Intact follicular maturation and defective luteal function in mice deficient for cyclin- dependent kinase-4.

Cell cycle progression of granulosa cells is critical for ovarian function, especially follicular maturation. During follicular maturation, FSH induces cyclin D2, which promotes G1 progression by activating cyclin-dependent kinase-4 (Cdk4). Because cyclin D2-deficient mice exhibit a block in follicular growth, cyclin D2/Cdk4 has been hypothesized to be required for FSH-dependent proliferation of granulosa cells. Here we investigate ovarian function in Cdk4-knockout mice we recently generated. Cdk4(-/-) females were sterile, but the morphology of their ovaries appeared normal before sexual maturation. The number of preovulatory follicles and the ovulation efficiency were modestly reduced in gonadotropin-treated Cdk4(-/-) mice. However, unlike cyclin D2-deficient mice, Cdk4(-/-) mice showed no obvious defect in FSH-induced proliferation of granulosa cells. Cdk4(-/-) ovaries displayed normal preovulatory expression of aromatase, PR, and cyclooxygenase-2. Postovulatory progesterone secretion was markedly impaired in Cdk4(-/-) mice, although granulosa cells initiated luteinization with induction of p450 side-chain cleavage cytochrome and p27(Kip1). Progesterone treatment rescued implantation and restored fertility in Cdk4(-/-) mice. Serum PRL levels after mating were significantly reduced in Cdk4(-/-) mice, suggesting the involvement of perturbed PRL regulation in luteal failure. Thus, Cdk4 is critical for luteal function, and some redundant protein(s) can compensate for the absence of Cdk4 in proliferation of granulosa cells.     

Thyroid dysfunction in megalin deficient mice

Megalin mediates transcytosis of thyroglobulin (Tg), the thyroid hormone precursor, resulting in its passage into the bloodstream. The process involves especially hormone-poor Tg, which may favour hormone secretion by preventing competition with hormone-rich Tg for proteolytic degradation. To gain more insight into the role of megalin, here we studied thyroid function and histology in megalin deficient mice compared with WT mice. As expected from the knowledge that megalin mediates Tg transcytosis, serum Tg levels were significantly reduced in homozygous (megalin-/-) mice, which, more importantly, were found to be hypothyroid, as demonstrated by significantly reduced serum free thyroxine and significantly increased serum thyroid stimulating hormone (TSH) levels. In heterozygous (megalin+/-) mice, in which megalin expression was normal, thyroid function was unaffected. Although the serological phenotype in megalin-/- mice was not associated with histological alterations or goiter, our results support a major role of megalin in thyroid hormone secretion.     

Pituitary hormone gene expression and secretion in human growth hormone-releasing hormone transgenic mice: focus on lactotroph function

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per microg total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased 2-fold in transgenic mice. Dopamine D2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled with an increase in D2 dopamine receptor gene expression and functional sensitivity in the pituitary result in normal plasma levels of PRL.     

Pituitary dysfunction in granulomatosis with polyangiitis

Purpose

Granulomatosis with polyangiitis (GPA) is a multisystem disease, characterized by necrotizing small-vessel vasculitis, which mainly affects the respiratory tract and the kidneys. Pituitary involvement in GPA is rare, present in about 1% of all cases of GPA. To date, only case reports or small case series have been published. Herein we report clinical features, imaging findings, treatment and outcomes in three patients with GPA-related pituitary dysfunction (PD).

Methods

A retrospective analysis of three cases of GPA-related PD was conducted, followed by systematic review of the English medical literature using PubMed.

Results

The three cases include three women aged between 32 and 37 years. PD was the presenting feature in one and two developed PD in the course of the disease. All patients had a pituitary lesion on MRI. Conventional treatment with high doses of glucocorticoids and cyclophosphamide led to resolution or improvement of the MRI abnormalities, whereas it was not effective in restoring PD. A systematic review identified 51 additional patients, showing that GPA can lead to partial or global PD, either at onset or, during the course of the disease. Secondary hypogonadism is the predominant manifestation, followed by diabetes insipidus (DI). Sellar mass with central cystic lesion is the most frequent radiological finding.

