Research Note: Heat stress affects immune and oxidative stress indices of the immune organs of broilers by changing the expressions of adenosine triphosphate-binding cassette subfamily G member 2, sodium-dependent vitamin C transporter-2, and mitochondrial calcium uniporter.

This study aimed to determine the mechanisms of heat-induced oxidative stress in the thymus and spleen of broilers. After 28 d, 30 broilers were randomly divided into the control (25°C ± 2°C; 24 h/d) and heat-stressed (36°C ± 2°C; 8 h/d) groups; the experiment lasted for 1 wk. The broilers in each group were euthanized, and some samples were collected and analyzed at 35 d. The results showed that the birds subjected to heat stress reduced the weight (P < 0.01) and the indices of thymus (P < 0.01), the activities of T-AOC (P < 0.01) and SOD (P < 0.05) of spleen, and levels of IL-10 (P < 0.05) and the GSH-PX (P < 0.05) in thymus and spleen, and increased the IL-6 content of thymus (P < 0.05), the MDA content (P < 0.01), and the reactive oxygen species (ROS) levels (P < 0.01) in thymus and spleen. Moreover, the expression of the IgG gene in the thymus and spleen of heat-stressed broilers was increased (P < 0.05); however, the expression of the IgM gene in the spleen was increased (P < 0.05), with no difference (P > 0.05) in the thymus of heat-stressed broilers compared with the control. Furthermore, the relative expression of adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) in the thymus and spleen both increased (P < 0.05). The sodium-dependent vitamin C transporter-2 (SVCT-2) (P < 0.01) and mitochondrial calcium uniporter (MCU) (P < 0.01) mRNA levels in the thymus of heat-stressed broilers increased, and the expression of ABCG2 (P < 0.05), SVCT-2 (P < 0.01), and MCU (P < 0.01) proteins in the thymus and spleen of heat-stressed broilers increased compared with the control group. This study confirmed that heat stress-induced oxidative stress in the immune organs of broilers, further reducing immune function.

Analysis of serum reproductive hormones and ovarian genes in pubertal female goats.

BACKGROUND: Age at puberty is an important factor affecting goat fertility, with endocrine and genetic factors playing a crucial role in the onset of puberty. To better understand the relationship between endocrine and genetic factors and mechanisms underlying puberty onset in goats, reproductive hormone levels were analyzed by ELISA and ultraperformance liquid chromatography-multiple reaction monitoring-multistage/mass spectrometry and RNA sequencing was performed to analyze ovarian genes. RESULTS: Serum follicle stimulating hormone, luteinizing hormone, estradiol, 11-deoxycortisol, 11-deoxycorticosterone, corticosterone, cortisone, and cortisol levels were found to be higher but progesterone were lower in pubertal goats as compared to those in prepubertal goats (P < 0.05). A total of 18,139 genes were identified in cDNA libraries, and 75 differentially expressed genes (DEGs) were identified (|log2 fold change|≥ 1, P ≤ 0.05), of which 32 were significantly up- and 43 were down-regulated in pubertal goats. Gene ontology enrichment analyses indicated that DEGs were mainly involved in "metabolic process," "signaling," "reproduction," and "growth." Further, DEGs were significantly enriched in 91 Kyoto Encyclopedia of Genes and Genomes pathways, including estrogen signaling pathway, steroid hormone biosynthesis, and cAMP signaling pathway. Bioinformatics analysis showed that PRLR and THBS1 were highly expressed in pubertal ovaries, and ZP3, ZP4, and ASTL showed low expression, suggesting their involvement in follicular development and lutealization. CONCLUSIONS: To summarize, serum hormone changes and ovarian DEGs expression were investigated in our study. Further studies are warranted to comprehensively explore the functions of DEGs in goat puberty.

TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of GoAstV, GPV, and GoCV.

