Rebecca S. Brogan

Effects of Food Deprivation on the GH Axis: Immunocytochemical and Molecular Analysis

Neuroendocrine mechanisms governing growth hormone (GH) secretion are sensitive to nutritional status since the normal pulsatile pattern of GH release is disrupted during conditions of food deprivation or malnutrition. A reasonable hypothesis for this occurrence is the alteration of somatostatin and GH-releasing hormone (GHRH) synthesis, storage and secretion. In this study, we investigated the effects of food deprivation on GH, GHRH, hypothalamic and pituitary galanin (GAL), and somatostatin through immunocytochemical and mRNA analysis. Adult male rats were subjected to 72 h of food deprivation, during which an average of 18% total body weight was lost. ICC studies were performed on brain sections from the rostral, middle and caudal regions of the median eminence of the hypothalamus using the avidin-biotin-peroxidase method. Immunocytochemical results were generated for the percent area and optical density (intensity) of immunostaining in the median eminence. Messenger RNA analyses were performed using sense and antisense riboprobes produced from cDNA clones for GH, GHRH, somatostatin and GAL. Food deprivation decreased somatostatin immunostaining in middle and caudal regions of the median eminence; similarly, food deprivation resulted in decreased GHRH immunostaining in rostral and middle sections of the median eminence of the hypothalamus. mRNA levels for somatostatin, GHRH and GH and GAL were also reduced by food deprivation. Our data suggest that suppressed GH secretion in food-deprived rats may reflect a general downregulation of the neuroendocrine and pituitary GH axis.

Physiological trade-offs of forming maggot masses by necrophagous flies on vertebrate carrion.

Necrophagous flies that colonize human and animal corpses are extremely efficient at locating and utilizing carrion. Adult flies deposit eggs or larvae on the ephemeral food resource, which signals the beginning of intense inter- and intra-species competition. Within a short period of time after egg hatch, large larval aggregations or maggot masses form. A period of intense larval feeding ensues that will culminate with consumption/decomposition of all soft tissues associated with the corpse. Perhaps the most distinctive feature of these feeding aggregations is heat production; that is, the capacity to generate internal heat that can exceed ambient temperatures by 30°C or more. While observations of maggot mass formation and heat generation have been described in the research literature for more than 50 years, our understanding of maggot masses, particularly the physiological ecology of the aggregations as a whole, is rudimentary. In this review, an examination of what is known about the formation of maggot masses is presented, as well as arguments for the physiological benefits and limitations of developing in feeding aggregations that, at times, can represent regions of intense competition, overcrowded conditions, or a microclimate with elevated temperatures approaching or exceeding proteotoxic stress levels.

Immunocytochemical and molecular analysis of the effects of glucocorticoid treatment on the hypothalamic-somatotropic axis in the rat.

Glucocorticoids are potent inhibitors of linear growth and growth hormone (GH) secretion when secreted or administered in pharmacological amounts in vivo. The mechanisms involved require further clarification although enhanced somatostatin tone has been suggested to play a role. In this study, we investigated the effects of excess glucocorticoids on pituitary GH, hypothalamic GHRH and hypothalamic somatostatin through immunocytochemical (ICC) and mRNA analysis. Twelve adult male rats were injected daily with dexamethasone (40 micrograms/day, i.p.) or saline for 4 days. ICC studies were performed on brain sections from the rostral, middle and caudal regions of the median eminence of the hypothalamus using the avidin-biotin-peroxidase method. Messenger RNA analyses were performed using sense and antisense riboprobes produced from GH, GHRH and somatostatin cDNAs. Immunocytochemical results were generated for the percent area and intensity (optical density) of immunostaining in the median eminence. Glucocorticoids increased somatostatin immunostaining of the rostral, middle and caudal regions of the median eminence while GHRH staining was only reduced in the rostral region of the median eminence and unchanged in the other hypothalamic regions. GH and somatostatin mRNA levels dramatically increased following glucocorticoid treatment concomitantly with a decrease in GHRH mRNA levels. Our data suggest that increased somatostatin synthesis and storage and a decrease in GHRH mRNA synthesis play a major role in the GH inhibitory effects of glucocorticoids.

Changes in development and heat shock protein expression in two species of flies (Sarcophaga bullata [Diptera: Sarcophagidae] and Protophormia terraenovae [Diptera: Calliphoridae]) reared in different sized maggot masses.

