来航鸡FOXL2基因的克隆与序列分析

为了研究来航鸡FOXL2基因的结构及功能,根据GenBank上登录的红色原鸡FOXL2基因序列设计引物,对来航鸡FOXL2基因进行了克隆、测序,并对该序列进行生物信息学分析。结果表明:克隆得到的来航鸡FOXL2基因全长为1 130 bp(GenBank登录号为JF708868),包含1个918 bp的完整开放式阅读框,编码305个氨基酸,其CDS编码区的核苷酸序列与红色原鸡、人、牛、野猪、家鼠、欧洲兔、蟾蜍、斑马鱼及罗非鱼的FOXL2基因对应序列的同源性分别为99.8%、80.0%、80.4%、80.3%、80.5%、81.5%、77.7%、71.8%、75.5%,推导的氨基酸序列同源性分别为99.7%、64.7%、64.7%、65.4%、63.5%、63.8%、79.7%、78.3%、79.9%;FOXL2编码蛋白主要存在于细胞核中,存在1个保守结构域,不含信号肽、跨膜结构域和剪切位点,并存在2个蛋白质激酶C磷酸化位点、1个cAMP和cGMP依赖的蛋白激酶磷酸化位点、2个N-糖基化位点、3个酪蛋白激酶Ⅱ磷酸化位点和4个N-豆蔻酰化位点,预测最佳抗原表位候选区域为45～49位(SQKPP)、147～152位(RPPPTH)和169～173位(SPPKY)。     

Regulation of Innate Immune Function in Bovine Oviduct Epithelial Cells in Culture: The Homeostatic Role of Epithelial Cells in Balancing Th1/Th2 Response

This study aimed to investigate the role of epithelial cells in regulating innate immunity in bovine oviduct epithelial cell (BOEC) culture. We studied the effect of Escherichia coli lipopolysaccharide (LPS) and its interaction with ovarian steroids, estradiol (E2) and progesterone (P4), and luteinizing hormone (LH) at concentrations observed during the preovulatory period on immune responses in BOEC culture. Immunohistochemistry of oviduct tissue showed intensive expression of Toll-like receptor-4 (TLR-4) and TLR-2 in epithelial cells. A dose of 10 ng/ml LPS stimulated TLR-4, cyclooxygenase-2 (COX-2), nuclear factor kappa B inhibitor A (NFKBIA), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) expression, indicating an early pro-inflammatory response. A dose of 100 ng/ml LPS did not induce expression of these genes but stimulated TLR-2, IL-10,IL-4 and microsomal prostaglandin E synthase-1 (mPGES-1) expression and PGE2 secretion, indicating an anti-inflammatory response. Ovarian steroids and LH completely block LPS (10 ng/ml)-induced TLR-4, IL-1β and TNF-α expression as well as LPS (100 ng/ml)-induced TLR-2 expression. Taken together, this study suggests the existence of an early signaling system to respond to infection in the BOEC. In addition, ovarian steroids and LH may play a critical role in inducing homeostasis and in controlling hyperactive pro-inflammatory responses detrimental to epithelial cells, sperm and the embryo.     

Role of ghrelin in regulating rabbit ovarian function and the response to LH and IGF-I

The aim of these in vivo and in vitro studies was to examine the role of ghrelin in the control of plasma hormone concentrations, the proliferation, apoptosis and secretory activity of ovarian granulosa cells and the response of these cells to hormonal treatments. Female rabbits were injected with ghrelin (10 μg/animal/day for one week before ovulation induced by 25 IU PMSG and 0.25 IU LHRH). On the day of ovulation, blood samples were collected and analyzed for concentrations of progesterone (P₄), testosterone (T), estradiol (E₂), estrone-sulphate (ES), insulin-like growth factor I (IGF-I) and leptin (L) by RIA. Some control and ghrelin-treated animals were killed in the periovulatory period, their ovaries were weighed and granulosa cells were isolated and cultured for 2 d. Cell proliferation (expression of PCNA) and apoptosis (expression of TdT) were evaluated by immunocytochemistry and TUNEL respectively. Secretion of P₄, T, E₂, IGF-I, and prostaglandin F (PGF) by granulosa cells cultured with and without LH or IGF-I (1, 10 or 100 ng/ml medium) was assessed by RIA. The remaining control and treated animals were kept until parturition, while the number, viability and body weight of pups were recorded.     