Goose astrovirus (GoAstV), goose parvovirus (GPV), and goose circovirus (GoCV) infections have similar symptoms, such as severe diarrhea, and cause serious economic losses to the goose industry globally. Therefore, it is necessary to develop a rapid and accurate method for the differential diagnosis of the 3 viruses. In this study, a TaqMan probe-based multiplex reverse transcription-qualitative polymerase chain reaction (RT-qPCR) method was established and optimized for simultaneous detection of the three viruses. Three pairs of specific primers and probes were designed considering the conserved sequences of ORF2, VP3, and Rep of GoAstV, GPV, and GoCV, respectively. Singleplex real-time RT-qPCR detected a minimum of 10 copies of these genes, while multiplex real-time RT-qPCR detected a minimum of 100 copies. The correlation coefficients exceeded 0.99, and the amplification efficiency was 80 to 100%. The assay had high sensitivity, specificity, and repeatability. In 85 tissue samples, GoAstV and GPV were the main pathogens and demonstrated co-infection. This assay provides a rapid, efficient, specific, and sensitive tool for the detection of GoAstV, GPV, and GoCV. This can facilitate disease management and epidemiological surveillance.

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of four feline diarrhea-associated viruses.

Since their recent discovery, the prevalence of novel feline enteric viruses, including feline bocavirus 1 (FBoV-1), feline astrovirus (FeAstV), and feline kobuvirus (FeKoV), has been reported in China. Co-infections of these viruses with feline parvovirus (FPV) are common causes of diarrhea in cats. Viral co-infections are difficult to identify because of their non-specific clinical signs. To detect and identify these viruses, a quick and specific pathogen-testing approach is required. Here, we establish a real-time PCR (qPCR) based on multiple TaqMan probes for the simultaneous detection of FBoV-1, FeAstV, FeKoV, and FPV. Specific primers and TaqMan fluorescent probes were designed to ensure specificity. The results showed that the detection limit of single qPCR was up to 10 copies, and the detection limit of multiplex qPCR was up to 100 copies, with correlation coefficients >0.995 in all cases. Clinical sample detection revealed a 25.19% (34/135) total rate of co-infection among the viruses and a 1.48% (2/135) quadruple infection rate. Thus, this multiplex qPCR approach can serve as a quick, sensitive, and specific diagnostic tool for FBoV-1, FeAstV, FeKoV, and FPV identification, and it may be utilized for routine surveillance of these emerging and reemerging feline enteric viruses.

The expression of IGFBP-5 in the reproductive axis and effect on the onset of puberty in female rats.

Insulin-like growth factor-binding protein-5 (IGFBP-5) has recently been shown to alter the reproductive capacity by regulating insulin-like growth factor (IGF) bioavailability or IGF-independent effects. The present study aimed to investigate the effect and mechanism of IGFBP-5 on the onset of puberty in female rats. Immunofluorescence and real-time quantitative PCR were used to determine the expression and location of IGFBP-5 mRNA and protein distribution in the infant's hypothalamus-pituitary-ovary (HPO) axis prepuberty, peripuberty, puberty and adult female rats. Prepubertal rats with IGFBP-5 intracerebroventricular (ICV) were injected to determine the puberty-related genes expression and the concentrations of reproductive hormones. Primary hypothalamic cells were treated with IGFBP-5 to determine the expression of puberty-related genes and the Akt and mTOR proteins. Results showed that Igfbp-5 mRNA and protein were present on the HPO axis. The addition of IGFBP-5 to primary hypothalamic cells inhibited the expression of Gnrh and Igf-1 mRNAs (P < 0.05) and increased the expression of AKT and mTOR protein (P < 0.01). IGFBP-5 ICV-injection delayed the onset of puberty, reduced Gnrh, Igf-1, and Fshß mRNAs, and decreased the concentrations of E2, P4, FSH,serum LH levels and the ovaries weight (P < 0.05). More corpus luteum and fewer primary follicles were found after IGFBP-5 injection (P < 0.05).

Ascorbic acid regulates mouse spermatogonial stem cell proliferation in a Wnt/ß-catenin/ROS signaling dependent manner.

