Conditions for Long-Term Culture of Cattle Undifferentiated Spermatogonia

Abstract

Continual and robust spermatogenesis relies on the actions of an undifferentiated spermatogonial population that contains stem cells. A remarkable feature of spermatogonial stem cells (SSCs) is the capacity to regenerate spermatogenesis following isolation from a donor testis and transplantation into a permissive recipient testis. This capacity has enormous potential as a tool for enhancing the reproductive capacity of livestock, which can improve production efficiency. Because SSCs are a rare subset of the undifferentiated spermatogonial population, a period of in vitro amplification in number following isolation from donor testicular tissue is essential. Here, we describe methodology for isolation of a cell fraction from prepubertal bull testes that is enriched for undifferentiated spermatogonia and long-term maintenance of the cells in both the feeder cell coculture and the feeder-free format. To achieve this method, we derived bovine fetal fibroblasts (BFF) to serve as feeders for optimizing medium conditions that promote maintenance of bovine undifferentiated spermatogonia for at least 2 mo. In addition, we devised a feeder-free system with BFF-conditioned medium that sustained bovine undifferentiated spermatogonia for at least 1 mo in vitro. The methodologies described could be optimized to provide platforms for exponential expansion of bovine SSCs that will provide the numbers needed for transplantation into recipient testes.

Glycolysis-Optimized Conditions Enhance Maintenance of Regenerative Integrity in Mouse Spermatogonial Stem Cells during Long-Term Culture

“…Thus, we recommend that both these modifications be employed in future studies utilizing primary cultures of mouse SSCs and anticipate that they will serve as the basis for developing culture conditions for SSCs from higher-order mammals. Indeed, we discovered in a previous study that long-term culture of cattle undifferentiated spermatogonia is enhanced in an atmosphere of reduced O 2 tension (Oatley et al., 2016). In addition, the findings of the current study highlight the importance of matching culture conditions to the bioenergetics of SSCs during long-term in vitro maintenance and provide the impetus for future studies to further refine the conditions to promote utilization of glycolysis.…”

Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene

“…Methodology for transplantation of SSCs that results in reestablishment of fertility is developed for rodents but, extension to higher order mammals has not been reported. For livestock, SSC transplantation has the potential for use as a tool to expand the availability of gametes from desirable (elite) sires, and thereby dramatically influence production efficiency, quality and other production traits in a population56. A key aspect for successful SSC transplantation is the requirement for recipient males with intact seminiferous tubules and somatic support cell populations, but lacking SSCs and other germ cells.…”

Biology and manipulation technologies of male germline stem cells in mammals

“…In the case of mice, the majority of testicular somatic cells can be excluded by differential plating with residual cells overwhelmed by proliferating SSCs . This procedure was thought to be applicable to large mammals . However, testis somatic cells from piglets show a much higher proliferation activity than those from mouse pups.…”