Engineered Hydroxyapatite Nanoadjuvants with Controlled Shape and Aspect Ratios Reveal Their Immunomodulatory Potentials.

Hydroxyapatite (HAP) has been formulated as adjuvants in vaccines for human use. However, the optimal properties required for HAP nanoparticles to elicit adjuvanticity and the underlying immunopotentiation mechanisms have not been fully elucidated. Herein, a library of HAP nanorods and nanospheres was synthesized to explore the effect of the particle shape and aspect ratio on the immune responses in vitro and adjuvanticity in vivo. It was demonstrated that long aspect ratio HAP nanorods induced a higher degree of cell membrane depolarization and subsequent uptake, and the internalized particles elicited cathepsin B release and mitochondrial reactive oxygen species generation, which further led to pro-inflammatory responses. Furthermore, the physicochemical property-dependent immunostimulation capacities were correlated with their humoral responses in a murine hepatitis B surface antigen immunization model, with long aspect ratio HAP nanorods inducing higher antigen-specific antibody productions. Importantly, HAP nanorods significantly up-regulated the IFN-γ secretion and CD107α expression on CD8+ T cells in immunized mice. Further mechanistic studies demonstrated that HAP nanorods with defined properties exerted immunomodulatory effects by enhanced antigen persistence and immune cell recruitments. Our study provides a rational design strategy for engineered nanomaterial-based vaccine adjuvants.

Metformin Prevents Follicular Atresia in Aging Laying Chickens through Activation of PI3K/AKT and Calcium Signaling Pathways.

Increased follicular atresia occurs with aging and results in reduced fecundity in laying chickens. Therefore, relieving follicular atresia of aging poultry is a crucial measure to maintain sustained high laying performance. As an antiaging agent, metformin was reported to play important roles in preventing aging in diverse animals. In this study, the physiological state of the prehierarchical follicles in the peak-laying hens (D280) and aged hens (D580) was compared, followed with exploration for the possible capacity of metformin in delaying atresia of the prehierarchical follicles in the aged D580 hens. Results showed that the capacity of yolk deposition within follicles declined with aging, and the point of endoplasmic reticulum- (ER-) mitochondrion contact decreased in the ultrastructure of the follicular cells. Meanwhile, the expression of apoptosis signaling genes was increased in the atretic small white follicles. Subsequently, the H2O2-induced follicular atresia model was established to evaluate the enhancing capacity of metformin on yolk deposition and inhibition of apoptosis in the atretic small white follicles. Metformin inhibited apoptosis through regulating cooperation of the mitochondrion-associated ER membranes and the insulin (PI3K/AKT) signaling pathway. Furthermore, metformin regulated calcium ion homeostasis to relieve ER-stress and inhibited release of mitochondrion apoptosis factors (BAD and caspase). Additionally, metformin activated PI3K/AKT that suppressed activation of BAD (downstream of the insulin signaling pathway) in the atretic follicles. Further, serum estrogen level and liver estrogen receptor-α expression were increased after dietary metformin supplementation in D580 hens. These results indicated that administration of dietary metformin activated the PI3K/AKT and calcium signaling pathway and enhanced yolk deposition to prevent chicken follicular atresia.

Improvement of eggshell quality by dietary N-carbamylglutamate supplementation in laying chickens.

