基質金屬蛋白酶-2, -9,其抑制因子及類固醇賀爾蒙在荷蘭牛及時排出胎膜之角色

牛胎盤被歸類為子葉狀 synepitheliochorial 類型，其結構形成直接連繫了胎 兒母體與名為胎盤節(placentomes)之特定區域，由胎兒子葉(fetal cotyledons)及母 體宮阜(maternal caruncles)交錯而成。胎盤節在小牛分娩後胎膜的排出扮演一個重 要的角色，其機制乃藉由調節基質金屬蛋白酶(matrix metalloproteinases, MMPs) 之活性影響細胞外間質架構，以及分娩末期之類固醇荷爾蒙之產生。MMPs 的作用會鬆 動胎盤上胎兒與母體交互作用以調節產後之胎膜分離。明膠酶(gelatinase)為 MMP 子 家族之一，其包含明膠酶 A (MMP-2)及明膠酶 B (MMP-9)，兩者皆涉及了細胞外基質 (extracellular matrix, ECM) 之降解。明膠酶活性調節會發生在幾個層面：基因轉 錄、轉譯後活性，以及 MMP 組織抑制因子(tissue inhibitors of MMP, TIMP) 之作用。 明膠酶在牛隻胎盤及時釋出之調節尚未定論，部份原因是由於明膠酶有多種形式存在， 包括酶元形式、活化形式，以及複合形式。為了描繪明膠酶調節正常牛隻胎盤及時釋出之角色，即牛隻未滯留胎盤之排出，使用 明膠酶譜(zymography)法來鑑定其明膠酶亞型，RT-PCR 則用於定量明膠酶基因表達和 TIMP-1 及 TIMP-2 之可能抑制作用。此外，類固醇荷爾蒙之濃度，包括助孕素 (progesterone)、雌二醇(estradiol)及皮質醇(cortisol)，皆利用 ELISA 試劑同時測 定。 十一頭荷蘭奶牛於小牛排出後十二小時內收集其胎盤組織，胎膜依不同區域分 別測定，包含尿囊層(allantoic layers)、絨毛膜層(allantoic layers)及子葉 (cotyledons)，樣品分別取樣自胎盤中央及周圍區域，以斷裂臍帶為橫切面，分胎兒 端、中段及胎盤端。體液包含來自臨產奶牛、出生小牛和臍帶之血液，以及胎兒液體 和初乳都將進行測定。明膠酶譜法結果顯示:在體液及胎盤組織中，相較於活化狀態或 NGAL-MMP-9 複 合物，MMP-2 及 MMP-9 之酶元形式為有最大量活性且最頻繁地被偵測到。ProMMP-9 於 中央胎層 (尿囊層和絨毛膜) 及子葉結構之活性高於周圍區域。此外，proMMP-2 於臍 帶斷裂端之活性高於胎盤端。在 RT-PCR 分析，MMP-2 及 TIMP-2 基因表達於胎膜中易被 檢測出，而 MMP-9 或 TIMP-1 在任一區域中皆無表達。在所有 MMP 及 TIMP 基因表中， 臍帶表達水平相較於胎膜各結構對明顯較低。MMP-2/GAPDH、TIMP-2/GAPDH 及 TIMP- 2/MMP-2 比值，在尿囊層、絨毛膜層和子葉結構之中央及周圍區域之間，或在不同臍帶 橫切面皆無顯著差異，然而在胎膜中則會高於臍帶。此外，胎膜中央及周圍區域之類 固醇荷爾蒙濃度，在尿囊層、絨毛膜層、子葉結構及臍帶切面皆無差異。另一方面， 尿囊層之助孕素濃度分別顯著 (P < 0.05) 高於絨毛膜層及子葉，並且絨毛膜層之助 孕素濃度亦高於子葉結構 (P < 0.05)。 總結而言，明膠酶譜法分析顯示:牛隻未滯留胎盤至臍帶附著之距離，其 proMMP-9 活性呈現一反向方梯度。此外，RT-PCR 結果證實， MMP-2 及 TIMP-2 為重要 影響因子，有助於胎膜於正常胎盤中釋出。在胎層 (尿囊層、絨毛膜層) 及子葉結構 之間，MMP-2 和 TIMP-2 表達之差異，顯示指出兩者於胎盤重塑之協調作用極為密切。 而牛胎膜類固醇荷爾蒙濃度之改變，可能反應了其小牛排出後及時協調胎膜釋放之角 色。The bovine placenta is classified into the cotyledonary synepitheliochorial type. The structure forms direct feto-maternal contact with specialized zones called placentomes, consisting of fetal cotyledons interdigitating with maternal caruncles. The placentomes play an essential role in the release of fetal membranes after calf expulsion by regulating the proteolytic activity of MMPs and the extracellular architecture, as well as steroid hormones at the end of parturition. Matrix metalloproteinases (MMP) mediate the detachment of fetal membrane postpartum through loosening the feto-maternal interaction of placenta. Gelatinases, a subfamily of MMP, include gelatinase A (MMP-2) and gelatinase B (MMP-9) and both participate in the degradation of extracellular matrix (ECM). The activity of gelatinase is regulated at several levels: gene level, post-translational activation, and the counteraction with tissue inhibitor of MMP (TIMP). Regulation of gelatinase in timely release of placenta in cattle is not yet conclusive partly due to the presence of multiple forms of gelatinases in situ, including proenzyme forms, active forms, and complex forms. To delineate the regulation of gelatinase in timely release of placenta in cattle, gelatin zymography was used to fingerprint the subtypes of gelatinases on the entire topography of cow non-retaining placenta and RT-PCR method was applied to estimate the mRNA expression of gelatinases and the counteracting TIMP-1 and TIMP-2. Also, the concentration of steroid hormones including progesterone, estradiol and cortisol were simultaneously determined using ELISA kits. Eleven Holstein cows were used to collect placental tissues within 12 h after calf expulsion. Different topographic regions on the fetal membrane were measured separately, including the allantoic layers, chorionic layers, and cotyledons, and