Functionalized chitosan hydrogel promotes osseointegration at the interface of3D printed titanium alloy scaffolds.

Autogenous bone transplantation is a prevalent clinical method for addressing bone defects. However, the limited availability of donor bone and the morbidity associated with bone harvesting have propelled the search for suitable bone substitutes. Bio-inspired scaffolds, particularly those fabricated using electron beam melting (EBM) deposition technology, have emerged as a significant advancement in this field. These 3D-printed titanium alloy scaffolds are celebrated for their outstanding biocompatibility and favorable elastic modulus. Thermosensitive chitosan hydrogel, which transitions from liquid to solid at body temperature, serves as a popular carrier in bone tissue engineering. Icariin (ICA), known for its efficacy in promoting osteoblast differentiation from bone marrow mesenchymal stem cells (BMSCs), plays a crucial role in this context. We developed a system combining a 3D-printed titanium alloy with a thermosensitive chitosan hydrogel, capable of local bone regeneration and integration through ICA delivery. Our in vitro findings reveal that this system can gradually release ICA, demonstrating excellent biocompatibility while fostering BMSC proliferation and osteogenic differentiation. Immunohistochemistry and Micro-CT analyses further confirm the effectiveness of the system in accelerating in vivo bone regeneration and enhancing osseointegration. This composite system lays a significant theoretical foundation for advancing local bone regeneration and integration.

Hypoxia stress induces hepatic antioxidant activity and apoptosis, but stimulates immune response and immune-related gene expression in black rockfish Sebastes schlegelii.

Dissolved oxygen concentrations both in the open ocean and coast have been declining due to the interaction of global climate change and human activity. Fish have evolved different adaptative strategies to cope with possibly damage induced by hypoxic environments. Black rockfish as important economic fish widely reared in the offshore sea cage, whereas related physiological response subject to hypoxia stress remained unclear. In this study, hepatic anti-oxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px]), aminotransferase (AST) and alanine aminotransferase (ALT) activities, lipid peroxidation (LPO), malondialdehyde (MDA) and glutathione (GSH) content, immunological parameters and the expression of apoptosis (bax, bcl2, p53, caspase3, xiap) and immune-related genes (c3, il-1ß, ccl25, saa, hap, isg15) of black rockfish were determined during hypoxia and reoxygenation to illustrate the underlying defense response mechanisms. Results showed that hypoxia stress remarkably increased hepatic LPO and MDA content, AST and ALT activity and proportion of pyknotic nucleus. Hepatic SOD, CAT and GSH-Px activity manifested similar results, whereas GSH levels significantly decreased under hypoxia stress. The apoptosis rate of hepatocyte increased during hypoxia stress and reoxygenation. Meanwhile, p53, caspase3, bax and xiap mRNAs and bax/bcl2 rations were significantly up-regulated under hypoxia stress. However, bcl2 mRNA was significantly down-regulated. Interestingly, hypoxia stress significantly increased NBT-positive cell percent, phagocytic index, respiratory burst and ACH50 activity, and lysozyme activity. The mRNA levels of c3, ilß, ccl25, saa, hap and isg15 were significantly up-regulated in the liver, spleen and head-kidney under hypoxia stress. The above parameters recovered to normal status after reoxygenation for 24 h Thus, hypoxia stress impairs hepatic antioxidant capacity, induces oxidative damage and apoptosis via the xiap-p53-bax-bcl2 and the caspase-dependent pathways, but enhances host immunity by regulating nonspecific immune indices and related genes expression to maintain homeostasis in black rockfish. These findings will help fully understand the hypoxia tolerance mechanisms of black rockfish and provide more data for offshore open ocean farming.

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus).

