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The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size. However, cell motility, a process commonly observed in a variety of tissues during development, may represent an additional mechanism to promote plasticity within the adult pituitary gland. To investigate this, we used multiphoton time-lapse imaging methods, GH-enhanced green fluorescent protein transgenic mice and sexual dimorphism of the GH axis as a model of divergent tissue demand. Using these methods to acutely (12 h) track cell dynamics, we report that ovariectomy induces a dramatic and dynamic increase in cell motility, which is associated with gross GH-cell network remodeling. These changes can be prevented by estradiol supplementation and are associated with enhanced network connectivity as evidenced by increased coordinated GH-cell activity during multicellular calcium recordings. Furthermore, cell motility appears to be sex-specific, because reciprocal alterations are not detected in males after castration. Therefore, GH-cell motility appears to play an important role in the structural and functional pituitary plasticity, which is evoked in response to changing estradiol concentrations in the female.

International audience; The effects of growth hormone on the uridine diphosphate glucuronosyltransferase functional state, biophysical membrane parameters (order parameters and rotational correlation frequency) and the composition in phospholipids were studied in male rat hepatic microsomes. Sham-operated and hypophysectomized animals were injected with two different dosages of growth hormone, mimicking either the male or female growth hormone secretion pattern. Half the animals received thyroxine and cortisol in concentrations chosen to compensate for the lack of thyroid hormones and glucocorticoids in hypophysectomized rats. Growth hormone treatment resulted in a decrease in the latency (that gives a quantification of uridine diphosphate glucuronosyltransferase functional state) of the glucuronidation activities towards various substrates (testosterone, androsterone, bilirubin and 4-nitrophenol). This decrease with growth hormone treatment was particularly evident in hypophysectomized animals that had received cortisol and thyroxine supplementation treatment. These modifications were strongly correlated with modifications in the microsomal membrane lysophospholipid content and to a lower extent with microsomal membrane fatty acid composition. The cytosolic phospholipase A(2)-dependent increase in the lysophospholipid content in the endoplasmic reticulum is probably a major determinant in the regulation of the functional state of glucuronoyltransferases in response to high dosage growth hormone treatment.

Recently, we have demonstrated in rainbow trout that environmental temperature may, independently of nutritional status, directly stimulate plasma growth hormone (GH) that is recognised as being an insulin-like growth factor (IGF) system regulator. The aim of this study was to determine whether temperature may directly regulate the IGF system or indirectly regulate it through plasma GH or nutritional status. For this purpose, rainbow trout were reared at 8, 12, or 16 degrees C and fed either ad libitum (similar nutritional status) to evidence the global effect of temperature, or with the same ration (1.2% body weight/day), to determine the temperature effect in fish with the same growth rate. Endocrine and autocrine/paracrine regulations of the IGF system were determined by measuring plasma IGF1 and IGF2, liver and muscle IGF1 and IGF2 mRNA as well as IGFRIa, IGFRIb mRNA, and the quantity of IGF type I receptor in muscle. Our results show that neither rearing temperature nor the nutritional status of fish affected the expression of both IGF receptor genes in muscle. Nevertheless, the quantity of IGF type I receptor determined by a binding study, appeared to be inversely proportional (P

Verslag van een consumentonderzoek naar de reacties van Nederlandse melkdrinkers, waarbij de melk van de aanduiding was voorzien dat deze geproduceerd was door met BST-behandelde koeien, en anderszijds bij een groep melkdrinkers, waarbij geen onderscheid was gemaakt tussen melk afkomstig van wel of niet behandelde koeien

