The influence of subclinical mastitis on the protein composition and protease activities of raw milk from lactating Thai-crossbred dairy cows

Background and Aim: Mastitis in dairy cattle is associated with a high rate of morbidity and death, which has major implications for milk production and quality. This study aimed to investigate the protein component and the activity of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in raw milk samples with different testing scores determined using the California mastitis test (CMT). Materials and Methods: Thirty cows were employed in the study, and milk from each quarter was tested for subclinical mastitis (SCM). According to the results of CMT, raw milk samples were classified into five categories: Healthy (score 0), trace (score T), weakly positive (score 1), distinctly positive (score 2), and strongly positive (score 3) for somatic cell count (SCC). The total milk protein was analyzed using the Bio-Rad protein assay, and the milk protein composition was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. In addition, gelatin zymography was used to evaluate changes in proteolytic abilities. Results: Milk samples with CMT scores of 1 and 3 had the highest total milk protein levels (32.25 ± 12.60 g/L and 32.50 ± 7.67 g/L, respectively), while the samples from healthy cows (CMT score 0) were only 6.75 ± 1.64 g/L. Globulin and lactoferrin were significantly increased in samples with a CMT score of 3 compared with those with other CMT scores. The bovine serum albumin level in samples with a CMT score of 2 was significantly (p < 0.05) higher than those with other CMT scores. No significant differences in casein abundance were found among samples with different CMT scores. Results from analysis of proteolytic activities demonstrated that the level of MMP-9 in samples with a CMT score of 3 was significantly (p < 0.05) higher than those with other CMT scores. Conclusion: The protein content and gelatinolytic activity of milk were drastically altered by the number of SCC, mainly due to SCM

小鼠子宮內膜Muc1於早期懷孕之調控與蛋白質表現之分析

摘要 Mucin 1（Muc1）為膜主體醣蛋白（integral membrane glycoprotein），表現於子宮等不同器官之分泌型或腺狀上皮細胞。子宮上皮細胞表面之Muc1表現消失有助於胎著床。此外，內源性18至24個核苷酸的非蛋白質編譯microRNAs（miRNAs）存在於多種有機物中，並藉由調節轉錄後基因表現，參予許多細胞表現調節過程。Muc1表現於本研究為一檢測指標，用以探討超級排卵（superovulation）時機對小鼠懷孕率之影響。正常情況下，與公鼠自然配種之母鼠其Muc1表現於懷孕第一天時最高，而後表現逐漸降低。然而，以即時聚合酶鏈鎖反應（real-time polymerase chain reaction, real-time PCR）分析發現，let-7a and let-7b miRNA表現則與Muc1相反。Luciferase分析顯示let-7a與let-7b miRNA直接與Muc1之3端UTR結合來調節Muc1表現。超級排卵常應用於增加卵子數目，然而對懷孕可能有不良影響。當不同動情週期（動情前期、動情期、動情後期與動情間期）之小鼠施予超級排卵後，動情前期（59%）與動情期（66%）之懷孕率顯著高於動情後期（23%）（P < 0.05）。動情前期與動情期表現較佳的配種率與懷孕率。我們推測可能與卵巢濾泡發育有關。因此，不同動情週期之小鼠經PMSG處理48小時後收集其卵巢。小鼠於動情前期與動情期施予超級排卵後之卵巢較動情後期與動情間期施打者有較多發育中濾泡與葛拉夫（Graafian）濾泡。相反的，動期後期與動期間期施予超級排卵之小鼠較動情前期與動情期者有較多的黃體（corpus luteum）（P < 0.05）。此外，動情前期與動情期組別之Muc1表現於懷孕第4天時顯著低於懷孕第1天（P < 0.05），然動情後期與動期間期組別並未有顯著差異。let-7a 與let-7b miRNA亦呈相反的表現方式。此外，亦利用二維膠體電泳（two-dimensional polyacrylamide gel electrophoresis，2-DE）分析懷孕第1天與第4天之小鼠子宮內膜上皮細胞之蛋白質表現。共有82個蛋白質點有表現上差異，並鑑定其中的52個蛋白質點。一些蛋白質如Vim、Gstm2、Hsp47、Hspa5（GRP78）與Ctsb可能與懷孕成功與否有關。總結來說，降低Muc1表現對於胚著床是必要的，且Muc1表現可被let-7a and let-7b miRNA調節。Muc1表現未減少，以及較少或較低品質之卵都可能造成懷孕率低落，可能導致不孕。這些發現或許可應用於改進或發展生殖醫療技術以增進懷孕率。