Expression of Mitochondria‐Associated Genes (PPARGC1A,NRF‐1, BCL‐2 and BAX) in Follicular Development and Atresia of Goat Ovaries

Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria‐dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria‐associated genes peroxisome proliferator‐activated receptor‐gamma, coactivator1‐alpha (PPARGC1A), nuclear respiratory factor‐1 (NRF‐1), B‐cell CLL/lymphoma 2 (BCL‐2) and BCL2‐associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria‐associated proteins (PGC‐1α, which are encoded by the PPARGC1A gene, NRF‐1, BCL‐2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC‐1α and NRF‐1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF‐1, increased BAX expression and increased ratio of BAX to BCL‐2 expression. These results suggested that changes of the mitochondria‐associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.     

FOXL2基因重组腺病毒载体的构建及鉴定

为了构建叉头框L2(Forkhead box L2,FOXL2)基因与绿色荧光蛋白(GFP)基因复制缺陷型腺病毒载体,试验将克隆的FOXL2基因与IRES-GFP片段通过酶切、纯化等方法共同连接到pShuttle-CMV载体中,再与pAdEasy-1腺病毒质粒在BJ5183大肠杆菌中进行同源重组,得到复制缺陷型AD-FOXL2腺病毒载体,再用PacⅠ酶线性化后转染HEK293细胞,观察绿色荧光表达,并测量病毒效价。结果表明:将AD-FOXL2腺病毒载体用PacⅠ酶线性化后,得到1个大于23 kb的大片段和1个4.5 kb的特异性小片段,证明同源重组成功;将所得的腺病毒载体转染HEK293细胞,可以观察到GFP的表达,证明包装成功,并测得病毒效价为1×10-8.61/0.1 mLTCID50。     

Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy

MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants—namely, MX1-a and MX1B. In ruminants, IFN-τ—a type I IFN—is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14–18, 25–40, 50–70, 80–100, and 130–150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14–18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.     

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.     

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.     

Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.     

过表达FOXL2基因对鸡胚性腺分化的影响

试验旨在研究FOXL2基因对鸡胚性腺分化的影响。本试验分为试验1组、试验2组和空白对照组（鸡胚数量分别为260、100、20枚），试验组通过胚盘下腔注射的方法分别将pLV-FOXL2慢病毒重组质粒、pLV空质粒注入胚胎期第2天的鸡胚，空白对照组不做处理并与试验组一起孵化至出雏，利用CHD1基因遗传性别鉴定的方法对出雏的雏鸡进行性别检测，分析其性腺解剖学、组织学结构变化，并利用免疫组化的方法检测性腺FOXL2和CYP19A1蛋白表达量。结果显示，试验1组遗传性别为公的23只，遗传性别为母的18只，表型性别为公的21只，表型性别为母的18只，其中有2只表型性别不典型，左侧性腺发生变化，朝卵巢结构转变；试验2组遗传性别为公的9只，遗传性别为母的12只，表型性别与遗传性别一致。阳性PCR检测结果显示，试验1组获得阳性个体10个，阳性率为24.4%（10/41）；试验2组获得阳性个体8个，阳性率为38.1%（8/21）。性腺解剖学结果显示，阳性pLV-FOXL2雄性鸡胚左侧性腺体积明显大于右侧性腺，表现膨松状态；组织切片结果显示，雄性鸡胚性腺具有典型的卵巢皮质层和髓质层结构；阳性pLV-FOXL2雌性鸡胚性腺的发育无明显变化。免疫组化结果显示，FOXL2和CYP19A1蛋白在试验1组左右侧睾丸中的表达量与空白对照组母鸡卵巢中的表达量相似，显著高于试验2组（P<0.05）。以上结果表明，FOXL2基因可能促进鸡雄性性腺的性反转，在鸡性腺分化和发育过程中发挥着重要的作用。     

Effect of Overexpression of FOXL2 Gene on Gonadal Differentiation in Chicken Embryo

