Spermatogonial Stem Cells
Spermatogenesis, an intricate and male-specific process in adult testes that produces a steady amount of spermatozoa for male fertility, is dependent on spermatogonial stem cells (SSCs).
SSCs comprise a subpopulation of undifferentiated spermatogonia which when removed from the testes show broader developmental plasticity. During embryonic development, primordial germ cells (PGCs) give rise to spermatogonia. SSCs depend upon a specific microenvironment known as a “niche” for survival and development. SSCs have some similarities to other stem cells, but they also have their own unique properties. SSCs are adult stem cells that have the ability to pass genetic information from the paternal generation to the progeny. SSCs are therefore very useful in animal genetics, breeding, and reproduction if gene transfection and homologous transplantation of SSCs are synthesized to produce transgenic animals with increased productivity and commercial value. SSCs are also an excellent model for studying the mechanics of stem cell self-renewal and differentiation.24.4.1 Isolation and Enrichment
of Spermatogonia from Domestic Animals
In domestic animal testes, SCs are extremely rare. As a result, for continued culture or manipulation of these cells, it is important to identify and enrich SSCs with high viability and purity.
Fluorescence-activated cell sorting (FACS) and magnetic- activated cell sorting (MACS) are routinely used for the isolation and enrichment of SSCs as they can differentiate SSCs from diverse testicular cell population with the help of several SSC-specific biological markers (Table 24.2).
24.4.2 In Vitro Culture of SSCs from Domestic Animals
SSCs remain in the basement membrane of seminiferous tubules in a three-dimensional (3D) microenvironment. In
Table 24.2 Spermatogonial stem cell markers used for isolation of SSCs in domestic animals
| Species | SSC markers |
| Cattle | UCHL1(PGP9.5), ZBTB16, DBA, THY1(CD90), NANOG2, POU5F1, Claudin-8 |
| Buffalo | UCHL1(PGP9.5), DBA, POU5F1 |
| Goat | UCHL1(PGP9.5), ZBTB16, THY1(CD90) |
| Sheep | ZBTB16, DBA |
| Pig | UCHL1(PGP9.5), ZBTB16, DBA, GFRα1 THY1(CD90), NANOG2, POU5F1, SSEA1 |
| Equid | ZBTB16, GFRα1, CSF1R |
in vitro condition, SSCs require a similar environment for optimal growth. SSCs can be identified, cultured in vitro, and sperm generated from them can be used in IVF or ICSI of oocytes.
In order to stimulate the proliferation of germ cells in culture, the right environment is required. Various culture conditions and growth agents, such as glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2), are supplemented in the cell culture media to keep SSCs growing and multiplying. Porcine SSCs are cultured with or without feeder cells in agarose-based 3D hydrogels and 2D culture plates.24.4.3 Applications of SSCs
24.4.3.1 Genome Editing Via SSCs
SSCs edited with TALEN or CRISPR/Cas9 and implanted into mice’s seminiferous tubules created spermatogenetic cell colonies that produced genetically modified sperm. As a result, gene editing can be used to create genetically altered model animals.
24.4.3.2 Generating Transgenic Animals
SSCs can be taken from postnatal animals, grown in vitro, and genetically targeted. SSCs that have been genetically modified can be differentiated into sperm in vitro or in vivo, which can be used to create transgenic embryos, ESCs, and progeny.
24.4.3.3 Restoration of Fertility
The use of SSC autotransplantation to treat infertility is a feasible approach for restoring fertility in male animals. Ablation of the endogenous germline is required for use as a breeding tool in animals; otherwise, the mixing of donor and recipient sperm production would occur after SSC transplantation. However, the process is the initial stage of development in livestock species.
24.5