Egg quality defects seriously reduce the quality grade and increase egg breakage in egg marketing activities. In this study, the effect of N-carbamylglutamate (NCG) on eggshell quality was investigated by evaluating calcium absorption and calcification in laying hens. A total of 30 newly hatched female Hy-Line chicks were randomly assigned to the control group (basal diet) and treatment group (basal diet supplemented with 1% NCG). At 25 wk, eggs from each group were obtained to assess egg quality parameters. Blood samples were collected for analysis of mineral, hormone, and amino acids levels at 16 h after laying egg. Uterine tissues were removed and fixed in 4% neutral paraformaldehyde solution or kept in liquid nitrogen for mineral determination, quantitative PCR, and Western blot. Results showed that the egg quality (eggshell thickness, strength and percentage, egg specific gravity, and eggshell effective thickness) was significantly increased while effective thickness of mastoid layer, width of mastoid gap, and mammillary knobs were significantly decreased by dietary NCG supplementation (P < 0.05). The levels of minerals (Ca, P, Fe, Mg, Na, K) in eggshell, plasma, and uterus were remarkably elevated (P < 0.05). Meanwhile, the concentrations of calcium metabolism-related hormones (17ß-estradiol, parathyroid hormone, and calcitonin) were increased in the NCG group (P < 0.05). Moreover, expression of calbindin 1, carbonic anhydrase 2, ovalbumin, ovotransferrin, ovocleidin-17, ovocleidin-116, and clusterin mRNAs, as well as calbindin 1 and ATP2A1 proteins in uterus, duodenum, and kidney, was all upregulated in hens fed with NCG (P < 0.05). In addition, the number of blood vessels in the uterus, height of uterine mucosal folds, villus length in endometrium, and areas of uterine mucosal folds were significantly increased in the NCG group (P < 0.05). In conclusion, dietary 1% NCG supplementation during 0 to 25 wk can improve eggshell quality through changes in endometrial morphology, expression of calcium metabolism-related genes, and secretion of related hormones to promote eggshell formation in the laying hens.

Effect of dietary N-carbamylglutamate on development of ovarian follicles via enhanced angiogenesis in the chicken.

N-carbamylglutamate (NCG), an analogue of N-acetyl-L-glutamate (NAG), can increase arginine synthesis in mammals and improve the reproductive performance. However, the effect of NCG on poultry laying performance is still unclear. This study investigated the effect of dietary NCG on development of chicken ovarian follicles. The dosage and timing for NCG administration were evaluated for its effect on follicular development. Results showed that supplementation with 1% NCG in the diet for 14 D led to accelerated development of growing follicles (over 60 µm in oocyte diameter) and significantly increased feed intake and feed efficiency. Plasma amino acids (AA) analysis showed that feeding with 1% NCG significantly increased of plasma AA levels. RNA-seq analysis revealed that NCG supplementation upregulated expression of genes related to angiogenesis and cell proliferation, but downregulated expression of apoptosis-related genes. Meanwhile, RT-qPCR and Western blot analysis validated the RNA-seq results. Moreover, NCG enhanced plasma NO level; upregulated expression of PKG-I, Raf1, and p-p38; and increased angiogenesis of the ovaries. In conclusion, dietary NCG (1% for 14 D) can promote development of ovarian follicles by increasing angiogenesis in ovaries of the chicken.

Melatonin Improves Laying Performance by Enhancing Intestinal Amino Acids Transport in Hens.

The high concentration of melatonin (MEL) in the intestinal mucosa suggests that it has a special physiological function in intestine. In hens, previous studies have shown that MEL treatment promoted egg-laying performance. Considering the importance of amino acids (AA) for egg formation, we hypothesized that MEL may enhance the intestinal absorption of AA from the feed, thus promoting egg laying performance. In this study, we supplemented the hens with MEL for two consecutive weeks. The results showed that, compared with control group, feeding with 0.625 mg MEL/kg diets gave rise to higher egg laying rate (by 4.3%, P = 0.016), increased eggshell thickness (by 16.9%, P < 0.01) and albumen height (by 4.5%, P = 0.042). Meanwhile, feeding with 0.625 and 2.5 mg MEL/kg diets could significantly increase serum levels of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, and proline. Furthermore, a 0.625 mg MEL/kg diets could significantly increase the expression of PepT1 (by 3949.9%), B0AT (by 6045.9%), b0, +AT (by 603.5%), and EAAT3 (by 412.7%) in the jejunum. Additionally, in the cultured intestinal crypt "organoids," treatment with 0.5 µM MEL could significantly enhance the expression of PepT1, b0, +AT and EAAT3 mRNAs by 35.4%, 110.0%, and 160.1%, respectively. Detection of MEL concentration in serum and intestinal fluid suggested that lower dosage of MEL feeding was mainly acted on intestine locally, and further increased intestinal antioxidases (GPx-3, SOD-1 or PRDX-3) mRNA expression. Taken together, we demonstrated that MEL feeding in laying hens could locally promote the expression and function of AA transporter in small intestine by up-regulating antioxidases expression, and finally elevate laying performance.

Grape Seed Proanthocyanidin Extract Prevents Ovarian Aging by Inhibiting Oxidative Stress in the Hens.

