IGF System Research Articles

ROLE of IGF-1 System in the Modulation of Longevity: Controversies and New Insights From a Centenarians' Perspective.

Human aging is currently defined as a physiological decline of biological functions in the body with a continual adaptation to internal and external damaging. The endocrine system plays a major role in orchestrating cellular interactions, metabolism, growth, and aging. Several in vivo studies from worms to mice showed that downregulated activity of the GH/IGF-1/insulin pathway could be beneficial for the extension of human life span, whereas results are contradictory in humans. In the present review, we discuss the potential role of the IGF-1 system in modulation of longevity, hypothesizing that the endocrine and metabolic adaptation observed in centenarians and in mammals during caloric restriction may be a physiological strategy for extending lifespan through a slower cell growing/metabolism, a better physiologic reserve capacity, a shift of cellular metabolism from cell proliferation to repair activities and a decrease in accumulation of senescent cells. Therefore, understanding of the link between IGF-1/insulin system and longevity may have future clinical applications in promoting healthy aging and in Rehabilitation Medicine.

The Role of Insulin-Like Growth Factors and Insulin-Like Growth Factor-Binding Proteins in the Nervous System.

The insulin-like growth factors (IGF-I and IGF-II) and their receptors are widely expressed in nervous tissue from early embryonic life. They also cross the blood brain barriers by active transport, and their regulation as endocrine factors therefore differs from other tissues. In brain, IGFs have paracrine and autocrine actions that are modulated by IGF-binding proteins and interact with other growth factor signalling pathways. The IGF system has roles in nervous system development and maintenance. There is substantial evidence for a specific role for this system in some neurodegenerative diseases, and neuroprotective actions make this system an attractive target for new therapeutic approaches. In developing new therapies, interaction with IGF-binding proteins and other growth factor signalling pathways should be considered. This evidence is reviewed, gaps in knowledge are highlighted, and recommendations are made for future research.

The insulin-like growth factor-1 system in the adult mammalian brain and its implications in central maternal adaptation.

Our knowledge on the bioavailability and actions of insulin-like growth factor-1 (IGF-1) has markedly expanded in recent years as novel mechanisms were discovered on IGF binding proteins (IGFBPs) and their ability to release IGF-1. The new discoveries allowed a better understanding of the endogenous physiological actions of IGF-1 and also its applicability in therapeutics. The focus of the present review is to summarize novel findings on the neuronal, neuroendocrine and neuroplastic actions of IGF-1 in the adult brain. As most of the new regulatory mechanisms were described in the periphery, their implications on brain IGF system will also be covered. In addition, novel findings on the effects of IGF-1 on lactation and maternal behavior are described. Based on the enormous neuroplastic changes related to the peripartum period, IGF-1 has great but largely unexplored potential in maternal adaptation of the brain, which is highlighted in the present review.

Oocyte-Secreted Factors Synergize With FSH to Promote Aromatase Expression in Primary Human Cumulus Cells.

The role of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) on aromatase regulation is poorly understood in humans. Determine GDF9 and BMP15 effects on FSH stimulation of estradiol production in primary human cumulus granulosa cells (GCs). We hypothesized that the combination of GDF9 and BMP15 potentiates FSH-induced aromatase expression. Primary human cumulus GCs in culture. University infertility center. GCs of 60 women undergoing in vitro fertilization were collected. Cells were treated with GDF9 and/or BMP15 (GB) in the presence or absence of FSH, dibutyryl cAMP, or SMAD inhibitors. Promoter activity, mRNA, protein, and estradiol levels were quantified. FSH and GB treatment increased CYP19A1 promoter activity, mRNA, and protein levels as well as estradiol when compared with cells treated with FSH only. GB treatment potentiated cAMP stimulation of aromatase and IGF2 stimulation by FSH. GB effects were inhibited by SMAD3 inhibitors and IGF1 receptor inhibitors. GB, but not FSH, stimulates SMAD3 phosphorylation. The combination of GDF9 and BMP15 potently stimulates the effect of FSH and cAMP on CYP19a1 promoter activity and mRNA/protein levels. These effects translate into an increase in estradiol production. This potentiation seems to occur through activation of the SMAD2/3 and SMAD3 signaling pathway and involves, at least in part, the effect of the IGF system.