Abstract Mucin 1 (Muc1), an transmembrane glycoprotein, is expressed on the apical surface of lumen and glandular epithelial cells in many different organs, including the uterus. The lessening of Muc1 on the surface of uterine epithelial cells is essential for embryo implantation. Also, the endogenous non-protein coding microRNAs (miRNAs) of 18-24 nucleotides exist in various organisms and participate a myriad of cellular processes through regulating gene expressions at post-transcriptional level. In this study, Muc1 expression was an indicator to examine the effects of timing of superovulation on pregnancy rate in mice. In normal situation, the expression of Muc1 in female mice mated with male mice naturally reached the highest level on day 1 of pregnancy and constantly decreased afterwards. However, the expression of let-7a and let-7b miRNA detected by real-time polymerase chain reaction (real-time PCR) showed the reverse patterns compared with Muc1 expression. The direct binding of let-7a and let-7b miRNA to the 3' UTR of Muc1 was shown by luciferase assay, indicating the regulation of Muc1 expression. Superovulation is a common application for increasing the number of oocytes, but it might impair the pregnancy. When superovulation was conducted at the different stage of estrous cycle in mice (i.e. proestrous, estrous, metestrous and diestrous stages), the pregnancy rate was significantly higher at the groups of proestrus (59%) and estrus (66%) than that at the groups of metestrus (23%) (P < 0.05). Proestrus and estrus exhibited the best mating and pregnancy rate. We suspected that this reason might be associated with the development of ovarian follicles. Therefore, mouse ovaries were collected at different stage of estrus cycle after PMSG treatment for 48 h. More number of developing and Graafian follicles were shown in ovarian sections of mice superovulated at proestrous and estrous stages compared with those superovulated at metestrous and diestrous stages. In contrast, the mice superovulated at metestrus and diestrus presented more number of corpus lutea than those superovulated proestrus and estrus (P < 0.05). Additionally, the expression of Muc1 was significantly low at the groups of proestrus and estrus on day 4 of pregnancy compared with the expression on day 1 of pregnancy (P < 0.05), but no significant difference was shown at the groups of metestus and diestrus. The reverse pattern of the expression of let-7a and let-7b miRNA was also presented. Furthermore, the protein expressions of mouse endometrial epithelial cells during day 1 and 4 of pregnancy were also analyzed using two-dimensional polyacrylamide gel electrophoresis (2-DE). Eighty two protein spots were shown differential expression (P < 0.05), and fifty two of them were identified. Some proteins might be associated with successful pregnancy, such as Vim, Gstm2, Hsp47, Hspa5 (GRP78), and Ctsb. In conclusion, the reduction of Muc1 expression is essential for embryo implantation, and the expression is regulated by let-7a and let-7b miRNA. The failure of decrease in Muc1 and less number or low quality of oocytes could result in the low pregnancy rate, which might be associated with infertility. These finding might be applied to improve and develop modern reproductive therapies for higher pregnancy rate.Category Chapter 1: Literature Review 1 Introduction 2 Part I. The factors involving in the successful pregnancy 4 1. Reproductive cyclicity (estrous cycle) 4 2. Fertilization and cleavage 7 3. Embryo implantation 8 4. Endometrium 9 Part II. Biomarkers related to endometrial receptivity and embryo implantation 9 1. Steroidal hormone 10 2. Adhesion molecule and embryo implantation 10 Part III. MicroRNAs and embryo implantation 17 1. History of microRNAs 17 2. Biogenesis of microRNAs 18 3. MicroRNAs and embryo implantation 19 Chapter 2: Study 1-Let-7-mediated Suppression of Mucin 1 Expression in Mouse Uterus During Embryo Implantation 20 1. Abstract 21 2. Introduction 22 3. Materials and Methods 24 3.1. Animals 24 3.2. Isolation