sampled from the central and peripheral areas of the placenta, respectively. The spontaneously ruptured umbilical cords were cross-sectioned as fetus end, middle and placenta end. Body fluids including blood samples from the parturient cows, their neonatal calves and umbilical cord, as well as fetal fluids and the first colostrum were measured. Results of gelatin zymography fingerprinting showed that the proenzyme forms of MMP-2 and MMP-9 were the most abundantly and frequently detected gelatinase species in both body fluid and placenta tissues than the active forms or the NGAL-MMP-9 complex. The activity of proMMP-9 in that central fetal layer (allantoic layers and chorionic layers) and cotyledon structure was higher than that in the peripheral area. In addition, proMMP-2 activity was higher in the rupture end than that of the placenta end of the umbilical cord. In RT-PCR analysis, expression of MMP-2 and TIMP-2 gene was easily detectable in fetal membranes but no expression of MMP-9 or TIMP-1 was detected in either region. Umbilical cord expressed a relatively low level of all MMP and TIMP genes when compared with fetal membranes. Neither, MMP-2/GAPDH, TIMP-2/GAPDH, nor TIMP-2/MMP-2 differed significantly between the central and peripheral regions in the allantoic layer, chorionic layer, or cotyledon structure, or among different umbilical cord sections, but was found higher in the proper fetal membrane than in the umbilical cord. In addition, concentrations of steroid hormones in the central and peripheral region of fetal membrane were not different in the allantoic layers, chorionic layers, cotyledons, and umbilical cord sections. On the other hand, progesterone concentration in allantoic layers was significantly (P < 0.05) higher than that in the chorionic layers and cotyledons, respectively, and the concentration of progesterone in the chorionic layers was also higher (P < 0.05) than the cotyledon structure. In conclusion, gelatin zymography analysis demonstrated an apparent gradient of proMMP-9 level on the non-retaining placenta of cows in a reverse fashion to the distance to the umbilical insertion point, and proMMP-2 level of umbilical cord was expressed in a reverse fashion to the distance to the rupture site, suggesting the differential activity of gelatinases in the detachment of fetal membrane at term. In addition, RT-PCR results demonstrated that MMP-2 and TIMP-2 as important local effectors contributing to fetal membranes in normal placenta release. The differential expression of MMP-2 and TIMP-2 between fetal layers (allantoic layer, chorionic layer) and cotyledon structure, suggested their closely coordination in placenta remodeling. Changes of steroid hormone levels in topographic subparts of bovine fetal membranes may reflect timely coordinated release following calf expulsion.Page ACKNOWLEDGMENTS………………………………………i ABSTRACT…………………………………………………ii TABLE OF CONTENTS………………………………………vii LIST OF TABLES……………………………………………xi LIST OF FIGURES……………………………………………xii CHAPTER 1: Literature review...…………………………………………1 1. Bovine placenta……………………2 1.1 Formation of the bovine placenta……………………3 1.2 Gross morphology of the bovine placenta ………………3 1.3 Placenta component……………………………………6 1.3.1 Fetal membrane……………………………………6 1.3.2 Placentome………………………………………8 1.4 Placenta remodeling during parturition……………………10 1.5 Placenta detachment…………………………………….………12 1.6 Retained fetal membrane………………………..