395 OVINE SPERMATOGONIA ISOLATION AND IN VITRO GROWTH ASSAY

Spermatogonia, as adult stem cells from the male reproductive system, are attracting strong interest from those studying male reproductive gamete preservation and developing new approaches in transgenic animals. Protein gene product 9.5 (PGP9.5) is a marker of sheep spermatogonia, which has been validated by J. R. Rodriguez-Sosa et al. (2006).The objective of this study was to develop an in vitro culture system for spermatogonial growth. Twenty 2- to 3-month-old rams were randomly selected at a local slaughter house for testicular tissue collection. Two-step enzymatic digestion methods were used for spermatogonia isolation from seminiferous tubules. In brief, mechanically isolated seminiferous tubules from testicular tissue were incubated in 1:1 1 mg mL-1 collagenase and hyaluronidase with 5 μg mL-1 DNase I for 20 min at 37°C. Most of the surrounding interstitial cells will fall off from seminiferous tubules by slightly pipetting. Seminiferous tubules were from cell suspensions after natural sedimentation in PBS and then were digested in 0.25% trypsin + 0.04% EDTAfor 5 to 7 min at 37°C to disassociate tubules into single cells. Data were analyzed with ANOVA procedures. Means of 5 specimens were presented. Of the total isolated cells, 19.7 ± 5.3% were identified as PGP9.5+cells, and 23.8 ± 3.6% were identified as c-kit+ cells. C-kit, the transmembrane tyrosine kinase receptor for stem cell factor, has been identified, which is expressed and functional in differentiating A1-A4 spermatogonia but not in spermatogonial stem cells (OhtaH2000). For in vitro culture of spermatogonia, DMEM supplemented with 1X ITS (insulin, transferrin, selenium), 100 μM β-mercaptoethano, 6 mM L-Glutamine, and 1X nonessential amino acids were used as basic culture medium. We have found that in primary spermatogonia culture, cells cultured together with testis somatic cells (sertoli cells) in basic medium supplied with 2.5% fetal bovine serum (FBS) were much more efficient than culturing with a supplement of 10% FBS. In primary culture, growing round-shaped cell colonies were visible from Day 5 in basic culture medium. In subculture, colonies were enzymically digested into smaller pieces from Day 8 to 10 and then placed onto mouse embryonic fibroblasts feeder layer in 2.5% FBS basic medium with the addition of 100 ng mL-1 glial cell-derived neurotropic factor, 10 ng mL-1 leukemia inhibitory factor, and 10 ng mL-1 basic fibroblast growth factor. PGP9.5+ spermatogonia cell colonies maintained their normal round shape until 4 to 5 passages. However, in subsequent passages, the colonies became flattened and cells gradually lost their interconnection and the growth pattern present in the early passages. Also in subsequent passages, cells began expressing more C-kit than PGP9.5. In conclusion, PGP9.5+ spermatogonia were successfully isolated from 2- to 3-month-old ram testis, and PGP 9.5 cell colonies were maintained and proliferated in the in vitro culture system up to 2 months.