Regulation of testicular proopiomelanocortin gene expression

The POMC gene is expressed in testicular Leydig cells, but its mRNA is about 150-200 nucleotides shorter in these cells than in the pituitary. For this reason this testicular mRNA has been termed POMC-like mRNA. The purpose of the present study was to define the ontogeny and regulation of POMC gene expression in rat testis. The level of POMC-like mRNA was very low in the testes of 15- and 20-day-old animals. A dramatic increase in mRNA concentration was observed between 20 and 25 days of age, and maximal levels were detected at 40 days. The ontogeny of testicular POMC gene expression correlates with the reported increases in Leydig cell numbers, immunostainable beta-endorphin in Leydig cells, and testicular LH receptors during development. Since these observations suggested that the expression of testicular POMC gene might be influenced by LH, we studied the effect of hypophysectomy and hCG treatment on testicular POMC-like mRNA. Total contents of testicular RNA and POMC-like mRNA decreased in parallel with the decline of testicular weight after hypophysectomy. Administration of hCG to rats 6 days after hypophysectomy prevented the regression of testes and the decrease in testicular POMC-like mRNA content. An increase in the total amount of testicular POMC-like mRNA was observed relative to that in hypophysectomized controls after 8 days of hCG injection. Similar results were obtained when hCG was administered to rats 13 days after hypophysectomy. The effect of glucocorticoid deprivation on testicular POMC-like mRNA was also studied. The POMC-like mRNA concentration did not increase in testis as it did in the anterior pituitary after adrenalectomy, suggesting that glucocorticoids are not a primary regulator of POMC-like mRNA in the testis. In summary, the ontogeny of expression of testicular POMC gene correlates closely with the maturation pattern of Leydig cells; and the expression of testicular POMC gene is regulated by gonadotropins and not by glucocorticoids. We conclude that the regulation of POMC-like mRNA in the testis is different from that in the pituitary.     

Genomic structure of the proopiomelanocortin gene and expression during acute low-water stress in channel catfish

Proopiomelanocortin (POMC) is an important gene involved in the stress response of the hypothalamic-pituitary-adrenal axis. It is a precursor of several peptide hormones including adrenocorticotropic hormone, melanocyte stimulating hormones, and beta-endorphin. Our study aims to determine genomic structure and expression of POMC gene during temporal stress in channel catfish (Ictalurus punctatus). The catfish POMC gene consisting of three exons and two introns has a similar structural organization to that of other species. The catfish and mammalian POMC promoters do not exhibit regions of conservation except that of one TATA box. Genomic Southern blot analysis indicated POMC is present as a single copy gene in the catfish genome. Real-time PCR allowed us to monitor temporal expression of the POMC mRNA in catfish pituitary during low-water stress. Plasma cortisol concentrations were also measured as an indicator of stress. Within 15 min after the onset of low-water stress, POMC mRNA expression was elevated 1.87-fold above the control value. The POMC mRNA level had declined after 30 min (1.29-fold) and 1h (1.1-fold) at which time stress was removed. After 1h recovery, a significant increase in the POMC mRNA expression was detected (2.44-fold, P<0.05) followed by a decline 2h later (1.52-fold) when the experiment was terminated. Plasma cortisol levels in stressed fish were significantly above the cortisol levels in control fish during stress application (t=15 min, t=30 min, and t=1h, P<0.05), which then returned to normal during recovery. We conclude that POMC and cortisol are both involved in the low-water stress response during which cortisol may serve as a negative regulator of POMC expression in catfish.     

Decreased type 2 corticotropin-releasing hormone receptor mRNA expression in the ventromedial hypothalamus during repeated immobilization stress.

Chronic or repeated stress results in reduction of food intake and body weight in rats. Stress-induced anorexia has been attributed to increased corticotropin-releasing hormone (CRH) function in the central nervous system. To explore possible roles of other neuropeptides and peripheral hormones involved in food intake and energy utilization during continuing stress, we examined the impact of repeated immobilization stress on expression of mRNAs coding for CRH, neuropeptide Y (NPY), galanin and pro-opiomelanocortin (POMC) mRNAs in such hypothalamic nuclei as the paraventricular nucleus (PVN), arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH), as well as plasma insulin and leptin concentrations. Changes in type 2 CRH receptor (CRHR-2) mRNA in the ventromedial hypothalamus (VMH), a possible target of anorectic CRH effect, were also examined. Rats were immobilized for 2 h daily for 6 days and sacrificed 24 h after the last immobilization. Immobilized rats had lower food intake and body weight and higher levels of PVN CRH mRNA than controls. Repeated immobiliza tion also lowered plasma insulin and leptin concentrations and VMH CRHR-2 mRNA levels. These results provide additional evidence linking VMH CRHR-2 mRNA levels to plasma leptin concentration. ARC NPY and DMH galanin mRNAs increased following repeated immobilization, while ARC POMC mRNA decreased. DMH NPY mRNA and ARC galanin mRNA were unaltered by immobilization. Since NPY and galanin are considered orexigenic, while the POMC-melanocortin-4 receptor system is apparently anorexigenic, the changes in neuropeptide mRNAs and VMH CRHR-2 mRNA may play counterregulatory roles against anorectic CRH effects.     