Insulin-like growth factor-II in adipocyte regulation: depot-specific actions suggest a potential role limiting excess visceral adiposity.

The IGF system has an important role in growth and development. IGF-II is a recognized fetal growth promoter. However, its physiological postnatal role remains uncertain, although it is maintained in the circulation at a substantially high level throughout life. IGF-II has been strongly linked to obesity in genetic studies, and more recent evidence suggests a metabolic role. We examined fat depot differences in IGF-II's action on differentiation and metabolism. We speculate a specific effect on visceral adipocytes in relation to the differential distribution of insulin receptors between visceral and subcutaneous fat depots. We used a previously established adipocyte, cell culture system of matched pairs of visceral and subcutaneous fat biopsies from 20 normal weight children undergoing routine surgery for nonmalignant, nonseptic conditions. Preadipocytes were differentiated for 14 days in the presence or absence of IGF-II. Oil Red O staining, Western blotting, and reverse transcription polymerase chain reaction techniques were employed to assess levels of adipogenesis markers and levels of the insulin receptor and insulin receptor isoforms. Our data indicate that IGF-II promotes preadipocyte differentiation in subcutaneous preadipocytes but showed a protective, opposing effect restricting visceral preadipocyte differentiation, confirmed by reductions in the differentiation markers peroxisome proliferator-activated receptor gamma and adiponectin and in triglyceride staining. Additionally, IGF-II reduced mRNA expression of the insulin receptor in adipocytes and downregulated insulin receptor isoform A and glucose transporter 4 abundance and corresponding glucose uptake in visceral adipocytes. In conclusion, IGF-II is a regulator of preadipocyte differentiation and metabolism by acting as a differential modulator of fat accumulation favoring less visceral fat deposition in children.

Effects of Chromeceptin on Insulin-like Growth Factor II in Human Osteoblastic and Osteosarcoma Cells

The insulin-like growth factor system is a complex regulatory system of insulin-like growth factors I and II (IGF-I and IGF-II) as well as their cell surface receptors and a family of insulin growth factor binding proteins (IGF-BPs). Despite extensive research on the IGF system in skeletal tissue, there is still not a complete understanding particularly of the role of IGF-II in the regulation of normal and pathological osseous cells in human skeletal biology. The purpose of this study was to further delineate the possible involvement of IGF-II in the regulation of human normal calvarial osteoblasts and G292 human osteosarcoma cellular activity using exogenously added IGF-II as well as a drug, Chromeceptin that has been shown in some other cellular systems to down regulate endogenous IGF-II levels.The results presented here show a similar dose dependent effect of Chromeceptin on inhibition of endogenous IGF-II levels (measured with an immunoassay) and cellular activity (measured with the MTT assay) in both the normal and osteosarcoma cells while small significant increases in activity with exogenously added IGF-II were observed only in the osteosarcoma cells. Moreover, IGF-BP1 levels (measured with an immunoassay) were shown to significantly increase in the G292 cells in a similar manner previously reported in other cell systems with only minor non-significant effects of exogenously added IGF-BP1 in the osteosarcoma cells studied here. These studies suggest that endogenous IGF-II is a critical regulator of activity in both normal and osteosarcoma cells and Chromeceptin can be an effective pharmacological agent for further studies on the role of the growth factor in human skeletal systems.

