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TITLE: IN VIVO ANTRAL FOLLICLE WALL BIOPSY: A NEW RESEARCH TECHNIQUE TO STUDY OVARIAN FUNCTION USING THE HORSE AS A MODEL Abstract Understanding hormone and growth factor receptor expression in the follicular wall associated with the intrafollicular milieu and systemic endocrine aspects in vivo is crucial for a complete discovery of mechanisms behind folliculogenesis in different species. Furthermore, the comprehension of the interrelationships existent among the preovulatory follicle (POF) and corpus luteum (CL) diameter and blood flow, and progesterone (P4) production during different seasons of the year are warranted to optimize fertility in different species. In this dissertation, four studies in mares are presented with the following general objectives: (i) develop and validate a minimally invasive in vivo technique to obtain antral follicle wall biopsy (FWB) and follicular fluid (FF) samples simultaneously and repeatedly from the same individuals, during different stages of follicle development in different seasons of the year; (ii) use the FWB technique to elucidate and compare the expression pattern of different receptors and proteins involved in folliculogenesis and ovulatory mechanisms during different seasons of the year; and (iii) use B-mode and color Doppler ultrasonography techniques to characterize POF and CL diameter and blood flow during two transitional ovulatory seasons (spring and fall), and elucidate the interrelationships among these variables with systemic P4 concentration. In the first study (Chapter 2), two experiments were conducted; in Experiment 1 (ex vivo), a proof of concept study was developed to check the feasibility of using the biopsy forceps to harvest FWB samples. Ovaries of six slaughtered mares were used, and each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from growing follicles during various developmental stages were harvested using a novel FWB transvaginal ultrasound-guided technique. In the second study (Chapter 3), antral follicle wall and FF samples were obtained from 10−, 20−, and 30−mm follicles during spring anovulatory (SAN) and spring ovulatory (SOV) seasons. Samples were collected in vivo from the same mares in both seasons, with the aims to study and compare the expression of proliferative, angiogenic, and pro-/anti-apoptotic receptors and proteins in the follicle wall among three follicle classes, and between the SAN and SOV seasons. In the third study (Chapter 4), FWB and FF samples were collected in vivo from dominant growing follicles during the SAN, SOV, summer (SU), and fall ovulatory (FOV) seasons, to study the expression patterns of different receptors and proteins involved in follicle development. In the fourth study (Chapter 5), the temporal relationships among diameter and blood flow of the POF and CL, and systemic P4 concentration during spring and fall transitional ovulatory seasons were studied. The main findings of the present studies were: (i) the histological appearance, organization, and thickness of the follicle wall layers (i.e. granulosa, theca interna, and theca externa) were not influenced by the harvesting techniques (biopsy forceps vs. scalpel blade); (ii) the overall recovery rates of FWB and FF samples were 97% and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size; (iii) follicles of the SOV season had a higher expression of proliferative, angiogenesis, and anti-apoptotic proteins compared to the SAN season; (iv) follicles of the FOV season had lower expression of proliferative, angiogenesis, and luteinizing hormone receptors; and (v) positive correlations were observed among POF and CL parameters, and P4 concentration. In conclusion, results demonstrated that the novel in vivo FWB technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function in mares. Furthermore, results presented herein showed that several proliferative, angiogenic, and pro-/anti-apoptotic proteins are differentially expressed within different layers of the antral follicle wall among different seasons of the year. Finally, results demonstrated that larger and well-vascularized POFs have the potential to produce larger CLs with greater blood flow, and subsequently greater P4 systemic levels. |
