Isolation Of Preantral Follicles Research Articles

Isolation of preantral follicles from nondomestic cats—viability and ultrastructural investigations

Abstract Large scale isolation of small preantral follicles (40–90 μm) from nondomestic cats (lion, puma, cheetah, jaguar, and three kinds of tigers) is described and compared with domestic cats. The viability of preantral follicles was estimated by trypan blue staining of granulosa cells and/or by 5-bromo-2′-deoxy-uridine (BrdU) incorporation into oocytes and granulosa cells during short term culture. Native and isolated preantral follicles were compared ultrastructurally. In nondomestic cats the mechanical dissection of ovaries provided 0–12 500 follicles per ovary with a viability of 20–50%, estimated by trypan blue staining. Even the follicles recovered from domestic cats, whose ovaries are considerable smaller than ovaries from all other felids, are characterised by only 28.7% viable follicles. The follicles from one Siberian tiger and three Indian lions were cultivated and their in vitro viability assessed by BrdU labelling. Lion follicles were comparable to domestic cat follicles with respect to BrdU incorporation. Tiger follicles were characterised by a decreased staining of granulosa cells. The ultrastructure of feline oocytes appears similar to that of most mammalian species and was only slightly affected during the isolation procedure. A central vesicular body was only observed in tiger oocytes.

Isolation and characterization of preantral follicles from foetal bovine ovaries.

A simple, mechanical method is described for the isolation of preantral follicles from bovine foetuses of 220-280 days of gestation. On average, 2918 + 621 (s.d.) preantral follicles were isolated per ovary. The isolated preantral follicles were characterized on the basis of the morphological appearance of the surrounding granulosa cells, the number of granulosa cell layers, and their diameter. The results show that primordial, primary, and secondary follicles differ morphologically and that they can be classified by their diameter.

Development of a combined new mechanical and enzymatic method for the isolation of intact preantral follicles from fetal, calf and adult bovine ovaries

The isolation of preantral follicles from the ovaries of bovine fetuses, calves and adult cows was performed using a simple, rapid mechanical and enzyme method. The ovaries were cut into small pieces with a tissue chopper. Then, the suspension was filtered successively through 500 and 100 μm nylon mesh filters. This simple mechanical procedure resulted in large numbers of isolated preantral follicles: 2,142 ± 254; 512 ± 92 and 298 ± 54 from the ovaries of bovine fetuses, calves and cows, respectively. In addition, the ovarian fragments between 100 and 500 μm were suspended in 10 ml of M199 Hepes medium plus 5% FCS and divided into 2 equal parts: one portion was used for collagenase treatment (200 U/ml) for 20 minutes, while the other served as a control. Collagenase treatment resulted in 841 ± 161; 216 ± 51 and 52 ± 17 preantral follicles from fetuses, calves and cows, respectively, compared with 312 ± 86; 52 ± 15 and 10 ± 2 in the control group. The use of collagenase with ovarian fragments selected by filtration as a method for increasing the rate of recovery of preantral follicles is described here.