Quercetin directly inhibits basal ovarian cell functions and their response to the stimulatory action of FSH.

Plants, fruits, and vegetables containing the bioflavonoid quercetin are widely used in food, beverages, and medicines; however, the effects of quercetin on reproductive processes and the possible mechanisms of quercetin action require extensive investigation. The aim of our study was to examine the direct effects of quercetin on basic ovarian cell functions and their response to follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I), known hormonal stimulators of reproduction. We analyzed the effects of quercetin alone (0, 1, 10, and 100 ng/ml) on cultured porcine ovarian granulosa cells or isolated ovarian follicles; or of quercetin (10 ng/ml) in combination with FSH (0, 0.01, 0.1, or 1 IU/ml) or IGF-I (0, 1, 10, or 100 ng/ml) on cultured porcine granulosa cells. The expression of proliferative (PCNA, cyclin B1) and apoptotic (BAX) markers, as well as markers for release of progesterone (P4), testosterone (T), and leptin (L), were measured by quantitative immunocytochemistry, Western immunoblotting, RT-qPCR, and EIA/RIA. Addition of quercetin reduced the accumulation of PCNA and cyclin B1, as well as their transcript levels, promoted the accumulation of BAX, decreased the release of P4 and L, and increased the release of T in cultured granulosa cells. In ovarian follicles, quercetin reduced the levels of both P4 and T. Exposure to FSH stimulated PCNA and decreased BAX accumulation, and increased the release of P4, T, and L. Quercetin inhibited and even reversed the effects of FSH. Like FSH, IGF-I also promoted granulosa cell proliferation and suppressed apoptosis. Quercetin did not modify IGF-I effects. These data suggest that the plant molecule quercetin can directly down-regulate basal ovarian cell functions (proliferation, apoptosis, and release of ovarian steroid and peptide hormones) and their response to the stimulatory activity of the upstream hormonal stimulator FSH.

Effect of quercetin on ovarian cells of pigs and cattle.

The bioflavonoid quercetin is a component of food with numerous biological effects, but its function in reproductive processes and mechanisms in various species remain unclear. The aim of this study was to examine the effect of quercetin on ovarian cells isolated from ovaries of two phytophagous mammalian species (i.e. pigs and cattle). There was analysis of the effect of quercetin (0, 1, 10, and 100 ng/mL) on cultured granulosa cells of pigs and cattle. Proliferation (PCNA) and apoptosis (bax) markers and release of progesterone (P4), testosterone (T), estradiol (E2), and IGF-I were quantified using quantitative immunocytochemistry, enzyme immunoassay, or radioimmunoassay. Treatments with quercetin reduced PCNA and bax accumulation and decreased P4 release from both granulosa cells of pigs and cattle. In cells of pigs, treatment with quercetin reduced T output, however, in cells of cattle quercetin increased T release. In cells of pigs, quercetin reduced IGF-I release. In cells of cattle, quercetin at smaller doses (1 or 10 ng/mL), promoted and at a large dose (100 ng/mL) reduced IGF-I secretions. There was no substantial E2 release from granulosa cells of pigs or cattle. These observations are the first to indicate there is a direct action of quercetin on basic ovarian cell functions (proliferation, apoptosis, and hormones release) which can be species-specific.

Direct effect of curcumin on porcine ovarian cell functions.

Curcuma longa Linn (L.) is a plant widely used in cooking (in curry powder a.o.) and in folk medicine, but its action on reproductive processes and its possible mechanisms of action remain to be investigated. The objective of this study was to examine the direct effects of curcumin, the major Curcuma longa L. molecule, on basic ovarian cell functions such as proliferation, apoptosis, viability and steroidogenesis. Porcine ovarian granulosa cells were cultured with and without curcumin (at doses of 0, 1, 10 and 100µg/ml of medium). Markers of proliferation (accumulation of PCNA) and apoptosis (accumulation of bax) were analyzed by immunocytochemistry. The expression of mRNA for PCNA and bax was detected by RT-PCR. Cell viability was detected by trypan blue exclusion test. Release of steroid hormones (progesterone and testosterone) was measured by enzyme immunoassay (EIA). It was observed that addition of curcumin reduced ovarian cell proliferation (expression of both PCNA and its mRNA), promoted apoptosis (accumulation of both bax and its mRNA), reduced cell viability, and stimulated both progesterone and testosterone release. These observations demonstrate the direct suppressive effect of Curcuma longa L./curcumin on female gonads via multiple mechanisms of action - suppression of ovarian cell proliferation and viability, promotion of their apoptosis (at the level of mRNA transcription and subsequent accumulation of promoters of genes regulating these activities) and release of anti-proliferative and pro-apoptotic progesterone and androgen. The potential anti-gonadal action of curcumin should be taken into account by consumers of Curcuma longa L.-containing products.

