Culture of Kenyan Goat (Capra hircus) Undifferentiated Spermatogonia in Feeder-Free Conditions

Abstract

The undifferentiated spermatogonial population in mammalian testes contains a spermatogonial stem cell (SSC) population that can regenerate continual spermatogenesis following transplantation. This capacity has the potential to be exploited as a surrogate sires breeding tool to achieve widespread dissemination of desirable genetics in livestock production. Because SSCs are relatively rare in testicular tissue, the ability to expand a population in vitro would be advantageous to provide large numbers for transplantation into surrogate recipient males. Here, we evaluated conditions that would support long-term in-vitro maintenance of undifferentiated spermatogonia from a goat breed that is endemic to Kenyan livestock production. Single-cell suspensions enriched for undifferentiated spermatogonia from pre-pubertal bucks were seeded on laminin-coated tissue culture plates and maintained in a commercial media based on serum-free composition. The serum-free media was conditioned on goat fetal fibroblasts and supplemented with a growth factor cocktail that included glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), stromal cell-derived factor (SDF), and fibroblast growth factor (FGF) before use. Over 45 days, the primary cultures developed a cluster morphology indicative of in-vitro grown undifferentiated spermatogonia from other species and expressed the germ cell marker VASA, as well as the previously defined spermatogonial marker such as promyelocytic leukemia zinc finger (PLZF). Taken together, these findings provide a methodology for isolating the SSC containing undifferentiated spermatogonial population from goat testes and long-term maintenance in defined culture conditions.

Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium

“…UCHL1 is related to the events of self-renewal and colocalization of SSCs and is specifically expressed in vivo in SSCs located in the basement membrane of seminiferous tubules, but not in differentiated GCs [ 43 , 44 ]. PLZF is a conserved marker of undifferentiated GCs in animal model including cattle, sheep, and goats [ 45 , 46 ]. PLZF is a transcriptional factor that represses the transcription of c-kit, which is a hallmark of SSC differentiation and thereby maintains the source of SSCs [ 47 ].…”

“…The expression of VASA, STELLA, SCP3 , and STRA8 has been related to later stages of GC maturation including post-migratory phase [ 61 , 63 ]. VASA gene encodes an RNA-binding protein and an ATP-dependent RNA helicase present in meiotic GCs and associated with the appearance of chromatoid bodies in spermatocytes and spermatids [ 46 , 63 ]. STELLA is believed to be involved in chromosomal organization and RNA processing and its expression has been detected during blastocyst stage after which it disappears, suggesting that STELLA is apparently not necessary for germline development [ 63 ].…”