2.1. of stem cells, is asymmetric. In

2.1. Stem cells

The
canonical definition of a stem cell is a cell with the ability to divide
indefinitely in culture and with the potential to give rise to mature
specialized cell types (Alison et al.,
2002). This style of cell division, characteristic of stem cells, is
asymmetric. In fact, when a stem cell divides, the daughter cells can either
enter a path leading to the formation of a differentiated specialized cell or
self-renew to remain a stem cell, thereby ensuring that a pool of stem cells is
constantly replenished in the adult organ. This is a necessary physiological
mechanism for the maintenance of the cellular composition of tissues and organs
in the body (Andrews, 2002; Bangso and Richard, 2004; Kanatsu-Shinohara et al., 2004). Stem cells can be
classified into Totipotent, Pluripotent and Multipotent cells. Totipotency is
the ability to form all cell types of the conceptus, including the entire fetus
and placenta.  Pluripotency is the
ability to form several cell types of all three germ layers (ectoderm, mesoderm
and endoderm) but not the whole organism. 
Multipotency is the ability of giving rise to a limited range of cells
and tissues appropriate to their location. The rigorous definition of a stem
cell requires that it possesses two properties: Self renewal and Unlimited
potency. Self renewal means the ability to go through numerous cycles of cell
division while maintaining the undifferentiated state. Unlimited potency means
the capacity to differentiate into any mature cell type.

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2.2. Spermatogonial stem
cells                                                                                                  

Spermatogonial stem cells are derived from growth-arrested
gonocytes of the newborn testis. Primordial germ cells are pluripotent and are
capable of forming all three primordial germ layers.  Germinal differentiation in the testis starts
from a small population of cells (2–3 × 104 per adult mouse testis)
designated Asingle (As) spermatogonia (de Rooij and
Grootegoed, 1998). Lining the basal membrane of the seminiferous tubule, they
show a characteristic morphology with a nucleus devoid of heterochromatin. A
series of mitotic divisions sequentially generate the Aaligneds
cells, which remain connected by cytoplasmic bridges. The latter in turn
generate the A1 to A4, Intermediate and B spermatogonia,
which eventually divide into preleptotene spermatocytes that will subsequently
enter the ?rst meiotic division. SSCs can undergo two types of cell divisions
namely symmetrical and asymmetrical. Daughter stem cells are always results of
symmetrical divisions while asymmetrical divisions give rise to both stem cells
and differentiating cells. Functionally, SSCs characterized by their ability to
re-establish permanent spermatogenesis upon transplantation into recipient
testes devoid of germ cells.

Unlike other stem cells, SSCs are distinct in that they
can transmit genetic information to the next generation. Unlike Embryonic
stem  cells, however, SSCs retain some
features of the original PGCs, including genome-wide demethylation, erasure of
genomic imprints and reactivation of X-chromosomes, the degree of which likely
reflects the developmental stages of the PGCs from which they are derived (Yu et al., 2008; Maser and Depinho, 2002).
The use of SSCs has several advantages over conventional methods based on
eggs/oocytes. First, SSCs are established from postnatal testes, whereas
Embryonic stem cells are derived from embryos. Second, SSCs have very stable
genetic and epigenetic properties, probably reflecting the features of SSCs as
committed stem cells. This is in contrast to Embryonic stem cells. Because they
are uncommitted to a specific lineage, Embryonic stem cells can easily
differentiate in to other lineages, but they may lose their germ-line
potential. Therefore, it can be considered that SSCs will become a target for
mutagenesis in many animal species. Unlike mice, most animals do not ovulate
large numbers of oocytes and they require a long period of time to reach sexual
maturity. These factors limit the genetic manipulation of such animal species.
Therefore, SSCs technology has an advantage in many animal species that produce
small numbers of offspring i.e. Livestock species.

2.3 Spermatogonial Stem Cells Transplantation in animals

            Brinster and Zimmerman (1994)
demonstrated that microinjection of a heterogeneous mouse testis cell
suspension directly into the seminiferous tubules of a genetically sterile
mouse resulted in donor spermatogenesis in the recipient. The next report was
that of Jiang and Short (1995) who demonstrated the successful transplantation
of rat testicular cells into the seminiferous tubules of rats that had been
treated with busulfan. The success of SSCs transplantation in rodents (Brinster
and Avarbock, 1994; Jiang and Short, 1995; Ogawa et al., 1999) has generated interest in its application for
commercial animal species (Honaramooz et
al., 2002; Honaramooz et al.,
2003; Herrid et al., 2006). This has
been highlighted by the production of live offspring of donor-origin after
transplantation in goat (Honaramooz et
al., 2003), chicken (Trefil et al.,
2009) and sheep (Herrid et al.,
2006). Reports of donor-origin sperm in recipient ejaculates in dog (Kim et al., 2008) and cattle (Stockwell et al., 2009), and colonization of recipient
testis with donor cells in pigs (Honaramooz 
et al., 2002) indicate
offspring may soon be produced in these species.

Among the eight
other large animal SSC transplant studies reviewed in Table 2.1, four reported
evidence of donor sperm in the ejaculate (goat, boar, dog, and sheep) and two
reported functional sperm (goat and sheep) that produced donor-derived progeny.

2.4 SSCs transplantation Protocol

SSCs
transplantation is a procedure which involves isolation of a mixed germ cell
population from donor testis and retrograde injection of isolated and enriched
cells into the testes of recipient.  SSCs
transplantation may be of three types- (a) Autologous; within same individual,
(b) Homologous/ syngeneic; within same species, (c) Xenogeneic; among animals
of different species. The whole transplantation procedure involves following
steps-1) Recipient preparation,   2) SSCs
Isolation from donor, 3) SSCs Enrichment, Characterization and Culture, 4)
Transplantation. Alternatively, Transfection and Cryopreservation may be done
for production of transgenic progeny and preservation of germplasm
respectively.

2.4.1
Depletion of endogenous germ cells (Recipient preparation)

Recepient
preparation is one of the major obstacles to the commercialization of SSCs transplantation
in animal species because it needs use of expensive irradiation techniques or
toxic chemotherapy drugs to deplete endogenous stem cells in the recipients to
enhance colonization of transplanted donor cells. Earlier researchers reported  reported SSC transplant in nonhuman primates
that were rendered infertile by testicular irradiation (Schlatt et al., 2002; Jahnukainen et al., 2011). Although, it has been
reported that the preparation of recipient’s testis with irradiation enhances
the efficiency of germ cell transplantation as well as increases the proportion
of donor sperm in recipient semen (Herrid et
al., 2009) yet till date, SSC transplantation into a chemotherapy-treated
large animal recipient has been reported only in the pig (Mikkola et al., 2006).