…………….15 2. Matrix metalloproteinase (MMPs………………………………19 2.1 MMP structure and classification………………………20 2.2 Regulation of MMPs……………………………………25 3. Tissue inhibitors of metalloproteinases (TIMPs)…………26 3.1 The involvement of MMP/TIMP in RFM………………………27 4. Steroid hormone on placenta……………………………………28 4.1 Steroid hormones and retained placenta……………………29 4.2 The relationship between steroid hormones and MMP……30 CHAPTER 2: Fingerprinting of gelatinase subtypes for different topographic regions on non-retaining placenta of Holstein cows…..………………………...………………..…...........31 1. Abstract………………………………….…..……...…………32 2. Introduction…………………………………….…………………33 3. Materials and Methods……….………..……….………………37 3.1Animals………………………………………………………………37 3.2 Bovine fluid samples collection and separation……....37 3.3 Bovine placenta tissues collection and preparation……39 3.4 Extraction protein of tissue samples……………………..39 3.5 Protein concentration determination……………………….40 3.6 Protein electrophoresis………………………..………...…40 3.7 Gelatin zymography……………..…….………...………….42 3.8 Statistical analyses………………………………….………43 4. Results………………………………………………..……..……44 4.1 Gelatinase production in the bovine fluids and placenta tissues 44 4.2 Expression of gelatinase subtype in placenta compartments and body fluids in arbitrary unit......….. 46 4.3 Expression of gelatinase subtypes in body fluid collected……..…...…. 48 4.4 Expression of gelatinase subtypes in fetal membrane of Holstein cows 50 4.5 Expression of gelatinase subtypes in different topographic locations…. 52 4.6 Expression of gelatinase subtypes in different topographic sections of umbilical cord…..……………………54 5. Discussion ………………..…...………………………..... 56 CHAPTER 3: Topographic expression of gelatinases, their inhibitor in spontaneously released term membrane on non-retaining placenta of Holstein cows…………………….…..……. 63 1. Abstract………………………………….…………..….... 64 2. Introduction………………………………………….……………65 3. Materials and Methods……………………………………….67 3.1 Animals and management…………………………….………….67 3.2 Dissection of fetal membranes ………………............67 3.3 Extraction of mRNA……………………………………….…...68 3.4 Reverse transcription (RT) and PCR…………………......69 3.5 Statistical analysis……………………………………………70 4. Results………………………………………………………....…71 4.1 Agarose gel images of mRNA expression of topographic subparts……71 4.2 Gene expression of GADPH, MMP-2 and TIMP-2 in bovine placenta.. 73 5. Discussion ……………………………………........………75   CHAPTER 4: Topographic profiles of steroid hormones in spontaneously released term fetal membranes of Holstein cows…………………….………………………….……………………78 1. Abstract…………………………...…..…….…………………79 2. Introduction…………………………….…………………………80 3. Materials and Methods…………………….……………………82 3.1 Animals and management……………….……………………….82 3.2 Dissection of fetal membranes………………….……………82 3.3 Tissue processing……………………………….………………83 3.4 Protein content determination.…………………….……...83 3.5 Determination of steroids hormones……………….………83 3.6 Statistical analyses………………………………….………85 4. Results……………………………………………………….……86 4.1 ELISA measurements……………………………….…………...86 4.2 Effects of central or peripheral region on the steroid hormone concentrations of fetal layers and cotyledon structure……….…………………….……... 88 4.3 Effects of regions, as well as fetal layers and cotyledon structure on the steroid hormone concentrations of placenta tissues………………..………..….90 5. Discussion ………………..…...………..……….……..…92 CONCLUSIONS……………………..…………………….………………94 APPENDICES………………………………………………………………96 REFERENCES……………………..……….……………………………10

