Biology of Reproduction, Vol 13, 423-447, Copyright © 1975 by Society for the Study of Reproduction

Rabbit Ovarian Follicles. I. Isolation Technique and Characterization at Different Stages of Development

¹ Division of Reproductive Biology, Department of Obstetrics and Gynecology, and Department of Pathology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19174

Clostridium histolyticum collagenase, a noncytotoxic enzyme which is active in complete tissue culture medium at neutral pH was used successfully for achieving partial or total dissection of rabbit follicles from surrounding ovarian stroma. The procedure involved a 20 min incubation of ovarian slices in collagenase, 150 U/ml of culture medium containing 95 percent TCM199, 5 percent heat-inactivated rabbit serum and antibiotics. After washing, harvesting of follicles proceeded as follows: 1) washing solutions were centrifuged at 350 rpm for 5 min, the resulting pellet yielding 40-60 small primary follicles; 2) remaining ovarian slices were mechanically freed of incompletely dissected follicles under a stereomicroscope; this procedure yielded 80-100 large primary and secondary follicles. Light and electron microscopy showed cytostructural preservation and localization of the zone of separation between follicles and adjacent ovarian stroma at the granulosa- or theca externa-interstitial gland, and less frequently, at the theca interna-externa junctions. Occasionally, contamination of isolated follicles with 2-4 interstitial cells/follicle section was noted. Harvested follicles were divided into 6 morphologically different classes and characterized according to their size, protein, estrogen (E) and progesterone (P) content: small primary (127.05 µm ± 2.45; 2.30 µg ± 0.48; E, 26.59 pg ± 6.63; P, 24.21 pg ± 5.89); large primary (280.81 µm ± 8.92; 4.53 µg ± 0.80; E, 46.40 pg ± 4.91; P, 55.40 pg ± 16.35; small secondary (619.31 µm ± 21.88; 8.80 µg ± 2.00; E, 58.50 pg ± 12.57; P, 74.00 pg ± 19.62); large secondary (812.13 µm ± 25.40; 32.25 µg ± 8.13; E, 76.99 pg ± 33.79; P, 105.19 pg ± 28.48); small (150 µm) and large atretic (478.00 µm ± 14.99; 15.73 µg ± 1.03; E, 66.59 pg ± 27.54; P, 104.33 pg ± 24.03). Total estrogen and progesterone follicular content increased progressively from small primary to large secondary follicles. Ultrastructurally, granulosa cells of harvested follicles displayed cytological features characteristic of active protein synthesis. Cytological features suggestive of steroid synthesis were predominantly seen in theca cells, increased progressively with follicular size and were particularly pronounced in large atretic follicles. Small atretic follicles contained a central core of lipid-rich cell debris and calcium deposits. A large perioocytic accumulation of calcospherules with frequent budding or fusion figures was often noted in large atretic follicles. This follicle isolation procedure was developed to provide baseline biochemical and morphological data at different stages of follicular development in preparation for the study of follicle maturation, regression and steroid secretion in organ culture.