Spermatogonia Stem Cell Dynamics Following Hormonal Induction, Ischemic Disturbance In Vivo or Proliferation under In Vitro Culture in Pre- and Postpubertal Bulls

The overall aim of the studies described herein was to evaluate bovine spermatogonial cell dynamics under various conditions. Results from these experiments will provide the basis for potential production of offspring following spermatogonial stem cell transfer. Experiment 1 evaluated gonadotropin administration effects at initiation of inhibin passive immunization in Jersey bull calves on testicular morphology and development. Primary treatments consisted of control (KLH) or immunization (INH) plus a combination of saline, FSH, or GnRH. Administration of FSH at the time of initial immunization against inhibin significantly increased number of germ cells (92.2 ± 9 x 106 cells) compared to INH-Saline bulls (54.9 ± 10 x 106 cells) with INH-GnRH bulls being intermediate (64.5 ± 9 x 106 cells; P < 0.05). These results suggested that gonadotropin administration at time of inhibin immunization increases number of germ cells in the testis. Experiment 2 evaluated transiently induced ischemia in testes of Jersey calves on morphology and development. Treatments consisted of control or banding for 2 h, 4 h, and 8 h periods. Transiently induced ischemia significantly decreased number of germ cells in 8 h (12.6 ± 5 x 106 cells) compared to 0 (38.1 ± 6 x 106 cells), 2 (31.9 ± 6 x 106 cells), and 4 h (33.4 ± 5 x 106 cells; P < 0.05). These results suggested that transiently-induced ischemia significantly decreases number of germ, Sertoli and Leydig cells in the testis. Experiment 3 evaluated spermatogonial stem cells (SSC) proliferation, isolated from prepubertal and adult bulls, during short term in vitro culture. Spermatogonia were cultured in the presence or absence of a feeder monolayer (FL or NF), FBS type (FBS-S or FBS-SF), and media type (ELSC or RSC) treatment combinations. Viable type A spermatogonia survived under in vitro conditions and were able to proliferate and form different types of colonies. Furthermore, co-culture spermatogonial cells with a feeder monolayer plus FBS-S enhanced colony number (may be due to increasing cell viability). At 15 days of culture, colonies from both types of bulls were positive to AP. Therefore, these finding provide the basis for potential production of offspring through in vitro genetic manipulation such as intracytoplasmic sperm injection (ICSI), round spermatid injection (ROSI), or following SSC transfer.