Spermatogonial stem cells (SSCs) provide a foundation for spermatogenesis, but the mechanism of SSC proliferation is still poorly understood. To investigate whether and how ascorbic acid (AA) regulates the growth of mouse SSCs in vitro, the SSCs were cultured in different concentration AA medium for 14 days. The proliferation, apoptosis and the reactive oxygen species (ROS) levels of SSCs in different AA groups were respectively detected. Moreover, the SSC activity in 40 µg/mL AA group and the control was tested by a transplantation assay. To explore the mechanism of AA regulating mouse SSCs proliferation, the dishevelled homolog 2 (DVL2) and nucleoredoxin (NRX) protein levels, the expression of axis inhibition protein 2 (Axin2), leucine-rich G-protein coupled receptor 5 (Lgr5), B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), c-myc and cyclin D1 genes in Wnt/ß-catenin pathway were respectively confirmed. The results showed that the adding concentration of AA did not affect the main shape of SSCs. A 40 µg/mL AA in culture medium promoted the proliferation, and decreased the ROS production and apoptosis rate of SSCs. Moreover, colonization efficiency in the seminiferous tubules of the recipient testis in 40 µg/mL AA group was higher compared with the control group by a transplantation assay. Finally, the appropriate ROS in the 40 µg/mL AA group further adjust the levels of DVL2 and NRX protein in the Wnt/ß-catenin pathway to maintain the nuclear intensity of ß-catenin, in turn, the expression of apoptosis gene Bax decreased, while the expression of Bcl2, Axin2, Lgr5, c-myc and cyclin D1 genes increased. The study confirmed that AA adjusts the endogenous ROS level to impact on SSC proliferation in a dose-dependent manner by Wnt/ß-catenin signaling pathway.

l-Theanine improves emulsification stability and antioxidant capacity of diacylglycerol by hydrophobic binding ß-lactoglobulin as emulsion surface stabilizer.

Diacylglycerol (DAG) is commonly used as fat substitute in food manufacture due to its functional properties, but DAG has poor emulsification and oxidation stability, which limits its wide application in food industry. In this work, fluorescence quenching data and thermodynamic parameters were analyzed to investigate the interaction mechanism between l-theanine (L-Th) and ß-lactoglobulin (ß-LG). DAG emulsion was prepared by using ß-lactoglobulin-theanine (ß-LG-Th) as surface stabilizer, and its emulsification and oxidation stability were evaluated. The results showed that the hydrophobic interaction played an important role on the conjugate of ß-LG and L-Th due to the negative values for ΔG, positive values for ΔH and ΔS at pH 4.0, pH 6.0 and pH 8.0. The DAG has been better embedded by using ß-LG-Th as surface stabilizer, and the droplet size was about 0.2 µm to 1.5 µm when the pH was 6.0, the ratio of L-Th to ß-LG was 1:1. ß-LG-Th as surface stabilizer for DAG can increase the ζ-potential and emulsion index, make the emulsion droplet size distribution more uniform. The l-theanine was better to be used to improve the emulsification stability and antioxidant capacity of DAG by binding ß-LG as surface stabilizer.

Knockdown of Ptprn-2 delays the onset of puberty in female rats.