of mouse endometrial epithelial cells 24 3.3. Western blotting 24 3.4. Real time reverse transcription-polymerase chain reaction (Real-time PCR) 25 3.5. Transfection and Luciferase Assay 26 3.6. In vitro embryo adhesion assay 27 3.7. Statistical analysis 27 4. Results 28 4.1.Expression of Muc1protein in mouse endometrial epithelial cells during early pregnancy 28 4.2.Expression of let-7a and let-7b in mouse endometrial epithelial cells during early pregnancy 29 4.3. Let-7a and let-7b directly target the 3'UTR of Muc1 30 4.4. Suppression of Muc1 by overexpression of let-7a orlet-7b 32 4.5. Reduction of embryo adhesion on the endometria by let-7a and let-7b 33 5. Discussion 34 Chapter 3: Study 2-Estrous Cycle Determines the Pregnancy Performances in Superovulated Mice 37 1. Abstract 38 2. Introduction 39 3. Materials and Methods 41 3.1. Animals 41 3.2. Determining the stage of estrous cycle and superovulation 41 3.3. Histological study of ovaries 41 3.4. Immunohistochemistry of uterine horns 42 3.5. Investigation of let-7a and let-7b miRNA expressions on endometrial epithelial cells by real-time PCR 42 3.6. Statistical analysis 43 4. Results 44 4.1. Pregnancy performances of superovulated mice was determined by the stage of estrous cycle 44 4.2. The effect of PMSG on ovarian follicle development 47 4.3. Abnormal expression of Muc1 in endometrial epithelial cells of mice superovulated at metestrus and diestrus 49 4.4. Abnormal expression of let-7a and let-7b miRNA in endometrial epithelial cells of superovulated mice at metestrus and diestrus 51 5. Discussion 52 Chapter 4: Study 3-Proteomic Analysis of Differentially Expressed Proteins in Mouse Endometrial Epithelial cells During Early Pregnancy 55 1. Abstract 56 2. Introduction 57 3. Materials and Methods 58 3.1 Animals 58 3.2 Sample collection 58 3.3 Two dimensional gel electrophoresis (2-DE) 58 3.4 Staining and imaging of the 2-DE gels 59 3.5 Analysis of differential protein expression 59 3.6 Protein identification by MALDI-TOF/MS and MALDI- TOF/TOF 60 3.7 Bioinformation analysis 60 3.8 Statistical analysis 60 4. Results 61 4.1 Proteomic analysis of protein expression in mouse endometrial epithelial cells during early pregnancy 61 4.2 Quantitative changes of protein expression in mouse endometrial epithelia during early pregnancy 62 4.3 Classification of the differentially expressed proteins 69 5. Disscussion 71 Chapter 5: Conclusion 73 Appendix 76 1. Western blotting 77 2. Immunohistochemistry (IHC) 81 3. Clone MUC1 3'UTR construct vector 82 References 8

Regulation of muc1 expression by miRNA 199a in mouse endometrial epithelium during implantation

胚胎著床為一複雜的過程，與囊胚直接和子宮上皮細胞接觸有關。於許多生物體中可發現微型RNA（miRNAs）的存在，其為21-24個核苷酸所構成的非編碼RNA。miRNAs被證實藉由調控基因表現參與許多細胞生理反應。本試驗中，欲探討著床過程中，著床相關之基因MUC1和miRNAs 199a於小鼠子宮上皮細胞之表現情形。MUC1為一種鑲嵌膜蛋白（integral membrane protein）表現於子宮及許多不同器官之腺體的上皮細胞中，並與胚胎著床過程中扮演重要的角色。減少子宮上皮細胞表面的MUC1表現為胚胎著床過程中所必需。試驗中進一步利用免疫組織化學染色法（Immunohistochemistry, IHC） 西方吸漬（western blot, WB） 即時聚合酶連鎖反應（Real-Time PCR） 轉染（Transfection） Dual-luciferase assay、免疫螢光染色法（Immunofluorescent, IFC） 及懷孕早期體外胚胎黏著試驗進行分析。於WB及IHC結果顯示，懷孕0.5天的小鼠子宮上皮細胞有較多MUC1表現，然而於4.5天時顯著下降。相反地，利用Real-Time PCR偵測懷孕小鼠子宮上皮細胞中miRNAs 199a的表現，發現於4.5天表量顯著下降。構築luciferase下游帶有MUC1-3’ UTR之載體，稱為pRL-MUC1，將其轉染至3T3細胞株中。利用 Dual-luciferase assay評估miRNAs 199a與MUC1-3’ UTR之結合情形，結果顯示，於轉染miRNAs 199a及pRL-MUC1組別中抑制luciferase的表現，然而，於轉染miRNAs 199a、pRL-MUC1與miRNAs 199a抑制子組別中可恢復luciferase的表現，因此認為miRNAs 199a藉由與MUC1-3’ UTR結合而調控MUC1之表現。另外，利用WB及IFC試驗證實，經由轉染後miRNAs 199a可抑制MUC1之表現。於懷孕早期體外胚胎黏著試驗中，發現轉染miRNAs 199a較無轉染miRNAs 199a之子宮內膜上皮細胞更具有使小鼠孵化的囊胚貼附於上皮細胞之能力，因此證實miRNAs 199a可直接調控MUC1之表現，並於懷孕早期具有抑制MUC1表現之功能。