Hypertrophy and altered activity of the adrenal cortex in Homer 1 knockout mice

Homer 1 gene products are involved in synaptic transmission and plasticity, and hence, distinct behavioral abnormalities, including anxiety- and depression-like behaviors, have been observed in Homer 1 knockout (KO) mice. Here we report that Homer 1 KO mice additionally exhibit a pronounced endocrine phenotype, displaying a profoundly increased adrenal gland weight and increased adrenal/body weight ratio. Histological examinations of Homer 1 deficient adrenal glands revealed an increased size of the adrenal cortex, especially the sizes of the zona fasciculata and zona glomerulosa. Moreover, the plasma corticosterone and aldosterone were higher in Homer 1 KO than wild-type (WT) mice while the plasma ACTH levels were not different between the genotypes. The in vivo ACTH test revealed that corticosterone and aldosterone plasma levels were higher in saline injected Homer 1 KO mice than in WT mice (saline injected mice served as controls for the respective groups of ACTH-injected animals), but the magnitude of steroid responses to ACTH was similar in both genotypes. In contrast, an in vitro experiment performed on isolated cells of adrenal cortex clearly showed increased production of both steroids in response to ACTH in Homer 1 KO cells, which is in line with an ~8-fold increase in the expression of ACTH receptor mRNA in the adrenal cortex of these mutants. These results, together with the detection of Homer 1 mRNA and protein in the adrenal cortex of WT mice, indicate that Homer 1 directly affects the steroidogenic function of the adrenal glands.     

Nestin expression in normal adrenal gland and adrenocortical tumors

Human adrenocortical cells have been shown to express cytokeratins and vimentin. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous system and that has been recently reported in rat adrenal gland as well. Using immunohistochemical and biochemical approaches, the present study demonstrates that nestin is constantly expressed in situ in the cortex of normal human adrenal glands. Nestin expressing cells were prevalently located in the zona reticularis but some positive cells could be spotted in the zona fasciculata as well. Moreover, patches of nestin-positive cells have been constantly detected on sections of cortical adenomas. In contrast, adrenal carcinomas displayed a variable number of nestin-immunoreactive cells that in some cases were virtually absent. Samples of renal clear cell carcinoma metastasis in the adrenals were also examined which did not show nestin-immunoreactivity. We propose that a positive nestin-immunoreaction could be useful in differential diagnosis of clear cell tumors in adrenal glands.     

APP processing and synaptic plasticity in presenilin-1 conditional knockout mice

We have developed a presenilin-1 (PS1) conditional knockout mouse (cKO), in which PS1 inactivation is restricted to the postnatal forebrain. The PS1 cKO mouse is viable and exhibits no gross abnormalities. The carboxy-terminal fragments of the amyloid precursor protein differentially accumulate in the cerebral cortex of cKO mice, while generation of beta-amyloid peptides is reduced. Expression of Notch downstream effector genes, Hes1, Hes5, and Dll1, is unaffected in the cKO cortex. Although basal synaptic transmission, long-term potentiation, and long-term depression at hippocampal area CA1 synapses are normal, the PS1 cKO mice exhibit subtle but significant deficits in long-term spatial memory. These results demonstrate that inactivation of PS1 function in the adult cerebral cortex leads to reduced Abeta generation and subtle cognitive deficits without affecting expression of Notch downstream genes.     

On the presence of chromaffin cells in the adrenal cortex: their possible role in adrenocortical function

Using light and electron microscopy, we have observed the presence of rays containing medullary tissue extending across the cortex of rat adrenal glands. Within these rays chromaffin cells, as well as collagen and nerve fibers, were present. It is suggested that these endocrine cells may have a paracrine function within the cortex, possibly via their secretory product.     

Keratin 5 knockout mice reveal plasticity of keratin expression in the corneal epithelium

We have recently demonstrated that the keratin K3 gene, which is active in the suprabasal human corneal epithelium, is missing in the genome of the mouse. We show that a normal K3 gene exists in a wide variety of mammals while in rodents the gene is converted to a pseudogene with a very strong sequence drift. The availability of K5-/- mice provides a unique opportunity to investigate type-specific keratin function during corneal differentiation in the absence of both K5 and K3. Here, we report that the deletion of K5, which in wild-type mice forms a cytoskeleton with K12, does neither cause keratin aggregation nor cytolysis in the cornea. This is due to the induction of K4 in corneal epithelial cells, normally restricted to corneal stem stem cells residing in the limbus. Using a combination of antibodies and RT-PCR, we identified additional keratins expressed in the mouse cornea including K23 which was previously thought to be specific for pancreatic carcinomas. This reflects an unexpected complexity of keratin expression in the cornea. Our data suggest that in the absence of mechanical stress, corneal differentiation does not depend on distinct keratin pairs, supporting a concept of functional redundancy, at least for certain keratins.     

