Effect of plasma from cyclic versus nutritionally induced anovulatory beef heifers on proliferation of granulosa cells in vitro.

The effect of plasma from cyclic versus nutritionally induced anovulatory beef heifers was evaluated on proliferation of bovine granulosa cells in vitro. Granulosa cells were obtained from small (1-5mm) follicles of cattle and cultured for 4 days. During the last 2 days of culture, cells were exposed to medium containing 0, 1 or 10% plasma from cyclic or anovulatory heifers in the presence or absence of IGF-I (100ng/ml). Cell numbers were determined. Regardless of source, increasing percentage of plasma to culture medium increased cell numbers. However, the plasma-induced increase was greater in granulosa cells exposed to cyclic heifer plasma versus anovulatory heifer plasma. In addition, concomitant treatment with IGF-I dramatically improved cell proliferation induced by anovulatory heifer plasma. These results indicate that plasma from cyclic heifers contain factors that are a greater stimulus to granulosa cell proliferation than plasma from anovulatory heifers. Systemic factors such as IGF-I may play a role in directly regulating granulosa cell proliferation in cattle.

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus.

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)i-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIA(i) locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene's involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas.

Protein kinase A and its role in human neoplasia: the Carney complex paradigm.

The type 1 alpha regulatory subunit (R1alpha) of cAMP-dependent protein kinase A (PKA) (PRKAR1A) is an important regulator of the serine-threonine kinase activity catalyzed by the PKA holoenzyme. Carney complex (CNC) describes the association 'of spotty skin pigmentation, myxomas, and endocrine overactivity'; CNC is in essence the latest form of multiple endocrine neoplasia to be described and affects the pituitary, thyroid, adrenal and gonadal glands. Primary pigmented nodular adrenocortical disease (PPNAD), a micronodular form of bilateral adrenal hyperplasia that causes a unique, inherited form of Cushing syndrome, is also the most common endocrine manifestation of CNC. CNC and PPNAD are genetically heterogeneous but one of the responsible genes is PRKAR1A, at least for those families that map to 17q22-24 (the chromosomal region that harbors PRKAR1A). CNC and/or PPNAD are the first human diseases to be caused by mutations in one of the subunits of the PKA holoenzyme. Despite the extensive literature on R1alpha and PKA, little is known about their potential involvement in cell cycle regulation, growth and/or proliferation. The presence of inactivating germline mutations and the loss of its wild-type allele in CNC lesions indicated that PRKAR1A could function as a tumor-suppressor gene in these tissues. However, there are conflicting data in the literature about PRKAR1A's role in human neoplasms, cancer cell lines and animal models. In this report, we review briefly the genetics of CNC and focus on the involvement of PRKAR1A in human tumorigenesis in an effort to reconcile the often diametrically opposite reports on R1alpha.

Ontogeny of corticosterone-inducible growth hormone-secreting cells during chick embryonic development.