綜合上述，miRNAs 199a可能於成功懷孕過程中扮演重要之角色。The embryo implantation process is complex, involving direct of the blastocyst with the luminal epithelium of receptive uterus. MicroRNAs (miRNAs) are 21-24 nucleotides non-coding RNA found in diverse organism. It has been shown that miRNAs participate in range of cellular process by means of regulating gene expression at post-transcriptional level. In this study, we have investigated miRNA 199a is spatiotemporally expressed in mouse uterus profiles during implantation coincident with expression of MUC1, a gene critical for implantation. MUC1 protein is an integral membrane protein that is expressed apically by simple secretory epithelium and glandular in many different organs which plays a fundamental role in embryo implantation. By means lossing of MUC1 on the surface of uterine epithelium cells are believed to be necessary for embryo implantation. In this study, an extensive analysis was performed by immunohistochemistry (IHC), western blot (WB), real-Time PCR, transfection, dual-luciferase assay, Immunofluorescent (IFC) and in vitro embryo adhesion assay during early pregnancy. The results generated from this present study indicate that MUC1 was much more on day 0.5 and decrease at day 4.5 of pregnancy base on observations from western blot and IHC and analyses, respectively. In contrast, miRNA 199a was expressed much more at day 4.5 of pregnancy after observed by real-time PCR. A reporter construct containing the MUC1-3' UTR located immediately downstream of luciferase, designated as pRL-MUC1, was transfected into 3T3 cell. The dual-luciferase assay was applied to evaluate the binding of miRNA 199a to the 3' UTR of MUC1. It was shown that the expression of luciferase was suppressed in the group of cells transfected with pRL-MUC1and miRNA 199a, and restored in the group of cells co-transfected with pRL-MUC1, miRNA199a and inhibitor of miRNA 199a, indicating the direct binding of miRNA 199a to the 3' UTR of MUC1 and regulatory effect of miRNA 199a on MUC1.By Using Western blot combined with, immunofluorescence staining and in vitro adhesion confirm that miRNA-199a can be down-regulation MUC1 expression with transfection. The hatching mouse blastocysts cultured on the endometrial epithelium which had been transfected with miRNA 199a precursor showed more adhesion ability than those cultured on the untransfected epithelium. We identified MUC1 as a direct target of miRNA 199a. By means, miRNA 199a down-regulated MUC1 expression at early pregnancy. The present data implicated that miRNA 199a might play a gateway role to a successful pregnancy.Table of contents Chinese abstract...………………………………………………………………………… i English abstract.……………………………….………………………………………...... ii Table of contents……………………………………………………..…………………… iv List of tables ……………………………..……………………………………………..... vii List of figures …………………………..………………………………………………... viii Chapter I. Introduction………………………………………………………………………...…… 1 II. Literature review……………………….……………………………………………… 3 1. Embryo implantation ………………………………………................................... 3 1.1 Preimplantation embryonic development …………………………............. 3 1.2 Apposition/Attachment and Invasion …………………………………...… 5 1.3 Decidualization ……………………………………...………….................. 7 2. Trophoblast-endometrial interaction ……………………………………………... 8 3. Steroidal hormone ………………………………………………………………... 9 4. Adhesion molecule correlated with implantation. ……………………………….. 10 4.1 Intergrins ………………………………………………………………....... 10 4.2 Heparan sulfate (HS) proteoglycan and HS-binding proteins ……………... 11 4.3 Selectins……………………………………………………………………. 