In vivo effects of 17α-ethinylestradiol, 17β-estradiol and 4-nonylphenol on insulin-like growth-factor binding proteins (igfbps) in Atlantic salmon

Feminizing endocrine disrupting compounds (EDCs) affect the growth and development of teleost fishes. The major regulator of growth performance, the growth hormone (Gh)/insulin-like growth-factor (Igf) system, is sensitive to estrogenic compounds and mediates certain physiological and potentially behavioral consequences of EDC exposure. Igf binding proteins (Igfbps) are key modulators of Igf activity, but their alteration by EDCs has not been examined. We investigated two life-stages (fry and smolts) of Atlantic salmon (Salmo salar), and characterized how the Gh/Igf/Igfbp system responded to waterborne 17α-ethinylestradiol (EE2), 17β-estradiol (E2) and 4-nonylphenol (NP). Fry exposed to EE2 and NP for 21 days had increased hepatic vitellogenin (vtg) mRNA levels while hepatic estrogen receptor α (erα), gh receptor (ghr), igf1 and igf2 mRNA levels were decreased. NP-exposed fry had reduced body mass and total length compared to controls. EE2 and NP reduced hepatic igfbp1b1, -2a, -2b1, -4, -5b2 and -6b1, and stimulated igfbp5a. In smolts, hepatic vtg mRNA levels were induced following 4-day exposures to all three EDCs, while erα only responded to EE2 and E2. EDC exposures did not affect body mass or fork length; however, EE2 diminished plasma Gh and Igf1 levels in parallel with reductions in hepatic ghr and igf1. In smolts, EE2 and E2 diminished hepatic igfbp1b1, -4 and -6b1, and stimulated igfbp5a. There were no signs of compromised ionoregulation in smolts, as indicated by unchanged branchial ion pump/transporter mRNA levels. We conclude that hepatic igfbps respond (directly and/or indirectly) to environmental estrogens during two key life-stages of Atlantic salmon, and thus may modulate the growth and development of exposed individuals.

Involvement of insulin-like growth factor in intraspecific variation in growth of Pacific oyster Crassostrea gigas during winter

Insulin-like growth factor I (IGF-I) plays a critical role in regulating proliferation, differentiation, growth, and survival in fish as well as in humans. Few studies have investigated the involvement of IGFs in bivalves. In Korea, the Pacific oyster Crassostrea gigas is an important aquaculture species, and its growth is dependent on environmental conditions. Intrinsic factors that can be used as indicators to assess oyster growth based on individual size are limited. This study analyzed IGF-I, IGF-I receptor (IGF-IR), and IGF binding protein (IGFBP) in adductor muscle of C. gigas in winter using immunoblotting and reverse transcriptase-polymerase chain reaction. Levels of IGF-I and the IGF-IR β subunit, and expression of C. gigas insulin receptor-related receptor and IGFBP messenger RNA were significantly higher in large individuals (LI) than small individuals (SI). The IGF-IR β subunit was detected in the adductor muscle of both groups; however, phosphorylation of the IGF-IR β subunit and extracellular signal-regulated kinase was greater in LI than SI. The condition index of C. gigas showed a similar pattern. The results of this study suggest that the IGF system is associated with growth rate differences in C. gigas during winter in Korea.

The influence of IGF-1 on fetal growth during pregnancy complicated by gestational diabetes mellitus

Insulin-like growth factor-1 (IGF-1) is an important part of IGF system. The structure and function of IGF-1 are similar to that of insulin. Thus, IGF-1 may participate both in the gestational diabetes mellitus (GDM) pathogenesis and the development of some complications. IGF-1, as one of the most important growth factors “in utero”, may influence the intrauterine growth leading to fetal macrosomia. The aim of the study was to present the structure and function of IGF-1 in terms of carbohydrate metabolism disturbances and to determine the role of IGF-1 on fetal growth during pregnancy complicated by GDM. Fetal growth is an extremely complex process and depends on many factors, including IGF-1 concentration.