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful and sensitive method used in gene expression analysis. Suitable reference genes, which are stable under all experimental circumstances and tissues significantly improve the accuracy of qRT-PCR data. In this study, the stability of six genes, namely, 18S ribosomal RNA (18s), beta-actin (actb), elongation factor 1-alpha (ef1α), glyceraldehyde-3-phosphate-dehydrogenase (gapdh), cathepsin D (ctsd), and beta-2-microglobulin (b2m) were evaluated as potential references for qRT-PCR analysis. The genes were examined in the hypothalamus-pituitary-ovary-liver (HPOL) axis throughout turbot ovarian development via using the geNorm, NormFinder and BestKeeper algorithms. Results showed that the most stable reference genes were ef1α, actb, and ctsd in the hypothalamus, pituitary, ovary and liver, respectively. The best-suited gene combinations for normalization were 18s, ef1α, and ctsd in the hypothalamus; actb, ctsd, and 18s in the pituitary; actb, and ctsd in the ovary; gapdh and ctsd in the liver. Moreover, the expression profile of estrogen receptor α (erα) manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development. However, no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development. In summary, these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of qRT-PCR data in HPOL axis-related gene expression analysis during turbot ovarian development.

Alternations in oxidative stress, apoptosis, and innate-immune gene expression at mRNA levels in subadult tiger puffer (Takifugu rubripes) under two different rearing systems.

Tiger puffer (Takifugu rubripes) is one of the major aquaculture fish species in China due to its high economic value. In this study, the transcriptions of hepatic antioxidant enzyme, stress, apoptosis, and immune-related genes of sub-adult tiger puffers (Takifugu rubripes) were evaluated under two different rearing systems [offshore sea cage aquaculture system (OSCS) and recirculating aquaculture system (RAS)]. Results showed that the mRNA expression levels of the antioxidant enzyme (mn-sod, cu/zn-sod, gpx, and gr) and stress-related (hsp70 and hsp90) genes of male tiger puffers reared in the OSCS were significantly higher than female fish reared in the OSCS and fish reared in the RAS. The anti-apoptotic gene bcl2 exhibited the similar results. By contrast, the mRNAs of the pro-apoptotic genes (p53, caspase8, caspase9, and caspase3) of male tiger puffers reared in the OSCS were significantly lower than female fish reared in the OSCS and fish reared in the RAS. Male tiger puffers reared in the OSCS displayed significantly higher complement components (c3) and inflammatory cytokine (il-6) mRNAs, whereas B-cell activating factor (baf) and tumor necrosis factor α (tnf-α) mRNAs remained unchanged. Meanwhile, the mRNA levels of pro-apoptotic (bax, caspase8) and immunity-related (c3, il-6 and il-7) genes of female tiger puffers reared in the OSCS were significantly lower and higher than female fish reared in the RAS, respectively. In conclusion, the hepatic antioxidant, anti-apoptosis, and innate immunity of tiger puffers reared in the OSCS were better than fish in the RAS, male tiger puffer obtained the best values. These results expand the knowledge on the combined RAS and OSCS alternative aquaculture model for tiger puffers and aid in their management in captive.

A new 3D printed titanium metal trabecular bone reconstruction system for early osteonecrosis of the femoral head.

Presently, biomechanical support therapy for the femoral head has become an important approach in the treatment of early osteonecrosis of the femoral head (ONFH). Previous studies have reported that the titanium metal trabecular bone reconstruction systems (TMTBRS) achieved satisfactory clinical results for the treatment of early femoral head necrosis. Electron beam melting technology (EBMT) is an important branch of 3D printing technology, which enables the construction of an interface that is required for support of bone in-growth. However, the effect of TMTBRS created using EBMT for clinical applications for early ONFH is still unknown. At present, there are no reports on this topic worldwide. The purpose of this study was to assess the safety of a new 3D printed TMTBRS implant and to evaluate its clinical efficacy in early ONFH.Thirty patients who underwent surgery for ONFH were selected. The stages of ONFH were classified according to the Association Research Circulation Osseus (ARCO) classification. They were followed-up and radiological examination was performed at 6, 12, and 24 months post-surgery to assess TMTBRS stability and bone growth in the bone trabecular holder portion surface. To evaluate hip function, postoperative Harris and Visual Analogue Scale (VAS) scores were used.The postoperative Harris score increased significantly and VAS score decreased significantly at the 12-month follow-up compared to the 24-month follow-up, wherein the Harris score declined slightly and the VAS score was slightly elevated with the aggravation of ONFH. With the passage of time, postoperative improvement rates were 100% for IIA, 70% for IIB, and 0% for IIC. Hip-preserving rates were 100% for IIA, 100% for IIB, and 50% for IIC.The effect of TMTBRS treatment for early ONFH in ARCO IIA and ARCO IIB is satisfactory. However, it is not recommended for a relatively large area of necrosis such as in ARCO IIC.