Recombinant bovine somatotropin (rbST) is a 22 kDa proteohormone, which can be used to increase milk production in dairy cows. It has been marketed since 1994 and while its use in food production is approved in several countries, such as the US, it is banned in the EU since 2000. To enforce the ban on rbST in the EU and to control for ‘rbST-free’ –labelling in the US, detection methods are required that identify whether rbST has been used. Existing rbST detection methods focus on the detection of rbST itself in bovine serum. The recombinant form of the hormone has one amino acid exchanged at the N-terminus of the protein. RbST can therefore be potentially discriminated from the endogenous bST by mass spectrometric methods. Other methods employ sandwich enzyme-linked immunosorbent assays (ELISAs) with antibodies having a higher affinity to rbST than bST. These methods, however mainly lack sensitivity, reproducibility or selectivity for rbST and are therefore not widely applied. Hence, no method has been implemented so far to monitor rbST abuse in dairy farming. Screening methods developed for veterinary drug residue control in the EU have to perform according to Commission Decision 2002/657/EC and have to identify at least 95 % of the treated animals. An alternative approach for rbST abuse detection is the analysis of rbST-dependent biomarkers. A biomarker is defined as an indicator of normal physiological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Therefore, the levels of rbST-dependent protein biomarkers are either up- or downregulated after administration of rbST and rbST-specific biomarker profiles can be used to detect its abuse. RbST exerts similar physiological actions in the cow’s body as the endogenous bST. Therefore, proteins involved in the regulatory circuit of bST have been chosen as candidate biomarkers, such as insulin-like growth factor-1 (IGF-1), IGF binding protein 2 (IGFBP2) and osteocalcin. Additionally to that, the administration of rbST induces anti-rbST antibodies in the cow’s body, which can be detected as biomarkers. This approach is according to the growth hormone (GH) abuse detection in sports doping control, where solely protein biomarker profiles are used to identify the abuse. Chapter 2 introduces protein biomarkers and how biomarkers can be used in sports doping and veterinary control to detect the abuse of illegal substances. The advantages of using biomarkers are that the biological effect of a substance usually lasts longer than the substance itself can be detected and therewith, the window of detection is expanded. Moreover, since different substances exert similar effects on physiological machineries for growth or production enhancement, biomarker-based-detection methods have the potential to detect a whole class of substances. Furthermore, low-dose mixtures of different banned substances, which might escape from direct detection of each individual substance used, could be still detected by the combined effect they exert. In this chapter, protein biomarker-based detection strategies are discussed against generic challenges in biomarker discovery and method development. Part I of the thesis concerns biomarker analysis in serum and plasma samples from cattle, which are analysed using laboratory-based equipment.A triplex flow cytometric immunoassay (FCIA), which combines the detection of three rbST-dependent biomarkers, viz. IGF-1, IGFBP2 and anti-rbST antibodies is demonstrated in Chapter 3. Serum samples from treated and untreated dairy cows from a single animal study were analysed using this triplex FCIA. Characteristic treatment-dependent responses for all three individual biomarkers were shown. These results were combined using the statistical model k-nearest neighbours (kNN). This model discriminated rbST-treated from untreated cows with a truepositive rate of 89.1 % and a true-negative rate of 97.7 %. This triplex FCIA was further extended with the biomarker osteocalcin and the resulting fourplex FCIA was used for biomarker profiling in serum samples from rbST-treated and untreated cows from two independent rbST treatment studies. In Chapter 4, different data analysis approaches were tested with the aim to detect the highest possible number of true-positive samples. The statistical model kNN was used on all 11 possible biomarker combinations and the combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95 % of the samples of treated cows starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12 % of the samples of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. It can be expected that rbST-dependent biomarkers also show a response upon other treatments. Therefore in Chapter 5, the fourplex FCIA for rbST abuse detection was applied to bovines treated with steroids, such as estradiol, dexamethasone and prednisolone. Each treatment resulted in a specific plasma biomarker profile for IGF-1, IGFBP2, osteocalcin and anti-rbST antibodies, which could be distinguished from the profile of untreated animals. Therefore, the fourplex biomarker FCIA is, apart from rbST, also capable of detecting treatment with other growth-promoting agents and clearly shows the potential of biomarker profiling as a screening method in veterinary control. Part II of the thesis focusses on protein biomarker analysis in milk samples and the change from laboratory-based to on-site analysis. In Chapter 6, the detection of anti-rbST antibodies in raw milk samples was demonstrated, which discriminated rbST-treated from untreated cows with a 67 % true-positive and 94 % true-negative rate. The laboratory-based assay was also applied to simulated tank milk and pasteurized milk samples. Using milk as a sample matrix for detection has the advantages of non-invasive sampling, and for tank milk analysis at the farm only one milk sample is needed to screen the whole farm for rbST (ab)use. As a next step in Chapter 7, this assay was translated to an on-site pre-screening platform including a cellphone. Using this on-site platform, samples can be tested at the point where they were taken. Only samples that are suspect are transported to a laboratory for further analysis. To this end, a cellphone-based fluorescence imaging platform for the detection of anti-rbST antibodies in milk extracts was developed, which is based on a microsphere fluorescence immunoassay. After performing the assay, the fluorescence is excited by UV LEDs embedded in a dedicated cellphone attachment and the emitted fluorescence light is imaged by the cellphone camera. The fluorescence micro-images were analysed using a custom-developed Android application running on the same cellphone and milk samples from rbST-treated and untreated cows were discriminated. Also in milk samples, the simultaneous detection of several biomarkers is advantageous as they can increase the confidence of a positive finding. Therefore in Chapter 8, a protein microarray-based platform for multiple rbST biomarker detection on a cellphone is presented, which detects anti-rbST antibodies and IGF-1 in milk samples. The 48 microspots on the microarray were labelled with Quantum Dots depending on the biomarker levels in the sample. Quantum Dot fluorescence was detected by the cellphone camera and the same opto-mechanical attachment as in Chapter 7 and images were analysed by custom software. RbST-treated clearly showed a treatment-dependent biomarker profile in milk that could be discriminated from the profile of untreated cows. Future research should focus on the simultaneous detection of different targets of interest in milk samples, such as hormones, allergens, antibiotics, contaminants and other substances, all at the same time using the microarray platform on the cellphone. Moreover, sample handling can be facilitated by the use of pre-fabricated microfluidic devices including all required assay reagents. With the work presented in this thesis, screening for rbST abuse in serum and milk becomes possible: in the laboratory and on-site. The future implementation of these testing platforms for rbST abuse detection is a major leap forward concerning the enforcement of the rbST ban in the EU and concerning the value of protein biomarker-based approaches in veterinary control.