Response of the insulin-like growth factor-1 (Igf1) system to nutritional status and growth rate variation in olive rockfish (Sebastes serranoides)

Growth performance in vertebrates is regulated by environmental factors including the quality and quantity of food, which influence growth via endocrine pathways such as the growth hormone (GH)/insulin-like growth factor somatotropic axis. In several teleost fishes, circulating concentrations of insulin-like growth factor-1 (Igf1) correlate positively with growth rate, and it has been proposed that plasma Igf1 levels may serve as an indicator of growth variation for fisheries and aquaculture applications. This study tested whether plasma Igf1 concentrations might serve as an indicator of somatic growth in olive rockfish (Sebastes serranoides), one species among dozens of rockfishes important to commercial and recreational fisheries in the Northern Pacific Ocean. Juvenile olive rockfish were reared under food ration treatments of 1% or 4% wet mass per d for 98 d to experimentally generate variation in growth. Juvenile rockfish in the 4% ration grew 60% more quickly in mass and 22% faster in length than fish in the 1% ration. Plasma Igf1 levels were elevated in rockfish under the 4% ration, and individual Igf1 levels correlated positively with growth rate, as well as with individual variation in hepatic igf1 mRNA levels. Transcripts encoding the Igf binding proteins (Igfbps) igfbp1a and igfbp1b were also at higher abundance in the liver of rockfish in the 1% ration treatment, while mRNAs for igfbp5a and igfbp5b were elevated in the skeletal muscle of 4% ration fish. These findings support the use of plasma Igf1 as a physiological index of growth rate variation in rockfish.

Insulin-Like Growth Factor (IGF) System in Liver Diseases

Hepatocyte differentiation, proliferation, and apoptosis are affected by growth factors produced in liver. Insulin-like growth factor 1 and 2 (IGF1 and IGF2) act in response to growth hormone (GH). Other IGF family components include at least six binding proteins (IGFBP1 to 6), manifested by both IGFs develop due to interaction through the type 1 receptor (IGF1R). The data based on animal models and/or in vitro studies suggest the role of IGF system components in cellular aspects of hepatocarcinogenesis (cell cycle progression, uncontrolled proliferation, cell survival, migration, inhibition of apoptosis, protein synthesis and cell growth), and show that systemic IGF1 administration can reduce fibrosis and ameliorate general liver function. In epidemiologic and clinicopathological studies on chronic liver disease (CLD), lowered serum levels, decreased tissue expression of IGF1, elevated production of IGF1R and variable IGF2 expression has been noted, from the start of preneoplastic alterations up to the developed hepatocellular carcinoma (HCC) stage. These changes result in well-known clinical symptoms of IGF1 deficiency. This review summarized the current data of the complex role of IGF system components in the most common CLD (nonalcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma). Better recognition and understanding of this system can contribute to discovery of new and improved versions of current preventive and therapeutic actions in CLD.

Molecular cloning and expression profiles of IGFBP-1a in common carp ( Cyprinus carpio ) and its expression regulation by growth hormone in hepatocytes

In this study, we cloned and determined IGFBP-1a cDNA from common carp (Cyprinus carpio) liver. The 1655 bp full-length cDNA consisted of a 96 bp 5-untranslated region (UTR), a 789 bp open reading frame encoding 262 amino acid residues and a 770 bp 3-UTR containing seven mRNA instability motifs. Northern blot revealed a 1.8 kb IGFBP-1a transcript. IGFBP-1a mRNA was widely distributed in all tissues examined and predominantly expressed in the liver. During embryogenesis, IGFBP-1a mRNA was firstly observed in blastula stage, and significant increases were observed in body segment stage, lens formation stage and blood cycling stage. After hatching, its expression increased more than twenty times. Furthermore, hypoxia could significantly up-regulate IGFBP-1a expression in the liver and brain. IGFBP-1a expression increased with ovarian maturation and decreased at regressed stage. In testis, IGFBP-1a mRNA maintained relatively higher levels at recrudescing and matured stages, while it sharply declined at regressed stage. In primary cultured hepatocytes, IGFBP-1a gene was greatly down-regulated by growth hormone via the MAPK and PI3 kinase signaling pathways. These results suggest that IGFBP-1a may be involved in the IGF system regulating growth, development and reproduction in common carp.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Influences Sensitivity to Anti-IGF System Agents Through the Translational Regulation of IGF1R.

Insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IGF2BP3) is an oncofetal protein that binds RNA, thereby influencing the fate of target transcripts. IGF2BP3 is synthesized de novo in cancer, where it promotes proliferation, drug resistance, and metastasis via both IGF2-dependent and IGF2-independent mechanisms. Ewing sarcoma (ES) is a rare bone and soft tissue tumor in which the IGF system plays a pivotal role. This study aimed to investigate the effect of IGF2BP3 on the regulation of the IGF system in ES. Among the components of the IGF axis, a direct significant correlation was identified between IGF2BP3 and IGF1R at mRNA and protein levels in two independent series of clinical specimens from patients with localized ES. After the formal demonstration of a direct association between IGF2BP3 and IGF1R mRNA using ribo-immunoprecipitation assay, we performed in vitro studies using A673 and TC-71 ES cell lines to demonstrate that IGF2BP3 loss promotes the downregulation of IGF1R and a decreased biological response to IGF1, represented by reduced migration and cell growth. Additionally, the compensatory activation of insulin receptor (IR) and its mitogenic ligand IGF2 is triggered in some but not all cell lines in response to IGF2BP3-mediated IGF1R loss. These findings have therapeutic implications because cells with a decreased expression of IGF2BP3/IGF1R axis but an increased expression of the IR/IGF2 loop display higher sensitivity to the dual inhibitor OSI-906 than do control cells. Therefore, studies on IGF2BP3, which was confirmed as a post-transcriptional regulator of IGF1R, provide a step forward in the identification of new mechanisms regulating the IGF system. In addition, our results demonstrate that the detection of IGF2BP3 expression should be combined with the assessment of the IGF1R/IR ratio to predict cell responses to anti-IGF1R/IR agents.

Luteinizing Hormone Facilitates Antral Follicular Maturation and Survival via Thecal Paracrine Signaling in Cattle

LH supplementation in assisted reproductive technology cycles improves the ongoing pregnancy rate in women with poor ovarian response (POR). However, our knowledge of the precise role of LH during the follicular phase of the menstrual cycle is incomplete. To explore the role of LH in the maturation of small antral follicles, we used an in vitro two-cell system that involved coculturing bovine granulosa cells (GCs) and theca cells (TCs) on a collagen membrane. Treatment of TCs with LH stimulated androgen production in TCs by inducing the expression of androgenic factors, subsequently increasing estrogen biosynthesis in GCs by providing androgen substrates, and inducing aromatase expression. LH stimulation of TCs induced functional LH receptor expression in GCs, a response modulated by the synthesis and action of estrogen. In the presence of TCs, LH stimulation of TCs and FSH stimulation of GCs increased the expression of IGF-1, IGF-2, and IGF-1 receptor in GCs. LH-induced expression of thecal IGF-1 protected GCs from apoptosis and promoted GC survival. Furthermore, LH stimulation of TCs increased FSH sensitivity in GCs. Thus, the LH-TC axis may be involved in the acquisition of LH dependence and the survival of small antral follicles by upregulating androgen/estrogen biosynthesis and activating the IGF system. The use of LH supplementation in ovarian stimulation may increase gonadotropin sensitivity in small antral follicles and promote follicular growth and survival by suppressing GC apoptosis and follicular atresia, resulting in multiple follicular development, even in patients with POR.

Effects of Lactogen 13, a New Probiotic Preparation, on Gut Microbiota and Endocrine Signals Controlling Growth and Appetite of Oreochromis niloticus Juveniles.