ABSTRACT Development of two species of necrophagous flies, Sarcophaga bullata Parker (Sarcophagidae) and Protophormia terraenovae (Robineau-Desvoidy) (Calliphoridae), was examined in different size maggot masses generated under laboratory conditions. Larvae from both species induced elevated mass temperatures dependent on the number of individuals per mass. The relationship was more evident for S. bullata, as larvae generated higher temperatures in every size maggot mass than P. terraenovae. Several development events were altered with increasing maggot mass size of flesh flies, and to a lesser extent blow flies, which corresponded with elevated temperatures. Duration of development of all feeding larval stages decreased with increased size of maggot mass. However, the length of development during puparial stages actually increased for these same flies. Puparial weights also declined with maggot mass size, as did the ability to eclose. The altered fly development was attributed to the induction of heat stress conditions, which was evident by the expression of heat shock proteins (23, 60, 70, and 90) in larval brains of both fly types.

Deficiency of Scavenger Receptor Class B Type I Negatively Affects Progesterone Secretion in Human Granulosa Cells

Our goal was to examine the effect of deficiency of the lipoprotein receptor, scavenger receptor class B type I (SR-BI), on progesterone secretion in human granulosa cells (HGL5). Scrambled or SR-BI small interfering RNA [knockdown (KD)] cells were exposed to dimethylsulfoxide [DMSO, vehicle for forskolin (Fo)], Fo, serum, high-density lipoprotein, low-density lipoprotein (LDL), or Fo plus lipoproteins or serum for 24 h. Progesterone secretion was lower in all of the SR-BI KD cells regardless of treatment. We examined progesterone secretion in SR-BI KD, LDL receptor KD, and double KD cells incubated with DMSO, Fo, LDL, or Fo + LDL for 6-24 h. As compared with scrambled cells, progesterone secretion was lower in SR-BI and double KD cells regardless of treatment; whereas progesterone secretion was only lower in LDL receptor KD cells incubated with LDL and Fo + LDL. We measured phosphorylation of hormone-sensitive lipase (pHSL) expression, intracellular total cholesterol (TC) mass, and progesterone secretion in scrambled and SR-BI KD cells incubated with DMSO or Fo for 2-24 h. The expression of pHSL was similar between the cells and conditions. The mean change in TC mass and progesterone secretion was lower in SR-BI KD cells exposed to DMSO and Fo. Incubating SR-BI KD cells with 22-hydroxy cholesterol did not overcome the reduction in progesterone secretion. At different time points, RNA expression of steroidogenic acute regulatory protein, side-chain cleavage, and 3β-hydroxysteroid dehydrogenase was significantly lower in SR-BI KD cells incubated with Fo. In conclusion, SR-BI protein deficiency, in part, might explain progesterone deficiency in some infertile women.

EXPRESSION OF THE INSULIN-LIKE GROWTH FACTOR AND INSULIN SYSTEMS IN THE LUTEINIZING MACAQUE OVARIAN FOLLICLE

OBJECTIVE To determine intrafollicular hormone levels and characterize the mRNA expression of the insulin-like growth factor (IGF) receptors, IGF binding proteins (IGFBP), and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells before and after an ovulatory hCG stimulus. DESIGN Experimental animal study. SETTING Academic medical center. ANIMAL(S) Adult rhesus macaques. INTERVENTION(S) Animals received exogenous FSH to promote the development of multiple preovulatory follicles. Follicles were aspirated before (0 hours) or 3, 6, 12, or 24 hours after an ovulatory hCG bolus. MAIN OUTCOME MEASURE(S) IGF1, IGF2, and insulin levels in follicular fluid were determined by radioimmunoassay. Messenger RNA (mRNA) levels in granulosa cells were determined by real-time reverse transcriptase-polymerase chain reaction. IGFBPs and PAPP-A in follicular fluid were determined by Western blot analysis and enzyme-linked immunosorbent assay. RESULT(S) IGF1, IGF2, and insulin in follicular fluid did not change during luteinization. IGF1R, IGFBP1, and IGFBP2 mRNAs were unchanged by hCG. IGF2R, IGFBP3, -5, and -6 and PAPP-A mRNA levels increased after hCG administration, while insulin receptor and IGFBP4 mRNA levels decreased after hCG administration. IGFBP3 and -6 and PAPP-A protein increased after hCG administration. CONCLUSION(S) Dynamic changes in the expression of the IGFBPs and PAPP-A suggest tight regulation of IGF action during ovulation and corpus luteum formation.