Recombinant bovine somatotropin (rbST) is a 22 kDa proteohormone, which can be used to increase milk production in dairy cows. It has been marketed since 1994 and while its use in food production is approved in several countries, such as the US, it is banned in the EU since 2000. To enforce the ban on rbST in the EU and to control for ‘rbST-free’ –labelling in the US, detection methods are required that identify whether rbST has been used. Existing rbST detection methods focus on the detection of rbST itself in bovine serum. The recombinant form of the hormone has one amino acid exchanged at the N-terminus of the protein. RbST can therefore be potentially discriminated from the endogenous bST by mass spectrometric methods. Other methods employ sandwich enzyme-linked immunosorbent assays (ELISAs) with antibodies having a higher affinity to rbST than bST. These methods, however mainly lack sensitivity, reproducibility or selectivity for rbST and are therefore not widely applied. Hence, no method has been implemented so far to monitor rbST abuse in dairy farming. Screening methods developed for veterinary drug residue control in the EU have to perform according to Commission Decision 2002/657/EC and have to identify at least 95 % of the treated animals. An alternative approach for rbST abuse detection is the analysis of rbST-dependent biomarkers. A biomarker is defined as an indicator of normal physiological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Therefore, the levels of rbST-dependent protein biomarkers are either up- or downregulated after administration of rbST and rbST-specific biomarker profiles can be used to detect its abuse. RbST exerts similar physiological actions in the cow’s body as the endogenous bST. Therefore, proteins involved in the regulatory circuit of bST have been chosen as candidate biomarkers, such as insulin-like growth factor-1 (IGF-1), IGF binding protein 2 (IGFBP2) and osteocalcin. Additionally to that, the administration of rbST induces anti-rbST antibodies in the cow’s body, which can be detected as biomarkers. This approach is according to the growth hormone (GH) abuse detection in sports doping control, where solely protein biomarker profiles are used to identify the abuse. Chapter 2 introduces protein biomarkers and how biomarkers can be used in sports doping and veterinary control to detect the abuse of illegal substances. The advantages of using biomarkers are that the biological effect of a substance usually lasts longer than the substance itself can be detected and therewith, the window of detection is expanded. Moreover, since different substances exert similar effects on physiological machineries for growth or production enhancement, biomarker-based-detection methods have the potential to detect a whole class of substances. Furthermore, low-dose mixtures of different banned substances, which might escape from direct detection of each individual substance used, could be still detected by the combined effect they exert. In this chapter, protein biomarker-based detection strategies are discussed against generic challenges in biomarker discovery and method development. Part I of the thesis concerns biomarker analysis in serum and plasma samples from cattle, which are analysed using laboratory-based equipment.A triplex flow cytometric immunoassay (FCIA), which combines the detection of three rbST-dependent biomarkers, viz. IGF-1, IGFBP2 and anti-rbST antibodies is demonstrated in Chapter 3. Serum samples from treated and untreated dairy cows from a single animal study were analysed using this triplex FCIA. Characteristic treatment-dependent responses for all three individual biomarkers were shown. These results were combined using the statistical model k-nearest neighbours (kNN). This model discriminated rbST-treated from untreated cows with a truepositive rate of 89.1 % and a true-negative rate of 97.7 %. This triplex FCIA was further extended with the biomarker osteocalcin and the resulting fourplex FCIA was used for biomarker profiling in serum samples from rbST-treated and untreated cows from two independent rbST treatment studies. In Chapter 4, different data analysis approaches were tested with the aim to detect the highest possible number of true-positive samples. The statistical model kNN was used on all 11 possible biomarker combinations and the combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95 % of the samples of treated cows starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12 % of the samples of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. It can be expected that rbST-dependent biomarkers also show a response upon other treatments. Therefore in Chapter 5, the fourplex FCIA for rbST abuse detection was applied to bovines treated with steroids, such as estradiol, dexamethasone and prednisolone. Each treatment resulted in a specific plasma biomarker profile for IGF-1, IGFBP2, osteocalcin and anti-rbST antibodies, which could be distinguished from the profile of untreated animals. Therefore, the fourplex biomarker FCIA is, apart from rbST, also capable of detecting treatment with other growth-promoting agents and clearly shows the potential of biomarker profiling as a screening method in veterinary control. Part II of the thesis focusses on protein biomarker analysis in milk samples and the change from laboratory-based to on-site analysis. In Chapter 6, the detection of anti-rbST antibodies in raw milk samples was demonstrated, which discriminated rbST-treated from untreated cows with a 67 % true-positive and 94 % true-negative rate. The laboratory-based assay was also applied to simulated tank milk and pasteurized milk samples. Using milk as a sample matrix for detection has the advantages of non-invasive sampling, and for tank milk analysis at the farm only one milk sample is needed to screen the whole farm for rbST (ab)use. As a next step in Chapter 7, this assay was translated to an on-site pre-screening platform including a cellphone. Using this on-site platform, samples can be tested at the point where they were taken. Only samples that are suspect are transported to a laboratory for further analysis. To this end, a cellphone-based fluorescence imaging platform for the detection of anti-rbST antibodies in milk extracts was developed, which is based on a microsphere fluorescence immunoassay. After performing the assay, the fluorescence is excited by UV LEDs embedded in a dedicated cellphone attachment and the emitted fluorescence light is imaged by the cellphone camera. The fluorescence micro-images were analysed using a custom-developed Android application running on the same cellphone and milk samples from rbST-treated and untreated cows were discriminated. Also in milk samples, the simultaneous detection of several biomarkers is advantageous as they can increase the confidence of a positive finding. Therefore in Chapter 8, a protein microarray-based platform for multiple rbST biomarker detection on a cellphone is presented, which detects anti-rbST antibodies and IGF-1 in milk samples. The 48 microspots on the microarray were labelled with Quantum Dots depending on the biomarker levels in the sample. Quantum Dot fluorescence was detected by the cellphone camera and the same opto-mechanical attachment as in Chapter 7 and images were analysed by custom software. RbST-treated clearly showed a treatment-dependent biomarker profile in milk that could be discriminated from the profile of untreated cows. Future research should focus on the simultaneous detection of different targets of interest in milk samples, such as hormones, allergens, antibiotics, contaminants and other substances, all at the same time using the microarray platform on the cellphone. Moreover, sample handling can be facilitated by the use of pre-fabricated microfluidic devices including all required assay reagents. With the work presented in this thesis, screening for rbST abuse in serum and milk becomes possible: in the laboratory and on-site. The future implementation of these testing platforms for rbST abuse detection is a major leap forward concerning the enforcement of the rbST ban in the EU and concerning the value of protein biomarker-based approaches in veterinary control.