Comparison of the effects of human and chicken ghrelin on chicken ovarian hormone release.

The aim of the present experiments was to examine the species-specific and cell-specific effects of ghrelin on chicken ovarian hormone release. For this purpose, we compared the effects of chicken and human ghrelin on the release of estradiol (E), testosterone (T), progesterone (P) and arginine-vasotocin (AVT) by cultured fragments of chicken ovarian follicles and on the release of T and AVT by cultured ovarian granulosa cells. In cultured chicken ovarian fragments, both human and chicken ghrelin promoted E release. T output was stimulated by chicken ghrelin but not by human ghrelin. No effect of either human or chicken ghrelin on P release was observed. Human ghrelin promoted but chicken ghrelin suppressed AVT release by chicken ovarian fragments. In cultured ovarian granulosa cells, human ghrelin inhibited while chicken ghrelin stimulated T release. Both human and chicken ghrelin suppressed AVT output by chicken granulosa cells. These data confirm the involvement of ghrelin in the control of ovarian secretory activity and demonstrate that the effect of ghrelin is species-specific. The similarity of avian ghrelin on avian ovarian granulosa cells and ovarian fragments (containing both granulosa and theca cells) suggests that ghrelin can influence chicken ovarian hormones primarily by acting on granulosa cells.

The role of metabolic state and obestatin in control of chicken ovarian hormone release.

The aim of the present study was to examine the role and interrelationships between calorie restriction and obestatin in the control of hormone release by chicken ovarian tissue. For this purpose, we compared the release of progesterone (P), testosteron (T), estradiol (E), and arginine-vasotocin (AVT) by ovarian fragments isolated from chicken subjected and not subjested to food restriction, as well as the response of these ovarian fragments to obestatin additions.It was observed that food restriction promoted release of P, reduced output of T, but did not affect basal E and AVT release. Obestatin addition reduced E, promoted AVT, and did not alter P and T release by ovarian tissue isolated from ad libitum fed chicken. In ovarian fragments of fasted hens it reduced E, promoted T, and did not influence P and AVT release.The present observations demonstrate (1) that obestatin can directly control the release of avian ovarian hormones - regulators of reproduction, (2) that metabolic state can control the release of these hormones, and (3) metabolic state can alter the response of ovarian hormones to obestatin.

Activation of epithelial proliferation induced by Eimeria acervulina infection in the duodenum may be associated with cholesterol metabolism.

Cell proliferation in the intestine is commonly occurred during infection and inflammation to replace damaged enterocytes, and cholesterol as an essential constituent of cell membrane, is required for cell proliferation and growth. Here we found that coccidium-challenged (CC) chickens showed severe damages in intestinal structure, a significant increase of cell proliferation, and an activation of genes expression involved in the innate immune response. Compared to control (CON), CC chickens showed a marked decrease of cholesterol (Tch) level in the circulating system, but a significant increase in local duodenum epithelium. Increase of LDLR protein combined with a significant decrease of CYP27A1 protein expression in duodenum epithelium may contribute to intestinal cholesterol accumulation in CC chickens. Moreover, we found miRNAs targeting to CYP27A1 gene participating in post-transcriptional regulation. Hence, these results provide a new insight for the intervention of epithelial proliferation and cholesterol metabolism in the gastrointestinal tracts.

Yucca schidigera extract can promote rabbit fecundity and ovarian progesterone release.