In the present study, we evaluated how Ptprn-2 (encoding tyrosine phosphatase, receptor type, N2 polypeptide protein) affects the onset of puberty in female rats. We evaluated the expression of Ptprn-2 mRNA and protein in the hypothalamus-pituitary-ovary axis of female rats using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence at infancy, prepuberty, puberty, peripuberty, and adulthood. We evaluated the effects of Ptprn-2 gene knockdown on different aspects of reproduction-related biology in female rats, including the expression levels of puberty-related genes in vivo and in vitro, the time to onset of puberty, the concentration of serum reproductive hormones, the morphology of ovaries, and the ultrastructure of pituitary gonadotropin cells. Our results demonstrated that PTPRN-2 was primarily distributed in the arcuate nucleus (ARC), periventricular nucleus (PeN), adenohypophysis, and the ovarian follicular theca, stroma, and granulosa cells of female rats at various stages. Ptprn-2 mRNA levels significantly varied between peripuberty and puberty (P < 0.05) in the hypothalamus and pituitary gland. In hypothalamic cells, Ptprn-2 knockdown decreased the expression of Ptprn-2 and Rfrp-3 mRNA (P < 0.05) and increased the levels of Gnrh and Kiss-1 mRNA (P < 0.05). Ptprn-2 knockdown in the hypothalamus resulted in delayed vaginal opening compared to the control group (n = 12, P < 0.01), and Ptprn-2, Gnrh, and Kiss-1 mRNA levels (P < 0.05) all decreased, while the expression of Igf-1 (P < 0.05) and Rfrp-3 mRNA (P < 0.01) increased. The concentrations of FSH and P4 in the serum of Ptprn-2 knockdown rats were lower than in control animals (P < 0.05). Large transverse perimeters and longitudinal perimeters (P < 0.05) were found in the ovaries of Ptprn-2 knockdown rats. There were fewer large secretory particles from gonadotropin cells in adenohypophysis tissue of the Ptprn-2 knockdown group compared to the control group. This indicates that Ptprn-2 knockdown can regulate levels of Gnrh, Kiss-1, and Rfrp-3 mRNA in the hypothalamus, regulate the concentration of serum FSH and P4, and alter the morphology of ovarian and gonadotropin cells, delaying the onset of puberty in female rats.

Effect of GABA-T on Reproductive Function in Female Rats.

This study explored the role of γ-aminobutyric acid transaminase (GABA-T) in the puberty and reproductive performance of female rats. Immunofluorescence technique, quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution of GABA-T and the expression of genes and hormones in female rats, respectively. The results showed that GABA-T was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and periventricular nucleus (PeN) of the hypothalamus, and in the adenohypophysis, ovarian granulosa cells and oocytes. Abat mRNA level at 28 d was lowest in the hypothalamus and the pituitary; at puberty, it was lowest in the ovary. Abat mRNA level was highest in adults in the hypothalamus; at infancy and puberty, it was highest in the pituitary; and at 21 d it was highest in the ovary. After vigabatrin (GABA-T irreversible inhibitor) was added to hypothalamus cells, the levels of Abat mRNA and Rfrp-3 mRNA were significantly reduced, but Gnrh mRNA increased at the dose of 25 and 50 µg/mL; Kiss1 mRNA was significantly increased but Gabbr1 mRNA was reduced at the 50 µg/mL dose. In prepubertal rats injected with vigabatrin, puberty onset was delayed. Abat mRNA, Kiss1 mRNA and Gnrh mRNA levels were significantly reduced, but Rfrp-3 mRNA level increased in the hypothalamus. Vigabatrin reduced the concentrations of GABA-T, luteinizing hormone (LH) and progesterone (P4), and the ovarian index. Lactation performance was reduced in adult rats with vigabatrin treatment. Four hours after vigabatrin injection, the concentrations of GABA-T and LH were significantly reduced in adult and 25 d rats, but follicle-stimulating hormone (FSH) increased in 25 d rats. In conclusion, GABA-T affects the reproductive function of female rats by regulating the levels of Gnrh, Kiss1 and Rfrp-3 in the hypothalamus as well as the concentrations of LH and P4.

Moderate hypoxia modulates ABCG2 to promote the proliferation of mouse spermatogonial stem cells by maintaining mild ROS levels.

The aim of this study was to investigate the effects of different oxygen (O2) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O2 concentrations (1%, 2.5%, 5%, and 20% O2) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O2 concentrations for 7 days, and there were no major morphologic differences between the O2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O2 treatment group. Compared with other O2 concentrations, the number of cells in G0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O2. Moreover, the intracellular ROS levels gradually increased with increasing O2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O2 was higher than in the other O2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro.