The present study was undertaken to determine the effect of GH administration on GH and IGF-I receptors in skeletal muscle compared with liver in growing pigs. Plasma IGF-I and GH-binding protein (GHBP) levels were also determined. Twelve Large White pigs (castrated males) were treated daily with 100 micrograms pituitary porcine GH (pGH) per kg body weight or vehicle for 41 days intramuscularly. Relative to controls, pGH administration increased plasma IGF-I concentrations by 3.3-fold. Administration of pGH had no effect on plasma GHBP levels. In liver, 125I-labelled bovine GH (bGH)-specific binding (P < 0.05) and GH receptor (GHR) mRNA levels (P < 0.05) were higher in pGH-treated than in control pigs. In longissimus dorsi (LD), 125I-labelled bGH specific binding did not differ significantly between the two groups while GHR mRNA levels (P < 0.05) were lower in pGH-treated than in control pigs. Administration of pGH had no effect on 125I-labelled bGH-specific binding and GHR mRNA levels in trapezius (TR). 125I-Labelled IGF-I-specific binding in liver was unaffected by pGH administration. Similarly, in liver, LD and TR, IGF-I receptor mRNA levels were not different between pGH-treated and control animals. It can be concluded that (1) GH binding and IGF-I receptor mRNA are not affected by GH in skeletal muscle, (2) GH influences GHR in a tissue-specific manner and (3) hepatic GHR and GHBP levels are not co-regulated.

Recently, in addition to IGF-1 and IGF-2 the existence of a third form of IGF, termed IGF-3, limited to fishes, to be present only in the gonads and encoded by a separate gene has been reported. However, no further data have been presented on IGF-3. The present study on tilapia (Oreochromis niloticus) uses quantitative real-time PCR specific for tilapia IGF-1 and IGF-3. The organ distribution of IGF-3 mRNA in adult fish and the early ontogeny of IGF-3 in male and female gonads were studied. The potential sensitivity of IGF-3 to GH was revealed by intraperitoneal injections of bream GH using IGF-1 as control gene. The effects of 17alpha-ethinylestradiol (EE2) exerted after feeding of high EE2 doses and exposure to low environmentally relevant EE2 doses on IGF-3 expression in testis and ovary during early development were determined. Low IGF-3 mRNA expression levels were detected in most organs studied, with the highest extra-gonadal amount in the pituitary. During development, the IGF-3 gene was significantly upregulated in male but downregulated in female gonad. Injections of GH elevated IGF-1 mRNA in male and female liver and ovary. IGF-3 did not respond to GH treatment neither in ovary nor in testis. Both EE2 treatments resulted in significant downregulations of IGF-3 mRNA in testis while ovarian IGF-3 mRNA did not respond. Thus, IGF-3 may be involved in reproduction of fishes most likely in the male gonad only. Whether IGF-3 also has some physiological significance in ovary or other organs should be the topic of further studies.

