转基因拟南芥中雌激素诱导的转录激活系统诱导条件的优化

[目的]探索雌激素诱导的转录激活系统（XVE）在拟南芥中高效表达外源蛋白的条件.[方法]构建XVE-AtNF-YA10：3×flag植物表达载体,转化拟南芥并获得了阳性植株;采用3种不同的诱导表达条件对外源蛋白的表达量进行检测.[结果] 1/2MS培养基生长10 d后的拟南芥转基因苗外源融合蛋白表达量最高,进一步研究表明1/2MS培养基生长10 d后的拟南芥转基因苗在受诱导后8、16、24和32 h外源融合蛋白均有表达,在16 h时表达量达到最高,并持续到32 h.[结论]AtNF-YA10：3×flag融合外源基因最优表达条件为1/2MS培养基生长10 d后的拟南芥转基因苗雌激素诱导16 h.     

Pregnancy recognition signaling mechanisms in ruminants and pigs

Maternal recognition of pregnancy refers to the requirement for the conceptus (embryo and its associated extra-embryonic membranes) to produce a hormone that acts on the uterus and/or corpus luteum (CL) to ensure maintenance of a functional CL for production of progesterone; the hormone required for pregnancy in most mammals. The pregnancy recognition signal in primates is chorionic gonadotrophin which acts directly on the CL via luteinizing hormone receptors to ensure maintenance of functional CL during pregnancy. In ruminants, interferon tau (IFNT) is the pregnancy recognition signal. IFNT is secreted during the peri-implantation period of pregnancy and acts on uterine epithelia to silence expression of estrogen receptor alpha and oxytocin receptor which abrogates the oxytocin-dependent release of luteolytic pulses of prostaglandin F2-alpha (PGF) by uterine epithelia; therefore, the CL continues to produce progesterone required for pregnancy. Pig conceptuses secrete interferon delta and interferon gamma during the peri-implantation period of pregnancy, but there is no evidence that they are involved in pregnancy recognition signaling. Rather, pig conceptuses secrete abundant amounts of estrogens between Days 11 to 15 of pregnancy required for maternal recognition of pregnancy. Estrogen, likely in concert with prolactin, prevents secretion of PGF into the uterine venous drainage (endocrine secretion), but maintains secretion of PGF into the uterine lumen (exocrine secretion) where it is metabolized to a form that is not luteolytic. Since PGF is sequestered within the uterine lumen and unavailable to induce luteolysis, functional CL are maintained for production of progesterone. In addition to effects of chorionic gonadotrophin, IFNT and estrogens to signal pregnancy recognition, these hormones act on uterine epithelia to enhance expression of genes critical for growth and development of the conceptus.     

Rat Uterine Oxytocin Receptor and Estrogen Receptor α and β mRNA Levels are Regulated by Estrogen Through Multiple Estrogen Receptors

Estrogen action is mediated through several types of receptors (ERs), such as ERα, ERβ and putative membrane ERs. Oxytocin receptor (OTR) and ER expression levels in the rat uterus are regulated by estrogen; however, which types of ERs are involved has not been elucidated. This study examined OTR, ERα and ERβ levels in ovariectomized rats treated with 17β-estradiol (E2), an ERα agonist (PPT), an ERβ agonist (DPN) or estren (Es). E2 and PPT increased OTR mRNA levels and decreased ERα and ERβ mRNA levels 3 and 6 h posttreatment. DPN decreased ERα and ERβ mRNA levels at 3 and 6 h, while OTR mRNA levels increased at 3 h and decreased at 6 h. OTR mRNA levels increased 3 h after the Es treatment and then declined until 6 h. ERα and ERβ mRNA levels decreased by 3 h and remained low until 6 h posttreatment with Es. The ER antagonist ICI182,780 (ICI) suppressed the increases in OTR mRNA levels induced 3 h after the Es treatment. However, ICI and tamoxifen (Tam) had no significant effect on ERα and ERβ mRNA levels in the Es-treated or vehicle-treated group. In intact rats, proestrus-associated increases in OTR mRNA levels were antagonized by both ICI and Tam. However, decreases in ERα and ERβ mRNA levels were not antagonized by Tam and ICI, respectively. Therefore, uterine OTR gene expression is upregulated by estrogen through the classical nuclear (or non-nuclear) ERs, ERα and ERβ, while the levels of these ERs are downregulated by estrogen through multiple pathways including Es-sensitive nonclassical ERs.     

