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Thesis Molecular, Hormonal and Clinical Aspects of Aglepristone In THESIS MOLECULAR, HORMONAL AND CLINICAL ASPECTS OF AGLEPRISTONE IN LUTEAL PHASE AND PLANNED CESAREAN SECTION IN BITCHES CHUNSUMON LIMMANONT A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (Veterinary Clinical Studies) Graduate School, Kasetsart University 2021 iii Chunsumon Limmanont 2021: Molecular, Hormonal and Clinical Aspects of Aglepristone in Luteal Phase and Planned Cesarean Section in Bitches. Doctor of Philosophy (Veterinary Clinical Studies), Major Field: Veterinary Clinical Studies, Faculty of Veterinary Medicine. Thesis Advisor: Associate Professor Kaitkanoke Sirinarumitr, Ph.D. 129 pages. Effects of aglepristone, progesterone receptor blocker, in non-pregnant and pregnant bitches were studied. Fetal head diameter (HD) measured by radiography (XR) and ultrasonography (US) in pregnant dogs and cats were also studied. Exp. 1: Radiographic measurements of fetal HD were larger than those measured by US in both 24 dogs (P < 0.0001) and 16 cats (P < 0.0001). Skull heads from XR and US were significantly correlated (r = 0.73 in dogs, r = 0.94 in cats, P < 0.0001). Linear regression formulas were y = 1.08x + 0.13 (R² = 0.73) in dogs, y = 0.96x + 0.30 (R² = 0.88) in cats (y = HD by XR and x = HD by US). Exp. 2: TM Aglepristone (Alizine , 10 mg/kg) was injected SC 5 days before OVH in 6 treated and 6 control bitches. ERα mRNA (P < 0.01) and OTR mRNA (P < 0.05) were increased after treatment. Of treated bitches, lower expression levels of ERα were observed in luminal epithelium, crypt and endometrial epithelium, but higher expression in endometrial stroma and myometrium (P < 0.05); however, PR expression was decreased only in luminal epithelium, crypt epithelium and glandular epithelium (P < 0.01). Exp. 3: There was no significant difference in age, body weight, HD, estradiol, progesterone, milk production and vaginal discharge from both control and treated with aglepristone at 15 mg/kg in pregnant groups. Apgar score in treatment group was significant higher than control group at the time of birth (P < 0.01). The OTR was significantly lower at placental site of control compared to placental and inter placental sites of treatment group (P < 0.05). In conclusion, Aglepristone is a useful medication for planned C-section in bitches on 61-62 days from ovulation date, and C-section can be performed 20-24 hours post injection. / / Student’s signature Thesis Advisor’s signature ACKNOWLEDGEMENTS I would like to sincerely acknowledge many persons who supported this research. Special acknowledgement to, Assoc. Prof. Dr. Kaitkanoke Sirinarumitr, my major thesis advisor, the numerous of her kindness always be taken for advising, supporting and promoting to me, Asst. Prof. Dr. Preeda Lertwatcharasarakul, my co advisor, his kindness and smile always be shared and taken for supporting my molecular laboratory practice, and Asst. Prof. Dr. Suppawiwat Ponglowhapan, my invited co advisor form Chulalongkorn University, for his advice, guidance and motivation. For this successful research, there were many persons who supported me, including my lecturers, colleagues, academic officers and laboratory technicians. All my subjects in the clinical trial, were attended by their lovely and enthusiastic owners. Place and some facilities were provided by the Department of Companion Animal Clinical Sciences, Kasetsart University Veterinary Teaching Hospital and Kasetsart University Veterinary Diagnosis Centre, Bangkhaen and Kamphaeng Saen campus, the Faculty of Veterinary Medicine, Kasetsart University and Obstetrics Gynecology and Reproductive Unit, the Faculty of Veterinary Science, Chulalongkorn University. This work was supported by Center for Advanced Studies in Agriculture and Food (CASAF), NRU-KU, Bangkok, Faculty of Veterinary Medicine, Kasetsart University. The last, no more words is adequate to thank my family including my parents, my elder brother, my elder sister, and their families for their sympathy and support. Chunsumon Limmanont May, 2021 i TABLE OF CONTENTS Page TABLE OF CONTENTS i LIST OF TABLES ii LIST OF FIGURES v LIST OF ABBREVIATIONS xii INTRODUCTION 1 OBJECTIVES 3 LITERATURE REVIEW 4 MATERIALS AND METHODS 34 RESULTS AND DISCUSSION 53 Results 53 Discussion 78 CONCLUSION AND RECOMMENDATIONS 87 LITERATURE CITED 88 APPENDICES 96 Appendix A Chemicals Preparation and Processing 97 Appendix B Experiment 1 100 Appendix C Experiment 2 103 Appendix D Experiment 3 118 CURRICULUM VITAE 129 ii LIST OF TABLES Table Page 1 Approximate timing of fetal development in pregnant dog and cat 17 2 Ultrasonographic diagnosis of pregnancy in dog and cat 18 3 The modified Apgar scoring system used in this study 22 4 The information of ERα, PR, OTR and GAPDH primers (Appendix Figure C4) 45 5 Mean ± SD of body weight (BW), litter size, pelvic diameter measured from radiography (XR), fetal HD from XR and ultrasonography (US), and percentage of fetal HD measured from ventro-drosal (VD) and lateral views of XR from the last trimester of pregnant in dogs and cats are presented. Numbers of sample (n) from each parameter are shown in parenthesis. 