Insulin-like growth factor (IGF)-I, which is crucially involved in fish growth, differentiation, and reproduction, occurs in tilapia pituitary. IGF-I peptide, which is probably produced in hypothalamic perikarya, is present in axons of the neurohypophysis, and IGF-I mRNA and peptide are present in the adenohypophysis in adrenocorticotrophic hormone cells, melanocyte-stimulating hormone cells, with interindividual differences in growth hormone cells, and, starting with puberty, in gonadotrophic hormone (GTH) cells. Subordinate males showed a high IGF-I but a lower beta-luteinizing hormone expression, while in dominant males the opposite was found. IGF-I from the GTH cells may act as auto/paracrine regulators of GTH cell proliferation and enhance GTH synthesis and release during puberty and reproduction, depending on the social status.

Berekeningen tonen dat BST-toepassing als managementinstrument tijdelijke tekorten in melkproduktie kan opvangen zonder dat ingrijpende investeringen nodig zijn

International audience; In fish, as in all poikilotherms, growth is strongly dependent on water temperature. Given that the GH/IGF system regulates growth, it could mediate the effects of temperature on fish growth. Indeed, before hatching, the higher embryonic growth rate in rainbow trout at high temperatures is associated with higher expression of the IGF2 gene in the whole embryo. Furthermore, post-natal growth fluctuations depend on water temperature and are associated with variations of plasma GH and IGF1. Although seasonal parameters such as photoperiod and nutritional status can also affect GH/IGF system activity, it has been shown that an increased temperature led to a specific increase of plasma GH. Moreover, this increase of plasma GH leads to higher plasma IGF1 levels in correlation with the growth rate. By contrast, plasma IGF2 levels as well as muscular levels of IGF1 and IGF2 mRNA are not specifically modified by temperature. Thus, seasonal fluctuations of water temperature affect growth rate through a direct action on plasma GH and IGF1 levels. The mechanisms of this effect are not yet elucidated, but could arise from modifications of metabolite levels (glucose, amino acids, fatty acids, etc.), which regulate GH secretion directly or indirectly through somatostatin of pancreatic or hypothalamic origin.

In fish, the GH/IGF system installs very early during development suggesting that this system could promote embryonic growth and development. In contrast to mammals, the embryonic growth rate of poikilotherms depends considerably on the incubation temperature. Therefore, the aim of this study was to determine if variations of embryo growth in response to temperature could be associated with modifications in the gene expression of the GH/IGF system. In this study, using whole mount in situ hybridisation, we demonstrated that embryo incubation temperature (4, 8, and 12 degrees C) did not change the timing of GH-1 and GH-2 mRNA expression in somatotroph cells (stage 24). Similarly, at hatching (stage 30), we did not observe an obvious difference in GH protein and GH-1 and GH-2 transcript amounts in relation to the incubation temperature. Furthermore, from stage 22 to 25, the highest temperature led to a specific up-regulation of IGF-2 (2-fold between 4 and 12 degrees C), and both IGF-RIa and IGFRIb mRNA (1.5-fold between 4 and 12 degrees C), while no difference was observed for IGF-1 mRNA. Conversely, at hatching, the highest temperature specifically down-regulated IGF-2 (3-fold between 4 and 12 degrees C) and both IGF receptor mRNAs (2 fold between 4 and 12 degrees C) present in the head, while no difference was observed in the trunk. Our results demonstrated that different incubation temperatures during trout embryonic development did not change the stage of somatotroph cell appearance. Before hatching, IGF-2 and both IGF receptors, but not IGF-1 mRNA, were specifically up-regulated by high temperatures and could be related to the enhancement of embryonic growth rate.

*INRA SCRIBE Rennes (FRA) Diffusion du document : INRA SCRIBE Rennes (FRA) Diplôme : Dr. d'Université