Oxidative stress is an important inducement in ovarian aging which results in fecundity decline in human and diverse animals. As a potent antioxidant, grape seed proanthocyanidin extract (GSPE) was investigated to ameliorate chicken ovarian aging in this study. Firstly, ovarian antioxidant capacity of hens at different ages (90, 150, 280, and 580 days old) was compared to elucidate its age-related changes. Subsequently, a D-gal-induced (2.5 mg/mL) aging ovarian model was established and the cultured ovarian tissues were treated with GSPE at 5 µg/mL for 72 h to evaluate the putative attenuating effects of GSPE on ovarian aging. Meanwhile, ovaries of D280 (young) and D580 (old) were treated with GSPE for 72 h in culture to verify the protective effects of GSPE on natural aging ovary. The results showed that GSPE could rescue the antioxidant capacity decline by increasing the antioxidase activities and their gene expression in either D-gal-induced or natural aging ovaries. Moreover, GSPE could maintain the homeostasis between cell proliferation and apoptosis in the D-gal-induced and natural aging ovaries, as well as alleviate D-gal-induced nucleus chromatin condensation in the ovarian granulosa cells. In conclusion, GSPE treatment can effectively prevent the ovarian aging process in hens by reducing oxidative stress.

Ameliorative Effect of Grape Seed Proanthocyanidin Extract on Cadmium-Induced Meiosis Inhibition During Oogenesis in Chicken Embryos.

Cadmium (Cd) is an environmental endocrine disruptor that has toxic effects on the female reproductive system. Here the ameliorative effect of grape seed proanthocyanidin extract (GSPE) on Cd-induced meiosis inhibition during oogenesis was explored. As compared with controls, chicken embryos exposed to Cd (3 µg/egg) displayed a changed oocyte morphology, decreased number of meiotic germ cells, and decreased expression of the meiotic marker protein γH2AX. Real time RT-PCR also revealed a significant down-regulation in the mRNA expressions of various meiosis-specific markers (Stra8, Spo11, Scp3, and Dmc1) together with those of Raldh2, a retinoic acid (RA) synthetase, and of the receptors (RARα and RARß). In addition, exposure to Cd increased the production of H2 O2 and malondialdehyde in the ovaries and caused a corresponding reduction in glutathione and superoxide dismutase. Simultaneous supplementation of GSPE (150 µg/egg) markedly alleviated the aforementioned Cd-induced embryotoxic effects by upregulating meiosis-related proteins and gene expressions and restoring the antioxidative level. Collectively, the findings provided novel insights into the underlying mechanism of Cd-induced meiosis inhibition and indicated that GSPE might potentially ameliorate related reproductive disorders.

[Study on volatile components of Lonicerae Japonicae Flos in bud stage extended type].

Volatile components of Lonicerae Japonicae Flos in bud stage extended type Beihua 1 were determined by the headspace solid-phase micro-extraction, compared with traditional cultivar Damaohua. There are fifty-two volatile compounds were identified and the relative content of the volatiles was calculated by the area normalization method. Thirty-nine compounds were found in Beihua 1, whereas thirty-three components in Damaohua. Total twenty identical compounds existed in Beihua 1 and Damaohua. The contents of alcohols and hydrocarbons of Beihua 1 were higher significantly than that of Damaohua, while significantly lower than that of Damaohua in ketones content. Besides, twenty components were only detected in Beihua 1, such as methyl nicotinate, hexadecanoic acid, methyl ester,acetophenone, nonanoic acid.

Supplementation with quercetin attenuates 4-nitrophenol-induced testicular toxicity in adult male mice.