The aim of the present in vivo study was to determine the effects of yucca powder extract added to the rabbit females feed mixtures on kindling and conception rate. Rabbit does of the experimental groups were fed with the standard diet enriched with supplement of yucca dry extract at doses of 5 g/100 kg feed (E1 group) or 20 g/100 kg feed (E2 group) for 50 days. In our preliminary in vivo results, we shown that conception rate was significantly higher in both experimental E1 and E2 groups (82.4% and 100.0%, respectively) than in the control group (47.1%). The kindling rate was also significantly higher in the experimental groups (70.6% and 100.0%, respectively) than in the control group (41.2%). The differences between control and yucca-treated groups in the number of liveborn, stillborn, and weaned pups per doe were not statistically significant. To understand possible endocrine mechanisms of yucca action on fertility rate, we have examined the influence of yucca extract additions on the release of steroid hormones by isolated and cultured rabbit ovarian fragments. Yucca additions promoted release of progesterone (at dose of 1 µg/mL, but not at doses of 10 and 100 µg/mL). Yucca addition at these doses did not affect testosterone or estradiol release. Our observations show the stimulatory effect of yucca consumption on rabbit fecundity, which can be due to its direct stimulatory influence on ovarian progesterone but not on testosterone or estradiol output.

Interrelationship between feeding level and the metabolic hormones leptin, ghrelin and obestatin in control of chicken egg laying and release of ovarian hormones.

The aim of the present experiment is to examine the role of nutritional status, metabolic hormones and their interrelationships in the control of chicken ovarian ovulatory and secretory activity. For this purpose, we identified the effect of food restriction, administration of leptin, ghrelin 1-18, obestatin and combinations of food restriction with these hormones for 3days on chicken ovulation (egg laying) rate and ovarian hormone release. The release of progesterone (P), testosterone (T), estradiol (E) and arginine-vasotocin (AVT) by isolated and cultured ovarian fragments was determined by EIA. It was observed that food restriction significantly reduced the egg-laying rate, T, E and AVT release and promoted P output by ovarian fragments. Leptin, administrated to ad libitum-fed chickens, did not change these parameters besides promoting E release. Nevertheless, administration of leptin was able to prevent the effect of food restriction on ovulation, T and E (but not P or AVT) release. Ghrelin 1-18 administration to ad libitum-fed birds did not affect the measured parameters besides a reduction in P release. Ghrelin 1-18 administration prevented the food restriction-induced decrease in ovarian T, E and AVT, but it did not change P output or egg laying. Obestatin administrated to control chicken promoted their ovarian P, E and inhibited ovarian AVT release but did not affect egg laying. It was able to promote the effect of food restriction on P, T and AVT, but not E release or egg laying. Our results (1) confirm an inhibitory effect of food restriction on chicken ovulation rate; (2) shows that food restriction-induced reduction in egg laying is associated with a decrease in ovarian T, E and AVT and an increase in ovarian P release; (3) confirm the involvement of metabolic hormones leptin, ghrelin and obestatin in the control of chicken ovarian hormones output; and (4) the ability of metabolic hormones to mimic/antagonize or prevent/promote the effects of food restriction on both egg laying and ovarian hormones demonstrates that nutritional status can influence ovarian ovulatory and endocrine functions via changes in metabolic hormones.

Involvement of microRNA Mir15a in control of human ovarian granulosa cell proliferation, apoptosis, steroidogenesis, and response to FSH.