Beneficial effects of PGC-1α in the substantia nigra of a mouse model of MPTP-induced dopaminergic neurotoxicity.

BACKGROUND: Mitochondrial dysfunction and oxidative stress are closely associated with the pathogenesis of Parkinson's disease. Peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) is thought to play multiple roles in the regulation of mitochondrial biogenesis and cellular energy metabolism. We recently reported that altering PGC-1α gene expression modulates mitochondrial functions in N-methyl-4-phenylpyridinium ion (MPP+) treated human SH-SY5Y neuroblastoma cells, possibly via the regulation of Estrogen-related receptor α (ERRα), nuclear respiratory factor 1 (NRF-1), nuclear respiratory factor 2 (NRF-2) and peroxisome proliferator-activated receptor γ (PPARγ) expression. In the present study, we aimed to further investigate the potential beneficial effects of PGC-1α in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated C57BL mice. METHODS: The overexpression or knockdown of the PGC-1α gene in the mouse model of dopaminergic neurotoxicity was performed using a stereotactic injection of lentivirus in MPTP-treated male C57BL/6 mice. Mice were randomly assigned to one of 6 groups (n=24 per group): normal saline (NS) intraperitoneal injection (i.p.) (con); MPTP i.p. (M); solvent of the lentivirus striatal injection (lentivirus control) + MPTP i.p. (LVcon+M); lentivirus striatal injection + MPTP i.p. (LV+M); LV-PGC-1α striatum injection + MPTP i.p. (LVPGC+M); and LV-PGC-1α-siRNA striatal injection + MPTP i.p. (LVsiRNA+M). Intraperitoneal injections of MPTP/NS were conducted two weeks after lentivirus injection. RESULTS: We found significant improvement in motor behavior and increases in tyrosine hydroxylase expression in the substantia nigra (SN) in the brains of mice in the LVPGC+M group. The opposite tendency was observed in those in the LVsiRNA+M group. The expression of superoxide dismutase (SOD) in the SN region was also consistent with the changes in PGC-1α expression. Electron microscopy showed an increasing trend in the mitochondrial density in the LVPGC+M group and a decreasing trend in the M and LVsiRNA+M groups compared to that in the controls. CONCLUSIONS: Our results indicated that PGC-1α rescues the effects of MPTP-induced mitochondrial dysfunction in C57BL mice.

Effects of duration of thermal stress on growth performance, serum oxidative stress indices, the expression and localization of ABCG2 and mitochondria ROS production of skeletal muscle, small intestine and immune organs in broilers.

The purpose of the current study was to investigate that effect of duration of thermal stress on growth performance, oxidative stress indices in serum, the expression and localization of ABCG2, and mitochondria ROS production in skeletal muscle, small intestine and immune organs, and then to further reveal correlations between indicators. At 28 days of age, sixty broilers were randomly divided into the control group (25 ±â€¯2 °C; 24 h/day) and the heat stress group (36 ±â€¯2 °C; 8 h/day lasted for 1 week or 2 weeks). Fifteen broilers per group were respectively euthanized, and some samples were respectively collected from the control and the heat stress groups at the end of the 1st week or the 2nd week of heat stress. A typical heat stress response has been observed at this temperature. Compared with the control group, the birds subjected to heat stress at the end of the 1st week reduced (P ＜ 0.05) body weight (BW), average daily feed intake (ADFI), average daily gain (ADG), the activity of serum antioxidant enzyme and content of glutathione (GSH), while increased (P ＜ 0.05) feed conversion ratio (FCR), serum corticosterone and malondialdehyde (MDA) levels. However, when the heat stress lasted for the end of the 2nd week, there was no significant difference (P ＞ 0.05) in ADFI, ADG, FCR and serum contents of corticosterone, MDA and GSH. Regardless of duration of thermal stress, the localization of ABCG2 protein had no change. Moreover, heat stress also did not affect (P ＞ 0.05) the IOD of the ABCG2 positive portion and the expression of the ABCG2 mRNA in the pectorales, crureus, duodenum, jejunum, ileum and spleen, while significantly increased (P ＜ 0.05) the corresponding tissues ROS production at the end of the 1st week of heat stress. In contrast, at the end of the 2nd week of heat stress, IOD of the ABCG2 positive portion and the expression of the ABCG2 mRNA in heat stress group significantly increased (P ＜ 0.05), while the corresponding tissues ROS production had no difference (P ＞ 0.05) compared to the control group. Collectively, duration of thermal stress affects growth performance, serum oxidative stress indices, and the expression of ABCG2 and the ROS production of broiler tissues in a time-dependent manner. There is a negative correlation between the expression of ABCG2 and the ROS production in the corresponding tissues under heat stress.

