Effects of N-Acetylimidazole on Oxytocin Binding in Bovine Mammary Tissue

Abstract

The effects of N-acetylimidazole on specific binding of oxytocin to microsomal fractions of bovine mammary gland were studied. N-acetylimidazole suppressed oxytocin binding, with time and concentration dependence. Decreased oxytocin binding activity appeared to be due to decreased affinity of the hormone for its receptor. Acetylation of oxytocin, rather than of oxytocin receptors, seemed to be responsible for the decreased binding.     

Dynamics of Insulin Action on Mammary Epithelium

WE have found that, without entering its target cells, insulin elicits biological effects by impinging on the cell membrane. Furthermore, prolonged contact between hormone and mammary cell membrane is not necessary; transient contact through collisions suffices to produce a biological response.     

Oxytocin Receptors in Bovine Mammary Tissue

Abstract

Oxytocin receptors were identified and characterized in bovine mammary tissue. [³H]-oxytocin was specifically bound to the 105,000 × g particulate fractions from 5 lactating cows and 5 non-lactating cows. Binding reached equilibrium by 50 min at 20°C and by 8 hr at 4°C. The half-time of displacement at 20°C was approximately 1 hr. ACTH, TRH, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive in the dose range tested at 20°C. The ability of other peptides to inhibit ³H-oxytocin binding was as follows: oxytocin > vasotocin > arginine - vasopressin >lysine - vaso-pressin > Pen¹ Phe² Thr⁴ - oxytocin. The Kd of the oxytocin receptor averaged 1.66 ± 1.19 nMol/L for lactating cows and 0.97± 0.49 nMol/L for non-lactating cows, respectively. The maximum number of binding sites was 0.14 ± 0.12 nM/mg protein and 0.15 ± 0.08 nM/mg protein for lactating cows and non-lactating cows, respectively. Identification and characterization of these receptors now makes it possible to study the dynamics of hormonal binding throughout various physiological states of the animal.     

Regulation of Autophagy in Bovine Mammary Epithelial Cells

The bovine mammary gland undergoes intensive remodeling during the lactation cycle, and the escalation of this process is observed during dry periods. The main type of cell death responsible for bovine mammary gland involution is apoptosis; however, there are also a lot of cells exhibiting morphological features of autophagy during drying off. Our in vitro and in vivo studies of bovine mammary gland physiology suggest that the enhanced process of autophagy, observed at the end of lactation and during dry periods, is the result of: (1) decreased level of lactogenic hormones (GH, IGF-I), (2) decreased GH-R and IGF-IRα expression, (3) increased expression of auto/paracrine apoptogenic peptides (IGFBPs, TGF-β1), (4) increased influence of sex steroids (17β-estradiol and progesterone) and (5) enhanced competition between the intensively developing fetus and the mother organism for nutritional and bioactive compounds. The above conditions may create a state of temporary malnutrition of mammary epithelial cells, which forces the cells to the induction of autophagy, as a mechanism for stabilizing intracellular supplies of energy and amino acids, especially during the enhanced activity of apoptogenic factors.

Addendum to:

Apoptosis and Autophagy in Mammary Gland Remodeling and Breast Cancer Chemotherapy

T. Motyl, B. Gajkowska, J. Zarzyńska, M. Gajewska and M. Lamparska-Przybysz

J Physiol Pharmacol 2006; 57:17-32     

Murine Mammary Tumor Virus Expression During Mammary Tumorigenesis in BALB/c Mice

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.     

乳铁蛋白肽基因的合成及其在动物乳腺中的表达

根据已发表的编码牛乳铁蛋白肽(LfcinB)25个氨基酸的mRNA序列,设计了4条用于合成牛乳铁蛋白肽全基因的引物,用重叠延伸PCR法合成149 bp的牛乳铁蛋白肽基因(包括牛乳铁蛋白信号肽序列、上下游酶切位点和终止密码子),并将此基因克隆到载体pI-B,获得了乳腺特异性表达质粒pI-BL,该质粒经生理盐水稀释后直接注入稳定泌乳期奶山羊和奶牛乳腺组织(注射量为400μg),以琼脂板溶圈法检测奶样抑菌活性。结果显示,注射后3~48 h,奶样有明显抑菌活性,其中3~9 h抑菌活性最高。     

奶山羊不同发育时期乳腺上皮细胞中内质网和线粒体的变化

为研究奶山羊乳腺的胚后形态发育,采用活细胞荧光标记法结合激光共聚焦显微技术,观察奶山羊乳腺发育中内质网和线粒体的变化.结果显示:腺上皮细胞内质网和线粒体在妊娠初期和中期数量较少,妊娠晚期开始逐步增加,泌乳中期达到高峰并维持在一个较高的水平上,退化期迅速下降,逐渐恢复到妊娠前状态;退化期乳腺上皮细胞内仍保持一定数量的细胞器,以维持细胞的正常机能和活动.     