Our study aimed to examine the role of micro RNA Mir15a in control of basic ovarian cell functions: proliferation, apoptosis, and secretory activity. In the first series of experiments, primary human ovarian granulosa cells were transfected with antisense construct blocking Mir15a (anti-Mir15a) and cultured without hormonal treatments. Accumulation of markers of proliferation (MAPK/ERK1,2 and PCNA) and apoptosis (caspase 3 and bax), and release of steroid hormones (progesterone, testosterone, and estradiol) were evaluated by immunocytochemical analysis and by enzyme immunoassay. In the second series of experiments, granulosa cells were transfected with gene construct encoding Mir15a precursor (pre-Mir15a) and cultured with and without follicle-stimulating hormone (FSH; 0, 1, 10, and 100 ng/ml). Expression of markers of proliferation (MAPK/ERK1,2) apoptosis (caspase 3), and steroidogenesis (release of progesterone, testosterone, and estradiol) were evaluated. Transfection of cells with anti-Mir15a resulted in a significant increase in accumulation of both proliferation and apoptosis markers, a reduction in progesterone and testosterone release, and an increase in estradiol release. Transfection of cells with pre-Mir15a had an opposite effect: it reduced accumulation of proliferation- and apoptosis-related proteins MAPK/ERK1,2 and caspase 3, and promoted release of progesterone and testosterone, but not estradiol. Moreover, pre-Mir15a reversed the effect of FSH on caspase 3, progesterone, and testosterone, but not on MAPK/ERK1,2 and estradiol. Our observations demonstrate involvement of Mir15a in control of multiple ovarian functions: proliferation, apoptosis, release of progesterone, androgen, and estrogen, and response to gonadotropin. Moreover, this is the first demonstration that miRNAs can affect response of cells to hormonal regulators. We propose that Mir15 could potentially be used for control of different reproductive processes.

Differences in egg deposition of corticosterone and embryonic expression of corticosterone metabolic enzymes between slow and fast growing broiler chickens.

Glucocorticoids (GCs) are vital for embryonic development and their bioactivity is regulated by the intracellular metabolism involving 11ß-hydroxysteroid dehydrogenases (11ß-HSDs) and 20-hydroxysteroid dehydrogenase (20-HSD). Here we sought to reveal the differences in egg deposition of corticosterone and embryonic expression of corticosterone metabolic enzymes between slow and fast growing broiler chickens (Gallus gallus). Eggs of fast-growing breed contained significantly higher (P<0.05) corticosterone in the yolk and albumen, compared with that of a slow-growing breed. 11ß-HSD1 and 11ß-HSD2 were expressed in relatively higher abundance in the liver, kidney and intestine, following similar tissue-specific ontogenic patterns. In the liver, expression of both 11ß-HSD1 and 11ß-HSD2 was upregulated (P<0.05) towards hatching, yet 20-HSD displayed distinct pattern showing a significant decrease (P<0.05) on posthatch day 1 (D1). Hepatic mRNA expression of 11ß-HSD1 and 11ß-HSD2 was significantly higher in fast-growing chicken embryos at all the embryonic stages investigated and so was the hepatic protein content on embryonic day of 14 (E14) for 11ß-HSD1 and on E14 and D1 for 11ß-HSD2. 20-HSD mRNA was higher in fast-growing chicken embryos only on E14. Our data provide the first evidence that egg deposition of corticosterone, as well as the hepatic expression of glucocorticoid metabolic enzymes, differs between fast-growing and slow-growing chickens, which may account, to some extent, for the breed disparities in embryonic development.

Effect of corticosterone on growth and welfare of broiler chickens showing long or short tonic immobility.

Tonic immobility (TI) test is commonly used to assess fear. Animals showing different TI durations demonstrate distinct behavior and biochemical responses to stress. However, less is known about how TI phenotype affects growth and welfare of domestic fowl. In this study, broiler chickens (Gallus gallus) were classified into short and long TI duration (STI and LTI) phenotypes and treated chronically with vehicle (CON) or corticosterone (CORT). STI broilers demonstrated significantly higher growth rate with higher breast muscle yield (P<0.05) and liver weight relative to BW tended to be lower (P=0.053), which was accompanied by higher serum concentration of CORT (P<0.05) and uric acid (P<0.01), but lower serum level of T4 (P=0.01). CORT severely reduced body weight, as well as the relative weight of muscle, bursa of Fabricius and spleen (P<0.001), but relative liver weight was increased (P<0.001). CORT-treated chickens had reduced serum CORT, elevated heterophile/lymphocyte ratio, and increased serum levels of total and free T3. STI broilers displayed more preening behavior (P<0.05), yet CORT elicited more walking behavior (P<0.05). No difference was observed in the welfare assessment scores between STI and LTI phenotypes under basal situation, while LTI chickens showed significantly increased incidence of pad dermatitis compared to STI under CORT exposure. The results suggest that STI broilers demonstrate better growth performance and higher adaptability to stress compared to LTI chickens.