Exogenous estradiol improves shell strength in laying hens at the end of the laying period

Background

Cracked shells, due to age related reduction of shell quality, are a costly problem for the industry. Parallel to reduced shell quality the skeleton becomes brittle resulting in bone fractures. Calcium, a main prerequisite for both eggshell and bone, is regulated by estrogen in a complex manner. The effects of estrogen, given in a low continuous dose, were studied regarding factors involved in age related changes in shell quality and bone strength of laying hens. A pellet containing 0.385 mg estradiol 3-benzoate (21-day-release) or placebo was inserted subcutaneously in 20 birds each of Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB) at 70 weeks of age. Eggs were collected before and during the experiment for shell quality measurements. Blood samples for analysis of total calcium were taken three days after the insertion and at sacrifice (72 weeks). Right femur was used for bone strength measurements and tissue samples from duodenum and shell gland were processed for morphology, immunohistochemical localization of estrogen receptors (ERα, ERβ), plasma membrane calcium ATPase (PMCA) and histochemical localization of carbonic anhydrase (CA).

Results

Estrogen treatment increased shell thickness of both hybrids. In addition, shell weight and shell deformation improved in eggs from the brown hybrids. The more pronounced effect on eggs from the brown hybrid may be due to a change in sensitivity to estrogen, especially in surface epithelial cells of the shell gland, shown as an altered ratio between ERα and ERβ. A regulatory effect of estrogen on CA activity, but not PMCA, was seen in both duodenum and shell gland, and a possible connection to shell quality is discussed. Bone strength was unaffected by treatment, but femur was stronger in LSL birds suggesting that the hybrids differ in calcium allocation between shell and bone at the end of the laying period. Plasma calcium concentrations and egg production were unaffected.

Conclusions

A low continuous dose of estrogen improves shell strength but not bone strength in laying hens at the end of the laying period.     

Alteration of the Endometrial EGF Profile as a Potential Mechanism Connecting the Alterations in the Ovarian Steroid Hormone Profile to Embryonic Loss in Repeat Breeders and High-producing Cows

Poor reproductive efficiency is a worldwide problem that has affected the dairy industry during the last several decades. In an attempt to explain the changes in reproductive physiology caused by high milk production, a model of elevated steroid metabolism in lactating dairy cows has been proposed. A slow increase in levels and low peak levels of estradiol (E₂) and progesterone (P₄) characterize endocrine changes in high producing cows. Similar changes have been reported in the repeat breeder cows. The abnormal changes in E₂ and P₄ concentrations of these cows may cause an improper uterine environment due to disturbed expression of growth factors and cytokines in the endometrium. This review focuses on the alteration in epidermal growth factor (EGF) profile in the endometrium during the estrous cycle. The normal cow has two peaks of EGF concentrations on days 2–4 and 13–14. Low concentrations of EGF on these days distinguished both high-producing and repeat breeder cows from normal cows. Alteration of the EGF profile could be found in 70 and 40% of the repeat breeder and high-producing cows, respectively. Treatment with a high dose of estradiol benzoate and an intravaginal progesterone-releasing device restored the normal EGF profile in about 70% of the affected cows. The cows having a normal EGF profile after treatment showed a higher pregnancy rate than the cows with the altered profile. Further studies to understand the etiology of the alteration in the EGF profile are needed to develop another treatment option and preventive management for this problem.     