Conclusion

GPA should be carefully considered in patients with a sellar mass and unusual clinical presentation with DI and systemic disease. Although conventional induction-remission treatment improves systemic symptoms and radiological pituitary abnormalities, hormonal deficiencies persist in most of the patients. Therefore, follow-up should include both imaging and pituitary function assessment.

     

Pituitary dysfunction in granulomatosis with polyangiitis

Clinical Rheumatology - Granulomatosis with polyangiitis (GPA) is a necrotizing granulomatous vasculitis of small vessels that affect the pituitary gland in less than 1% of cases being...     

Increased myocardial collagen and ventricular diastolic dysfunction in relaxin deficient mice: a gender-specific phenotype

OBJECTIVE: To investigate cardiac phenotypes in mice deficient in the peptide hormone relaxin by gene targeting. METHODS: Echocardiography and cardiac catheterization were performed on male and female relaxin deficient (Rlx(-/-)) mice as well as heterozygous (Rlx(+/-)) and wildtype (Rlx(+/+)) littermates aged between 8 and 24 months. Collagen expression and content in the heart were analysed by real-time PCR, hydroxyproline assay and histology. RESULTS: Heart rate, blood pressures, left ventricular (LV) dimensions, fractional shortening and maximal and minimal dP/dt did not differ significantly between the three genotypes of either gender at any age. However, 8-10-month-old Rlx(-/-) males exhibited a greater transmitral flow velocity (A-wave) at the late LV diastolic phase. Male Rlx(-/-) mice aged between 12 and 24 months had significantly higher LV end-diastolic pressures, a 30% increase in atrial weight and 10-30% increases in lung and liver weights. Male mice also showed an age-dependent increase (P<0.01) in LV collagen content that was more pronounced in Rlx(-/-) than control littermates (P<0.01). Procollagen type-1 expression was also significantly higher in the LV of Rlx(-/-) males compared with either Rlx(+/-) or Rlx(+/+) males at 6, 9 and 12 months of age. Age-matched female Rlx(-/-) mice did not display any of these cardiac phenotypes seen in Rlx(-/-) males. CONCLUSIONS: Male Rlx(-/-) mice had impeded LV diastolic filling and increased atrial weights, most likely due to an increase in ventricular collagen content and chamber stiffness. These phenotypes in the Rlx(-/-) males were not observed in Rlx(-/-) females, indicating the importance of other gender-related factors in cardiovascular function.     

Pituitary enlargement with suprasellar extension in functional hyperprolactinemia due to lactotroph hyperplasia: a pseudotumoral disease

Beside the well characterized PRL-secreting adenomas, a wide spectrum of functional hyperprolactinemic states exists. We describe here five women, 21-38 yr old, all suspected of having a PRL-secreting adenoma because of a pseudotumoral appearance of the pituitary on computerized tomographic (CT) scan or magnetic resonance imaging (MRI). Four had oligomenorrhea with or without galactorrhea, one had amenorrhea with galactorrhea, and two complained of infertility. In the same patient, basal plasma PRL levels were variable on different days, sometimes normal (mean +/- SEM, 11.3 +/- 1.5 micrograms/L), sometimes elevated (49 +/- 7 micrograms/L), but in all cases, a PRL response of large amplitude to TRH (6- to 8-fold increase in the basal value) was observed. Basal plasma levels of estradiol were within luteal phase normal values (0.41 +/- 0.13 pmol/L), while progesterone levels were low (1.92 +/- 0.47 nmol/L). CT scan or MRI showed an intrasellar mass with suprasellar extension, suggesting a tumoral process. However, the signal intensity was homogeneous, and on coronal views, the suprasellar extension was pyramidal and symmetrical, and the pituitary stalk was always in the midline. The five patients were operated on by the transsphenoidal route, but no adenoma was found. Surgical biopsies were taken in four cases, and lactotroph hyperplasia, i.e. enlarged cell cords consisting mainly of PRL cells, was found in three of them. One case displayed a continuum between areas of lactotroph hyperplasia and adenomatous PRL cells. We conclude that functional hyperprolactinemia may mimic on CT scan or MRI a PRL-secreting adenoma.     