Systemic and local bactericidal potentiality in late lactation Holstein-Friesian cows following a combined antibiotics and Enterococcus faecium SF68 dry-cow treatment

Antibiotic dry-cow treatment contributes a major part to the total use of antibiotics in dairy herds. Enterococcus faecium strain SF68 (SF68) was of human origin but has been authorized in EU as probiotic feed additive. In the present study, one of the front and rear quarters of twelve late lactation Holstein-Friesian cows were infused once with a commercial antibiotic dry-cow formula (antibiotics quarter) on the first milk-stasis day (d 1), when the contrallateral quarters were infused with 5 × 108-CFU SF68 plus half-dose antibiotic dry-cow formula (SF68/antibiotics quarter) meanwhile. Gelatinase level and cellular reactive oxygen species (ROS) production capacity were measured for blood and quarter secretion. The results showed that the count of blood total leukocytes minorly decreased on d 3 only but the microscopic somatic cell count (MSCC) continuously increased up to d 7, especially in SF68/antibiotics quarters. Plasma level of gelatinase A remained similar up to d 7 but gelatinase B was not detectable in plasma throughout the study. The level of gelatinase A in quarter secretion increased up to d 7 but gelatinase B increased even more drastically, especially in SF68/antibiotics quarters. The ROS production capacity of blood leukocytes increased temporarily only on d 3, but that of milk cells continuously increased up to d 7, especially in SF68/antitiotics quarters. Overall, late lactation Holstein-Friesian cows were systemically adaptable to the combined antibiotics and SF68 dry-cow treatment, while the local bactericidal potentiality in mammary gland was actively responsive to additional SF68 intramammary treatment

Modifications of the defense and remodeling functionalities of bovine neutrophils inside the mammary gland of milk stasis cows received a commercial dry-cow treatment

An appropriate length of milk stasis between two consecutive lactations of dairy cows is crucial for sustainable milk production. This dry period of cows allows extensive remodeling and sufficient cell renewal in mammary gland. Nevertheless, early dry period is one of the most risky stages in cow lactation cycle to intramammary infection. Dry-cow treatment through teats is, therefore, widely practiced at the commencement of milk stasis. Neutrophils are the most abundant cellular components in cow mammary secretion during early dry period, which in turn attribute to the meanwhile elevation of somatic cell counts and matrix metalloproteinase-9 (MMP-9) level. This study used bovine peripheral neutrophils as a cell model to examine the mode of modifications in their defense and remodeling functionalities after infiltration into mammary gland during early dry period. Results indicate a dose-dependent suppression of phorbol 12-myristate 13-acetate-stimulated free radical production and induction of MMP 9 degranulation in bovine peripheral neutrophils exposure to the d 7-dry secretion of cows received dry cow treatment at d 0 in milk stasis. Meanwhile, an enhancement of plasminogen activation and TNF-alpha shedding on bovine peripheral neutrophils were also observed. These two cellular events might be involved in the functional modifications on infiltrated neutrophils during early dry period. In conclusion, the opposite trend of modifications in the defense and matrix remodeling functionalities of neutrophils inside the mammary gland of cows at early dry period reflect the collaboration of infiltrated neutrophils for promoting extensive glandular remodeling at minimum compromise of local defense during the acute involution period without apparent disturbance by dry cow treatment. (C) 2011 Elsevier B.V. All rights reserved

Modifications of the defense and remodeling functionalities of bovine neutrophils inside the mammary gland of milk stasis cows received a commercial dry-cow treatment

An appropriate length of milk stasis between two consecutive lactations of dairy cows is crucial for sustainable milk production. This dry period of cows allows extensive remodeling and sufficient cell renewal in mammary gland. Nevertheless, early dry period is one of the most risky stages in cow lactation cycle to intramammary infection. Dry-cow treatment through teats is, therefore, widely practiced at the commencement of milk stasis. Neutrophils are the most abundant cellular components in cow mammary secretion during early dry period, which in turn attribute to the meanwhile elevation of somatic cell counts and matrix metalloproteinase-9 (MMP-9) level. This study used bovine peripheral neutrophils as a cell model to examine the mode of modifications in their defense and remodeling functionalities after infiltration into mammary gland during early dry period. Results indicate a dose-dependent suppression of phorbol 12-myristate 13-acetate-stimulated free radical production and induction of MMP 9 degranulation in bovine peripheral neutrophils exposure to the d 7-dry secretion of cows received dry cow treatment at d 0 in milk stasis. Meanwhile, an enhancement of plasminogen activation and TNF-α shedding on bovine peripheral neutrophils were also observed. These two cellular events might be involved in the functional modifications on infiltrated neutrophils during early dry period. In conclusion, the opposite trend of modifications in the defense and matrix remodeling functionalities of neutrophils inside the mammary gland of cows at early dry period reflect the collaboration of infiltrated neutrophils for promoting extensive glandular remodeling at minimum compromise of local defense during the acute involution period without apparent disturbance by dry cow treatment