Like many poecilotherms, salmonids exhibit seasonal variations of growth rate in relation with seasonal temperatures and plasma GH level. However, temperature alters other parameters like food intake, which may directly modify the level of plasma GH. In order to determine whether temperature regulates plasma GH levels independently of nutritional status, fish were reared at 8, 12, or 16 degrees C and either fed ad libitum (fish with different food intake) to determine the global effect of temperature, or with the same ration (1.2%/body weight) to observe the temperature effect in fish with the same growth rate. Plasma insulin level was inversely proportional to the temperature (8, 12, and 16 degrees C) in fish fed ad libitum (12.1+/-0.3 ng/ml, 10.9+/-0.3 ng/ml, 9.5+/-0.4 ng/ml; P0.001) and in restricted fish (14.0+/-0.3 ng/ml, 11.3+/-0.3 ng/ml, 10.0+/-0.2 ng/ml; P0.0001), probably due to a prolonged nutrient absorption, and delayed recovery of basal insulin level at low temperature. Conversely, temperature did not affect plasma T3 level of fish fed ad libitum (2.5+/-0.2 ng/ml, 2.4+/-0.1 ng/ml, 2.5+/-0.1 ng/ml at 8, 12, and 16 degrees C) while fish fed with the same ration present less T3 at 16 degrees C than at 8 degrees C (1.83+/-0.1 ng/ml versus 1.2+/-0.1 ng/ml; P0.001) throughout the experiment; these observations indicate that different plasma T3 levels reflect the different nutritional status of the fish. The levels of GH1 and GH2 mRNA, and GH1/GH2 ratio were not different for whatever the temperature or the nutritional status. Pituitary GH content, of fish fed ad libitum did not exhibit obvious differences at 8, 12, or 16 degrees C (254+/-9 ng/g bw, 237+/-18 ng/g bw, 236+/-18 ng/g bw), while fish fed with the same ration have higher pituitary GH contents at 16 degrees C than at 8 degrees C (401+/-30 ng/g bw versus 285+/-25 ng/g bw; P0.0001). Interestingly, high temperature strongly increases plasma GH levels (2.5+/-0.3 ng/ml at 8 degrees C versus 4.8+/-0.6 ng/ml at 16 degrees C; P0.0001) to the same extent in both experiments, since at a given temperature average plasma GH was similar between fish fed ad libitum or a restricted diet. Our results, demonstrate that temperature regulates plasma GH levels specifically but not pituitary GH content, nor the levels of GH1 and GH2 mRNA. In addition no differential regulation of both GH genes was evidenced whatever the temperature.

The present study reports the hormone profile [growth hormone (GH) and thyroid hormones] in a fast- and slow-growing strain of rainbow trout under different feeding regimes. An examination as to whether growth rate could be related to the endogenous levels of these growth-promoter hormones was also undertaken. Fish were fed to satiation to establish routine feeding behaviour; others were submitted to a restricted ration level (2% of body weight) to nullify the feed intake effect, thereby allowing a comparison of the general metabolism of the fish, over a period of 16 weeks. Finally, fish were submitted to a 30-day fasting period, which functioned as a test to amplify the capacity of the animals to maintain high levels of circulating hormones. Differences in growth rate between the two rainbow trout strains, when fed to satiation, could not be associated with differences in hormonal status (pituitary and plasma GH, plasma T 3 and T 4 ). Nonetheless, fish of the fast-growing strain exhibited significantly higher circulating GH values than slow-growing fish, after the fasting period. Additional studies on both the thyroid hormones and GH receptor systems, and the cellular response to these hormones are warranted to further characterize this aspect of the endocrine regulation of body growth in the two rainbow trout strains.

International audience

Verslag van een consumentonderzoek naar de reacties van Nederlandse melkdrinkers, waarbij de melk van de aanduiding was voorzien dat deze geproduceerd was door met BST-behandelde koeien, en anderszijds bij een groep melkdrinkers, waarbij geen onderscheid was gemaakt tussen melk afkomstig van wel of niet behandelde koeien

Various hormones were analyzed during the course of a reproductive cycle in the cichlid fish Oreochromis niloticus: plasma levels of the gonadal steroids 17beta-estradiol (E2), testosterone (T), 17, 20beta-OH progesterone (17,20beta-P), gonadotropin (taGtH), and plasma and pituitary concentrations of prolactin (tiPRL(I) and tiPRL(II)) and growth hormone (tiGH). Two categories of fish were sampled and sacrificed on days 1 and 3 postspawning and at 3-day intervals thereafter: typical incubating females (INC), and nonincubating females (NI), deprived of their eggs just after spawning. Such deprivation is known to suppress maternal behavior and to accelerate ovarian development and especially vitellogenesis, thus shortening the mean interspawning interval. In both groups, variations of the plasma concentrations of E2 and T appeared to depend on ovarian stages, and differences between groups appeared to reflect underlying differences in the kinetics of ovarian development. The observation of noticeable levels of 17,20beta-P in plasma before spawning, when high values of taGtH could also be detected in NI females, suggests the implication of this progestin in the control of final maturation events, as in some other teleosts. Moreover, 17,20beta-P, which was still detected a few days after spawning, but at low concentrations and only in the plasma of INC females, might play a role at the beginning of the reproductive cycle in incubating females (maternal behavior and/or slowing down of ovarian growth). The pituitary and plasma profiles of both tiPRLs isoforms appeared to depend mainly on the kinetics of ovarian development in each group of fish, suggesting a role during the beginning of vitellogenesis. However, the variance of plasma tiPRL(II), which was significantly enhanced during maternal behavior in INC females, also suggests an implication of this hormone in the control of that behavior. Concerning tiGH, comparison of the plasma profiles in INC and NI fish also suggest an influence on the control of maternal behavior, but a main effect of starvation of INC during mouthbrooding cannot be excluded.

