Giorgi Berishvili

Environmentally Relevant Concentrations of 17α-Ethinylestradiol (EE2) Interfere With the Growth Hormone (GH)/Insulin-Like Growth Factor (IGF)-I System in Developing Bony Fish

The aim of this study was to evaluate whether effects of environmental estrogens on fish growth and reproduction may be mediated via modulating the growth hormone (GH)/insulin-like growth factor I (IGF-I) system. To this end, developing male and female monosex populations of tilapia were exposed to 17alpha-ethinylestradiol (EE2) at 5 and 25 ng EE2/l water from 10-day postfertilization (DPF) until 100 DPF. Under exposure to both EE2 concentrations, sex ratio shifted toward more females and body length, and weight were significantly reduced in males. The growth-reducing effect was associated with significant changes in hepatic IGF-I expression, both in males and females and with significant alterations of IGF-I mRNA and GH mRNA in the brain. The changes in IGF-I and GH mRNA were accompanied by altered estrogen receptor alpha (ERalpha) expression in brain and liver. These findings point to an influence of estrogenic exposure on the endocrine GH/IGF-I axis. In addition, the EE2 treatment resulted in significant changes of ERalpha and IGF-I expression in ovaries and testis, suggesting that the estrogens interact not only with the endocrine but also with the autocrine/paracrine part of the IGF-I system. Overall, our results provide evidence that EE2 at environmentally relevant concentrations is able to interfere with the GH/IGF-I system in bony fish and that the impairing effects of estrogens reported on fish growth and reproductive functions may rather result from a cross talk between the sex steroid and the IGF-I system than be toxicological.

Ethinylestradiol differentially interferes with IGF-I in liver and extrahepatic sites during development of male and female bony fish

Growth and sexual development are closely interlinked in fish; however, no reports exist on potential effects of estrogen on the GH/IGF-I-axis in developing fish. We investigate whether estrogen exposure during early development affects growth and the IGF-I system, both at the systemic and tissue level. Tilapia were fed from 10 to 40 days post fertilization (DPF) with 17alpha-ethinylestradiol (EE(2)). At 50, 75, 90, and 165 DPF, length, weight, sex ratio, serum IGF-I (RIA), pituitary GH mRNA and IGF-I, and estrogen receptor alpha (ERalpha) mRNA in liver, gonads, brain, and gills (real-time PCR) were determined and the results correlated to those of in situ hybridization for IGF-I. Developmental exposure to EE(2) had persistent effects on sex ratio and growth. Serum IGF-I, hepatic IGF-I mRNA, and the number of IGF-I mRNA-containing hepatocytes were significantly decreased at 75 DPF, while liver ERalpha mRNA was significantly induced. At 75 DPF, a transient decline of IGF-I mRNA and a largely reduced number of IGF-I mRNA-containing neurons were observed in the female brain. In both sexes, pituitary GH mRNA was significantly suppressed. A transient downregulation of IGF-I mRNA occurred in ovaries (75 DPF) and testes (90 DPF). In agreement, in situ hybridization revealed less IGF-I mRNA signals in granulosa and germ cells. Our results show for the first time that developmental estrogen treatment impairs GH/IGF-I expression in fish, and that the effects persist. These long-lasting effects both seem to be exerted indirectly via inhibition of pituitary GH and directly by suppression of local IGF-I in organ-specific cells.

Insulin-like growth factor-3 (IGF-3) in male and female gonads of the tilapia: Development and regulation of gene expression by growth hormone (GH) and 17α-ethinylestradiol (EE2)

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.