The effect of icariin on immunity and its potential application.

Icariin (ICA) is a major bioactive monomer belonging to flavonoid glycosides attracted from Epimedium, being a classic tonic agent in traditional Chinese medicine. ICA commonly presents multiple effects such as regulating sex hormones, relieving atherosclerosis and antioxidant activity, etc. Recently, more and more studies have demonstrated the application of ICA in autoimmune diseases such as rheumatoid arthritis, bronchial asthma, multiple sclerosis and systemic lupus erythematosus due to its anti-inflammatory. Additionally, ICA also has the anti-tumor activities. Multiple targets and mechanisms of ICA are reported which relates to regulate lymphocytes balance, anti-inflammatory/inflammatory cytokines, signal pathways like NF-kappaß and Erk-p38-JNK, lymphocyte transcription factors and other targets such as TLRs, STAT and PTEN, etc. In this review, we have updated the advance in this field and these studies have suggested that ICA has a potential to treat immunological and inflammatory diseases.

Mitochondrial Effects of PGC-1alpha Silencing in MPP+ Treated Human SH-SY5Y Neuroblastoma Cells.

The dopaminergic neuron degeneration and loss that occurs in Parkinson's disease (PD) has been tightly linked to mitochondrial dysfunction. Although the aged-related cause of the mitochondrial defect observed in PD patients remains unclear, nuclear genes are of potential importance to mitochondrial function. Human peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α) is a multi-functional transcription factor that tightly regulates mitochondrial biogenesis and oxidative capacity. The goal of the present study was to explore the potential pathogenic effects of interference by the PGC-1α gene on N-methyl-4-phenylpyridinium ion (MPP+)-induced SH-SY5Y cells. We utilized RNA interference (RNAi) technology to probe the pathogenic consequences of inhibiting PGC-1α in the SH-SY5Y cell line. Remarkably, a reduction in PGC-1α resulted in the reduction of mitochondrial membrane potential, intracellular ATP content and intracellular H2O2 generation, leading to the translocation of cytochrome c (cyt c) to the cytoplasm in the MPP+-induced PD cell model. The expression of related proteins in the signaling pathway (e.g., estrogen-related receptor α (ERRα), nuclear respiratory factor 1 (NRF-1), NRF-2 and Peroxisome proliferator-activated receptor γ (PPARγ)) also decreased. Our finding indicates that small interfering RNA (siRNA) interference targeting the PGC-1α gene could inhibit the function of mitochondria in several capacities and that the PGC-1α gene may modulate mitochondrial function by regulating the expression of ERRα, NRF-1, NRF-2 and PPARγ. Thus, PGC-1α can be considered a potential therapeutic target for PD.

Expression of follicle-stimulating hormone receptor (FSHR), protein kinase B-2 (AKT2) and adapter protein with PH domain, PTB domain, and leucine zipper (APPL1) in pig ovaries.