In the present study, Nile tilapia Oreochromis niloticus was used as experimental model to study the molecular effects of a new probiotic preparation, Lactogen 13 (Lactobacillus rhamnosus IMC 501® encapsulated with vegetable fat matrices by spray chilling and further indicated as probiotic microgranules), on growth and appetite during larval development. Probiotic microgranules were administered for 30days to tilapia larvae starting from first feeding. Molecular analysis using high-throughput sequencing revealed that the probiotic could populate the gastrointestinal tract and modulate the microbial communities by significantly increasing the proportion of Lactobacillus as well as reducing the proportion of potential pathogens such as members of the Family Microbacteriaceae, Legionellaceae, and Weeksellaceae. Morphometric analysis evidenced that body weight and total length significantly increased after probiotic treatment. This increase coincided with the modulation of genes belonging to the insulin-like growth factors (igfs) system and genes involved on myogenesis, such as myogenin, and myogenic differentiation (myod). Alongside the improvement of growth, an increase of feed intake was evidenced at 40days post-fertilization (dpf) in treated larvae. Gene codifying for signals belonging to the most prominent systems involved in appetite regulation, such as neuropeptide y (npy), agouti-related protein (agrp), leptin, and ghrelin were significantly modulated. These results support the hypothesis that gastrointestinal (GI) microbiota changes due to probiotic administration modulate growth and appetite control, activating the endocrine system of tilapia larvae.

Insulin-Like Growth Factor Binding Proteins and IGFBP Proteases: A Dynamic System Regulating the Ovarian Folliculogenesis.

The aim of the present article is to update our understanding of the expression of the insulin-like growth factor binding proteins (IGFBPs), IGFBP proteases and their implication in the different processes of ovarian folliculogenesis in mammals. In the studied species, IGFs and several small-molecular weight IGFBPs (in particular IGFBP-2 and IGFBP-4) are considered, respectively, as stimulators and inhibitors of follicular growth and maturation. IGFs play a key role in sensitizing ovarian granulosa cells to FSH action during terminal follicular growth. Concentrations of IGFBP-2 and IGFBP-4 in follicular fluid strongly decrease during follicular growth, leading to an increase in IGF bioavailability. Inversely, atresia is characterized by an increase of IGFBP-2 and IGFBP-4 levels, leading to a decrease in IGF bioavailability. Changes in intrafollicular IGFBPs content are due to variations in mRNA expression and/or proteolytic degradation by the pregnancy-associated plasma protein-A (PAPP-A), and likely participates in the selection of dominant follicles. The identification of PAPP-A2, as an IGFBP-3 and -5 protease, and stanniocalcins (STCs) as inhibitors of PAPP-A activity extends the IGF system. Studies on their implication in folliculogenesis in mammals are still in the early stages.

Does IGF-1 play a role in the biology of ovarian cancer?

The aim of the study was to investigate serum concentrations of the insulin-like growth factor-1 in women with ovarian cancer and healthy controls, and to compare free IGF-1 levels with selected clinical and pathological param-eters. Correlation analysis was used to measure the following: IGF-1 concentration and Ca125; IGF-1 level and the height of the OC patients. The study included 70 patients with OC and 50 healthy controls. Serum concentrations of free IGF-1 were measured in all subjects. Routine diagnostic tests (CBC and USG and Ca125) were performed. Significantly higher serum concentrations of free IGF-1 were found in the study group as compared to controls. No statistically significant relationships between IGF-1 serum concentrations and tumor differentiation, histological type, and disease stage were detected. No statistically significant correlations between IGF-1 and Ca125 level or between IGF-1 and growth of OC patients were found. Serum IGF-1 participates in the etiopathogenesis of ovarian cancer in menstruating women, while local synthesis of this factor and other components of the autocrine loop of the IGF-1 system play a greater role in their post-menopausal peers.

Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insulin receptor (INSR) and type-I insulin-like growth factor receptor (IGF1R), have not, to our knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.-Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stévant, I., Griffeth, R. J., Pitetti, J.-L., Andric, S. A., Hu, M.-C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

Effects of Parental Aging During Embryo Development and Adult Life: The Case ofNothobranchius furzeri

Studies on parental aging are a very attractive field, although it is poorly understood how parental age affects embryonic development and adult traits of the offspring. In this study, we used the turquoise killifish Nothobranchius furzeri, as is the vertebrate with shortest captive lifespan and an interesting model. The embryos of N. furzeri can follow two distinct developmental pathways either entering diapause or proceeding through direct development. Thus, this embryonic plasticity allows this model to be used to study different factors that could affect their embryonic development, including parental age. The first goal of the present study was to investigate whether parental aging could affect the embryo development. To do this, we collected F1 embryos from two breeder groups (old parents and young parents). We monitored the duration of embryonic development and analyzed genes involved in dorsalization process. The second goal was to investigate if embryonic developmental plasticity could be modulated by an epigenetic process. To this end, the expression of DNMTs genes was examined. Our data support the hypothesis that diapause, occurring more frequently in embryos from old parents, is associated with increased expression of DNMT3A and DNMT3B suggesting an epigenetic control. Finally, we analyzed whether parental age could affect metabolism and growth during adult life. Morphometric results and qPCR analysis of genes from IGF system showed a slower growth in adults from old breeders. Moreover, a gender-specificity effect on growth emerged. In conclusion, these results may contribute to the better understanding of the complex mechanism of aging.

58 Evidence of Metabolic Compartmentalization or Zonation in the Bovine Placenta: Significance for the Regulation of Placental Function and Fetal Growth

The concept of “metabolic zonation” in the liver proposes that opposing or complementary metabolic pathways are carried out within distinct regions of the liver lobule to maintain optimal metabolic homeostasis. As bovine placentomes morpho-topographically differ in mass and shape within the pregnant tract, an event more pronounced in cloned-derived concepti, the aims of this study were (a) to investigate the occurrence of metabolic zonation in the bovine placenta by placentome shape and size, and (b) to compare placenta metabolic functions between control and cloned concepti, which may relate to the abnormal conceptus growth after cloning. Bovine pregnancies established by superovulation and AI (Control group, n = 4) or by cloning by nuclear transfer (Cloned group, n = 6) were slaughtered on Day 225 of gestation, with the harvesting of maternal and conceptus tissues and fluids [Gerger et al. 2016 Reprod. Fertil. Dev. 29, 950-967; 10.1071/RD15215]. Placentomes were excised, weighed, measured, and classified by shape as types A, B, C or D. Total RNA from each placentome type per group was extracted, reverse transcribed, and used for the RT-qPCR analysis of 27 key genes related to glycolysis, gluconeogenesis, fructolysis, fructogenesis, pentose phosphate pathway (PPP), fatty acid synthesis, cholesterol synthesis, glucose transporters, apoptosis, and components of the IGF system. Data were analysed by ANOVA, the Tukey test, and a simple correlation test. Day-225 cloned concepti were 2-fold larger than controls, with 64% of placentomes being larger than the largest in the Control group. A linear positive correlation between mass, size and shape was observed in the control group, with 83% of the normal-shaped placentomes (A and B) representing 85% of the total placenta weight. Conversely, abnormal-shaped placentomes (C and D) represented 39% in the cloned group and 32% total placenta mass, for fewer type A and more type C placentomes in clones. No differences were detected in expression for key metabolic-related genes between placentas of clones and controls, irrespective of the placentome type. However, differences were verified between placentome types, with an increase in gene expression in type C in relation to type A placentomes for glucose transporters (2.2-fold), glycolysis (1.7-fold), PPP (2.1-fold), fructogenesis (1.3- to 1.6-fold), Igf2 (2.1-fold) and Igf1r (2.2-fold), for a 2.5-fold decrease in Igf2r, with no difference in fatty acid and cholesterol synthesis or apoptosis. The differential expression pattern observed only between placentome types demonstrates a potential metabolic zonation in the bovine placenta. Therefore, morpho-physiologic differences observed between clones and controls could be due to changes in size and proportions of placentome types, and not due to changes in tissue unit function per se. The more significant proportion of type C placentomes in the placenta of clones may be associated with increased fructose synthesis due to increased glucose supply, in a nutrient surplus that promotes conceptus growth.