Preantral Follicle Manipulation and Germ Cell Characterization

Jeong M. Lim

Department of Food and Animal Biotechnology, Seoul National University, 56-1 Sillim-Dong, Seoul 151-921, Republic of Korea

Tel: +82-2-880-4806; Fax: +82-2-874-2555; E-mail: limjm@snu.ac.kr

Biotechnology has been developed from the basic knowledge of biology, medicine, and animal and plant science, thus multidisciplinary approach is absolutely required for efficient developing biotechnical methodologies and materials. The final end-products derived from biotechnological skills contribute to improving human welfare which is related with discovering novel drugs, inventing medical materials and devices, and developing novel technologies to overcome various catastrophic diseases. We have mainly attempted to develop cell-based biotechnology, which includes the culture, cryopreservation and micromanipulaton of preantral follicles, follicular oocytes and embryos. Korean Stem Cell Research Center (KSCRC) also supported the establishment of research framework and other required management for efficiently carrying out this study. To date, the first set of preliminary experiments titled aimed at establishing a basic manipulation system of preantral follicles was completed. Randomized, prospective study using animal model was conducted in this preliminary study and, as experimental model animal, two-week-old female F1 (C57BL6/DBA2) mice were employed. Preantral follicles of primary, early secondary and late secondary follicles were retrieved by either a mechanical or an enzymatic method. The collected follicles were cultured in vitro for various durations and in vitro-growth of preantral follicles were sequentially monitored. The maturation of intrafollicular oocytes retrieved from cultured follicles was further assessed and the fertilizability of the oocytes matured was monitored after in vitro-insemination. As results, a mechanical method retrieved more preantral follicles than an enzymatic method. Regardless of retrieval methods, the number of early secondary follicles was larger than that of primary and late secondary follicles. Retrieval of primary follicle by an enzymatic method was not possible. The follicles cultured in vitro commonly underwent a step-by-step growth consisting of the follicular, diffuse, psudoantral and degenerative stages, but the retrieval method greatly influenced the follicular growth: to reach the pseudoantral stage, primary, early secondary and late secondary follicles retrieved by the mechanical method required 11, 10 and 7 days, respectively. In the case of the early and secondary follicles retrieved from an enzymatic method, however, 9 and 6 days were necessary, respectively. When the intrafollicular oocytes collected from the psudoantral stage of cultured follicles were cultured in vitro, various durations were required for final maturation (developed to metaphase II stage) of oocytes. The optimal maturation time of follicular oocytes was 11 days, 7-9 days and 7 days for primary, early secondary and late secondary follicles, respectively. When microscopic observation was made for cultured oocytes, general decrease in oocyte diameter was detected in oocytes derived from all categories of follicles compared with in in vivo-derived oocytes. The enzymatic retrieval typically reduced zona thickness of oocytes compared with in vivo-ovulated oocytes, while the mechanical method did not reduce. Pronuclear formation after in vitro insemination with epididymal semen was possible in all mature oocytes derived from in vitro-cultured follicles of different categories. In conclusion, preantral follicles underwent step-by-step growth in vitro and retrieval method greatly affected in vitro-growth of preantral follicles and follicular oocytes.

            Next, we characterized chicken primordial germ cells (PGCs) for the first time. This study was undertaken to develop novel markers for chicken PGCs, which are of potentially enormous value in transgenic research. Gonadal cells collected from 5.5-day-old chicken embryos were cultured in a DMEM-based medium, and the PGC colonies formed during the primary culture period were subcultured three times. Characterization of the PGCs with the candidate marker reagents was performed on the mixed cell population 2 h after seeding, after the primary culture period (day 10), and after the third passage (day 40). Mouse embryonic stem (ES) cells were used as controls. The cytochemical reagents investigated included periodic acid-Schiff (PAS) stain; antibodies to stage specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4); antibody to epithelial membrane antigen (EMA)-1, antibodies to integrins α6 and β1; several lectins (STA, DBA, ConA, and WGA), and double staining with antibodies to SSEA-1, SSEA-3, SSEA-4, integrin α6, or integrin β1 then with the lectin STA. Densitometric quantification was used to identify PGC-specific markers. The results showed that chicken PGCs were stained selectively by PAS and by antibodies to SSEA-1, SSEA-3, SSEA-4, EMA-1, integrin α6, and integrin β1. The control mouse ES cells reacted with PAS, anti-SSEA-1 and anti-EMA-1 antibodies, as well as with antibodies to integrins α6 and β1, but not with antibodies to SSEA-3 and SSEA-4. Chicken PGCs reacted with the lectins STA and DBA, but mouse ES cells reacted with STA and WGA. The results of double staining of PGC colonies subcultured three times showed that the intensity of staining was not altered by concomitant use of the marker reagents. This study demonstrated that, in addition to PAS and antibodies to SSEA-1 and EMA-1, new specific markers of chicken PGCs are recognized by the lectins STA and DBA and by antibodies to SSEA-3, SSEA-4, integrins α6 and β1. Double staining using these newly developed markers might be the method of choice for rapid characterization of chicken PGCs.