Normal muscle glucose uptake in mice deficient in muscle GLUT4

Skeletal muscle insulin resistance is a major characteristic underpinning type 2 diabetes. Impairments in the insulin responsiveness of the glucose transporter, Glut4 (Slc2a4), have been suggested to be a contributing factor to this disturbance. We have produced muscle-specific Glut4 knockout (KO) mice using Cre/LoxP technology on a C57BL6/J background and shown undetectable levels of GLUT4 in both skeletal muscle and heart. Our aim was to determine whether complete deletion of muscle GLUT4 does in fact lead to perturbations in glucose homoeostasis. Glucose tolerance, glucose turnover and 2-deoxyglucose uptake into muscle and fat under basal and insulin-stimulated conditions were assessed in 12-week-old KO and control mice using the oral glucose tolerance test (OGTT) and hyperinsulinaemic/euglycaemic clamp respectively. KO mice weighed ～17% less and had significantly heavier hearts compared with control mice. Basally, plasma glucose and plasma insulin were significantly lower in the KO compared with control mice, which conferred normal glucose tolerance. Despite the lack of GLUT4 in the KO mouse muscle, glucose uptake was not impaired in skeletal muscle but was reduced in heart under insulin-stimulated conditions. Neither GLUT1 nor GLUT12 protein levels were altered in the skeletal muscle or heart tissue of our KO mice. High-fat feeding did not alter glucose tolerance in the KO mice but led to elevated plasma insulin levels during the glucose tolerance test. Our study demonstrates that deletion of muscle GLUT4 does not adversely affect glucose disposal and glucose tolerance and that compensation from other transporters may contribute to this unaltered homoeostasis of glucose.     

Young MLP deficient mice show diastolic dysfunction before the onset of dilated cardiomyopathy

Targeted deletion of cytoskeletal muscle LIM protein (MLP) in mice consistently leads to dilated cardiomyopathy (DCM) after one or more months. However, next to nothing is known at present about the mechanisms of this process. We investigated whether diastolic performance including passive mechanics and systolic behavior are altered in 2-week-old MLP knockout (MLPKO) mice, in which heart size, fractional shortening and ejection fraction are still normal. Right ventricular trabeculae were isolated from 2-week-old MLPKO and wildtype mice and placed in an apparatus that allowed force measurements and sarcomere length measurements using laser diffraction. During a twitch from the unloaded state at 1 Hz, MLPKO muscles relengthened to slack length more slowly than controls, although the corresponding force relaxation time was unchanged. Active developed stress at a diastolic sarcomere length of 2.00 microm was preserved in MLPKO trabeculae over a wide range of pacing frequencies. Force relaxation under the same conditions was consistently prolonged compared with wildtype controls, whereas time to peak and maximum rate of force generation were not significantly altered. Ca2+ content of the sarcoplasmic reticulum (SR) and the quantities of Ca2+ handling proteins were similar in both genotypes. In summary, young MLPKO mice revealed substantial alterations in passive myocardial properties and relaxation time, but not in most systolic characteristics. These results indicate that the progression to heart failure in the MLPKO model may be driven by diastolic myocardial dysfunction and abnormal passive properties rather than systolic dysfunction.     

Pituitary tumorigenesis in prolactin gene-disrupted mice

Targeted disruption [knockout (KO)] of the mouse prolactin (PRL) gene created an animal model of primary isolated PRL deficiency in which there is no detectable PRL bioactivity. Pituitary glands of young adult female PRLKO mice were hyperplastic, and many cells had expanded cytoplasms with granular accumulations of an N-terminal peptide encoded by the disrupted PRL gene (KO/10 peptide). Confocal imaging showed that the pituitaries in PRL(+/+) and PRL(+/-) females contained dense accumulations of apparently Golgi-associated immunoreactive PRL. PRLKO female mice (15-18 months old) developed hyperemic pituitary adenomas. The pituitary tumors in PRLKO mice synthesized the KO/10 peptide, which implies that the tumors arise from the lactotroph lineage. Anchorage-independent growth was observed among pituitary cells from PRLKO mice, aged 8 months or older, but not in cells from 3-month-old PRLKO mice. GH cells appeared to be normal in PRLKO pituitaries, but were displaced by the hyperplastic and hypertrophic growth of KO/10-positive cells. Bromocriptine suppressed mean pituitary weight in 8-month-old PRLKO mice compared with vehicle-treated PRLKO animals (20 +/- 0.01 and 60 +/- 10 mg; P < 0.01). We infer that pituitary lactotrophs of PRLKO mice suffer from a dual pathology that includes hypertrophy resulting from endoplasmic reticulum expansion and hyperplasia, with adenomatous transformation, in part as a consequence of disrupted dopaminergic feedback regulation.     