We reported that corticosterone administration into the albumen of fertile chicken eggs on embryonic day (e) 11 induces an increase in the population of GH-secreting cells. The present study evaluated the ontogeny, dose response, localization, and persistence of the glucocorticoid-induced increase in the somatotroph population during chicken embryonic development. Corticosterone (0, 0.02, 0.2, and 2 microg in 300 microl saline) was injected into separate eggs on e9, e10, e11, and e12, and the population of GH-secreting cells was assessed 2 days later using reverse hemolytic plaque assays. Corticosterone treatment on e9 or e10 was unable to increase the population of GH-secreting cells on e11 or e12. In contrast, 0.2 and 2 microg of corticosterone on e11 increased the population of GH-secreting cells on e13 (P < 0.05, n = 3 experiments) to 8.2 +/- 0.6 and 6.4 +/- 0.5% of all cells, respectively, relative to controls (2.4 +/- 0.2%). For e14 embryos treated on e12, only the 2 microg dose increased the proportion of GH-secreting cells (6.4 +/- 0.6%) relative to controls (3.6 +/- 0.4%). In a second experiment, 0, 0.02, 0.2, 2, and 20 microg of corticosterone were injected on e0, e8, e9, e10, e11, and e12, and the population of GH-secreting cells was assessed on e13 in all groups. No dose of corticosterone was effective when given on e0, e8, e9, or e10. The 0.2 microg and 2 microg doses increased the population of GH-secreting cells (7.6 +/- 0.9% and 6.7 +/- 0.8%, respectively) relative to controls (2.3 +/- 0.4%) when injected on e11 (P < 0.05, n = 4 experiments). The 2-microg dose also increased GH cell abundance when injected on e12 (5.6 +/- 0.4%), relative to controls (2.7 +/- 0.5%). Treatment with 20 microg on e11 and e12 induced the greatest responses (10.3 +/- 1.1% and 8.7 +/- 0.9%, respectively). However, in subsequent experiments, administration of 20 microg on e11 resulted in embryonic death by e18. In a third set of experiments, two groups of eggs were injected either with 2 microg of corticosterone in saline or saline alone on e11, and the number of GH-secreting cells was estimated on e13, e16, e19, and the day of hatch (d1). The population of GH-secreting cells in corticosterone treated embryos was significantly higher than in saline treated embryos only on e13 (7.1 +/- 0.8% and 2.7 +/- 0.3%, respectively). No significant differences were observed on e16 (12.4 +/- 1.5% and 13.6 +/- 1.2%), e19 (19.0 +/- 1.0% and 18.2 +/- 1.7%) and d1 (23.8 +/- 2.1% and 25.1 +/- 1.8%) between corticosterone treated and control embryos, respectively. In a fourth set of experiments, whole mount in situ hybridization indicated that injection of corticosterone on e11 induced GH messenger RNA expression in the caudal part of the pituitary gland on e13, where somatotrophs are located normally later in development. We conclude that corticosterone administration in ovo can increase the population of GH-secreting cells in the caudal anterior pituitary only during a small window of development between e11 and e13 and that this premature increase of GH-secreting cells does not affect the percentage of GH-secreting cells later in development.

Identification of the somatostatin receptor subtypes involved in regulation of growth hormone secretion in chickens.

The effects of somatostatin (SRIF) are mediated through five distinct G-protein-coupled receptors (SSTR1-5). In the present study, pituitary cells from 6-week-old chickens were subjected to reverse hemolytic plaque assays for growth hormone (GH) in the presence of SSTR subtype specific nonpeptidyl agonists. A SSTR2 selective agonist (L-779,976) potently inhibited both basal and GH-releasing hormone (GHRH)-stimulated GH release at low nanomolar concentrations. A SSTR5 agonist (L-817,818) inhibited GH release only under basal conditions and in a subpopulation of somatotrophs. In contrast, a SSTR4 selective agonist (L-803,087) used at high nanomolar concentrations modestly stimulated GH release under basal conditions but did not influence GHRH-stimulated GH secretion. The SSTR1 and SSTR3 specific agonists did not affect GH secretion under any condition tested. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis using a partial cDNA for chicken SSTR2 showed relatively high levels of SSTR2 mRNA in the anterior pituitary (both in the caudal and cephalic lobes) and brain and detectable levels in liver, muscle, heart and small intestine. These results indicate that SSTR2 is the primary mediator of the inhibitory effects of SRIF on GH secretion in chickens.

Nutritionally induced anovulation in beef heifers: ovarian and endocrine function preceding cessation of ovulation.