54 6 Mean ± SD and range of serum estradiol and progesterone levels on the ovariohysterectomy day from both control and treatment groups 56 7 Thickness (mean ± SD) of the endometrial stroma, myometrial stroma, luminal epithelium, glandular epithelium, and density of stromal gland 57 8 Mean ± SD and range of age and body weight from control and treatment groups 62 9 Mean ± SD and range of serum estradiol (E2) and progesterone (P4) levels at 20-24 hours before C-section and at C-section time from both control and treatment groups 65 10 Mean ± SD and range of Apgar score at 0, 5, 15 and 30 minutes after birth of puppies from control and treatment groups. The superscript indicates a significant difference between groups (P < 0.05). 66 iii LIST OF TABLES (Continued) Table Page 11 Mean ± SD and range of the gene expression of ERα, PR and OTR at placental and inter placental sites from control and treatment groups. The superscript indicates a significant difference between groups (P < 0.05). 69 12 Mean ± SD and range of the H-score of ERα at epithelium, glandular (uterine gland), stroma and myometrium from placental and inter placental site in control and treatment groups. The superscript indicates a significant difference between placental and inter placental site within and between groups (P < 0.05). 71 13 Mean ± SD and range of the H-score of PR at epithelium, glandular (uterine gland), stroma and myometrium from placental and inter placental site of control and treatment groups. The superscript indicates a significant difference between placental and inter placental site within and between groups (P < 0.05). 72 Appendix Table B1 The average of fetal HD measured by radiography (XR) and ultrasonography (US) in dogs (cm). 101 B2 The average of fetal HD measured by radiography (XR) and ultrasonography (US) in cats (cm). 102 C1 Age, body weight and serum estradiol and progesterone level at OVH time from control group 107 C2 Age, body and serum estradiol and progesterone level at treatment (1) and OVH time (2) from treatment group 108 iv LIST OF TABLES (Continued) Appendix Table Page C3 Thickness of the endometrial stroma, myometrial stroma, luminal epithelium, glandular epithelium, and density of stromal gland from control group 109 C4 Thickness of the endometrial stroma, myometrial stroma, luminal epithelium, glandular epithelium, and density of stromal gland from treatment group 110 C5 Comparison of relative gene expressions between control and treatment groups 117 D1 Age, body weight (BW) and serum estradiol and progesterone level before (1) and C-section time (2) from control group 122 Age, body weight (BW) and serum estradiol and progesterone D2 level at treatment (1) and C-section time (2) from treatment group 123 D3 Hematology and blood chemistry profiles from bitches at 20- 24 hours before C-section 124 D4 Fetal HD and their averages (cm) are measured from control group by ultrasonography at 20-24 hours before C-section. 125 D5 Fetal HD and their averages (cm) are measured from treatment group by ultrasonography at 20-24 hours before C-section. 125 D6 Apgar score evaluation at 0, 5, 15 and 30 minutes after birth from puppies in control group 127 D7 Apgar score evaluation at 0, 5, 15 and 30 minutes after birth from puppies in treatment group 128 v LIST OF FIGURES Figure Page 1 Hypothalamic-Pituitary-Ovarian Axis of hormones in bitch 4 2 Schematic diagram of changes of hormone concentrations; luteinizing hormone, estrogen, progesterone during the estrus cycle in non-pregnant and pregnant bitch including pregnancy diagnosis 7 3 Cross-section of the canine uterine horn; H&E staining 8 4 Components of a choriovitelline placenta and chorioallantoic placenta 10 5 Canine fetal-placental unit approximated 58 days of gestation. 11 6 Drawing of canine zonary endotheliochorial deciduate placenta (A) and transverse section at zone of attachment to endometrial (B) 13 7 Histology of canine utero-placental section from C-section and ovariohysterectomy (OVH) bitch at gestation 61 days form ovulation date; H&E staining 14 8 Intracellular Hormone Receptors pathway; in the cytoplasm, the receptor (R) bound to a heat shock protein (hsp). When hormone (H) passes to cytoplasm, and binds to the receptor and hsp. 24 9 Plasma membrane hormone receptors; the drawing example of non-soluble hormones (H) and receptor (R) on the plasma membrane of cells. Hormone binding to receptor activates a G- protein, following activates adenylyl cyclase, converting ATP to cAMP. cAMP is a second messenger that mediates a cell- specific response. Phosphodiesterase that breaks down cAMP inhibits the signal. 26 10 Cellular and molecular mechanism of progesterone at the target cell. 28 vi LIST OF FIGURES (Continued) Figure Page 11 Chemical structures of progesterone, mifepristone and aglepristone 29 12 Molecular mode mechanism of aglepristone to explain the anti- hormonal effect by reinforcing the receptor-hsp90 interaction.
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