plus T, or vehicle. ResultsWe show that androgen receptor blockade in normal female monkeys results in a more than twofold increase in MEP, indicating that endogenous androgens normally inhibit MEP. Moreover, we show that addition of a small, physiological dose of T to standard estrogen therapy almost completely attenuates estrogen-induced increases in MEP in the ovariectomized monkey, suggesting that the increased breast cancer risk associated with estrogen treatment could be reduced by T supplementation. Testosterone reduces mammary epithelial estrogen receptor (ER) &agr; and increases ER&bgr; expression, resulting in a marked reversal of the ER&agr;/&bgr; ratio found in the estrogen-treated monkey. Moreover, T treatment is associated with a significant reduction in mammary epithelial MYC expression, suggesting that Ts antiestrogenic effects at the mammary gland involve alterations in ER signaling to MYC. ConclusionsThese findings suggest that treatment with a balanced formulation including all ovarian hormones may prevent or reduce estrogenic cancer risk in the treatment of girls and women with ovarian failure.

Clinical Uses of Insulin-like Growth Factor I

Dr. Carolyn Bondy (Developmental Endocrinology Branch, National Institute of Child Health and Human Development): Insulin-like growth factor I (IGF-I; somatomedin-C) is an anabolic polypeptide that is structurally homologous to insulin [1]. Its actions are mediated primarily by the IGF-I receptor, which is structurally and functionally homologous to the insulin receptor. The ligand-binding domains of these receptors are sufficiently different that each binds its cognate hormone with about ten times more affinity than does the related ligand [2]. The signal-transducing, tyrosine kinase domains of the two receptors, however, are very similar [2] and activate common intracellular pathways [3]. Thus, it appears that the difference in physiologic effects of insulin and IGF-I are not due primarily to intrinsic differences in signaling capacities of their receptors [4]. Furthermore, with a few notable exceptions, both receptors are widely expressed, with some tissues apparently expressing hybrid receptors that combine insulin and IGF-I receptor subunits [5, 6]. Because insulin and IGF-I are subject to very different regulatory influences and have markedly different patterns of secretion and circulating profiles, hormone bioavailability is probably an important factor in determining the different roles served by IGF-I and insulin. Recombinant human IGF-I recently became available for clinical studies, allowing, for the first time, direct investigation of the metabolic and anabolic effects of IGF-I and its relations with insulin and growth hormone. Our view of the regulatory relations among IGF-I, growth hormone, and insulin is outlined in Figure 1. Growth hormone and insulin stimulate the constitutive secretion of IGF-I from the liver [7] and IGF-I, in turn, suppresses growth hormone and insulin secretion, even under euglycemic conditions [8-11]. In contrast to the highly regulated secretory patterns and fluctuating serum profiles of growth hormone and insulin, circulating IGF-I levels are relatively stable. This stability is due to its constitutive pattern of secretion and to the fact that most circulating IGF-I is bound to high-affinity IGF-binding proteins, which prolong the half-life and titrate the supply of this hormone to its receptors [12, 13]. Six IGF binding proteins have been identified, but clinical data are most abundant for IGF-binding protein-3. This IGF-binding protein binds IGF-I and another component, the acid-labile subunit, and forms a high molecular weight ternary complex, which constitutes the primary reservoir of circulating IGF-I. Circulating levels of this complex are positively regulated by growth hormone. Insulin-like growth factor-binding protein-1 binds a smaller fraction of the total circulating IGF-I, but this fraction may be disproportionately influential in terms of the effects of IGF-I on intermediary metabolism, because IGF-binding protein-1 levels are potently suppressed by insulin. Figure 1. Relations between insulin-like growth factor I (IGF-I) and IGF-binding proteins, growth hormone (GH), and insulin. Originally, the somatomedin hypothesis [1] suggested that circulating IGF-I mediates most of the effects of growth hormone on linear growth. Recently, however, growth hormone was found to stimulate the local production of IGF-I in several tissues in addition to the liver in rodents [1], and thus local autocrine or paracrine effects of IGF-I appeared to be important for normal growth. There is, however, little evidence for growth hormone-stimulated IGF-I synthesis in human tissues other than the liver, and the apparent success of systemic IGF-I treatment in producing linear growth in growth hormone-resistant children, discussed in the following section by Dr. Underwood, suggests that neither local IGF-I production nor direct anabolic effects of growth hormone are essential for statural growth in children. Local autocrine/paracrine growth processes in humans might be regulated by another member of the insulin family of peptides. Insulin-like growth factor II is structurally closely related to IGF-I [1] and binds the IGF-I receptor with high affinity, but unlike IGF-I it is not regulated by growth hormone. In rodents, IGF-II expression is abundant during embryonic development but is largely suppressed after birth. In humans, however, IGF-II levels are equal to or greater than IGF-I in the circulation and in many tissues during adulthood [1, 14-16]. Growth hormone and IGF-I have continuing roles in fuel metabolism and in the maintenance of musculoskeletal mass in adults. Many of the changes in body composition, such as increasing adiposity and decreasing muscle mass, that occur during aging correlate specifically with decreasing levels of these hormones [17]. Several clinical situations exist in which the anabolic or metabolic effects of growth hormone, IGF-I, or both may prove to have substantial therapeutic benefit. Starvation, cachexia, hyperalimentation, and