Proliferation and Differentiation of Bovine Type A Spermatogonia During Long-Term Culture1

Abstract

The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2-4 wk at 32 degrees C or 37 degrees C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37 degrees C than at 32 degrees C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

“…In general, the colonies are able to regenerate new colonies upon subculturing [9]. Since in our study condition we just performed the primary culture, we could not see meaningful propagations of colonies at the end of 2 weeks culture period (Table 3).…”

“…To study the biochemical and colonogenic characteristics of SSCs, large enough populations of SSCs must be isolated. One way to reach this goal is to propagate these cells in vitro and various attempts to achieve this have been carried out [9,10]. Kanatsu-Shinohara et al [11] by using a combination of growth factors and a special stem cell medium, by way of continuous subculturing during 4-5 months, could achieve an expansion of SSCs in the order of 10-12-fold.…”

Expression of stem cell markers: OCT4, KIT, ITGA6, and ITGB1 in the male germinal epithelium

“…In rodents, SG KIT (-) were identified as the truly stem cells (type A-single, which is similar to A-dark SG in men), whereas KIT (+) SG corresponded to the progenitor cells (type A-aligned, which correspond to human A-pale SG) [Shinohara et al 1999;Schrans-Stassen et al 1999;Izadyar et al 2003;Singh et al 2011]. The KIT receptor has, indeed, been considered as a marker of differentiation as the Erk 1/ 2 pathway can be activated by stem cell factor in KIT expressing SG to stimulate their proliferation [Singh et al 2011].…”

Male germ cell transplantation in livestock

“…Improving culture conditions for male germ line stem cells is still under intense study, as evidenced by recent reports of improved culture systems for mouse germ cells (Jeong et al 2003;Kanatsu-Shinohara et al 2003c), including an efficient long-term culture system for mouse spermatogonial stem cells (Kubota et al 2004a(Kubota et al , 2004bKanatsu-Shinohara et al 2005). In cattle, type A spermatogonia have been cultured for up to 1 month (Izadyar et al 2003b).…”