Studies on the adrenal cortex of hypophysectomized rats: A model for abnormal cellular atrophy and death

Summary Biochemical and ultrastructural studies indicate that the atrophy of adrenal cortex in hypoyhysectomized rats involves the following changes: (1) One to two days after hypophysectomy, there is loss of template activity resulting from cumulative DNA-damage and heterochromatinization.In vivo ACTH-administration led to recuperation of these cells, indicating damage during hypophysectomized state to be reversible. (2) If the duration of hypophysectomy is prolonged, some of the cells become irreversibly damaged and can no longer recuperate afterin vivo ACTH administration. (3) The period of most rapid cell death is from the third to seventh day after hypophysectomy. The cause of cell death is probably due to membrane damage in the absence of protein synthesis, leading to lysis of the cells. Lysozomes and macrophages are apparently not involved.Supported by U.S.P.H.S. grants AM-5384 and AM-13724 and taken in part from dissertations submitted by Chan and by Mostafapour to Wayne State University in partial fulfillment towards the Ph.D. degree.An invited article.     

Expression of the gene for the alpha-subunit of inhibin in human adrenocortical tumours

BACKGROUND: To determine whether the pathogenesis of human adrenocortical tumours is associated with variations of inhibin expression, we assayed the mRNA of the alpha-subunit of inhibin in 5 normal adrenals and 48 adrenocortical tumours, including 10 paediatric tumours. RESULTS: mRNA of alpha-subunit of inhibin was detected in all adrenocortical tissues. It was similarly abundant in the three pathological groups of adult tumours (benign, suspect and malignant) and in normal adrenal tissues, irrespective of the hormonal pattern. However, in paediatric tumours, the levels of the mRNA for the alpha-subunit of inhibin were significantly higher than those in adult tumours (p < 0.01). CONCLUSION: Inhibin is more abundant in the foetal than in the adult adrenal cortex and therefore these data suggest that the paediatric tumours may have a foetal pattern.     

The biology of caveolae: lessons from caveolin knockout mice and implications for human disease

Caveolae, plasma membrane invaginations that serve as membrane organizing centers, are found in most cell types, but are enriched in adipocytes, endothelial cells, and myocytes. Three members of the caveolin family (Cav-1, -2, and -3) are essential for the formation of caveolae. Specialized motifs in the caveolin proteins function to recruit lipids and proteins to caveolae for participation in intracellular trafficking of cellular components and operation in signal transduction. Mutations in the gene encoding CAV-1 are associated with the development and progression of breast cancers, whereas mutations in the CAV-3 gene result in Rippling Muscle Disease and a form of Limb-Girdle Muscular Dystrophy. The generation of caveolin-null mice has confirmed the essential role of these proteins in caveolae biogenesis and in the pathophysiology of diverse tissues. Caveolin-null mice provide new animal models for studying the pathogenesis of a number of human diseases, including cancer, diabetes, atherosclerosis, restrictive lung disease and pulmonary fibrosis, cardiomyopathy, muscular dystrophy, and bladder dysfunction.     

Cyclin D2 and p27 are tissue-specific regulators of tumorigenesis in inhibin alpha knockout mice

Inhibins are heterodimeric (alpha:betaA and alpha:betaB) endocrine, paracrine, and autocrine factors of the TGFbeta superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin alpha (Inhalpha) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inhalpha knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1-->S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inhalpha mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inhalpha knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inhalpha lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin alpha single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin alpha knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inhalpha double knockouts. After gonadectomy, 50% of cyclin D2, Inhalpha double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin alpha double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inhalpha single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inhalpha knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland.     

Retention and egg production of Microphallus pygmaeus in mice: the influence of the adrenal cortex

Fewer adult Microphallus pygmaeus are recovered from the small intestine of adrenalectomized male mice than from sham-operated counterparts. Preinfection intraperitoneal injection of 2 IU or 4 IU ACTH daily for 7 days increases the initial primary worm burden in male mice. Preinfection intramuscular injection of 325 micrograms corticosterone daily for 7 days increases the initial worm burden and alters the subsequent rate of decline and duration of the primary infection. However, egg production is unaffected. The changes in parasite numbers are attributed to the immunosuppressive action of corticosterone.     

The broken mouse: the role of development, plasticity and environment in the interpretation of phenotypic changes in knockout mice

With the advent of gene knockout technology has arisen the problem of how to interpret the resulting phenotypic changes in mice lacking specific genes. This problem is especially relevant when applied to behavioral phenotypes of knockout mice, which are difficult to interpret. Of particular interest are the roles of development and compensatory changes, as well as other factors, such as the influence of the gene knockout on nearby genes, the effect of the genetic background strain, maternal behavioral influences, and pleiotrophy.     