The aim of this study was to investigate the effect of FOXL2 gene on gonadal differentiation in chicken embryos.This test was divided into the experiment groups 1,2 and blank control group(chicken embryos were 260,100 and 20,respectively).In the experimental group,pLV-FOXL2 lentivirus recombinant plasmids and pLV empty plasmids were injected into the chicken embryo on the second day of the embryonic stage by subpaneoidal injection.The blank control group was not treated and incubated with the experimental groups until the chick was hatched.CHD1 gene genetic sex identification method was used to detect the sex of chicks,the changes of gonadal anatomy and histological structure were analyzed,and the expression levels of FOXL2 and CYP19A1 proteins were detected by immunohistochemistry.The results showed that in the experiment group 1,there were 23 male and 18 female of genetic sex,21 male and 18 female of phenotypic sex,and two of them had atypical phenotypic gender,with changes in the left gonadal gland toward ovarian structure.The phenotypic sex was consistent with the genetic sex in experiment group 2,with 9 male and 12 female.Positive PCR results showed that 10 positive individuals were obtained in the experiment group 1,with a positive rate of 24.4% (10/41),and 8 positive individuals were obtained in the experiment group 2,with a positive rate of 38.1% (8/21).The anatomical structure of the gonad showed that the volume of the left gonad was significantly larger than that of the right gonad in the positive plV-FOXL2 male embryos.The results of tissue sections showed that the gonad of male chicken embryo had typical structure of ovarian cortex and medulla.There was no significant change in the development of gonad in female embryos with positive pLV-FOXL2.Immunohistochemical results showed that the expression levels of FOXL2 and CYP19A1 proteins in the left and right testicle of experiment group 1 were similar to that in the hen ovary of the blank control group,and were significantly higher than that in experiment group 2 (P<0.05).These results suggested that FOXL2 gene might promote the sexual inversion of chicken gonads and played an important role in the differentiation and development of chicken gonads.     

Ovarian localization of 11β-hydroxysteroid dehydrogenase (11βHSD): effects of ACTH stimulation and its relationship with bovine cystic ovarian disease

Cystic ovarian disease (COD) is an important cause of infertility in cattle, and ACTH has been involved in regulatory mechanisms related to ovarian function associated with ovulation, steroidogenesis, and luteal function. Here, we examined the localization of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and 11βHSD2 proteins in the ovary of healthy cows and animals with spontaneous and ACTH-induced COD and the in vitro response of the follicular wall exposed to ACTH. After stimulation by ACTH, we documented changes in 11βHSD expression and cortisol secretion by the follicular wall of large antral and follicular cysts. Follicular cysts showed a higher constitutive expression of both enzymes, whereas ACTH induced an increase in 11βHSD1 in tertiary follicles and follicular cysts and a decrease in 11βHSD2 in follicular cysts. Moderate expression of 11βHSD1 was observed by immunohistochemistry in granulosa of control animals, with an increase (P < 0.05) from primary to secondary, tertiary, and atretic follicles. The level of immunostaining in theca interna was lower than that in granulosa. The expression of 11βHSD2 was lower in the granulosa of primary follicles than in that of secondary, tertiary, and atretic follicles and was lower in the theca interna than in the granulosa. In ACTH-induced and spontaneously occurring follicular cysts, differences from controls were observed only in the expression of 11βHSD1 in the granulosa, being higher (P < 0.05) than in tertiary follicles. These findings indicate that follicular cysts may be exposed to high local concentrations of active glucocorticoids and indicate a local role for cortisol in COD pathogenesis and in regulatory mechanisms of ovarian function.     

Prevalence,O-genotype and Shiga toxin (Stx) 2 subtype of Stx-producing Escherichia coli strains isolated from Argentinean beef cattle

The aims of this study were to investigate prevalence, O-genotype, and virulence gene profile including Shiga toxin (Stx) 2 gene-subtype of Stx-producing Escherichia coli (STEC) in beef cattle from the Bahía Blanca in Argentina. Rectal swabs were collected from 283 beef cattle in 2012. stx genes were detected in 90 (32%) out of the 283 rectal swabs by stx gene-specific PCR assay. The positive cases were 13 with stx1, 58 with stx2, and 19 with both stx1 and stx2. Among 90 stx gene-positive samples, 45 STEC strains were isolated, which included 3 stx1, 34 stx2, and eight stx1 and stx2 genes positive isolates. O-genotyping grouped 45 STEC strains into 19 different O-genotypes such as Og8, Og145, Og171, Og185 (4 from each), Og22, Og153, Og157 (3 from each) and others. Various stx2 gene-subtypes were identified in 42 STEC strains: 13 positive cases for stx2a, 11 for stx2c, 3 for stx2g, 10 for stx2a and stx2d, 4 for stx2a and stx2c, and 1 for stx2b, stx2c and stx2g. efaI gene, generally prevalent in clinical strains, was detected in relatively high in the STEC strains. These data suggest that stx2a and stx2c were distributed not only in O145 and O157 but also in minor O-genotypes of STEC in Argentina.     

The role of rpoS,hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H₂O₂, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGΔ3 (SGΔrpoSΔhmpΔssrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGΔ3 mutant did not cause any mortality after inoculation with either 1 × 10⁶ or 1 × 10⁸ colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGΔ3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGΔ3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGΔ3 could be a promising candidate for a live Salmonella vaccine against FT.