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Steinberg, Vera (2006): Expression von lokalen Regulationsfaktoren im Ovar des Schweins. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

The aim of the study was to further investigate the importance of local other and to detect possible differences of expression and localisation in poly- and monoovulatory species. Therefore ovaries of sexually mature gilts were collected at the local abattoir and follicles, which were determined to be in the follicular phase of oestrous cycle, ovarian stromal tissue and corpus luteum (CL) were prepared. In two experiments mRNA expression of gonadotropin receptors (LHR, FSHRregulatory factors during the final follicle growth in pig, to clarify relations among each), Aromatase (P450 Aro) and growth factors (fibroblast growth factor (FGF-1, FGF-2, FGF-7) and their receptors (FGFR-1IIIc, FGFR-2IIIb, FGFR-2IIIc), vascular endothelial growth factor (VEGF) and receptors (VEGFR-1, VEGFR-2), angiopoietin-tie-system (Angp-1, Angp-2, Tie-1, Tie-2) and nitric-oxid-synthase (eNOS, iNOS) were measured by qRT-PCR. An immunohistochemically determination and localisation was carried out for FGF-2, VEGF and VEGFR-1. Molecular size of FGF-2 was determined by westernblot. mRNA expression of the selected factors in pig total follicles and expression intensity in comparison to stromal tissue and CL was checked in experiment 1. Experiment 2 was concerned with the exact regulation of growth factors in the follicle compartments granulosa cells (GC) and theca interna (TI) during final follicular growth. A classification of follicles was performed according to follicular fluid oestradiol-17β (E2)-, progesterone and prostaglandin F2α content into four groups (2-3mm, 4-6mm, >7mm, post LH). Expression of FSHR decreased during follicle growth, while LHR and Aromatase increased. mRNA of FGF-family members was strongly expressed in stromal tissue. This was confirmed immunohistochemically by dominant localisation of FGF-2 protein in stromal cells. 18kDa and 22kDa isoforms of FGF-2 were overriding determined in stromal tissue. VEGF was located in GC and TI but not in stromal tissue. The receptor, VEGFR-1 was found in the endothelial- and smooth muscle cells of blood vessels. VEGF mRNA expression decreased after LH-peak. VEGFR-2 was upregulated in large follicles, VEGFR-1 after LH-peak. Angp-2/Angp-1 ratio was highest in small follicles, lowest in large follicles. After LH-peak the ratio rose again. Expression of eNOS was opposite with iNOS, which was downregulated in large follicles. FSH plays an important part in recruiting a folliclepool at time of luteolysis, while the further developing follicles become LH dependent. The number of LHR is an important selection criteria for the follicles. LH and E2 cause GC proliferation by mediators like FGF-7 and enhance angiogenesis by stimulating VEGF. While the follicle growth-up fast the pressure of oxygen in the follicular antrum decreases, whereby angiogenetic factors like VEGF and angiopoietins get stimulated and provide an adequate blood supply for the follicles. Activated by LH and VEGF eNOS is producing NO, which controls E2 synthesis and leads to a better blood flow in the TI through vasodilatation. Increasing E2 concentrations in large follicles stimulate Angp-1, which stabilise the now sufficient bloodvessel network in the TI. After the LH-peak Angp-2 destabilises the blood vessels, which is important for the process of ovulation and the succeeding angiogenesis in CL buildup. For follicle selection an important parameter seems to be the localisation of VEGF and FGF in the follicle. VEGF acts in the follicle and therefore follicles which express more VEGF and receptors have a developmental advantage. In cows this fact is suggested as one basic in selection of the one dominant follicle. FGF-2 in the pig is mainly expressed in stromal tissue and able to activate FGFR in the TI and increases angogenesis in a synergistic way with VEGF and its receptors. FGF-2 in stromal tissue consequently has influence on a few follicles and is maybe an important component in selection of follicles during final follicular growth in polyovulatory species.