Food restriction, ghrelin, its antagonist and obestatin control expression of ghrelin and its receptor in chicken hypothalamus and ovary.

The purpose of the present study was to identify the role of age, nutritional state and some metabolic hormones in control of avian hypothalamic and ovarian ghrelin/ghrelin receptor system. We examined the effect of food restriction, administration of ghrelin 1-18, ghrelin antagonistic analogue (D-Lys-3)-GHRP-6, obestatin and combinations of them on the expression of ghrelin and ghrelin receptor (GHS-R1a) in hypothalamus and ovary of old (23months of age) and young (7months of age) chickens. Expression of mRNAs for ghrelin and GHS-R1a in both hypothalamus and largest ovarian follicle was measured by RT-PCR. It was observed that food restriction could promote the expression of ghrelin and GHS-R1a in hypothalamus and ovary of the old chickens, but in the young chickens it reduced expression of ghrelin and did not affect expression of GHS-R1a in the ovary. Administration of ghrelin 1-18 did not affect hypothalamic or ovarian ghrelin mRNA, but significantly increased the expression of GHS-R1a in hypothalamus, but not in ovary. (D-Lys-3)-GHRP-6, significantly stimulated accumulation of ghrelin, but not GHS-R1a mRNA in hypothalamus or ghrelin or GHS-R1a in the ovary. Ghrelin 1-18 and (D-Lys-3)-GHRP-6, when given together, were able either to prevent or to induce effect of these hormones. Obestatin administration increased expression of ghrelin gene in the hypothalamus, but not expression of hypothalamic GHS-R1a, ovarian ghrelin and GHS-R1a. Furthermore, obestatin was able to modify effect of both ghrelin and fasting on hypothalamic and ovarian mRNA for ghrelin GHS-R1a. Our results (1) confirm the existence of ghrelin and its functional receptors GHS-R1a in the chicken hypothalamus and ovary (2) confirm the age-dependent control of ovarian ghrelin by feeding, (3) demonstrate, that nutritional status can influence the expression of both ghrelin and GHS-R1a in hypothalamus and in the ovary (3) demonstrates for the first time, that ghrelin can promote generation of its functional receptor in the hypothalamus, but not in the ovary, (4) show that ghrelin1-18 and (D-Lys-3)-GHRP-6 could not only be antagonists in the action on chicken hypothalamus and ovaries, but also independent regulators and even agonists, and (5) provide first evidence for action of obestatin on hypothalamic ghrelin and on the response of hypothalamic and ovarian ghrelin/GHS-R1a system to food restriction. These data indicate the involvement of both hypothalamic and ovarian ghrelin/GHS-R1 systems in mediating the effects of nutritional status, ghrelin and obestatin on reproductive processes.

Early-age feed restriction affects viability and gene expression of satellite cells isolated from the gastrocnemius muscle of broiler chicks.

BACKGROUND: Muscle growth depends on the fusion of proliferate satellite cells to existing myofibers. We reported previously that 0-14 day intermittent feeding led to persistent retardation in myofiber hypertrophy. However, how satellite cells respond to such nutritional insult has not been adequately elucidated. RESULTS: One-day-old broiler chicks were allocated to control (Con, ad libitum feeding), intermittent feeding (IF, feed provided on alternate days) and re-feeding (RF, 2 days ad libitum feeding after 12 days of intermittent feeding) groups. Chickens were killed on Day 15 and satellite cells were isolated. When cultured, satellite cells from the IF group demonstrated significant retardation in proliferation and differentiation potential, while RF partly restored the proliferation rate and differentiation potential of the satellite cells. Significant up-regulation of insulin like growth factor I receptor (IGF-IR) (P<0.05) and thyroid hormone receptor α (TRα) (P<0.05), and down-regulation of growth hormone receptor (GHR) (P<0.01) and IGF-I (P<0.01) mRNA expression was observed in freshly isolated IF satellite cells when compared with Con cells. In RF cells, the mRNA expression of IGF-I was higher (P<0.05) and of TRα was lower (P<0.01) than in IF cells, suggesting that RF restored the mRNA expression of TRα and IGF-I, but not of GHR and IGF-IR. The Bax/Bcl-2 ratio tended to increase in the IF group, which was reversed in the RF group (P<0.05), indicating that RF reduced the pro-apoptotic influence of IF. Moreover, no significant effect of T3 was detected on cell survival in IF cells compared with Con (P<0.001) or RF (P<0.05) cells. CONCLUSIONS: These data suggest that early-age feed restriction inhibits the proliferation and differentiation of satellite cells, induces changes in mRNA expression of the GH/IGF-I and thyroid hormone receptors in satellite cells, as well as blunted sensitivity of satellite cells to T3, and that RF partially reverses these effects. Thus, a moderate nutritional strategy for feed restriction should be chosen in early chick rearing systems.

Effects of repeated injection of kisspeptin-10 on the initiation of egg-laying in juvenile quail.

The kisspeptin (Kp) signaling pathway plays an essential role in the onset of reproduction in mammals. To investigate the effects of Kp on the initiation of egg laying in birds, juvenile female quail were given daily intraperitoneal injections of 300µl saline (control, Con), or 10nmol (low dosage, L) or 100nmol (high dosage, H) kisspeptin-10 (Kp-10) dissolved in 300µl saline for 3 weeks. The ratio of egg laying of quail in the L and H groups was notably increased compared to that of the Con group (P<0.01), which paralleled earlier ovarian growth and increases in circulating estrogen (E(2)) concentrations. In the hypothalamus, gonadotropin-releasing hormone-I (GnRH-I) mRNA expression was markedly up-regulated, whereas the level of gonadotropin-inhibiting hormone mRNA was down-regulated by high-dose Kp-10 (P<0.05). In the pituitary gland, expression of GnRH receptor type II, but not type I mRNA was significantly up-regulated by high-dose Kp-10 administration (P<0.05). Moreover, compared with the Con group, follicle-stimulating hormone (FSH) gene expression in the pituitary was significantly decreased in the L and H groups (P<0.05), whereas luteinizing hormone (LH) mRNA expression was significantly increased in the H, but not the L group (P<0.05). These results indicate that repeated peripheral Kp-10 injections can advance the sexual maturation of female quail by regulating the activities of the hypothalamus-pituitary-gonadal axis.

In ovo leptin administration affects hepatic lipid metabolism and microRNA expression in newly hatched broiler chickens.

BACKGROUND: A leptin-like immunoreactive substance has been found in chicken eggs and has been implicated in serving as a maternal signal to program offspring growth and metabolism. In the present study, we investigated the effects of in ovo leptin administration on hatch weight, serum and hepatic concentrations of metabolites and hormones, as well as on the expression of genes involved in hepatic lipid metabolism and the predicted microRNAs (miRNAs) targeting the affected genes. To this end we injected fertile eggs with either 0.5 µg of recombinant murine leptin or vehicle (PBS) before incubation. RESULTS: Prenatally leptin-exposed chicks showed lower hatch weight, but higher liver weight relative to the body weight, compared to the control group. In ovo leptin treatment increased the hepatic content and serum concentration of leptin in newly hatched chickens. The hepatic contents of triglycerides (TG) and total cholesterol (Tch) were decreased, whereas the serum levels of TG, Tch and apolipoprotein B (ApoB) were increased. The hepatic mRNA expression of sterol regulator element binding protein 1 (SREBP-1c), SREBP-2, hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and cholesterol 7α-hydroxylase 1 (CYP7A1) was significantly up-regulated, as was the protein content of both SREBP-1c and SREBP-2 in hepatic nuclear extracts of leptin-treated chickens. Moreover, out of 12 miRNAs targeting SREBP-1c and/or HMGCR, five were significantly up-regulated in liver of leptin-treated chicks, including gga-miR-200b and gga-miR-429, which target both SREBP-1c and HMGCR. CONCLUSIONS: These results suggest that leptin in ovo decreases hatch weight, and modifies hepatic leptin secretion and lipid metabolism in newly hatched broiler chickens, possibly via microRNA-mediated gene regulation.

Chicken FTO gene: tissue-specific expression, brain distribution, breed difference and effect of fasting.

Fat mass and obesity-associated (FTO) gene is widely expressed in central and peripheral tissues of mammals, and exhibits a range of functions, especially in energy balance. However, basic knowledge of FTO in the chicken is lacking. Therefore, we studied the tissue distribution, age and breed dependent changes, brain localization, as well as the impact of fasting on FTO mRNA expression in the chicken. FTO mRNA was expressed in all the tissues studied, and generally, with high expression in hypothalamus, liver, visceral fat and cerebellum. However it exhibited breed-specific patterns: in broilers, the highest expression was seen in the liver, while in layers, hypothalamus and cerebellum showed relatively higher FTO mRNA expression. One-week-old broilers expressed markedly higher FTO mRNA in liver compared with the layers of the same age (P<0.01), while the breed difference was reversed in visceral fat and cerebellum (P<0.05). Compared with newly hatched chicks (one week of age), adult layers expressed higher FTO mRNA in liver and visceral fat, while adult broilers showed higher expression in hypothalamus and cerebellum. In situ hybridization demonstrated distribution of FTO mRNA in paraventricularis magnocellularis (PVN), nucleus ventromedialis hypothalami (VMN), nucleus lateralis hypothalami (LHy), nucleus dorsomedialis hypothalami (DMN) of the hypothalamus and nucleus habenularis medialis (HM) and stratum cellulare externum (SCE) of the thalamus. Breed-specific expression of FTO mRNA was shown in PVN, but not in VMN, with higher abundance in broilers compared to layers. The decrease in FTO mRNA levels after 24h of fasting was seen only in VMN of layer chickens. These results may provide some intriguing hints for further investigation of FTO function in the chicken.

Effects of kisspeptin-10 on progesterone secretion in cultured chicken ovarian granulosa cells from preovulatory (F1-F3) follicles.

The effect of kisspeptin-10 (Kp-10) on the secretion of progesterone (P(4)) was investigated in cultured granulosa cells from F(1) to F(3) follicles of hens. The results showed that granulosa cells were stained with clear signals for kisspeptin using immunocytochemistry with the specific antibody against Kp-10. Among 10, 100 and 1000 nM concentrations tested, 100 nM Kp-10 treated for 24h significantly increased P(4) secretion in granulosa cells from F(1) to F(3) follicles. After 24h and 48 h of treatment, 100 nM Kp-10 showed a significant increase in P(4) secretion, while after 72 h of treatment P(4) secretion was markedly decreased by Kp-10 compared to the control group (P<0.05). F(1) and F(2/3) cells treated with 100 nM Kp-10 for 24h showed significantly increased viability (P<0.05) and which was in parallel to a marked increase in P(4) secretion (P<0.01). Real-time PCR results showed that the gene expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc) and the enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in F(1) and F(2/3) granulosa cells was significantly up-regulated by 24h-100 nM Kp-10 treatment (P<0.05 versus P<0.01, respectively). However, there was no significant difference in StAR, P450scc and 3ß-HSD protein content between control and the Kp-10 treated group (P>0.05). These results indicate that Kp-10 stimulates P(4) secretion in cultured chicken granulosa cells, which was associated with an up-regulation in StAR, P450scc and 3ß-HSD gene transcription.

Leptin stimulates hepatic activation of thyroid hormones and promotes early posthatch growth in the chicken.