Molecular characterization and quantification of estrogen receptors in turbot (Scophthalmus maximus).

Estrogens regulate various reproductive processes via estrogen receptor (ER)-mediated signaling pathway in vertebrates. In this study, full-length sequences coding for ERα, ERß1 and ERß2 were isolated from female turbot (Scophthalmus maximus) by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR). The nucleotide and amino acid sequences of turbot ERs showed high homologies with the corresponding sequences of other fish species and significant homology with the Japanese flounder (Paralichthys olivaceus) and the European sea bass (Dicentrarchus labrax). Turbot ERs contained six typical nuclear receptor-characteristic domains and exhibited high evolutionary conservation in the functional domains. Quantitative real-time polymerase chain reaction analysis revealed that the erα and erß (ß1, ß2) mRNAs were abundant in the liver and ovary, respectively. Furthermore, hepatic mRNA levels of erα and vitellogenin (vtg) were found increased gradually from pre-vitellogenesis to late-vitellogenesis stages, with the highest values observed at the late-vitellogenesis stage, and then decreased from migratory-nucleus to atresia stages. However, mRNA levels of erα in the ovary remained unchanged during ovarian development. Hepatosomatic index, gonadosomatic index, serum estradiol-17ß and the mRNA levels of erß1 and erß2 in the ovary manifested results similar to the expression of erα mRNAs in the liver. These findings indicated that ERα is mainly involved in hepatic vitellogenesis, and ERßs may play crucial roles to regulate ovarian development in turbot. Overall, this study improves understanding of the physiological functions of turbot ERs, which will be valuable for fish reproduction and broodstock management.

Naringin enhances osteogenic differentiation through the activation of ERK signaling in human bone marrow mesenchymal stem cells.

OBJECTIVES: Naringin has been reported to regulate bone metabolism. However, its effect on osteogenesis remains unclear. The aim was to investigate the effect of naringin on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) through the activation of the ERK signaling pathway in osteogenic differentiation. MATERIALS AND METHODS: Annexin V-FITC assay and MTT assay were used to measure the effect of naringin on cytotoxicity and proliferation of hBMSCs, respectively. Alkaline phosphatase activity analysis, Alizarin Red S staining, Western blotting, and real-time PCR assay were used to evaluate both the potential effect of naringin on osteogenic differentiation and the role of ERK signaling pathway in osteogenic differentiation. RESULTS: Our results showed that naringin had no obvious toxicity on hBMSCs, and could significantly promote the proliferation of hBMSCs. Naringin also enhanced the osteogenic differentiation of hBMSCs and increased the protein and mRNA expression levels of osteogenic markers such as Runx-2, OXS, OCN, and Col1 in a dose-dependent manner. In addition, we found that the enhancing effect of naringin on osteogenic differentiation was related to the activation of phosphor-ERK, with an increase in duration of activity from 30 min to 120 min. More importantly, both the enhancing effect of naringin on osteogenic differentiation and the activity effect of naringin on ERK signaling pathway were reversed by U0126 addition. CONCLUSION: Our findings demonstrated that naringin promoted proliferation and osteogenesis of hBMSCs by activating the ERK signaling pathway and it might be a potential therapeutic agent for treating or preventing osteoporosis.

Ameliorative effect of vitamin E on hepatic oxidative stress and hypoimmunity induced by high-fat diet in turbot (Scophthalmus maximus).