Distinct organ-specific up- and down-regulation of IGF-I and IGF-II mRNA in various organs of a GH-overexpressing transgenic Nile tilapia

Several lines of GH-overexpressing fish have been produced and characterized concerning organ integrity, growth, fertility and health but few and contradictory data are available on IGF-I that mediates most effects of GH. Furthermore, nothing is known on IGF-II. Thus, the expression of both IGFs in liver and various extrahepatic sites of adult transgenic (GH-overexpressing) tilapia and age-matched wild-type fish was determined by real-time PCR. Both IGF-I and IGF-II mRNA were found in all organs investigated and were increased in gills, kidney, intestine, heart, testes, skeletal muscle and brain of the transgenics (IGF-I: 1.4–4-fold; IGF-II: 1.7–4.2-fold). Except for liver, brain and testis the increase in IGF-I mRNA was higher than that in IGF-II mRNA. In pituitary, no significant change in IGF-I or IGF-II mRNA was detected. In spleen, however, IGF-I and IGF-II mRNA were both decreased in the transgenics, IGF-I mRNA even by the 19-fold. In agreement, in situ hybridisation revealed a largely reduced number of IGF-I mRNA-containing leukocytes and macrophages when compared to wild-type. These observations may contribute to better understanding the reported impaired health of GH-transgenic fish. Growth enhancement of the transgenics may be due to the increased expression of both IGF-I and IGF-II in extrahepatic sites. It is also reasonable that the markedly enhanced expression of liver IGF-II mRNA that may mimick an early developmental stage is a further reason for increased growth.

Growth hormone (GH) treatment acts on the endocrine and autocrine/paracrine GH/IGF-axis and on TNF-α expression in bony fish pituitary and immune organs

There exist indications that the growth hormone (GH)/insulin-like growth factor (IGF) axis may play a role in fish immune regulation, and that interactions occur via tumour necrosis factor (TNF)-α at least in mammals, but no systematic data exist on potential changes in GH, IGF-I, IGF-II, GH receptor (GHR) and TNF-α expression after GH treatment. Thus, we investigated in the Nile tilapia the influence of GH injections by real-time qPCR at different levels of the GH/IGF-axis (brain, pituitary, peripheral organs) with special emphasis on the immune organs head kidney and spleen. Endocrine IGF-I served as positive control for GH treatment efficiency. Basal TNF-α gene expression was detected in all organs investigated with the expression being most pronounced in brain. Two consecutive intraperitoneal injections of bream GH elevated liver IGF-I mRNA and plasma IGF-I concentration. Also liver IGF-II mRNA and TNF-α were increased while the GHR was downregulated. In brain, no change occurred in the expression levels of all genes investigated. GH gene expression was exclusively detected in the pituitary where the GH injections elevated both GH and IGF-I gene expression. In the head kidney, GH upregulated IGF-I mRNA to an even higher extent than liver IGF-I while IGF-II and GHR gene expressions were not affected. Also in the spleen, no change occurred in GHR mRNA, however, IGF-I and IGF-II mRNAs were increased. In correlation, in situ hybridisation showed a markedly higher amount of IGF-I mRNA in head kidney and spleen after GH injection. In both immune tissues, TNF-α gene expression showed a trend to decrease after GH treatment. The stimulation of IGF-I and also partially of IGF-II expression in the fish immune organs by GH indicates a local role of the IGFs in immune organ regulation while the differential changes in TNF-α support the in mammals postulated interactions with the GH/IGF-axis which demand for further investigations.

Seawater and freshwater challenges affect the insulin-like growth factors IGF-I and IGF-II in liver and osmoregulatory organs of the tilapia

Contradictory studies suggest IGF-I in fish liver and gills is involved in osmoregulation, but nothing is known about the kidney and intestines role nor about IGF-IIs role in any organ. Tilapia were transferred from freshwater (FW) to seawater (SW) for 1week (wk) and retransferred to FW for another week. At 4h, 1d, 2d, 3d and 1wk after SW-transfer and FW-retransfer IGF-I, IGF-II and growth hormone receptor (GHR1) mRNA were measured by real-time PCR. Hepatic IGF-I, IGF-II and GHR1 mRNA were downregulated in parallel after SW-transfer, recovered and were again downregulated after FW-retransfer. In gills, IGF-I, IGF-II and GHR1 were upregulated synchronously after SW-transfer and, partially also after FW-retransfer. The renal genes were downregulated after SW-transfer and partially upregulated after FW-retransfer. Persisting upregulation in intestinal IGF-I mRNA occurred after FW-retransfer. Thus, endocrine and auto/paracrine IGF-I and IGF-II seem to be involved in fish osmoregulation in an organ-specific manner.

