Two Methods of Vitrification Followed by In Vitro Culture of the Ovine Ovary: Evaluation of the Follicular Development and Ovarian Extracellular Matrix

The aim of this study was to evaluate the influence of two vitrification techniques on the extra cellular matrix (ECM) and ovarian follicular development. The ovarian cortex was fragmented (9 mm³) and divided into six groups, viz. fresh control, cultured control, vitrified by the Ovarian Tissue Cryosystem (OTC) method, conventional solid surface vitrification (SSV) method, OTC/cultured and SSV/cultured. Follicles from all the fragments were analysed for morphology, development and viability. The ECM was evaluated based on the condition of collagen and reticular fibres and the immunolocalization of type I collagen and fibronectin. After 7 days of culture, the tissue vitrified by OTC revealed a higher percentage (p < 0.05) of morphologically normal (30.66%) and viable (60.00%) follicles when compared with those vitrified using the SSV technique (21.33% and 23.00%). In all the fragments cultured, regardless of the vitrification method, a significantly higher percentage of developing follicles was observed when compared with the non‐cultured tissue. Analysis of the type I collagen showed increased immunostaining after the in vitro culture in the vitrified fragments. In conclusion, the OTC is better for preserving the follicular viability and morphology and maintaining the integrity of the extracellular matrix components of the ovine ovary.     

Immunolocalization of angiogenic growth factors in the ovine uterus during the oestrus cycle and in response to Steroids

The vascular changes associated with endometrial maturation in preparation for embryo implantation depend on numerous growth factors, known to regulate key angiogenic events. Primarily, the vascular endothelial growth factor (VEGF) family promotes vascular growth, whilst the angiopoietins maintain blood vessel integrity. The aim was to analyse protein levels of VEGFA ligand and receptors, Angiopoietin‐1 and 2 (ANG1/2) and endothelial cell receptor tyrosine kinase (TIE‐2) in the ovine endometrium in the follicular and luteal phases of the oestrus cycle and in response to ovarian steroids. VEGFA and its receptors were localized in both vascular cells and non‐vascular epithelium (glandular and luminal epithelium) and stroma cells. VEGFA and VEGFR2 proteins were elevated in vascular cells in follicular phase endometrium, compared to luteal phase, most significantly in response to oestradiol. VEGFR1 was expressed by epithelial cells and endothelial cells and was stimulated in response to oestradiol. In contrast, Ang‐1 and Ang‐2 proteins were elevated in luteal phase endometrium compared to follicular phase, and in response to progesterone, evident in vascular smooth muscle cells and glands which surround TIE‐2‐expressing blood vessels. Our findings indicate that VEGFA is stimulated by oestradiol, most predominantly in follicular phase endometrium, and Ang‐1 and 2 are stimulated by progesterone and were increased during the luteal phase of the oestrus cycle, during the time of vascular maturation.