The beneficial effects of quercetin on reproductive damage elicited by 4-nitrophenol (PNP) were studied in adult male mice. A six-week treatment of weekly intraperitoneal injections of PNP (50 mg/kg) resulted in severe damage to the seminiferous tubules, a remarkable increase in both hydroxyl radical and malondiadehyde production, and notably decreased glutathione peroxidase and superoxide dismutase activities. Moreover, PNP treatment induced germ cell apoptosis, inhibited Bcl-xl expression, and then activated Bax expression and the caspase-3 enzyme. Exposure to PNP also increased XBP-1 and HO-1 mRNAs levels. However, simultaneous supplementation with quercetin (75 mg/kg) attenuated the toxicity induced by PNP through renewal of the antioxidant enzyme's status, alleviating apoptosis by regulating the expressions of Bax and Bcl-xl, XBP-1 and HO-1mRNAs, and the regulation of caspase-3 activity. Taken together, these findings indicated that the antioxidant quercetin displays a potential preventive effect on PNP-induced oxidative damage in mouse testes and may represent an efficient supplement to attenuate reproductive toxicity from environmental toxicants in order to ensure reproductive health and sperm production.

Ameliorative effect of grape seed proanthocyanidin extract on thioacetamide-induced mouse hepatic fibrosis.

The present study was designed to examine the effect of the grape seed proanthocyanidin extract (GSPE) on developing hepatic fibrosis that was induced by thioacetamide (TAA) in mice. Administration of TAA for 9 weeks led to a serious necrosis and apoptosis of the parenchymal cells, which resulted in an accumulation of excessive collagen in the liver and an increase of transformed hepatic stellate cells (HSCs). In addition, the mRNA expression of transforming growth factor ß1 (TGF-ß1), α-smooth muscle actin (α-SMA), as the marker of the activated HSCs, and α1-(I)-collagen were all up-regulated significantly when compared with the control. However, combined oral administration of GSPE at 100 mg/kg suppressed the mRNA expression of TGF-ß1 and α-SMA, with decreased collagen accumulation as demonstrated by histomorphological evaluation and quantitative RT-PCR. The mRNA expression of the pro-inflammatory factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), was remarkably enhanced by TAA treatment. However, their levels displayed a down-regulated trend beyond simultaneous GSPE treatment. Moreover, GSPE administration markedly suppressed lipid peroxidation. In conclusion, as a plant antioxidant, GSPE manifested effective hepatocellular protective action to ameliorate the developing liver fibrosis induced by chronic TAA administration in mice.

Protective effect of quercetin on cadmium-induced oxidative toxicity on germ cells in male mice.

Cadmium is a toxic heavy metal that is widely distributed in the environment. As a critical process, oxidative toxicity mediates the morphological and functional damages in germ cells after cadmium exposure. In this study, the protective effect of quercetin on cadmium-induced oxidative toxicity was investigated in mouse testicular germ cells. After oral administration of cadmium chloride at 4 mg/kg body weight for 2 weeks, damages in spermatozoa occurred in the early stage of spermatogenesis. Cadmium treatment significantly decreased the testicular antioxidant system, including decreases in the glutathione (GSH) level, superoxide dismutase (SOD), and GSH peroxidase (GSH-Px) activities. Moreover, exposure to cadmium resulted in an increase of hydrogen peroxide production and lipid peroxidation in testes. In addition, cadmium provoked germ cell apoptosis by upregulating expression of the proapoptotic proteins Bax and caspase-3 and downregulating expression of the antiapoptotic protein Bcl-XL. However, combined administration of a common flavonoid quercetin at 75 mg/kg body weight significantly attenuated cadmium-induced germ cell apoptosis by suppressing the hydrogen peroxide production and lipid peroxidation in testicular tissue. Simultaneous supplementation of quercetin markedly restored the decrease in GSH level and SOD and GSH-Px activities elicited by cadmium treatment. Additionally, quercetin protected germ cells from cadmium-induced apoptosis by downregulating the expression of Bax and caspase-3 and upregulating Bcl-XL expression. These results indicate that quercetin, due to its antioxidative and antiapoptotic characters, may manifest effective protective action against cadmium-induced oxidative toxicity in mouse testicular germ cells.

Quercetin attenuates cadmium-induced oxidative damage and apoptosis in granulosa cells from chicken ovarian follicles.