This study was conducted to examine the effects of vitamin E on growth performance, oxidative stress and non-specific immunity of turbot (Scophthalmus maximus) fed with high-fat diet. Results showed that high-fat diet significantly increased hepatosomatic index, viscerosomatic index, hepatic malondialdehyde level and decreased catalase and superoxide dismutase activities, whereas final weight, specific growth rate and survival rate remained unchanged. Meanwhile, nitro blue tetrazolium positive leucocytes of head kidney, respiratory burst activity in head-kidney macrophage, phagocytic index and serum lysozyme activity were significantly reduced after feeding with high-fat diet. Furthermore, fish fed with high-fat diet promoted higher expression of heat shock protein (hsp70, hsp90), and inhibited expression of complement component 3 (c3) in the liver and tumor necrosis factor-α (tnf-α), interleukine 1ß (il-1ß), toll like receptor 22 (tlr-22) in the spleen and head-kidney, respectively. However, simultaneous supplementation with 480 mg kg-1 vitamin E protected turbot against high-fat diet-induced hepatic oxidative stress, hypoimmunity through attenuating lipid peroxidation, renewing antioxidant enzymes activities and nonspecific immune responses, and modulating the expression of stress protein (hsp70, hsp90) and immune-related genes (c3, tnf-α, il-1ß, tlr-22). In conclusion, the obtained results indicate the vitamin E as a wildly used functional feed additive contributes potentially to alleviate high-fat diet-induced hepatic oxidative stress and hypoimmunity, maintain the health, and improve the broodstock management for turbot.

Acute hyperthermic responses of heat shock protein and estrogen receptor mRNAs in rainbow trout hepatocytes.

Heat shock proteins (HSPs) are induced upon elevated temperature in fishes. HSPs also function as molecular chaperones for cellular proteins, including steroid hormone receptors. Estrogen receptors (ERs) are critical for the hormone signaling necessary during the liver production of the yolk precursor protein vitellogenin in oviparous vertebrates. Considering the possible regulatory role of HSPs on the ER signaling pathway, the present study characterized the mRNA expression of all known isoforms of HSP70 (hsp70a, hsp70b), HSP90 (hsp90a1a, hsp90a1b, hsp90a2a, hsp90a2b, hsp90b1, hsp90b2), and ERs (erα1, erα2, erß1, erß2) in Rainbow Trout hepatocytes following an acute heat shock (1h at 25°C) compared to a control treatment (12°C). The results showed that the mRNA levels of hsp70a, hsp70b, hsp90a1b, hsp90a2a, and hsp90b2 were significantly increased after heat shock, while erα1 mRNA levels were significantly reduced by this treatment. hsp90a1a, hsp90a2b, hsp90b1, erα2, erß1 and erß2 were unaffected by this acute hyperthermic treatment. Comparatively, the responses of the two hsp70 isoforms were much greater than the hsp90 isoforms. Acute heat shock treatment of hepatocytes followed by a 24h exposure to 17ß-estradiol (E2) exposure also resulted in decreased expression of erα1 mRNA, but not vitellogenin (vtg) mRNA. This study showed that some hsp70 and hsp90 isoforms display a robust response to an acute hyperthermic treatment in Rainbow Trout hepatocytes. Among the transcripts measured here, the erα1 isoform uniquely showed significantly decreased mRNA levels upon acute heat treatment.

What is the optimal approach for tranexamic acid application in patients with unilateral total hip arthroplasty?