Angus x Hereford heifers were used to determine endocrine and ovarian function preceding nutritionally induced anovulation. Six heifers were fed to maintain body condition score (M), and 12 heifers were fed a restricted diet (R) until they became anovulatory. Starting on d 13 of an estrous cycle, heifers were given PGF2alpha every 16 d thereafter to synchronize and maintain 16 d estrous cycles. Ovarian structures of M and R heifers were monitored by ultrasonography daily from d 8 to ovulation (d 1 of the subsequent cycle) until R heifers became anovulatory. Concentrations of LH and FSH were quantified in serum samples collected every 10 min for 8 h on d 2 and 15 (48 h after PGF2alpha), and estradiol and IGF-I were quantified in daily plasma samples from d 8 to 16 during the last ovulatory cycle (Cycle -2) and the subsequent anovulatory cycle (Cycle -1). During the last two cycles before anovulation, M heifers had 50% larger (P < .0001) ovulatory follicles than R heifers and 61% greater (P < .0001) growth rate of the ovulatory follicles. There was a treatment x cycle x day effect (P < .001) for concentrations of estradiol. The preovulatory increase in estradiol occurred in the R and M heifers during Cycle -2 but only in M heifers during Cycle -1. A treatment x cycle x day effect (P < .05) influenced LH concentrations. During Cycle -2, LH concentrations were similar for M and R heifers, but during Cycle -1, M heifers had greater LH concentrations than did R heifers. Concentrations of FSH were greater (P < .05) in R than M heifers after induced luteolysis when R heifers failed to ovulate. There was a treatment x cycle interaction (P < .05) for IGF-I concentrations, and M heifers had 4.7- and 8.6-fold greater IGF-I concentrations than did R heifers during Cycle -2 and -1, respectively. We conclude that growth rate and diameter of the ovulatory follicle, and concentrations of LH, estradiol, and IGF-I are reduced before the onset of nutritionally induced anovulation in beef heifers.

Protein kinase A subunit expression is altered in Bloom syndrome fibroblasts and the BLM protein is increased in adrenocortical hyperplasias: inverse findings for BLM and PRKAR1A.

Bloom syndrome is a genetic disorder associated with chromosomal instability and a predisposition to tumors that is caused by germline mutations of the BLM gene, a RecQ helicase. Benign adrenocortical tumors display a degree of chromosomal instability that is more significant than benign tumors of other tissues. Cortisol-producing hyperplasias, such as primary pigmented nodular adrenocortical disease (PPNAD), which has been associated with protein kinase A (PKA) abnormalities and/or PRKAR1A mutations, also show genomic instability. Another RecQ helicase, WRN, directly interacts with the PRKAR1B subunit of PKA. In this study, we have investigated the PRKAR1A expression in primary human Bloom syndrome cell lines with known BLM mutations and examined the BLM gene expression in PPNAD and other adrenal tumor tissues. PRKAR1A and other protein kinase A (PKA) subunits were expressed in Bloom syndrome cells and their level of expression differed by subunit and cell type. Overall, fibroblasts exhibited a significant decrease in protein expression of all PKA subunits except for PRKAR1A, a pattern that has been associated with neoplastic transformation in several cell types. The BLM protein was upregulated in PPNAD and other hyperplasias, compared to samples from normal adrenals and normal cortex, as well as samples from cortisol- and aldosterone-producing adenomas (in which BLM was largely absent). These data reveal an inverse relationship between BLM and PRKAR1A: BLM deficiency is associated with a relative excess of PRKAR1A in fibroblasts compared to other PKA subunits; and PRKAR1A deficiency is associated with increased BLM protein in adrenal hyperplasias.

Phosphodiesterase 11A expression in the adrenal cortex, primary pigmented nodular adrenocortical disease, and other corticotropin-independent lesions.