The attenuating effect of quercetin on cadmium-induced oxidative damage and apoptosis was investigated in cultured granulosa cells from chicken ovarian follicles. Results showed that exposure to 5 µM CdCl(2) induced a decrease in granulosa cell number and viability, caused chromatin condensation and DNA fragmentation. Moreover, cadmium treatment markedly increased malondialdehyde level and decreased glutathione peroxidase and superoxide dismutase activities. Furthermore, cadmium provoked higher BAX expression, inhibited expression of BCL2 and X-linked inhibitor of apoptosis protein (XIAP) and activated caspase-3. However, simultaneous supplementation with 1 µg/ml quercetin protected granulosa cells against cadmium-induced cytotoxicity through attenuating lipid peroxidation, renewing antioxidant enzymes activities and alleviating apoptosis by modulating XIAP, BAX and BCL2 expression, and inhibiting caspase-3 activity. Therefore, these results suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in granulosa cells through attenuating lipid peroxidation, elevating intracellular antioxidant status and inhibiting apoptosis to ensure reproductive health.

Quercetin attenuates oxidative damage induced by treatment of embryonic chicken spermatogonial cells with 4-nitro-3-phenylphenol in diesel exhaust particles.

Quercetin, an antioxidant flavonoid, is considered beneficial for human and animal health. In this study, the protective effect of quercetin on oxidative damage to testicular cells was studied in embryonic chickens after treatment with 4-nitro-3-phenylphenol (PNMPP) derived from diesel exhaust particles. Testicular cells were challenged with PNMPP (10(-8)-10(-6) M) alone and in combination with quercetin for 48 h. The results showed that quercetin manifested no deleterious effect on spermatogonial cells up to 1.0 microg/ml. Exposure to PNMPP (10(-6) M) induced condensed nuclei and vacuolated cytoplasm and reductions in testicular cell viability and spermatogonial cell numbers (p<0.05). It also induced lipid peroxidation by an elevation of thiobarbituric acid reactive substances and decreased glutathione peroxidase activity and superoxide dismutase activity (p<0.05). Simultaneous supplementation with quercetin restored these parameters to the same levels as in the control. These data indicate that quercetin protects spermatogonial cells from oxidative damage in embryonic chickens intoxicated with PNMPP.

Ginsenosides promote proliferation of granulosa cells from chicken prehierarchical follicles through PKC activation and up-regulated cyclin gene expression.

The effect of GS (ginsenosides) on proliferation of chicken GCs (granulosa cells) from prehierarchical SYF (small yellow follicles) was evaluated, and involvement of the PKC (protein kinase C) signalling pathway as well as mRNA expression of cyclins and CDK (cyclin-dependent kinase) were investigated. Whole SYF or GCs isolated from SYF were cultured in Medium 199 supplemented with 0.5% FCS (fetal calf serum). After 16 h, the cells were challenged with GS alone or in combination with PKC inhibitor H7 or activator PMA (phorbol 12-myristate 13-acetate) for 24 h in serum-free medium. Results showed that in both whole follicles and pure GCs monolayer culture system, GS (0.1-10 microg/ml) significantly increased the number of GCs in SYF in a dose-dependent manner, and this stimulatory effect was inhibited by H7, but enhanced by PMA. Meanwhile, the PCNA-LI (proliferating cell nuclear antigen labelling index) of GCs displayed similar changes with the cell number. Mechanism of GS action was further evaluated in cultured GCs separated from SYF. Western blot analysis showed that 10 microg/ml GS increased PKC translocation from cytoplasm to the plasma membrane of the GCs to become the active state. This effect was blocked by H7. Furthermore, GS up-regulated the expression of cyclin D1/CDK6 and cyclin E/CDK2 mRNAs in GCs; however, inhibition of PKC with H7 attenuated this stimulatory effect. These results indicated that GS could stimulate proliferation of chicken GCs through activated PKC-involved up-regulation of cyclin D1/CDK6 and cyclin E/CDK2 genes, subsequently promoting development of the chicken prehierarchical follicles.

Protective effect of quercetin on the reproductive toxicity of 4-nitrophenol in diesel exhaust particles on male embryonic chickens.