Bacterial Interactions with Necrophagous Flies

ABSTRACT Animal remains represent ephemeral resources that provide nutrients to a wide range of organisms. On death, vertebrate carrion is immediately colonized with a variety of microorganisms (typically obligate or facultatively anaerobic bacteria from the air, from insects, or from the corpse itself), which produce odors through the breakdown of tissues, the alteration of volatile chemicals present in the environment, or both. Within minutes, certain necrophagous flies are attracted by these chemical signals, resulting in waves of oviposition and larviposition activity. Although there are certainly detrimental (pathogenic) bacteria in the milieu, there is significant evidence suggesting that the presence of bacteria in or on the corpse seems to aid in larval development and pupariation. This may be because of a change in larval nutrition, with the bacteria either being used as a food source themselves or making nutrients more available to larvae. Maggots also produce and secrete or excrete antimicrobial molecules that are effective in killing certain bacteria. It is unclear whether this is a defensive mechanism, a selective measure to enhance the survival of bacteria beneficial to the larva, or a combination of both. Significant research is still needed to fully appreciate the potential role that these bacteria—insect interactions have in conferring a competitive advantage for surviving in a carrion community.

EGF-Like Ligands Mediate Progesterone's Anti-Apoptotic Action on Macaque Granulosa Cells

ABSTRACT A local autocrine/paracrine role for progesterone is an absolute requirement for corpus luteum formation in primates. Despite this, the mechanism(s) remain obscure, although existing data suggest an anti-apoptotic action to be central. There are a limited number of progestin-regulated gene targets identified in the luteinizing primate follicle, suggesting that a small number of important genes may mediate progesterone action. Possible gene targets could be the epidermal growth factor (EGF) family members amphiregulin (AREG) and epiregulin (EREG). Using macaques undergoing controlled ovarian stimulation cycles, we show that the phosphorylation of EGF receptor (EGFR), ERK 1/2, and AKT increases 6 h after an ovulatory human chorionic gonadotropin (hCG) stimulus and remains activate through 24 h. Immunoreactive EREG and AREG ligands in the follicular fluid both increased in a time frame commensurate with EGFR phosphorylation. The mRNA expression of AREG and EREG in nonluteinized granulosa cells (NLGC) was induced in culture with hCG, an effect blocked by progesterone receptor (PGR) antagonists. Overexpression of PGR B in NLGC and treatment with a nonmetabolizable progestin did not increase either gene, indicating both progesterone and luteinizing hormone/CG are necessary. Addition of EGF and EGF-like ligands did not promote steroidogenesis in vitro by granulosa cells in the presence of gonadotropin, but were able to partially reverse RU486-induced cell death. These data suggest that progesterone promotes the expression of AREG and EREG, which in turn maintain viability of luteinizing granulosa cells, representing one possible mechanism whereby progesterone promotes corpus luteum formation in the primate.

Oviposition Restraint and Developmental Alterations in the Ectoparasitic Wasp, Nasonia vitripennis, When Utilizing Puparia Resulting From Different Size Maggot Masses of Lucilia illustris, Protophormia terraenovae, and Sarcophaga bullata

ABSTRACT Adult females of the ectoparasitoid Nasonia vitripennis (Walker) are capable of distinguishing between hosts of different quality, and then correspondingly adjust clutch sizes and sex ratios of the offspring. In this study, we examined whether the size of the maggot mass, and presumably the developmental temperature, influenced the suitability of the resulting fly pupal and pharate adult stages as hosts for N. vitripennis. Three sizes of maggot masses (100; 500; and 1,000 individuals per mass) were selected for use to generate hosts based on previous studies characterizing developmental and heat shock response differences for the flies. For all host species tested (Lucilia illustris, Protophormia terraenovae, and Sarcophaga bullata), the rate of parasitism by N. vitripennis decreased with increasing maggot mass size. When successful parasitism did occur, parasitoid development increased in duration, clutch sizes decreased, mortality from egg hatch to adult emergence elevated, male biased sex ratios were produced, and adult wasp body sizes were truncated with increasing fly larval density. These wasp life history features are consistent with reductions in host quality. Host quality reductions corresponded to production of heat shock proteins 23, 60, and 70. Heat shock protein synthesis appeared to occur at the expense of normal protein production because total hemolymph protein concentrations decreased with increased larval density in maggot masses. These observations argue that use of N. vitripennis in criminal investigations to estimate periods of insect activity or a minimum post mortem interval must take into account the maggot mass history of the hosts used by the wasp.

Spatial characterization of proteolytic enzyme activity in the foregut region of the adult necrophagous fly, Protophormia terraenovae

The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion.