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.

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.

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.

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.

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.

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.

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.

Age-dependent role of steroids in the regulation of growth of the hen follicular wall.

BACKGROUND: The ovaries are the primary targets of senescence effects in mammalian and avian species. In the present study, relationships between reproductive aging, sex steroids and the growth pattern of the pre-ovulatory follicle wall were investigated using young hens with long clutch (YLC), old hens with long clutch (OLC), old hens with short clutch (OSC), and old hens with interrupted long clutch (OILC). METHODS: Experiment 1: Hens were sacrificed 1.5 and 14.5 h after ovulation. Experiment 2: YLC and OILC hens were sacrificed 3.5 h after treatments with LH and/or aminoglutethimide (AG), an inhibitor of steroid synthesis. Volumes of pre-ovulatory follicles (F1-F5) and plasma concentrations of ovarian steroids were determined. Experiment 3: Granulosa and theca cells from F3 follicles of OSC and/or YLC hens were exposed in vitro to estradiol-17beta (E2), testosterone (T) and LH and the proliferative activity of the cells was examined using CellTiter 96 Aqueous One Solution Assay. RESULTS: In YLC and OLC groups, the total volume of F1-F5 follicles rose between 1.5 and 14.5 h after ovulation (P < 0.01), negatively correlating with the plasma level of E2 (P < 0.01). There was no growth of pre-ovulatory follicles in the middle of the ovulatory cycle in the OSC group, with a positive correlation being present between E2 and the follicular volume (P < 0.05). In young hens, AG caused a rise in the total follicular volume. This rise was associated with a fall in E2 (r = -0.54, P < 0.05). E2 enhanced proliferation of granulosa cells from YLC and OSC groups. The proliferative activity of granulosa and theca cells of YLC hens depended on the interaction between T and LH (P < 0.01). CONCLUSIONS: These data indicate for the first time that the growth pattern of pre-ovulatory follicles during the ovulatory cycle changes in the course of reproductive aging. E2 seems to play a dual role in this adjustment; it stimulates the growth of the follicular wall in reproductive aged hens, whereas it may inhibit this process in young birds. T and LH are apparently involved in the growth regulation during the pre-ovulatory surge in young hens.

Identification of microRNAs controlling human ovarian cell steroidogenesis via a genome-scale screen.

The aim of our studies was to identify miRNAs affecting the release of the major ovarian steroid hormones progestagen, androgen and estrogen by human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different gene constructs encoding human pre-miRNAs on release of progesterone, testosterone and estradiol was evaluated by enzyme immunoassay. In addition, effect of two selected antisense constructs blocking corresponding miRNA on progesterone release was tested. Efficiency of transfection (incorporation transfection reagent) and silencing of marker substances (GAPDH mRNA, GAPDH and CREB-1) were validated by fluorescent microscopy, real-time reverse transcription-PCR analysis and immunocytochemical analysis. Thirty-six out of 80 tested miRNA constructs resulted in inhibition of progesterone release in granulosa cells, and 10 miRNAs promoted progesterone release. Transfected of cells with antisense constructs to two selected miRNAs blocking progesterone release induced increase in progesterone output. Fifty-seven miRNAs tested inhibited testosterone release, and only one miRNA enhanced testosterone output. Fifty-one miRNAs suppressed estradiol release, while none of the miRNAs tested stimulated it. This is the first demonstration that miRNAs can control reproductive functions resulting in enhanced or inhibited release of ovarian progestagen, androgen and estrogen. We hypothesize that such miRNA-mediated effects could be potentially used for regulation of reproductive processes, including fertility, and for treatment of reproductive and other steroid-dependent disorders.

Maternal low-protein diet programmes offspring growth in association with alterations in yolk leptin deposition and gene expression in yolk-sac membrane, hypothalamus and muscle of developing Langshan chicken embryos.

The present study was aimed to investigate the mechanism underlying the influence of maternal low-protein (LP) diet on offspring growth in the chicken. One hundred and twenty Chinese inbred Langshan breeder hens were allocated randomly into two groups fed diets containing low (10%, LP) or normal (15%) crude protein levels. Low dietary protein did not affect the body weight of hens, but significantly decreased the laying rate and egg weight. The yolk leptin content was significantly lower in eggs laid by LP hens, while no differences were detected for yolk contents of corticosterone, tri-iodothyronine (T3) or thyroxine. Despite significantly lower hatch weight, the LP offspring demonstrated obviously higher serum T3 concentration, which is in accordance with the faster post-hatch growth rate achieving significantly heavier body weight and pectoralis major muscle weight 4 weeks post-hatching. Expression of 20-hydroxysteroid dehydrogenase (20-HSD) mRNA in the yolk-sac membrane was significantly down-regulated at embryonic day 14, whereas that of transthyretin and leptin receptor (LepR) was not altered. Moreover, hypothalamic expression of 20-HSD, glucocorticoid receptors, thyrotropin-releasing hormone and LepR mRNA was significantly up-regulated in the LP group compared with their control counterparts. In the pectoralis major muscle, significantly higher expression of insulin-like growth factor (IGF)-I and IGF-I receptor mRNA was observed in LP embryos. The present study provides evidence that maternal LP diet programmes post-hatch growth of the offspring. The associated alterations in yolk leptin deposition as well as in yolk-sac membrane, fetal hypothalamus and muscle gene expression may be involved in mediating such programming effect in the chicken.

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.

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.

Galanin immunoreactivity increased in chicken supraoptic neurons after activation of the vasotocin system at oviposition.

The avian arginine vasotocin (AVT) synthesized in the hypothalamic magnocellular neurons and released from the posterior pituitary is known to be involved in the regulation of uterine contractions for oviposition in chickens. However, regulation of AVT synthesis and release within the magnocellular hypothalamus has not been elucidated. Galanin, the oviposition inducing factor in the oviduct of the hen, has been demonstrated to have sexually dimorphic stimulatory action in oxytocin- and vasopressin neurons in the mammalian hypothalamus. In this study, galanin and AVT immunoreactivity was investigated around the time of oviposition in the supraoptic nucleus (SON) to determine if galanin modulates AVT synthesis and/or release. Within SON neurons increased AVT immunoreactivity before oviposition and the decreased AVT immunoreactivity after oviposition implied function-related peptide release. The significantly increased number of galanin neurons co-localizing with AVT immediately after oviposition suggests that galanin is involved in the negative feedback to limit AVT release in the SON. Thus, these data support the idea that AVT in the SON is involved in central regulation of oviposition and that AVT release could be modulated by the neuropeptide galanin.

Effect of dietary daidzein on egg production, shell quality, and gene expression of ER-alpha, GH-R, and IGF-IR in shell glands of laying hens.

Our previous studies demonstrated that dietary daidzein improves egg production in ducks during the late period of the laying cycle. The present study was aimed to investigate the effect of daidzein in laying hens, with more focus on eggshell quality. The expression of ER-alpha, GH-R, and IGF-IR mRNA in shell glands was determined to identify the target genes of daidzein action and to reveal the relationship between shell quality and profiles of gene expression in shell glands of laying hens. 1000 ISA hens, at 445 days of age, were allotted at random to two groups and given the basal diet with or without 10 mg of daidzein per kg diet for 9 weeks. Daidzein supplement significantly increased the egg laying rate and the feed conversion ratio. The eggshell thickness increased, while the percentage of cracked eggs decreased in daidzein-treated hens. Serum E2 and phosphate concentrations were not altered, but the level of serum Ca2+ and the tibia bone mineral density were significantly increased in the daidzein-treated group compared with their control counterparts. In parallel with the significant increase of oviduct weight, significant down-regulation of GH-R and IGF-IR mRNA and a trend of decrease in ERalpha mRNA expression in shell glands were observed in daidzein-treated hens. The results indicate that dietary daidzein improves egg laying performance and eggshell quality during the late (postpeak) laying stage of hens, which is associated with modulations in gene expression in the shell gland.

Developmental changes of FSH-R, LH-R, ER-beta and GnRH-I expression in the ovary of prepubertal ducks (Anas platyrhynchos).

Normal ovarian development is dependent on stimulation of the gonadotropic hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), as well as some regulatory factors locally produced in ovary, e.g. 17beta-estradiol (E2) and gonadotropin-releasing hormone (GnRH), mediated by their respective receptors. In order to elucidate the potential roles of LH, FSH, E2 and GnRH-I during early follicular development in prepubertal ducks, mRNA expression of LH-R, FSH-R, ER-beta and GnRH-I in ovaries of 1-day-old (D1), 30-day-old (D30), 60-day-old (D60) and 90-day-old (D90) ducks was measured with semi-quantitative RT-PCR using beta-actin as an internal standard. The ovary index (the ratio of ovary weight/body mass) did not change from D30 to D90, while the ovary weight and serum E2 levels rose progressively, indicating the prepubertal development of the ovary. Ovarian expression of FSH-R, LH-R, ER-beta and GnRH-I mRNA changed greatly during this period. Abundance of FSH-R and ER-beta mRNA went up gradually from D1 to D60, followed by a decline on D90. LH-R and GnRH-I mRNA expression increased from D1 to D90, reaching a peak at D90. These results indicate that the developing ovary is highly responsive to the regulation of FSH during the early stage, while close to the onset of sexual maturation, the ovary is likely more responsive to LH. In addition, the expression of GnRH-I and ER-beta mRNA in the ovary suggest that GnRH-I and E2 are involved in the regulation of prepubertal follicular development in the ovary of ducks.

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.

Sexually dimorphic immunoreactivity of galanin and colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus).

The bed nucleus of the stria terminalis medialis (BSTM) of adult chickens (Gallus gallus domesticus) was previously shown to synthesize arginine vasotocin (AVT) in males only and coincides spatially and temporally with steroid activity regulating male reproductive behavior. Galanin has been shown to be a potent modulator of the behavioral and neuroendocrine responses in the mammalian BSTM and in other sexually dimorphic brain regions. In the present study of adult chickens the morphological relationship of AVT and galanin was examined by immunohistochemical analysis of two limbic structures, the BSTM and the lateral septum (SL). The analysis also included the hypothalamic nuclei supraopticus (SON) and paraventricularis (PVN). In males galanin and AVT were both synthesized in the BSTM, while in females neither galanin nor AVT was present. Furthermore, in the males galanin and AVT were colocalized in the majority of neurons within BSTM and in fibers of the SL. In both sexes galanin neurons in the PVN were scattered between the distinct clusters of AVT neurons and there was no colocalization of galanin and AVT in single PVN neurons. Furthermore, AVT immunoreactivity was significantly higher in the SON than in the PVN in both sexes. In the SON, galanin was colocalized with AVT in significantly more neurons in hens than in males (P

Ovulatory cycle-related alterations in the thecal growth and membrane protein content of thecal tissue of hen preovulatory follicles.

In the hen ovary, each preovulatory follicle in the hierarchy, irrespective of its size and the level of its maturity is exposed to the preovulatory LH surge in each ovulatory cycle of an egg laying sequence. In the present study, the thecal weight and membrane protein content of theca layers at different stages of hen ovulatory cycle were assessed. Hens were killed 2 h (stage I), 9 h (stage II), 16 h (stage III), and 23 h (stage IV) after oviposition. The first (F1), second (F2), third (F3), fourth (F4) and fifth (F5) largest yellow follicles were utilized. In all follicles except F1, the thecal weight rose considerably between stages I and III (P < 0.05) followed by a slight cessation of the thecal growth at stage IV. The mean content of the theca membrane protein in F1-F5 follicles was lowest at stage III, increasing at stage IV (P < 0.05), although, in the case of individual follicles the difference was significant (P < 0.05) in F3 follicles only. Estradiol-17beta levels in the plasma were lowest (but not significant) at stage III, and a fourfold increase in the plasma progesterone concentration occurred at stage IV. These findings demonstrate for the first time the ovulatory cycle-related alterations in the thecal weight and membrane protein content in the hen preovulatory follicles. Data suggest that the preovulatory rise in ovarian steroid hormones is probably involved in transient termination of the growth and induction of differentiation of the theca in preovulatory follicles as they pass from one category to the next.

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.

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.