利用多重筛选机制提高山羊乳腺上皮细胞基因打靶的效率

构建了山羊β-酪蛋白基因座基因打靶载体pGBC—GFP—neo,载体包含了正负筛选标记基因neo和tk,以及无启动子的GFP基因。打靶载体线性化后用脂质体包裹转染山羊乳腺上皮细胞,利用G418和GANC进行抗性细胞克隆的药物筛选,共得到抗性细胞克隆51个,对抗性克隆利用激素诱导β-酪蛋白基因启动子指导GFP表达,得到GFP表达阳性细胞克隆17个,对其中4个生长状态良好的克隆PCR检测验证后用于核移植,克隆胚发育率为59．5％,部分发育到桑椹胚或囊胚。     

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.     

Structures Indicative of Keratinization in Lactating Cells of Bovine Mammary Gland

Keratohyalin granule-like aggregates and tangles of filaments similar to epidermal prekeratin fibrils have been observed in lactating cells of bovine mammary epithelium. The concomitant occurrence of such structures, which are characteristic of early stages of keratinization, with typical secretory products such as casein micelles demonstrates that keratinization can take place in functional secretory cells. The observations are discussed in relation to current concepts of keratinization in epidermal and in non-epidermal epithelia.     

甲状腺素T4对体外高温培养奶牛乳腺上皮细胞生长和凋亡的影响

以体外培养的奶牛乳腺上皮细胞为模型,采用台盼蓝染色绘制生长曲线,细胞流式检测细胞凋亡,以正常培养温度(38℃)为对照,研究体外高温培养条件下(42℃),添加不同浓度(0.01、01和1mol/L)甲状腺素(thyroxine,T4)对细胞生长和凋亡的影响.结果表明,不同浓度的T4在38℃有促进奶牛乳腺上皮细胞生长的趋势,但是变化不显著(P>0.05),而T4对缓解高温所造成的乳腺上皮细胞的生长抑制作用也不显著(P>0.05);不同的T4都能够极显著缓解42℃培养1 h和3 h的奶牛乳腺上皮细胞的凋亡(P<0.01),并且对缓解42℃培养3 h的细胞凋亡效果更加明显,但是对42℃培养5 h和8 h的细胞,仅1 μmol/L的T4能够极显著缓解其凋亡(P<0.01).结果提示,T4对高温造成的奶牛乳腺上皮细胞的生长抑制没有明显的缓解作用,但能缓解高温诱导的奶牛乳腺上皮细胞凋亡.     

Effects of Chain Length and Saturability of Fatty Acids on Phospholipids and Proteins in Plasma Membranes of Bovine Mammary Gland

Free fatty acids (FFAs) in plasma are essential substrates for de novo synthesis of milk fat, or directly import into mammary cells. The physico-chemical properties of mammary cells membrane composition affected by FFAs with different chain lengths and saturability are unclear yet. Employing GC, FTIR and fluorescence spectroscopy, the adsorption capacity, phospholipids content, membrane proteins conformation, lipid peroxidation product, and free sulfhydryl of plasma membranes (PMs) interacted with different FFAs were determined. The mammary cells PMs at 38 and 39.5 °C showed different adsorption capacities: acetic acid (Ac) > stearic acid (SA) > β-hydroxybutyric acid (BHBA) > trans10, cis12 CLA. In the FTIR spectrum, the major adsorption peaks appeared at 2920 and 2850 cm^?1 for phospholipids, and at 1628 and 1560 cm^?1 for membrane proteins. The intensities of PMs-FFAs complexes were varied with the FFAs species and their initial concentrations. The β-sheet and turn structures of membrane proteins were transferred into random coil and α-helix after BHBA, SA and trans10, cis12 CLA treatments compared with Ac treatment. The quenching effects on the fluorescence of endogenous membrane protein, 1, 8-ANS, NBD-PE, and DHPE entrapped in PMs by LCFA were different from those of short chain FFAs. These results indicate that the adsorption of FFAs could change membrane protein conformation and polarity of head group in phospholipids. This variation of the mammary cells PMs was regulated by carbon chain length and saturability of FFAs.     