Oestrogen and insulin-like growth factors during the reproduction and growth of the tilapia Oreochromis niloticus and their interactions

Oestrogens and insulin-like growth factors (Igfs) play both a central role in the regulation of reproduction and growth and can interact especially in species showing a clear-cut sex-linked growth dimorphism (SGD) like in tilapia. Aromatase is essential in ovarian differentiation and oogenesis since it controls oestrogen synthesis. During tilapia sex differentiation, aromatase cyp19a1a expression increases from 9 days post-fertilization (dpf), resulting in high oestradiol level. High temperature, exogenous androgens or aromatase inhibitors override genetic sex differentiation inducing testes development through the suppression of cyp19a1a gene expression and aromatase activity. Supplementation with 17ß-oestradiol (E2) of gonadectomized juveniles induced a sustained and higher E2 plasma level than in intact or gonadectomized controls and both sexes showed reduced growth. Juvenile and mature females treated with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione had 19% lower E2 plasma level compared to controls and they showed a 32% increased growth after 28 days of treatment. Altogether, these data suggest that E2 inhibits female growth leading to the SGD. Regarding Igf-1, mRNA and peptide appeared in liver at ∼ 4 dpf and then in organs involved in growth and metabolism, indicating a role in early growth, metabolism and organogenesis. Gonad igf-1 showed an early expression and the peptide could be detected at ∼ 7 dpf in somatic cells. It appeared in germ cells at the onset of ovarian (29 dpf) and testicular (52 dpf) meiosis. In testis, Igf-1 together with steroids may regulate spermatogenesis whereas in ovary it participates in steroidogenesis regulation. Igf-1 and Igf-2 promote proliferation of follicular cells and oocyte maturation. Igf-3 expression is gonad specific and localized in the ovarian granulosa or testicular interstitial cells. In developing gonads igf-3 is up-regulated in males but down-regulated in females. In contrast, bream Gh injections increased igf-1 mRNA in male and female liver and ovaries but gonadal igf-3 was not affected. Thus, local Igf-1 and Igf-2 may play crucial roles in the formation, development and function of gonads while Igf-3 depending on the species is involved in male and female reproduction. Furthermore, precocious ethynylestradiol (EE) exposure induced lasting effects on growth, through pituitary gh inhibition, local suppression of igf-1 expression and in testis only down-regulation of igf-3 mRNA. In conclusion, SGD in tilapia may be driven through an inhibitory effect due to E2 synthesis in female and involving Igfs regulation.

The advantage of absolute quantification in comparative hormone research as indicated by a newly established real-time RT-PCR: GH, IGF-I, and IGF-II gene expression in the tilapia, Oreochromis niloticus.

Abstract: We have developed a real‐time RT‐PCR that absolutely quantifies the gene expression of hormones using the standard curve method. The method avoids cloning procedures by using primer extension to create templates containing a T7 promoter gene sequence. It is rapid since neither separate reverse transcriptions nor postamplification steps are necessary, and its low detection level (2 pg/μg total RNA) allows precise absolute quantification. Using the method, we have quantified the gene expression of GH, IGF‐I, and IGF‐II in the tilapia.

A survey on the expression of IGF-I in the early developing bony fish with special emphasis on the tilapia, Oreochromis niloticus

Abstract: The present study investigates the expression of IGF‐I in the early developing tilapia (Oreochromis niloticus). IGF‐I was detected very early in ontogeny (4‐5 days postfertilization, DPF), first in liver and in organs involved in growth and metabolism, thus suggesting a high physiological impact of IGF‐I in growth, metabolism, and organogenesis.