IGF-I retards proper development of acinar structures formed by bovine mammary epithelial cells via sustained activation of Akt kinase

Insulin-like growth factor-I is involved in mammary gland development, promoting proliferation and inhibiting apoptosis of mammary epithelial cells (MECs). Mitogenic actions of IGF-I are mainly mediated by the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. We have found that in the presence of IGF-I bovine BME-UV1 MECs cultured on reconstituted basement membrane form large spheroids with disrupted polarity and no cavity in the center. These cells showed enhanced phosphorylation of Akt, decreased level of cleaved caspase-3, and sustained proliferative activity throughout the 16-d period of 3-dimensional culture. Inhibition of the PI3K/Akt pathway by a specific inhibitor of PI3K, LY294002, resulted in the restoration of the normal acinar phenotype. However, this effect was noted only when LY294002 was added in the second week of 3-dimensional culture, which corresponded with the time of cell cycle arrest and polarity formation under control conditions. Normal development of acini was also obtained when BME-UV1 cells were treated simultaneously with IGF-I and 17β-estradiol. The addition of 17β-estradiol regulated Akt activation, enabling the subsequent initiation of polarization processes. 17β-Estradiol also increased the level of IGFBP-3 protein in MECs cultured on Matrigel in the presence of IGF-I. The presented results indicate important interactions between signaling pathways activated by estrogen and IGF-I, which regulate alveologenesis in bovine mammary gland.     

Ganoderic acid DM, a natural triterpenoid, induces DNA damage, G1 cell cycle arrest and apoptosis in human breast cancer cells

Ganoderic acid DM (GADM) is a triterpenoid isolated from Ganoderma lucidum, a well-known edible medicinal mushroom. In the present study, we found that GADM effectively inhibited cell proliferation and colony formation in MCF-7 human breast cancer cells, which was much stronger than that of MDA-MB-231 breast cancer cells. GADM both concentration- and time-dependently mediated G1 cell cycle arrest and significantly decreased the protein level of CDK2, CDK6, cycle D1, p-Rb and c-Myc in MCF-7 cells. Moreover, GADM obviously induced DNA fragmentation and cleavage of PARP which are the characteristics of apoptosis and decreased the mitochondrial membrane potential in MCF-7 cells. Besides, we also showed that GADM elicited DNA damage as measured by comet assay which is a sensitive method for DNA damage detection. γ-H2AX, a marker of DNA damage, was also slightly up-regulated after treated with GADM for 6h, suggesting that the G1 cell cycle arrest and apoptosis induced by GADM may be partially resulted from GADM-induced DNA damage. These results have advanced our current understandings of the anti-cancer mechanisms of GADM.     

早期给予雌激素对去卵巢兔动脉斑块及血脂、单核细胞趋化蛋白1的影响

目的观察早期不同剂量雌激素替代治疗对高脂饮食诱导的去卵巢雌兔动脉粥样硬化及血脂、单核细胞趋化蛋白1的影响。方法健康雌性新西兰白兔28只，随机分为假手术组、去卵巢组、去卵巢＋小剂量雌激素替代治疗组（苯甲酸雌二醇200μg）和去卵巢＋大剂量雌激素替代治疗组（苯甲酸雌二醇1mg）。术后给予高脂饲料喂养，测定血清总胆固醇、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、雌二醇和单核细胞趋化蛋白1浓度，并分离主动脉标本行组织形态学观察。结果与假手术组比较，去卵巢组高脂喂食后血清总胆固醇、低密度脂蛋白胆固醇、单核细胞趋化蛋白1均明显升高，血清高密度脂蛋白胆固醇、雌二醇水平下降；而雌激素替代治疗组的上述指标与假手术组相似；与小剂量雌激素替代治疗组比较，大剂量雌激素替代治疗组12周后雌二醇水平较高、单核细胞趋化蛋白1水平较低。比较主动脉斑块面积，去卵巢组大于假手术组，而雌激素替代治疗两组明显小于去卵巢组和假手术组，但雌激素替代治疗两组之间差异无显著性。主动脉斑块面积与高脂喂养12周后血清低密度脂蛋白胆固醇、总胆固醇以及单核细胞趋化蛋白1水平呈明显正相关（r分别为0．765，0．803，0．829，P均〈0．01），与血清高密度脂蛋白胆固醇（r=-0．441，P〈0．05）、雌二醇水平呈负相关（r=-0．755，P〈0．01）。结论早期雌激素替代治疗可改善血脂代谢、降低单核细胞趋化蛋白1水平、减少主动脉斑块面积。雌激素的调脂、抗炎作用可能与其抗动脉粥样硬化作用有关。