Follicle-stimulating hormone (FSH) regulates oogenesis and spermatogenesis by binding to its receptor (FSHR) on target cells in the ovary and testis, respectively. The signaling cascades activated after ligand binding are extremely complex and have been shown to include protein kinase A and phosphatidylinositol 3-kinase/protein kinase. The adapter protein APPL1 (adapter protein with PH domain, PTB domain, and leucine zipper), which is an assortment of other signaling proteins, was previously identified to interact with the FSH receptor (FSHR) and the protein kinase B (AKT) pathway. APPL1 plays an important role in promoting cell survival within the preovulatory follicle granulosa layer. Here, we aimed to evaluate the FSHR, AKT2, and APPL1 gene and protein expression levels in the ovaries of different prolific porcine breeds (Wannan Black [WB] and Large White [LW] pigs) using immunohistochemistry and qRT-PCR, respectively. Our results showed that FSHR, AKT2, and APPL1 mRNA levels were significantly higher (P < 0.05) in the ovaries of WB pigs than in the ovaries of LW pigs. Additionally, the FSHR, AKT2, and APPL1 proteins were mainly found distributed in the granulosa cells and oocytes. This study showed that high levels of FSHR, AKT2, and APPL1 were expressed in the ovaries of high prolific breed pigs.

[Cloning and Ion Transportation of Cryptosporidium andersoni ATP-binding Cassette 1 Gene].

Objective: To investigate the transportation of intracellular and extracellular K(+), Ca(2+), Na(+) and Mg(2+) under the function of Cryptosporidium andersoni ATP-binding cassette (CaABC) 1 gene. Methods: CaABC1 gene was amplified by PCR using specifically designed primers. The eukaryotic expression plasmid pEGFP-C1-CaABC1 was constructed, and transfected into mouse intestinal epithelial cells via liposome transfection. The blank (with no transfection) and control groups (transfected with empty plasmid pEGFP-C1) were also set. Changes in intracellular and extracellular K(+), Ca(2+), Na(+) and Mg(2+) concentrations were examined by the ion concentration assay kit. Results: PCR amplification resulted in a 544 bp product. The recombinant plasmid pEGFP-C1-CaABC1 was successfully constructed. Green fluorescence was seen in the control and transfected groups, but not in the blank group. The concentrations of K(+), Ca(2+), Na(+) and Mg(2+) in intracellular fluid were （5.51 ± 0.51）, （1.98 ± 0.06）, （108.33 ± 1.33） and （0.93 ± 0.03） mmol/L in the blank group; （6.25 ± 0.70）, （1.90 ± 0.13）, （107.73 ± 1.79） and （0.87 ± 0.05） mmol/L in the control group; and （14.84 ± 0.90）, （3.40 ± 0.14）, （127.64 ± 1.49） and （1.72 ± 0.20） mmol/L in the transfected group. The concentrations of K+, Ca2+, Na+ and Mg2+ in extracellular fluid were （12.72 ± 0.83）, （3.72 ± 0.03）, （116.83 ± 1.04） and (2.02 ± 0.18) mmol/L in the blank group; （10.11 ± 0.90）, （3.58 ± 0.06）, （115.89 ± 1.86） and (1.71 ± 0.41) mmol/L in the control group; and （5.77 ± 0.21）, （1.29 ± 0.18）, （96.21 ± 1.19） and (0.64 ± 0.02) mmol/L in the transfected group. There were significant differences in K+, Ca2+ and Mg2+ concentrations between the transfected group and the control group. Conclusion: CaABC1 participates in the transportation of K+, Ca2+ and Mg2+.

Overexpression of PGC-1α Influences Mitochondrial Signal Transduction of Dopaminergic Neurons.