PURPOSE: In the total hip arthroplasty (THA), the optimal administration route of tranexamic acid (TXA) remains controversial. This study was designed to investigate the impact of topical injection of TXA on blood loss during primary unilateral THA as well as short-term safety and adverse side effects compared with intravenous administration of TXA. MATERIAL AND METHODS: In this study, 75 patients who underwent unilateral THA were randomly divided into 3 groups receiving intra-articular TXA (IA group), intravenous TXA (IV group) or no TXA (control group C). Blood loss, postoperative drainage, covert blood loss, total blood volume, the number of blood transfusions after surgery and transfusion rate, incidence of deep venous thrombosis and pulmonary embolism were recorded and evaluated in the three groups after 1 week and 1 month. RESULTS: There were significant differences in the quantity of postoperative drainage, covert blood loss, total blood volume, the number of blood transfusions after surgery and transfusion rates between the three groups (P < 0.05), but blood loss during surgery showed no significant differences among the three groups (P > 0.05). In the IV group, 1 patient suffered from deep venous thrombosis of the lower limbs and in the C group, 2 patients suffered from superficial venous thrombosis of the lower limbs 2 and 4 days after surgery, respectively. In the IA group no complications occurred during the follow-up period. CONCLUSION: Preoperative intravenous TXA and postoperative topical TXA significantly reduced postoperative blood loss and transfusion rates among the patients who underwent primary unilateral THA and the short-term safety was good. The data suggest that topical injection of TXA is safer and more effective, without postoperative complications.

[Application of minimally invasive, decompression bone graft implantation combined with metal trabecular bone reconstruction system for early stage osteonecrosis of femoral head].

OBJECTIVE: To observe the application effect of minimally invasive decompression, bone graft implantation and metal trabecular bone reconstruction system for early stage osteonecrosis of femoral head and discuss the treatment of hip-salvage operation in early stage osteonecrosis of femoral head; METHODS: From January 2010 to June 2011, 50 patients (62 hips) Which were osteonecrosis of femoral head of early stake,were treated with minimally invasive decompression, bone graft implantation and metal trabecular bone reconstruction system, including 31 males (40 hips), 19 females (22 hip) with an average age of 36.2 years old ranging from 22 to 54 years old. The course of disease was from 6 to 15 months (averaged 10.5 months). Among them, 19 cases (23 hips) were steroid-induced, 25 cases (33 hips) were alcohol-induced, 6 cases (6 hips) were idiopathic; According to ARCO stage, 28 hips were at stage I, 34 hips were at stage II. All of them were diagnosed as femoral head necrosis by imaging examination before operation. Then each patient was followed to assess by Harris hip score, curative effect, and conduct the femoral head survival analysis during the postoperation. RESULTS: All patients had finished operation, the operation time was between 30 and 85 min, intraoperative blood loss was 50 to 220 ml, and 47 cases (58 hips) were follow-up from 24 to 46 months with an average of 34.05 months. As compared with preoperative, the Harris hip score at the last follow-up was improved, the difference was statistically significant (P<0.01). The Harris hip score, curative effect and survival time of femoral head in ARCO stage I was superior to these in ARCO Stage II, the difference was statistically significant (P< 0.05). CONCLUSION: Effect of minimally invasive decompression,bone graft implantation combine with the metal trabecular bone reconstruction system for early stage osteonecrosis of femoral head was good,it could significantly improve the Harris hip score, increase the femoral head survival time, delay the hip replacement, and performance better in ARCO stage I.

[SURGICAL HIP DISLOCATION APPROACH FOR TREATMENT OF FEMORAL HEAD FRACTURE].

