Endocrine Abstracts (2004) 7 P181

The CAM technique and follicle development

AI Qureshi1, G Bano2, S Whitehead1, SS Nussey2 & HD Mason3


1Department of Basic Medical Sciences, St George's Hospital Medical School, London, UK; 2Department of Cellular & Molecular Medicine, St George's Hospital Medical School, London, UK; 3Departments of Clinical Developmental Sciences and Basic Medical Sciences, St George's Hospital Medical School, London, UK.


Background

Current techniques to study the early stages of follicular development (i)in vitro(/i) are handicapped by the spontaneous wholesale transition of primordial to primary follicles and by primary follicle arrest. We have circumvented this problem by culturing fragments of ovarian cortical tissue on the chorioallantoic membrane (CAM) of chick embryos and shown it to be successful for the (i)in ovo(/i) culture of cortical slices from a variety of species (P57, 194th Meeting of Society for Endocrinology).

Aim

The present study assesses the nature of follicle growth and atresia in lamb ovarian cortex cultured (i)in vitro(/i).

Methods

White fertilised chicken eggs were incubated at 37-38 degC and 55% relative humidity. On day 5-6 of incubation, lamb cortical fragments (8mm3) were placed on the CAM and at day 10 tissue was retrieved for routine histology. Paraffin embedded tissue was serially sectioned at 5-6 microns and stained with haematoxylin and eosin. Using image capture software (Nikon ACT-1 version 2.20) all follicles were counted, staged and classified as atretic or healthy.

Results

Fetal chick survival and cortical tissue vascularisation rates were 60.8% and 33.3% respectively. Of 236 follicles in successfully vascularised lamb cortex, 61.0% were primordial, 17.8% primary and 21.2% secondary. This contrasts non-implanted cortex in which 86.4% of follicles were primordial, 10.6% were primary and 3.0% were secondary (n=471). The ratios of primordial to primary and primary to secondary follicles in vascularised lamb tissue were 3.4 and 0.8 respectively compared with 8.1 and 3.6 in non-implanted tissue. The rate of atresia in vascularised tissue was 15.3% versus >99.9% in implanted tissue that failed to vascularise.

Conclusion

This data confirms that the use of this method is not associated with wholesale transition of primordial to primary follicles and allows normal follicular development to the secondary stage with low rates of atresia.

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