Parkinson's disease (PD) is a common neurodegenerative disease in the elderly. Mitochondrial dysfunction plays an important role in the pathogenesis of PD. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a powerful transcription factor, interacting with multiple transcription factors and widely involving in the regulation of mitochondrial biogenesis, oxidative stress, and other processes. The present study investigated the neuroprotective effects and signal transduction mechanisms of the overexpression of PGC-1α on N-methyl-4-phenylpyridinium ion (MPP(+))-induced mitochondrial damage in SH-SY5Y cell, establishing the cell model of overexpression of PGC-1α and the cell model of PD by using adenoviral vectors and MPP(+). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) assay was used to investigate the effects of MPP(+) and adenovirus on the cell viability of SH-SY5Y cells and the cell viability of experimental groups. Western blot and real-time PCR analysis were used to detect the expression of PGC-1α. Flow cytometry and ELISA were used to detect mitochondrial membrane potential and the level of cytochrome C, respectively. The level of intracellular ATP and H2O2 was measured by multifunctional fluorescence microplate. Western blot analysis and real-time PCR were used to observe the expression of estrogen-related receptor α (ERRα), peroxisome proliferator-activated receptor γ (PPARγ), nuclear respiratory factor (NRF)-1, and NRF-2. Confocal fluorescence analysis was used to observe subcellular localization of PGC-1α in SH-SY5Y cells under the intervention of MPP(+). The expression of PGC-1α messenger RNA and protein significantly increased in Adv-PGC-1α + GFP groups, compared with the control and Adv-GFP groups (P < 0.01). The overexpression of PGC-1α could increase mitochondrial membrane potential, reduce the release of mitochondrial cytochrome C, inhibit H2O2 production, and improve the level of ATP in SH-SY5Y cells. The trend of expression of ERRα, PPARγ, and NRF-1 was more consistent with PGC-1α, the most remarkable change is ERRα, but the expression of NRF-2 has no significant changes. Under the gradually increasing concentration of MPP(+), microscale PGC-1α gradually appeared in the cytoplasm of SH-SY5Y cells. The overexpression of PGC-1α can inhibit MPP(+)-induced mitochondrial damage in SH-SY5Y cells, and PGC-1α may realize the neuroprotective effects via the ERRα, PPARγ, and NRF-1 pathway.

Isolation and characterization of a non-specific endoglucanase from a metagenomic library of goat rumen.

A cellulase gene (cel28a) was isolated from a rumen microbial metagenome library of goat rumen microorganisms, cloned into E. coli, and expressed in active form. The gene has a length of 1596 bp obtained using a genome walking Kit and encodes a protein of 509 amino acids with a calculated MW of 55 kDa. The deduced amino acid sequence was homologous with cellulases belonging to the glycosyl hydrolase family 5 (GH5). The expressed protein showed activity toward carboxymethylcellulose (CMC) and xylan, suggesting non-specific endoglucanase activity. The optimal conditions for endoglucanase and xylanase activities were 50 °C and pH 5.0. The metal ions (Ca(2+), Fe(2+), Mn(2+) and Co(2+)) stimulated the cellulase activity of cel28a, while the other metal ions and chemicals (Ni(2+), Mg(2+), Zn(2+), Cu(2+), SDS and EDTA) inhibited the cellulase activity. Further examination of substrate preference showed a higher activity with CMC, oat spelt xylan and birchwood xylan than with filter paper and microcrystalline cellulose, again suggesting that the protein was an endoglucanase with xylanase activity.

Cloning and Iron Transportation of Nucleotide Binding Domain of Cryptosporidium andersoni ATP-Binding Cassette (CaABC) Gene.

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of Ca(2+), Mg(2+), K(+), and HCO3 (-) in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

Establishment of recipient model for spermatogonial stem cells transplantation in Kunming mice.

The objective of this study was to establish a recipient model for spermatogonial stem cells (SSCs) transplantation in the Kunming mice after different doses busulfan treatment. The results showed that the most optimal dose of busulfan was 20mg/kg and the most appropriate time for transplantation was 5-7 wk after busulfan treatment. Then, the cloned fragments existed in the testis of recipient mice after 20mg/kg busulfan treatment and the offspring with enhanced green fluorescent protein (EGFP) were produced by the transplanting SSCs. Hence, we established the effective recipient model for donor-derived SSCs transplantation in Kunming mice.