The 4-nitrophenol (PNP) in diesel exhaust particles (DEP) has been identified as a vasodilator and is a known degradation product of the insecticide parathion. In this study, the protective effect of quercetin, a potent oxygen free radical scavenger and metal chelator, against the oxidative damage of PNP on cultured testicular cells was studied in male embryonic chickens. Testicular cells from Day 18 embryos were cultured in serum-free McCoy's 5A medium and challenged with quercetin (1.0 microg/ml) alone or in combinations with PNP (10(-7)-10(-5) M) for 48 h. The oxidative damage was estimated by measuring cell viability, content of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and glutathione peroxidation (GSH-Px) activity. The results showed that exposure to PNP (10(-5) M) induced condensed nuclei, vacuolated cytoplasm and a decrease in testicular cell viability and spermatogonial cell number. Exposure to PNP induced lipid peroxidation by elevation of the content of MDA. Exposure to PNP also decreased GSH-Px activity and SOD activity. However, simultaneous supplementation with quercetin restored these parameters to the same levels as the control. Consequently, PNP induced oxidative stress in spermatogonial cells, and dietary quercetin may attenuate the reproductive toxicity of PNP to restore the intracellular antioxidant system in the testicular cells of embryonic chickens.

Sex-specific effects of androgen and estrogen on proliferation of the embryonic chicken hypothalamic neurons.

Effects of androgen and estrogen on proliferation of hypothalamic neurons were evaluated by a chicken hypothalamic neuron-glia coculture model. Hypothalamic cells were dispersed from 17-day-old embryos and challenged with testosterone (T) and 17beta-estradiol (E2) alone or combined with androgen receptor antagonist flutamide, estrogen receptor antagonist tamoxifen, or aromatase inhibitor letrozole for 48 h. The neuron number was counted and the proliferating cells were identified by immunocytochemistry of proliferating cell nuclear antigen (PCNA) and 5-bromo-2-deoxyuridine (BrdU) incorporation. Results showed that both E2 and T stimulated proliferation of hypothalamic neurons. E2 showed more intensive effect on females and this promoting effect was abrogated by tamoxifen. T played more intensive effect on males and the effect was inhibited by flutamide, tamoxifen, or letrozole. The above results indicated that E2 stimulated neuron proliferation through estrogenic actions with more sensitive effect on females and T promoted neuron proliferation through both androgenic and estrogenic actions with more intense effect on males. These observations suggested that steroid hormones influence the proliferation of hypothalamic neurons in a sexually dimorphic manner during the development of chicken embryos.

Ginsenosides promote proliferation of chicken primordial germ cells via PKC-involved activation of NF-kappaB.

The effect of ginsenosides on proliferation of chicken primordial germ cells (PGCs) was evaluated and involvement of nuclear factor (NF)-kappaB in the signaling pathway was investigated. PGCs were isolated from the genital ridge of 3.5-4 day embryos and cultured in Medium 199 supplemented with 5% FCS and 10 ng/ml LIF. PGCs subcultured on chicken embryonic fibroblast feeder were challenged with ginsenosides alone or in combination with PKC inhibitor H(7) or activator phorbol 12-myristate 13-acetate (PMA) for 24h. Moreover, the translocation of NF-kappaB and degradation level of IkappaBalpha were investigated by Western blot analysis. Results show that PGCs were identified by periodic acid-Schiff, alkaline phosphatase histochemistry as well as c-kit, SSEA-1 and Oct-4 immunocytochemistry. Treatment with ginsenosides at 1-100 microg/ml significantly increased the number and area of PGC colonies in a dose-dependent manner. However, this proliferating effect was obviously attenuated by combined treatment of H(7) (10(-7)-10(-5)M). Similarly, PKC staining of PGC colonies was more intensive after ginsenosides treatment compared with the control group. In addition, treatment with ginsenosides at 1-10 microg/ml stimulated the translocation of NF-kappaB (p65). However, the NF-kappaB translocation and the degradation of IkappaBalpha were significantly blocked by combined treatment with 10(-6)M H(7). These results indicated that ginsenosides promote proliferation of chicken PGCs through activation of PKC-involved NF-kappaB signaling pathway.

Quercetin protects spermatogonial cells from 2,4-d-induced oxidative damage in embryonic chickens.