A variety of adrenal tumors and bilateral adrenocortical hyperplasias (BAH) leading to Cushing syndrome (CS) may be caused by aberrant cAMP signaling. We recently identified patients with a micronodular form of BAH that we have called "isolated micronodular adrenocortical disease" (iMAD) in whom CS was associated with inactivating mutations in phosphodiesterase (PDE) 11A ( PDE11A). In the present study, we examined PDE11A expression in normal adrenocortical tissue, sporadic tumors, and hyperplasias without PDE11A mutations, and primary pigmented nodular adrenocortical disease (PPNAD) and adenomas from patients with PRKAR1A and a single tumor with a GNAS mutation. The total number of the tumor samples that we studied was 22. Normal human tissues showed consistent PDE11A expression. There was variable expression of PDE11A in sporadic adrenocortical hyperplasia or adenomas; PPNAD tissues from patients with PRKAR1A mutations expressed consistently high levels of PDE11A in contrast to adenomas caused by GNAS mutations. Phosphorylated CREB was the highest in tissues from patients with iMAD compared to all other forms of BAH and normal adrenal tissue. We conclude that PDE11A is expressed widely in adrenal cortex. Its expression appears to be increased in PPNAD but varies widely among other adrenocortical tumors. PRKAR1A expression appears to be higher in tissues with PDE11A defects. Finally, sequencing defects in PDE11A are associated with a high state of CREB phosphorylation, just like PRKAR1A mutations. These preliminary data suggest that these two molecules are perhaps regulated in a reverse manner in their control of cAMP signaling in adrenocortical tissues.

Pituitary size fluctuation in long-term MR studies of PROP1 deficient patients: A persistent pathophysiological mechanism?

Inactivating PROP1 gene alterations are responsible for over 50% of familial combined pituitary hormone deficiency cases. Pituitary enlargement followed by regression and subnormal pituitary size has been documented in a number of PROP1 deficient patients. Data derived from PROP1 deficient mice (Ames dwarfs) have revealed some of the underlying cellular mechanisms. Nevertheless, long-term magnetic resonance imaging (MRI) findings in two PROP1 deficient patients suggest the evolution of pituitary pathology as more complex and persistent than previously described. Patient A had enlarged pituitary gland (pituitary height: 9-10 mm), demonstrated by serial MRI carried out from age 5 to 8.5 yr, small pituitary gland (4 mm) at age 10 yr and pituitary enlargement (11 mm) at age 19 yr. Patient B had a pituitary gland of normal size at age 7 yr (5 mm), whereas at age 14.3 and 16.3 yr, an enlarged pituitary gland was disclosed (10 and 11 mm, respectively). Both series of events are suggestive of a persistent pathophysiological mechanism in the pituitary gland of patients with PROP1 gene defects. Therefore, long-term pituitary follow-up by MRI in such patients may be necessary even in the case of a small or normal pituitary gland. It must be noted that current data from the Ames dwarf mouse cannot fully explain the observed pituitary size fluctuation.

Nutritionally induced anovulation in beef Heifers: ovarian and endocrine function during realimentation and resumption of ovulation.

Nutritionally induced anovulatory and cyclic Angus x Hereford heifers were used to evaluate follicular growth and concentrations of hormones and metabolites during anovulation and resumption of ovulation. Anovulatory heifers were fed to gain 0.6 (LGAIN) or 1.5 (HGAIN) kg/day until resumption of ovulation, and heifers with normal estrous cycles were fed a maintenance diet (M). Follicles >/= 4 mm in diameter were measured by daily ultrasonography in HGAIN and LGAIN heifers during one follicular wave before realimentation (Wan) and in two waves (W-2, W-1) immediately before the wave resulting in first ovulation or luteinization (W0). Ovaries of M heifers were evaluated to determine the day of ovulation of the second-wave dominant follicle (DF). Resumption of ovulation after realimentation occurred 23 days earlier in HGAIN than in LGAIN. Maximum diameter, growth rate, and persistence of dominant follicles increased, while persistence of first subordinate follicles decreased between anovulation and resumption of ovulation in anovulatory heifers. Concentrations of LH in serum were similar for HGAIN and LGAIN and gradually increased during realimentation. The increase in estradiol before the first ovulation was less in realimented heifers compared with cyclic heifers. Concentrations of insulin-like growth factor-I (IGF-I) in HGAIN and LGAIN gradually increased during realimentation but were lower than concentrations of IGF-I in cyclic heifers at ovulation. Increased diameter, growth rate, and persistence of the DF were associated with increased concentrations of LH, estradiol, and IGF-I during the transition from nutritionally induced anovulation to resumption of ovulatory cycles.