Cloning and Expression of Bovine imc-415 cDNA from Mammary Gland

Bovine imc-415 cDNA was cloned from mammary gland using RACE PCR; it coded for 245 amino acids. The deduced amino acid sequence of mouse and human showed about 94% identity. Expression of bovine imc-415 increased about 40% in involuted mammary tissues compared with lactating tissues.     

牛乳腺上皮细胞的分离培养及其生物学特性

采用胶原酶消化法和胰蛋白酶选择性消化法分离、培养和纯化牛乳腺上皮细胞。形态学观察表明,培养的细胞具有典型的上皮细胞形态特征;染色体分析结果表明,培养的细胞具有正常的染色体数目。通过荧光免疫细胞染色方法鉴定了培养的细胞表达上皮细胞特异的角蛋白5和8。该细胞在添加胰岛素、氢化可的松以及羊催乳素的无血清培养液中诱导培养时,用RT-PCR方法检测到了β-酪蛋白基因的转录。这些结果表明,分离培养的细胞是乳腺上皮细胞,这些细胞在诱导培养的条件下能够转录表达β-酪蛋白。     

Murine Mammary Carcinomas Induced by Intravaginal Applications of Methylcholanthrene in Minimal Dosages

Mammary cancer occurred in five of 80 ICR Swiss mice and two of 52 phenotypically normal pituitary dwarf strain mice following intravaginal applications of 20-methylcholanthrene in doses totalling 2.0 to 5.5 mg. Twenty-two of these 132 methylcholanthrene-treated mice had lower genital tract cancer. One acetone-treated Swiss mouse developed mammary cancer. No genital tract or mammary cancer occurred in mice receiving less than 2.0 mg. methylcholanthrene. The carcinogen was administered when mice were young; mammary cancers appeared between 65 and 167 days after methylcholanthrene application was begun. This study provides additional examples of carcinogenesis in organs remote from sites of carcinogen treatment with carcinogen doses close to threshold levels for treated tissues.     

Characterization of Plasma Membrane Proteins from Lactating Bovine Mammary Gland

Properties of proteins in the light (<d 1.03 on Ficoll and then <d 1.14 on sucrose) and heavy (d 1.03/1.05 on Ficoll and then <d 1.14 on sucrose) plasma membranes (PM) isolated from lactating bovine mammary gland were investigated. The PMs consisted of 57 ~ 62% of protein and 38 ~ 43% lipid. The lipid/protein ratio was 0.77 in the light PM and higher than 0.61 of the heavy PM. However, no appreciable differences were found between the light and heavy PMs with respect to polypeptide, amino acid, and carbohydrate compositions. The protein moiety contained approximate 5 ~ 6% carbohydrate: fucose 8 ~ 9, mannose 11 ~ 12, galactose 16, N-acetylglucosamine 32,N-acetylgalactosamine 12 ~ 15, and sialic acid 17~20 mol%. PM protein was high in the content of aspartic acid, glutamic acid, and leucine and low in proline, cystine, methionine, and histidine. On double immunodiffusion both PMs formed precipitin lines against milk fat globule membrane (MFGM) antiserum. Electrophoretic analysis on sodium dodecyl sulfate-polyacrylamide gel revealed the presence of many minor polypeptides and three major glycopeptides (PAS-I, II, and III) of molecular weights of 115,000, 94,000, and 82,000. The glycoprotein profiles of the PMs were different from those of MFGM, except that PAS-II and -III of PM corresponded to PAS-3 and -4 of MFGM, respectively.     

Bovine Mammary Gene Expression Profiling during the Onset of Lactation

Background

Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards.

Methodology/Principal Findings

To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE) on bovine mammary tissue at three time points (on approximately day 35 before parturition (−35 d), day 7 before parturition (−7 d) and day 3 after parturition (+3 d)). Approximately 6.2 million (M), 5.8 million (M) and 6.1 million (M) 21-nt cDNA tags were sequenced in the three cDNA libraries (−35 d, −7 d and +3 d), respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥2 or ≤−2 and a false discovery rate (FDR) of ≤0.001, a total of 812 genes were significantly differentially expressed at −7 d compared with −35 d (stage I). Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with −7 d (stage II), and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with −35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes.

Conclusions

The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cell''s resources towards lactation.