Quercetin, an antioxidant flavonoid, is considered beneficial to human and animal health. In this study, the protective effects of quercetin in relation to oxidative damage of testicular cells were studied by analysis of the intracellular antioxidant system after treatment of embryonic chickens with hypoxanthine-xanthine oxidase (HX-XO) or 2,4-dichlorophenoxyacetic acid (2,4-D). Testicular cells from Day 18 embryos were challenged with quercetin alone or in combinations with HX-XO or 2,4-D for 48 h in culture. The results showed that quercetin manifested no deleterious effects on spermatogonial cells at concentrations up to 1.0 microg/ml. Exposure to HX-XO or 2,4-D (50 microg/ml) induced condensed nuclei and vacuolated cytoplasm and a decrease in testicular cell viability and spermatogonial cell number. Membrane integrity was damaged by elevated lactate dehydrogenase leakage. Exposure to HX-XO or 2,4-D also elicited lipid peroxidation by elevation of thiobarbituric acid reactive substances and decreased glutathione content and superoxide dismutase activity. However, simultaneous supplementation with quercetin restored these parameters to the levels in the controls. Consequently, HX-XO and 2,4-D induced oxidative stress in spermatogonial cells; however, dietary quercetin may attenuate the negative effects of environmental toxicants and restore the antioxidant system in testicular cells.

Activation of protein kinases A and C promoted proliferation of chicken primordial germ cells.

Many growth factors or cytokines regulate cell proliferation via different intracellular signaling pathways. The mechanisms remained quite unclear in avian primordial germ cells (PGCs). In the present study, two major protein kinases, PKA and PKC, were investigated to be involved in signal transduction of PGC proliferation. PGCs were isolated from genital ridge of 3.5-day chicken embryos and primary culture was performed with 5% fetal calf serum (FCS)-supplemented medium 199. After culture for 24 h, PGCs were subcultured on chicken embryonic fibroblast feeder (CEF) and the cells were characterized by histochemical stainings of alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) reagent as well as immunocytochemical stainings of c-kit and stage-specific embryonic antigen-1 (SSEA-I). In addition, cells were challenged with adenylate cyclase activator forskolin (FRSK) and PKC activator phorbol-12-myristate-13-acetate (PMA) alone or in combinations with PKA inhibitor H(89) and PKC inhibitor H(7), respectively. Results showed that subcultured PGCs on CEF displayed positive histochemical and immunocytochemical stainings for ALP, PAS, c-kit and SSEA-I and manifested intensive proliferating activity by colony formation. Downstream activation of PKA by FRSK (10(-7) to 10(-5)M) significantly promoted the proliferation of PGCs by increasing colony number (ALP-stained) in a dose-dependant manner. PMA (10(-8)M) also increased PGC colony number (P<0.05). However, the proliferating effects elicited by FRSK or PMA could be inhibited by the respective protein kinase inhibitor H(89) or H(7). Therefore, the above results suggest that activation of intracellular protein kinases A and C by external factors may promote proliferation of cultured PGCs and PKA represents the most likely mediator of PGC proliferation in embryonic chickens.

Prostaglandin involvement in follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles.

The aim of the present study was to evaluate the role of prostaglandin (PG) on proliferation of granulosa cells from prehierarchical small yellow follicles (SYF) of buff laying hens. The granulosa layers were separated by mechanic method and dispersed into single cells. After 16 h pre-incubation in 0.5% FCS medium, the medium was replaced with serum-free medium, which was supplemented with 10 microg/ml insulin, 5 microg/ml transferrin and 3 x 10(-8)M selenite. Cells were challenged with PGE1 and FSH for 24 h and then assessed for proliferation. The results showed that PGE(1) (0.1-10 ng/ml) had a similar proliferating effect as FSH on granulosa cells, and these stimulating effects were restrained by the PGE receptor antagonist SC19220 at 10(-7) to 10(-5)M. Prostaglandin synthase antagonist indomethacin (10(-7) to 10(-5)M) suppressed FSH-induced increase in the number of granulosa cells in a dose-dependent manner. Downstream activation of protein kinase A by forskolin-activated adenylate cyclase resulted in elevated proliferation of granulosa cells, an effect unobserved by phorbol-12-myristrate-13-acetate-activated protein kinase C. In addition, PGE1-stimulated proliferation of granulosa cells was hindered by H89 (PKA inhibitor) but not by H7 (PKC inhibitor). Furthermore, the proliferating cell nuclear antigen labeling index (PCNA-LI) of granulosa cells displayed similar changes with the number of cells. These results indicated that PGE1 promoted the proliferation of granulosa cells from SYF and was also involved in mediating FSH-stimulated intracellular PKA signal transduction.