National audience

152 ref. 6 graph. Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access; International audience

The present study was conducted to determine the influence of food restriction on growth hormone receptor (GHR) in porcine skeletal muscle (longissimus dorsi and trapezius) and liver in relationship to plasma growth hormone binding protein (GHBP). At 76 d of age, pigs were allotted to one of three groups. In Group R100kg-196d, pigs were fed 70% of control food intake and slaughtered at 100 kg. Control pigs had free access to food and were slaughtered either at the same weight (Group C100kg-161d) or at the same age (Group C130kg-196d) as R100kg-196d pigs. Plasma insulin-like growth factor-I concentrations tended to be lower in food-restricted pigs than in control pigs at 40 kg (P < 0.1) and 70 kg (P < 0.05). At slaughter, there were no significant differences among the three groups. In liver, 125 I-labeled bovine GH specific binding was higher (P < 0.05) in food-restricted pigs than in control pigs, and GHR mRNA level was higher (P < 0.05) in food-restricted pigs than in C100kg-161d pigs. In trapezius, 125 1-labeled bovine GH specific binding was lower (P < 0.05) in food-restricted pigs than in C130kg-196d pigs, and the level of GHR mRNA was higher (P < 0.01) in food-restricted pigs than in control pigs. The levels of GHR in longissimus dorsi were not affected by food restriction. The level of plasma GHBP was lower (P < 0.05) in food-restricted pigs than in C130kg-196d pigs. These data indicate that nutritional status regulates GHR in a tissue-specific manner and that there is no simple relationship between plasma GHBP and hepatic GHR in pigs.

L'influence de l'hormone de croissance (gh) sur les activites de glucuronidation hepatiques est etudiee chez des rats males, hypophysectomises ou non, et traites ou non avec du cortisol et de la thyroxine de facon a compenser l'absence d'hormones thyroidiennes et de glucocorticoides chez les animaux hypophysectomises. Deux doses de gh ont ete utilisees, la plus faible permettant de mimer un rythme de secretion de gh de type male, la plus forte permettant de mimer un rythme de secretion de type femelle. La gh agit de facon variable sur l'expression des enzymes de glucuronidation du paranitrophenol, de l'androsterone, de la testosterone, de la bilirubine et de l'oestrone, selon l'activite consideree. La gh diminue particulierement l'activite de glucuronidation de la bilirubine, vraisemblablement a un niveau transcriptionnel ou post-transcriptionnel. Cette influence ne s'apparente pas a un processus de feminisation par une exposition continue a l'hormone comme c'est le cas pour d'autres enzymes de metabolisation hepatiques, particulierement certaines monooxygenases a cytochrome p-450 ou certaines glutathion s-transferases (gsts), qui ont ete etudiees en parallele. Cette etude en parallele a par ailleurs montre l'existence d'une forte similitude de comportement de l'activite de glucuronidation de la bilirubine et de l'expression de la sous-unite 6 des gsts en fonction de l'hypophysectomie et des traitements utilises. La signification et les bases moleculaires de cette relation restent a etablir. La gh modifie egalement l'etat fonctionnel des enzymes de glucuronidation qui depend de l'environnement membranaire de ces enzymes, en interaction avec le traitement par le cortisol et la thyroxine. Cet effet est en relation avec une augmentation des lysophospholipides dans la membrane microsomale. En revanche, la faible influence de la gh sur differents parametres de fluidite membranaire, qui sont dependants de la composition en acides gras des microsomes elle-meme modifiee par la gh et l'hypophysectomie, ne semble pas etre en relation avec l'effet de cette hormone sur l'etat fonctionnel des enzymes de glucuronidation. La gh influence donc l'expression des enzymes de glucuronidation mais aussi leur etat fonctionnel, via la composition lipidique de la membrane microsomale. Les consequences sur le fonctionnement d'autres proteines membranaire reste a determiner, L'influence de l'hormone de croissance (gh) sur les activites de glucuronidation hepatiques est etudiee chez des rats males, hypophysectomises ou non, et traites ou non avec du cortisol et de la thyroxine de facon a compenser l'absence d'hormones thyroidiennes et de glucocorticoides chez les animaux hypophysectomises. Deux doses de gh ont ete utilisees, la plus faible permettant de mimer un rythme de secretion de gh de type male, la plus forte permettant de mimer un rythme de secretion de type femelle. La gh agit de facon variable sur l'expression des enzymes de glucuronidation du paranitrophenol, de l'androsterone, de la testosterone, de la bilirubine et de l'oestrone, selon l'activite consideree. La gh diminue particulierement l'activite de glucuronidation de la bilirubine, vraisemblablement a un niveau transcriptionnel ou post-transcriptionnel. Cette influence ne s'apparente pas a un processus de feminisation par une exposition continue a l'hormone comme c'est le cas pour d'autres enzymes de metabolisation hepatiques, particulierement certaines monooxygenases a cytochrome p-450 ou certaines glutathion s-transferases (gsts), qui ont ete etudiees en parallele. Cette etude en parallele a par ailleurs montre l'existence d'une forte similitude de comportement de l'activite de glucuronidation de la bilirubine et de l'expression de la sous-unite 6 des gsts en fonction de l'hypophysectomie et des traitements utilises. La signification et les bases moleculaires de cette relation restent a etablir. La gh modifie egalement l'etat fonctionnel des enzymes de glucuronidation qui depend de l'environnement membranaire de ces enzymes, en interaction avec le traitement par le cortisol et la thyroxine. Cet effet est en relation avec une augmentation des lysophospholipides dans la membrane microsomale. En revanche, la faible influence de la gh sur differents parametres de fluidite membranaire, qui sont dependants de la composition en acides gras des microsomes elle-meme modifiee par la gh et l'hypophysectomie, ne semble pas etre en relation avec l'effet de cette hormone sur l'etat fonctionnel des enzymes de glucuronidation. La gh influence donc l'expression des enzymes de glucuronidation mais aussi leur etat fonctionnel, via la composition lipidique de la membrane microsomale. Les consequences sur le fonctionnement d'autres proteines membranaires reste a determiner