12 4.4 Galectins …………………………………………………………………… 12 4.5 Trophinin-tastin-bystin complex ……….………………………………….. 13 4.6 Cadherins ……….…………………………………………………………. 13 4.7 Basigin ……….……………………………………………………………. 14 4.8 Intracellular adhesion molecule (ICAM)-1……….………………………... 15 4.9 Mucin glycoprotein ……….……………………………………………….. 15 5. Other factors endometrium ………………………………………………………. 17 5.1. Growth factors …………………………………………………………….. 17 5.2. Cytokines………………………………………………………………….. 19 5.3. Prostaglandins (PGs)………………………………………………………. 20 6. The origin of microRNA………………………………………………………….. 20 6.1. MicroRNA biogenesis and function………………………………………. 21 6.2. MicroRNAs and embryo implantation…………………………………….. 22 III. Materials and methods………………………………………………………………... 24 1. Animals…………………………………………………………………………… 24 2. Sample and tissue collection……………………………………………………… 24 3. Extraction MUC1…………………………………………………………………. 24 4. Epithelial cell culture……………………………………………………………... 25 5. Western blotting…………………………………………………………………... 25 6. Immunohistochemistry (IHC)………………………….….……………………… 26 7. Clone MUC1 3'UTR construct vector……………………………………………. 27 8. Transfection and luciferase assay…………………….….………………………... 30 9. RNA Isolation and Real-Time PCR………………….……….…………………... 31 10. Transfection and Immuncytochemistry…………………………………………... 33 11. Transfection and western blotting………………………………………………… 34 12. In vitro embryo adhesion assay…………………………………………………… 35 IV. Results ………………...……………………………………………………………… 36 1. Experiment 1……………………………………………………………………… 36 2. Experiment 2……………………………………………………………………… 39 3. Experiment 3……………………………………………………………………… 40 4. Experiment 4……………………………………………………………………… 45 5. Experiment 5……………………………………………………………………… 49 V. Discussion and Conclusion……………………………………………………............. 51 VI. References ………………………………………………………………..…………... 54 VII. Appendix ……………………………………………………………………...…….. 6

Understanding the Ovarian Interrelationship with Low Antral Follicle Counts (AFC) in the In Vivo Bos indicus Cow Model: Unilateral and Bilateral Main AFC as Possible Biomarkers of Ovarian Response to Hormonal Synchronisation

The antral follicle count (AFC) is a test in which the number of oocyte-containing follicles that are developing in both ovaries are visually counted. The count of these follicles strongly relates to the population of the growing follicle reserve on the ovaries. However, the importance of the main number of antral follicle populations (mAFC) in mono-ovulatory animal species has yet to be completely elucidated. Moreover, the investigation of the ovarian interrelationship with unilateral mAFC (main number of antral follicle populations appearing on only one side of the ovary) and bilateral mAFC (main number of antral follicle populations appearing in equivalent numbers on both sides of the ovary) and how understanding this interrelationship can offer possible indicators of ovarian response to hormonal induction have not yet been investigated in mono-ovulatory Bos indicus beef cows. The aim of this study is to investigate the different ovarian interrelationships of mAFC (unilateral and bilateral mAFC) at the time of exogenous hormonal stimulation on the total number of AFC (left and right ovaries) at the beginning of the hormonal protocol for ovarian stimulation and ovarian response at the completion of exogenous hormonal stimulation as well as their usefulness as possible biomarkers of successful hormonal stimulation in Bos indicus beef cattle. Beef cows (n = 104) with low total numbers of AFC (4.7 &plusmn; 2.4 follicles) were stimulated with a gonadotropin-releasing hormone-progesterone-prostaglandin F2&alpha;-based protocol. At the beginning of the hormonal protocol, ovarian