Corticotropin-releasing factor receptors and pituitary adrenal responses during immobilization stress

The regulation of pituitary and brain CRF receptors and corticotroph responses during stress were studied in rats subjected to prolonged immobilization. Plasma ACTH levels showed the characteristic biphasic changes, with a rapid 23-fold increase in 15 min, followed by a decrease to about twice the basal levels after 6-h immobilization. In contrast, plasma corticosterone levels were markedly elevated throughout the duration of the stress. Pituitary CRF receptor content, measured by binding of [125I]Tyr-ovine CRF to pituitary membrane-rich fractions, was unchanged after 2.5 h, but was reduced by 28 +/- 2.7% (+/- SE) and 47.6 +/- 1.1% after 18 and 48 h of immobilization, respectively. These results were confirmed by autoradiography in slide-mounted frozen pituitary sections. In contrast, no changes in CRF receptor content were observed in brain areas, including olfactory bulb, frontoparietal cortex, hippocampus, amygdala, and lateral septum. A concomitant decrease in immunoreactive (ir) CRF content in the median eminence of rats immobilized for 48 h is consistent with the hypothesis that increased release of CRF into the portal circulation occurs during chronic stress. Despite pituitary CRF receptor loss and reduced in vitro responses to CRF, the increases in plasma ACTH and corticosterone in vivo after ether exposure or CRF injection were greater and more prolonged in rats immobilized for 48 h than in nonimmobilized controls. The decrease in pituitary CRF receptors was accompanied by decreased CRF-stimulated cAMP and ACTH release in cultured pituitary cells from 48-h restrained rats. However, concomitant incubation of cells with CRF and vasopressin restored cAMP and ACTH responses to control levels, suggesting that the simultaneous release of both regulators from the hypothalamus determines the plasma ACTH level. These findings indicate that the decrease in plasma ACTH during the adaptation phase to stress is accompanied by decreases in pituitary CRF receptors. However, the enhanced pituitary response to a superimposed stress or CRF injection implies that the decrease in plasma ACTH levels during prolonged stress may be due to adaptive changes at the central level. These findings emphasize the importance of the integrated actions of CRF and other regulators in the control of the pituitary adrenal-axis during stress.     

Prolactin modulates hypothalamic preproenkephalin,but not proopiomelanocortin,gene expression during lactation

The aim of this study was to examine prolactin (PRL) regulation of preproenkephalin and proopiomelanocortin (POMC) gene expression in the hypothalamus during lactation. In the first experiment, lactating rats were deprived of pups for 3, 6, 12, or 24 h. Preproenkephalin mRNA levels were decreased in the arcuate nucleus (ARC) to 60 or 53% of suckled levels and in the ventromedial nucleus to 70% of suckled levels after 12 or 24 h but were unchanged in the striatum. POMC mRNA levels in the ARC and periarcuate area were increased to 165% of suckled levels within 3 h and remained elevated two- to threefold for 24 h. Subcutaneous administration of bromocriptine to suckled dams markedly suppressed circulating PRL levels and decreased preproenkephalin mRNA signal levels to 38 and 50% of control levels in the arcuate and ventromedial nuclei, respectively. Intravenous administration of oPRL completely reversed this effect. By contrast, bromocriptine with or without administration of ovine PRL (oPRL) did not alter POMC mRNA signal levels in the ARC. Administration of oPRL to pup-deprived dams increased preproenkephalin mRNA levels in the arcuate and ventromedial nuclei and reduced POMC mRNA levels in the ARC to levels similar to suckled control levels. In conclusion, POMC neurons in the ARC appear to be refractory to PRL regulation in the presence of a suckling stimulus, and other components of the suckling stimulus may contribute to the suppression of POMC mRNA levels during lactation. By contrast, PRL provides a regulatory influence for the suckling-induced increase in preproenkephalin mRNA signal levels in arcuate and ventromedial nuclei.