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19(Ink4d) and p27(Kip1), in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G(2) and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19(Ink4d) and p27(Kip1) cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.     

Premature ovarian aging in mice deficient for Gpr3

After becoming competent for resuming meiosis, fully developed mammalian oocytes are maintained arrested in prophase I until ovulation is triggered by the luteotropin surge. Meiotic pause has been shown to depend critically on maintenance of cAMP level in the oocyte and was recently attributed to the constitutive Gs (the heterotrimeric GTP-binding protein that activates adenylyl cyclase) signaling activity of the G protein-coupled receptor GPR3. Here we show that mice deficient for Gpr3 are unexpectedly fertile but display progressive reduction in litter size despite stable age-independent alteration of meiotic pause. Detailed analysis of the phenotype confirms premature resumption of meiosis, in vivo, in about one-third of antral follicles from Gpr3-/- females, independently of their age. In contrast, in aging mice, absence of GPR3 leads to severe reduction of fertility, which manifests by production of an increasing number of nondeveloping early embryos upon spontaneous ovulation and massive amounts of fragmented oocytes after superovulation. Severe worsening of the phenotype in older animals points to an additional role of GPR3 related to protection (or rescue) of oocytes from aging. Gpr3-defective mice may constitute a relevant model of premature ovarian failure due to early oocyte aging.     

Cardiac morphogenetic defects and conduction abnormalities in mice homozygously deficient for connexin40 and heterozygously deficient for connexin45

Connexin40 (Cx40) and connexin45 (Cx45) are involved in both cardiac morphogenesis and propagation of electrical activity. We found that Cx40/Cx45 double deficiency (Cx40(-/-)/Cx45(+/-)) causes a variety of cardiac defects leading to high mortality during embryonic development and at birth. The majority of Cx40(-/-)/Cx45(+/-) embryos and postnatal mice suffered from atrioventricular septal defects. Additional cardiac abnormalities, e.g., ventricular septal defects and abnormal myocardial arrangement, occurred at lower abundance. Electrocardiograms of Cx40(-/-)/Cx45(+/+) and Cx40(-/-)/Cx45(+/-) mice revealed prolongation of P-wave, PQ interval and QRS duration compared to controls. Interestingly, in Cx40(-/-)/Cx45(+/-) mice, PQ interval and QRS duration were significantly prolonged compared to Cx40(-/-)/Cx45(+/+) mice. We conclude that the gap junctional proteins Cx40 and Cx45 have overlapping and partially compensatory functions with regard to heart morphogenesis and cardiac conduction. Cx45 might be one of the genetic modifiers that can cause variations in the phenotype of connexin40-deficient animals. Our findings may be particularly relevant for understanding molecular factors contributing to human congenital cardiac diseases.     

Pituitary adenomas in mice transgenic for growth hormone-releasing hormone.

It has been shown that mice transgenic for human GH-releasing hormone (GRH) develop hyperplasia of pituitary somatotrophs, lactotrophs, and mammosomatotrophs, cells capable of producing both GH and PRL, by 8 months of age. We now report that GRH transgenic mice 10-24 months of age develop pituitary adenomas, which we characterized by histology, immunohistochemistry, in situ hybridization, and electron microscopy. Of 13 animals examined, all developed GH-immunoreactive neoplasms that had diffuse positivity for GH mRNA by in situ hybridization. Eleven also contained PRL immunoreactivity; in situ hybridization demonstrated focal PRL mRNA in 3 of 5 immunohistochemically positive tumors. Alpha-Subunit was positive by immunohistochemistry in 8 adenomas, and TSH beta was localized in tumor cells of 5 adenomas. The adenomas had variable ultrastructural appearances, ranging from cells that resembled somatotrophs or mammosomatotrophs to cells with features of the glycoprotein hormone cell line. These findings provide conclusive evidence that protracted GRH stimulation of secretory activity can result in proliferation, hyperplasia, and adenoma of adenohypophysial cells.