ultrasound scans were performed to evaluate AFC from both ovaries of cows. Beef cows were divided into two groups, unilateral (n = 74) and bilateral mAFC (n = 30), according to the ovarian interrelationship. At the completion of the hormonal stimulation, ovarian ultrasound scans were performed to evaluate the dominant follicle (DF) and cows with DF &gt; 8.5 mm in diameter emerging on their ovaries were defined as having experienced a response to hormonal stimuli. There was a difference of 19.1% between Bos indicus cows bearing unilateral mAFC that produced an increase in ovarian response (odds ratio = 2.717, p &lt; 0.05) compared to the responsive rate of cows displaying bilateral mAFC (82.4% vs. 63.3%). In unilateral mAFC, cows bearing mAFC ipsilateral to the ovary of dominant follicle (DF) had a higher responsive rate than cows bearing mAFC contralateral to the DF ovary (50.0% vs. 32.4%, p &lt; 0.05). In mAFC ipsilateral to the DF ovary, pregnancy rates were greatest in cows bearing mAFC and DF on the right ovary compared with cows bearing mAFC and DF on the left ovary (25.0% vs. 9.1%, p &lt; 0.05). In primiparous and multiparous cows, unilateral mAFC occurs with a greater (p &lt; 0.05) frequency than bilateral mAFC (69.0% and 72.0% vs. 31.0% and 28.0%, respectively). In unilateral mAFC, primiparous cows bearing mAFC ipsilateral to the DF ovary had a greater responsive rate than primiparous cows bearing mAFC contralateral to the DF ovary (55.0% vs. 20.0%, p &lt; 0.05). In mAFC ipsilateral to the DF ovary, responsive and pregnancy rates were greatest (p &lt; 0.05) in multiparous cows bearing mAFC and DF on the right ovary compared with multiparous cows bearing mAFC and DF on the left ovary (58.1% and 22.6% vs. 25.8% and 3.2%, respectively). Furthermore, there was a positive correlation between the mean diameter of AFC at the time of the exogenous hormonal trigger and the mean diameter of DF at the completion of hormonal synchronisation (p &lt; 0.05). Our findings emphasise that the ovarian interrelationship with unilateral mAFC at the time of the hormonal trigger might be a promising biomarker for predicting success in ovarian response to hormonal stimulation of mono-ovulatory Bos indicus beef cows with low AFCs

In Vivo Follicular and Uterine Arterial Indices as an Indicator of Successful Hormonal Stimulation for Inactive Ovaries in Repeat-Breeder Crossbred Dairy Cows Using a Short-Term Progesterone-Based Programme

An investigation of vascularity of ovarian and uterine arteries after hormonal treatment for inactive ovaries using the short-term progesterone-based programme had not yet been explored in repeat-breeder crossbred dairy cows. To investigate the in vivo follicular and uterine arterial indices as an indicator of successful hormonal stimulation for inactive ovaries in repeat-breeder crossbred dairy cattle, 59 cows with inactive ovaries were induced with a 5-day progesterone-based protocol. At the completion of hormonal synchronisation, cows were divided into two groups according to the size of the largest follicle (LF) on their ovary: small (≤10.0 mm) and large (>10.0 mm) LFs. Vascularities of LF and uterine artery (UtA) were evaluated using a colour Doppler tool. Cows that presented with large LF had greater follicular and UtA vascular indices (p p p < 0.001) between follicular size and LF and UtA vascular indices. Our findings highlighted that in vivo LF and UtA vascular indices at the completion of hormonal stimulation might be a promising indicator for predicting success in ovarian response to hormonal stimulation for inactive ovaries of infertile crossbred dairy cows