Histological studies of influences of pregnancy and lactation on the adrenal cortex in mice

Summary Histological and histometric studies were made of the adrenal cortex of the mouse during pregnancy and lactation. In earlier pregnancy the cortex histologically shows almost no changes, except that the X zone disappears during this period of the first pregnancy. In later pregnancy and at parturition the fasciculata cells are less vacuolated in appearence and relatively large in size, and their nuclei are remarkably enlarged in volume. After parturition the histophysiological findings of the fasciculata cells are different between lactating and non-lactating mice. In lactating mice the fasciculata cells show a marked increase in vacuolation and a striking enlargement of the size. Their nuclei are also hypertrophie. In non-lactating mice, the fasciculata cells are almost the same both in vacuolation and in size as those of normal females, although their nuclei remain somewhat large in volume. These results are discussed particularly in relation to the thymic changes dealt with in a previous paper (Ito and Hoshino 1962): the histophysiological findings of the fasciculata would give an explanation for the thymic changes during pregnancy and lactation.     

Increased neuronal excitability, synaptic plasticity, and learning in aged Kvbeta1.1 knockout mice

BACKGROUND: Advancing age is typically accompanied by deficits in learning and memory. These deficits occur independently of overt pathology and are often considered to be a part of "normal aging." At the neuronal level, normal aging is known to be associated with numerous cellular and molecular changes, which include a pronounced decrease in neuronal excitability and an altered induction in the threshold for synaptic plasticity. Because both of these mechanisms (neuronal excitability and synaptic plasticity) have been implicated as putative cellular substrates for learning and memory, it is reasonable to propose that age-related changes in these mechanisms may contribute to the general cognitive decline that occurs during aging. RESULTS: To further investigate the relationship between aging, learning and memory, neuronal excitability, and synaptic plasticity, we have carried out experiments with aged mice that lack the auxiliary potassium channel subunit Kvbeta1.1. In aged mice, the deletion of the auxiliary potassium channel subunit Kvbeta1.1 resulted in increased neuronal excitability, as measured by a decrease in the post-burst afterhyperpolarization. In addition, long-term potentiation (LTP) was more readily induced in aged Kvbeta1.1 knockout mice. Finally, the aged Kvbeta1.1 mutants outperformed age-matched controls in the hidden-platform version of the Morris water maze. Interestingly, the enhancements in excitability and learning were both sensitive to genetic background: The enhanced learning was only observed in a genetic background in which the mutants exhibited increased neuronal excitability. CONCLUSIONS: Neuronal excitability is an important determinant of both synaptic plasticity and learning in aged subjects.     

Regulable expression of inhibin A in wild-type and inhibin alpha null mice

Exogenous regulation of protein expression creates the potential to examine the consequences of homeostatic Dysregulation in many physiological systems and, when used in transgenic mice, provides the capability of restoring a gene product to its knockout background without antigenicity issues. In this study, we used a mifeprisone-inducible system (the GeneSwitch system) to regulate the expression of inhibin A from the liver of mice. Inhibin is a heterodimeric protein (alpha/beta) wherein one of its subunits (beta) is capable of homodimerizing to form its physiological antagonist, activin (beta/beta). Inhibin is also expressed in two forms, A and B, as determined by the subtype of beta-subunit that dimerizes with the alpha-subunit (alpha/betaA or alpha/betaB). To utilize the GeneSwitch system, transgenic transactivator mice with liver-specific expression of a mifepristone-activated chimeric nuclear receptor (GLVP) were crossed with transgenic target mice containing a GVLP-responsive promoter upstream of polio-virus IRES (internal ribosome entry site)-linked sequences coding for the alpha- and beta-subunits of inhibin A. This intercross produced "bigenic" mice capable of regulable expression of inhibin A from the liver. Overexpression of inhibin A in wild-type mice produced a phenotype wherein males had decreased testis size and females had a block in folliculogenesis at the early antral stage, findings similar to activin type IIA receptor (ActRIIA) null mice. These phenotypes were most likely due to suppressed serum FSH, confirming that the liver-derived inhibin A was secreted into the serum to down-regulate pituitary FSH levels. Furthermore, the generation of bigenic mice in the inhibin alpha null background allowed for the induction of inhibin A in inhibin alpha null male mice with subsequent rescue of these mice from their gonadal tumor-induced lethal phenotype. This work demonstrates the in vivo production of a heterodimeric hormone from a single inducible promoter to study its therapeutic and physiological effects. In addition, these studies are the first example of an inducible system being used to prevent a lethal knockout phenotype in an animal model.