OBJECTIVE: To discuss the value of surgical hip dislocation approach in the treatment of femoral head fracture. METHODS: A retrospectively analysis was made on the clinical data of 15 patients with femoral head fractures treated through surgical hip dislocation approach between January 2010 and February 2013. There were 11 men and 4 women with an average age of 30.8 years (range, 15-63 years). The causes included traffic accident injury in 9 cases, falling injury from height in 5 cases, and sports injury in 1 case. According to Pipkin typing, 2 cases were rated as type I, 7 cases as type II, 1 case as type III, and 5 cases as type IV. The interval of injury and operation was 2-10 days (mean, 4.1 days). Reduction was performed in 10 patients within 6 hours after injury, and then bone traction was given for 4-6 weeks except 5 patients who received reduction in the other hospital. RESULTS: Primary healing of incision was obtained in all patients after surgery without complications of dislocation and lower limbs deep venous thrombosis. The mean follow-up time was 29.9 months (range, 25-36 months). During follow-up, there was no infection, breakage of internal fixation, or nonunion of femoral greater trochanter fracture. In 3 patients having necrosis of the femoral head, 2 had no obvious symptoms [staging as IIa and IIb respectively according to Association Research Circulation Osseous (ARCO) staging system], and 1 (stage IIIb) had nonunion of the femoral neck fracture, who underwent total hip arthroplasty (THA). In 4 patients having myositis ossificans (2 cases of grade I, 1 case of grade II, and 1 case of grade III based on Brooker grading), no treatment was given in 3 cases and the focus was removed during THA in 1 case. According to the Thompson-Epstein scale at last follow-up, the results were excellent in 9 cases, good in 3 cases, fair in 1 case, and poor in 2 cases, and the excellent and good rate was 80%. CONCLUSION: Surgical hip dislocation approach can not only protect the residual vessels of the femoral head but also fully expose the acetabulum and femoral head, which is the ideal approach for the treatment of femoral head fractures.

Effect of dietary vitamin E on the growth performance and nonspecific immunity in sub-adult turbot (Scophthalmus maximus).

This study investigated the growth performance and non-specific immunity in sub-adult turbot fed with graded levels of vitamin E (0, 120, 240, 480 and 960 mg kg(-1)) for 15 weeks. Results showed that the final weight, specific growth rate, nitro blue tetrazolium positive leucocytes of head kidney, phagocytic index, serum lysozyme activity and superoxide dismutase activity significantly increased with increasing vitamin E levels. The highest values were recorded in the diet with 480 mg kg(-1) vitamin E. However, no significant differences in the hepatosomatic index, viscerosomatic index and survival rate were found among all dietary treatment. Furthermore, the expression levels of complement component 3 (C3), tumor necrosis factor-α (TNF-α) and interleukine 1ß (IL-1ß) were significantly upregulated in the fish feed with the vitamin E-supplemented diets. Compared with the basal diet, the diet supplemented with 480 mg kg(-1) vitamin E significantly augmented the mRNA expression of IL-1ß, TNF-α in the spleen and head-kidney, C3 in the liver, respectively. In conclusion, the obtained results indicate the basal diet supplemented with moderate dietary vitamin E (480 mg kg(-1)) increased the growth, nonspecific immune responses, and expression levels of some immune-related genes in sub-adult turbot. These observations suggest that optimal dietary vitamin E can promote the growth, maintain the health and improve the broodstock management for turbot.

miR-27 inhibits adipocyte differentiation via suppressing CREB expression.

miR-27 plays a negative role in the regulation of adipogenesis. However, the molecular mechanism still remains to be clarified. In the present study, we found that miR-27 inhibits adipogenesis partially by repressing the early adipogenic transcription factor cAMP response element-binding protein by directly targeting its 3' untranslated region. In addition, we demonstrated that tumor necrosis factor-α (TNF-α) treatment up-regulates miR-27 through the NF-κB pathway. Furthermore, anti-miR-27 reduces the TNF-α-induced inhibition of adipogenesis. Simultaneously, the levels of miR-27 expression were decreased in mature adipocytes of obese mice when compared with lean mice. Our data revealed a novel mechanism of miR-27 in the regulation of adipogenesis.

Baicalein ameliorates inflammatory-related apoptotic and catabolic phenotypes in human chondrocytes.

Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis and a major concern for aging societies worldwide. In OA, pro-inflammatory cytokines such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α can trigger the caspase cascade to promote apoptosis and activate NF-κB to induce catabolic factors in chondrocytes. Here, the anti-apoptotic and anti-catabolic effects of baicalein on human OA chondrocytes treated by a mixture of IL-1ß and TNF-α (IT) were investigated in vitro. In cultured chondrocytes, baicalein pretreatment attenuated apoptosis in a concentration-dependent manner in response to IT stimulation. Mechanistically, the anti-apoptotic effects of baicalein result from inhibition of nitric oxide production and downstream caspase signaling pathway. Moreover, administration of baicalein significantly reduced matrix metalloproteinase (MMP) 3 and MMP13 secretion in chondrocytes stimulated with IT. The anti-catabolic effects of baicalein were further demonstrated by the recovery of the glycosaminoglycan and type II collagen (COLII) deposition by histological analysis in IT-treated mouse articular cartilage explants. These findings suggest that baicalein might be a promising novel therapeutic agent for OA by virtue of its suppression of apoptosis and MMP secretion in OA chondrocytes.

Effect of epidermal growth factor on follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles.

The development of ovarian follicular cells is controlled by multiple circulating and local hormones and factors, including follicle-stimulating hormone (FSH) and epidermal growth factor (EGF). In this study, the stage-specific effect of EGF on FSH-induced proliferation of granulosa cells was evaluated in the ovarian follicles of egg-laying chickens. Results showed that EGF and its receptor (EGFR) mRNAs displayed a high expression in granulosa cells from the prehierarchical follicles, including the large white follicle (LWF) and small yellow follicle (SYF), and thereafter the expression decreased markedly to the stage of the largest preovulatory follicle. SYF represents a turning point of EGF/EGFR mRNA expression during follicle selection. Subsequently the granulosa cells from SYF were cultured to reveal the mediation of EGF in FSH action. Cell proliferation was remarkably increased by treatment with either EGF or FSH (0.1-100 ng/ml). This result was confirmed by elevated proliferating cell nuclear antigen (PCNA) expression and decreased cell apoptosis. Furthermore, EGF-induced cell proliferation was accompanied by increased mRNA expressions of EGFR, FSH receptor, and the cell cycle-regulating genes (cyclins D1 and E1, cyclin-dependent kinases 2 and 6) as well as decreased expression of luteinizing hormone receptor mRNA. However, the EGF or FSH-elicited effect was reversed by simultaneous treatment with an EGFR inhibitor AG1478. In conclusion, EGF and EGFR expressions manifested stage-specific changes during follicular development and EGF mediated FSH-induced cell proliferation and retarded cell differentiation in the prehierarchical follicles. These expressions thus stimulated follicular growth before selection in the egg-laying chicken.

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.

Effect of prostaglandin on luteinizing hormone-stimulated proliferation of theca externa cells from chicken prehierarchical follicles.

The effect of prostaglandin (PG) on proliferation of chicken theca externa cells from prehierarchical small yellow follicles (SYF) was evaluated and involved signaling pathways as well as mRNA expression of cAMP response element binding protein (CREB1), cyclins (CCND1 and CCNE1) and cyclin-dependent kinases (CDKs) were investigated. Results showed that PGE(1) (1-100 ng/ml) manifested a similar proliferating effect as LH on theca externa cells, and this stimulating effect was restrained by the prostaglandin receptor antagonist SC19220 at 10(-7) to 10(-5)M. Moreover, prostaglandin synthase inhibitor indomethacin (10(-7) to 10(-5)M) suppressed LH-induced increase in the cell number. In addition, PGE(1)-stimulated cell proliferation was also predominantly hindered by H(89) (PKA inhibitor) instead of H(7) (PKC inhibitor). Meanwhile, BrdU incorporation experiment displayed similar changes with the cell number. Furthermore, H(89), SC19220 and indomethacin abolished the PGE(1)-stimulated increase in the expression of CREB1, CCND1/CDK6 and CCNE1/CDK2 mRNAs, indicating that cAMP/PKA/CREB1 signaling cascade was involved in PGE(1)-stimulated DNA synthesis. In conclusion, PG could promote proliferation of theca externa cells from prehierarchical follicles through changes in cyclin D1/CDK6, cyclin E1/CDK2 and CREB1 mRNA expression via cAMP/PKA and CREB1 signaling cascade. These results suggest that PG may promote development of chicken prehierarchical follicles and is related to dominant follicle selection in laying hens.