Specific binding sites for porcine somatotropin (pST) have been identified in pig liver microsomal membranes. Little information, however, is available about the size and number of ST receptor (ST-R) forms present. Therefore, the present study was conducted to characterize ST-R in pig liver using two approaches. In the first set of experiments, cross-linking of [125I]bST (bovine ST) to microsomal membranes, followed by gel electrophoresis under reducing conditions, revealed the presence of a predominant protein of 107 kDa and four other proteins of 71, 52, 40, and 26 kDa. In a second set of experiments, ST-R were partially purified using affinity chromatography. Binding studies indicated that there was an approximately 1,800-fold purification compared to liver homogenate. Two specific proteins of 107 and 40 kDa were detected after crosslinking of [125I]bST to partially purified ST-R. Northern blot analysis revealed that these proteins arise by posttranslational modification of a single 4.2-kilobase somatotropin receptor messenger RNA transcript. Although the present study indicates that several forms of ST-R are present in pig liver, it is not clear what physiological role these different ST-R play in mediating the hepatic effects of pST. It is evident, however, that the smaller proteins are generated from the 107-kDa protein, which is the predominant isoform present in liver microsomal membranes.

Berekeningen tonen dat BST-toepassing als managementinstrument tijdelijke tekorten in melkproduktie kan opvangen zonder dat ingrijpende investeringen nodig zijn

The calculated profitability of using Bovine Somatotropin (BST) on typical dairy farms in The Netherlands ranges from Dfl.160 to 300 per cow per year, assuming 1985 prices and circumstances, and ignoring the costs of BST. A 20% increase in milk production and no change of the feed/milk relation were used for the calculations. BST is more profitable on intensive farms or on farms with more opportunities for alternative uses of land, buildings and labour. The quota system, however, leads to a considerable reduction of profitability. At a national level, and with an unchanged milk price, a 28% adoption rate of BST would increase national income about Dfl.120 million. However, the cost of BST or any decrease in milk price could reduce this amount, even to below zero. It is apparent that some dairy farmers who apply BST will earn more income whereas others will lose income.

Afhankelijk van de bedrijfsgrootte worden de baten berekend van het gebruik van het rundergroeihormoon BST per koe per jaar

In dit onderzoek is nagegaan wat het effect is van actieve immunisatie tegen somatostatine op de melkproduktie van de geit, door 6 geiten, halverwege de dracht geimmuniseerd met een conjugaat van somatostatine en humaan alfa-glubuline, te vergelijken met 6 andere geiten van dezelfde jaargang met een vergelijkbare melkproduktie en gewicht die alleen met globuline waren geimmuniseerd. Somatostatine, een 14 aminozuren tellend peptide, is o.a. betrokken bij de regeling van de groeihormoon-afgifte door de hypofyse, waarop het remmend werkt

In 2 proeven werd het effect van behandeling bij melkkoeien met recombinant bovine somatotropine bestudeerd met geheel of gedeeltelijke weidegang. In beide proeven werden 4 perioden van 28 dagen onderscheiden: een voorperiode van 28 dagen om de proefdiergroepen samen te kunnen stellen en een hoofdperiode van 3 x 28 dagen, waarin de dieren om de 28 dagen behandeld werden met BST. Na de weideperioden werd met een deel van de proefdieren de proef op stal nog 3 maanden voortgezet

In 2 proeven werd het effect van behandeling bij melkkoeien met recombinant bovine somatotropine bestudeerd met geheel of gedeeltelijke weidegang. In beide proeven werden 4 perioden van 28 dagen onderscheiden: een voorperiode van 28 dagen om de proefdiergroepen samen te kunnen stellen en een hoofdperiode van 3 x 28 dagen, waarin de dieren om de 28 dagen behandeld werden met BST. Na de weideperioden werd met een deel van de proefdieren de proef op stal nog 3 maanden voortgezet