Hepatic iodothyronine deiodinases (Ds) are involved in the conversion of thyroid hormones (THs) which interacts with growth hormone (GH) to regulate posthatch growth in the chicken. Previous studies suggest that leptin-like immunoreactive substance deposited in the egg may serve as a maternal signal to program posthatch growth. To test the hypothesis that maternal leptin may affect early posthatch growth through modifying hepatic activation of THs, we injected 5.0µg of recombinant murine leptin into the albumen of breeder eggs before incubation. Furthermore, chicken embryo hepatocytes (CEHs) were treated with leptin in vitro to reveal the direct effect of leptin on expression and activity of Ds. In ovo leptin administration markedly accelerated early posthatch growth, elevated serum levels of total and free triiodothyronine (tT3 and fT3), while that of total thyroxin (tT4) remained unchanged. Hepatic mRNA expression and activity of D1 which converts T4 to T3 or rT3 to T2, were significantly increased in leptin-treated chickens, while those of D3 which converts T3 to T2 or T4 to rT3, were significantly decreased. Moreover, hepatic expression of GHR and IGF-I mRNA was all up-regulated in leptin-treated chickens. Males demonstrated more pronounced responses. A direct effect of leptin on Ds was shown in CEHs cultured in vitro. Expression and activity of D1 were increased, whereas those of D3 were decreased, in leptin-treated cells. These data suggest that in ovo leptin administration improves early posthatch growth, in a gender-specific fashion, probably through improving hepatic activation of THs and up-regulating hepatic expression of GHR and IGF-I.

Effect of inhibitor and activator of ghrelin receptor (GHS-R1a) on porcine ovarian granulosa cell functions.

It was previously shown, that ghrelin and its agonistic analogue, ghrelin 1-18, can be a stimulator of ovarian cell functions (promoter of proliferation, inhibitor of apoptosis and stimulator of hormones release). The aim of our studies was to compare the action of two ghrelin analogues - ghrelin 1-18, activator of ghrelin receptors (GHS-R1a), and (D-Lys3)-GHRP-6, its inhibitor, on porcine ovarian granulosa cell functions. Effects of (D-Lys3)-GHRP-6 added at doses of 0, 1, 10 or 100 ng/ml on the expression of markers of proliferation (PCNA, cyclin B1, MAPK/ERK1,2), apoptosis (bax, p53, caspase 3) and release of steroid hormones (progesterone, testosterone, estradiol) were examined. In addition, some effect of ghrelin 1-8 on some of these parameters (expression of MAPK/ERK1,2, bax, p53) were verified. It was shown, that (D-Lys3)-GHRP-6 promotes all markers of granulosa cell proliferation, inhibits all markers of apoptosis and stimulates the release of all three steroid hormones. Similar effects of (D-Lys3)-GHRP-6 (inhibitor of GHS-R1a) and ghrelin 1-18 (its stimulator) suggest that the examined effects of these substances on porcine ovaries are not mediated by GHS-R1a. Both chemical analogues could be potentially useful for stimulation of reproductive processes, at least in in vitro conditions.

The cAMP analogue, dbcAMP affects release of steroid hormones by cultured rabbit ovarian cells and their response to FSH, IGF-I and ghrelin.

The aim of the present study was to examine possible involvement of cAMP-dependent intracellular mechanisms in control of ovarian cell steroidogenesis and its response to hormonal regulators. For this purpose, we examined the influence of administration of dbcAMP, a cAMP analogue (50 microg/animal) in vivo, on release of progesterone, testosterone and estradiol by isolated ovarian fragments, as well their response to hormonal regulators of ovarian steroidogenesis-FSH, IGF-I and ghrelin (all added at doses of 100 ng/ml). It was observed, that administration of dbcAMP resulted reduction in progesterone and testosterone, but not of estradiol release by isolated ovarian fragments. In ovarian tissue isolated from control animals, additions of hormones were able to reduce release of progesterone (FSH, IGF-I and ghrelin) and increase release of testosterone (ghrelin) but did not change estradiol output. Previous administration of dbcAMP modified action of exogenous hormones: it inverted inhibitory action of FSH, IGF-I and ghrelin on progesterone release to stimulatory action and induced stimulatory action of IGF-I on testosterone release and stimulatory effect of FSH on estradiol output. The present observations confirm involvement of peptide hormones FSH, IGF-I and ghrelin in the control of rabbit ovarian steroid hormones release and demonstrate the involvement of cAMP-dependent intracellular mechanisms in down-regulation of rabbit ovarian steroidogenesis and in modification, but not in mediating effect of FSH, IGF-I and ghrelin on ovarian steroid hormones release.