Spermatogonial Stem Cell Culture, Transfection, and Intra-testicular Transplantation as a Preliminary Tool Towards Obtaining Genetic Modification of Kenyan Galla Goat

Abstract

Spermatogonial stem cells (SSCs) are the germline stem cells responsible for continuous spermatogenesis. The continuous production of spermatozoa relies on the capacity of SSCs to undergo self-renewal to maintain a reservoir for future production. SSC has been previously isolated from testes and transplanted to homologous recipients, successfully re-establishing donor-derived spermatogenesis. This unique characteristic of SSC can be exploited as a reproductive tool in livestock production to propagate desirable genetics through SSC transplantation to surrogate sires. However, the initial population of SSC isolated from the testis is usually low; therefore, there is a need to optimize methodologies for their in vitro propagation to generate enough numbers for their use in these reproductive technologies. Surrogate sires are ideal recipients for SSC transplantation since they do not possess an endogenous germline layer, but they have functional somatic cell structural support. The aim of the current study was (a) to do Systematic review of literature for in vitro culture of spermatogonial stem cells (SSC) and their applications in livestock species, (b) to establish long-term SSC culture system for indigenous Galla goats in Kenya and characterize the SSC through morphology, immunochemistry, and molecular markers, (c) to optimize gene transfection protocols for the in vitro cultured SSC and (d) to transfer (transplant) SSC to germline intact prepubertal bucks and evaluate their ability to colonize the recipient seminiferous tubules. The literature search on spermatogonial stem cell culture was performed. Relevant data were screened and extracted. There was limited data on in vitro culture of SSC from goats and also none of the studies had been done on livestock in Africa. The SSC was isolated from prepubertal goat testes via a two-step enzymatic digestion method followed by testicular cell enrichment for SSC through a multiparameter selection approach. The isolated SSC had a viability mean of 77.4 ± 1.2 %. The multiparameter selection yielded a population of cells enriched for SSC with higher in vitro colony formation, cells of uniform size, cultures with very few somatic cells, and a majority (69.20 ± 1.0 %) of the cells stained positive for promyelocytic leukaemia zinc finger factor (PLZF), which is a specific SSC marker to ascertain their stem status through immunocytochemistry and real-time polymerase chain reaction (qPCR). The single enriched procedure of differential plating on gelatin-coated plates results in about 25.62±1.76% cell population of PLZF-staining cells. The study demonstrated a thriving goat SSC culture and proliferation on a feeder-free system with goat fetal fibroblasts (GFF) pre-conditioned Stempro medium for 45 days. Notably, the immunostaining of the feeder-free cultured SSC germ cell clumps invariably expressed PLZF staining, which indicated maintenance of undifferentiated spermatogonial phenotype through immunochemistry and RT-PCR. The goat SSC culture also exhibited typical germ cell clump morphology similar to what has previously been reported in rodent SSC. The cultured SSC were transfected with enhanced green fluorescent protein (eGFP) reporter gene plasmid bound to cytomegalovirus (CMV) promoter and delivered to the cell cytosol through lipofectamine reagents and electroporation. The use of Lipofectamine™ stem reagent carrier had a higher number of SSC colonies expressing the eGFP gene (25.25%) compared to Lipofectamine™ 2000 carrier molecule (22.25%). Electroporation of the SSC resulted in the highest transfection efficiency of 15% with a viability rate of 50% cells. The high voltage of electroporation resulted in SSC death. The two transfection methods yielded promising results for utilizing the techniques for gene transfer of genetic material into goat SSC. However, further studies are required to modify the parameters and transfection conditions for improvement of the overall transfection efficiency and the viability of cells after transfection. The SSC are the only cells with the ability to migrate to the basement membrane of the seminiferous tubules and colonize the membrane through the occupation of stem cell niches. An enriched population of cultured and eGFP transfected SSC has successfully been transplanted to prepubertal buck testes through mediastinum testis in an ultrasound-guided injection. The presence of eGFP-expressing cells in seminiferous tubules of recipient testis following transplantation in prepubertal bucks indicates that the ultrasound-guided transplantation of donor cells was successful, but whether these cells would persist long enough to colonize seminiferous tubules and donor-derived genotype was not evaluated. There was no inflammatory reaction or infection to transplantation of SSC in recipient bucks. The establishment of a robust long-term culture system for SSC can unlock possibilities of their use for transplantation technology in livestock production and a generation of transgenic animals. In conclusion, the study was the first report of a combined serum-free feeder-free in vitro culture system of goat SSC. The devised goat SSC culture system also marks the first report of culturing SSC in livestock in Africa. The established conditions can be used as a benchmark for further studies in the long-term expansion of goat SSC that will provide enough numbers for SSC application in transgenesis and surrogate sire breeding technology. The established protocols for transfection parameters, contribute significantly to knowledge of successful gene manipulation of SSC in generating transgenic animals with better traits for disease resistance, fertility, or production.