insulin-dependent diabetes mellitus are all associated with a state of functional growth hormone resistance in which, despite normal or high growth hormone levels, circulating IGF-I levels are low and do not respond to growth hormone treatment. A common factor in these conditions is under-insulinization of the liver, which impairs normal IGF-I and IGF-binding protein synthesis. Recent clinical trials evaluated the short-term metabolic effects of IGF-I administration in calorically deprived adult volunteers, as described by Dr. Clemmons, and in insulin-dependent diabetic patients, as described by Dr. Bach. Another area in which IGF-I may have important therapeutic benefit is the hyperglycemic disorders characterized by insulin resistance. In the short term, recombinant IGF-I reduces blood glucose and triglyceride levels in obese patients with noninsulin-dependent diabetes mellitus [11]. These salutary effects have been attributed to improved insulin sensitivity due to suppression of growth hormone and insulin secretion by IGF-I and to the direct, insulin-like metabolic effects of IGF-I. A few studies have reported that recombinant IGF-I treatment improves the hyperglycemia of patients with extreme insulin resistance caused by genetic defects in the insulin receptor, thus suggesting that IGF-I may act through its own receptor to regulate blood glucose [18-21]. Not all insulin-resistant patients respond well to IGF-I treatment, however, as reported by Drs. Guler and Skarulis in a following section. Insulin-like Growth Factor I in Growth Hormone-Resistant Short Stature Dr. Louis Underwood (Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina): We are treating two kinds of patients with short stature secondary to growth hormone insensitivity: patients with Laron-type dwarfism, now called the Laron syndrome [22], and growth hormone-deficient patients in whom large amounts of growth-attenuating antibodies have developed after treatment with growth hormone. Normally, growth hormone binds to the growth hormone receptor to induce hepatic IGF-I production, which in turn stimulates growth and feeds back at the level of the pituitary and hypothalamus to suppress growth hormone secretion (Figure 2). Patients with the Laron syndrome lack functional growth hormone receptors and thus do not respond to growth hormone; their IGF-I levels are very low, growth is slow, and circulating growth hormone levels are high because of decreased feedback suppression of growth hormone by IGF-I (Figure 2). Patients with a deletion of the growth hormone gene may recognize growth hormone as a foreign protein, and large numbers of antibodies may develop that attenuate or obliterate their response to it. Figure 2. Diagram of the growth hormone (GH) and insulin-like growth factor I (IGF-I) growth axis in healthy persons (left), those with the Laron syndrome (middle), and those with a deletion of the gene-encoding growth hormone (right). We studied a boy with the Laron syndrome [23] who was very short (111 cm at 9 years) and had the physical appearance of a person with growth hormone deficiency. Basal serum levels of growth hormone were elevated (10 to 12 g/L) and increased to 40 to 60 g/L after pharmacologic stimulus. His serum IGF-I level was low (5 to 6 g/L; normal for age, 100 g/L), and he had no increase in serum IGF-I after injections of growth hormone. He received growth hormone therapy for 6 months without an increase in growth rate. We admitted him to our Clinical Research Center for 5 weeks and ensured a constant dietary intake. In the second week, he was given three injections of growth hormone at therapeutic doses, and in weeks 3 and 4 he received continuous infusion of recombinant IGF-I (Genentech, San Francisco, California). This treatment was followed by 1 week of postinfusion observation. He showed no metabolic responses to growth hormone, but he had a marked decrease in urinary excretion of urea and in serum urea nitrogen with IGF-I infusion. His urinary calcium level increased and his urinary phosphate and sodium excretion levels decreased [24]. These all are fairly typical growth hormone-like effects and are similar to those that would occur in patients with growth hormone deficiency who are sensitive to growth hormone. Because of the insulin-like effects of IGF-I, he tended to become hypoglycemic when he was infused overnight in a fasting state. However, in the postprandial state, his glucose increased to high levels and his insulin level was suppressed, the latter because of a direct effect of IGF-I on insulin secretion. He was treated with subcutaneous injections of recombinant IGF-I (120 g/kg every 12 hours). After IGF-I injection, serum IGF-I concentrations were in the normal range for at least 7 hours. In general, however, acute metabolic responses to subcutaneous injections are less pronounced than are those observed with intravenous infusion. He has been treated with IGF-I for nearly 2 years and has grown at

Insulin-like growth factor 1 is essential for normal dendritic growth†

This study evaluated somatic and dendritic growth of neurons in the frontoparietal cortex of Igf1–/– brains. Pyramidal neuron density was increased by ∼25% (P = .005) and soma size reduced by ∼10% (P < .001). Golgi staining revealed that cortical layer II–III neurons exhibited a significant reduction in dendritic length and complexity in Igf1 null mice. Dendritic spine density and presumably synaptic contacts were reduced by 16% (P = .002). Similar findings were obtained for cortical layer V and piriform cortex pyramids. Supporting a reduction in synapses, synaptotagmin levels were reduced by 30% (P < .02) in the Igf1 null brain. Investigation of factors critically involved in dendritic growth and synaptogenesis showed an ∼50% reduction in cortical CDC42 protein expression (P < .001) and an ∼10% reduction in brain cholesterol levels (P < .01) in Igf1 null mice. Evidence is presented that Igf1 deletion causes disruptions in lipid and microtubule metabolism, leading to impaired neuronal somatic and dendritic growth. Published 2003 Wiley‐Liss, Inc.