Redesigning Animal Agriculture

Brinster et al., 2003). Treatment of pigs
and rams with busulfan between 5 and 17
weeks of age resulted in a significant reduction
of testis weight and germ cell number
but also in systemic toxicity. In litter-bearing
animals such as pigs, in utero treatment
is effective as previously described in the
mouse (Brinster et al., 2003; Honaramooz
et al., 2005). Administration of busulfan
to pregnant sows at d 98 and 108 of gestation
(a period of high proliferation of fetal
gonocytes) resulted in depletion of germ
cells with no observed adverse effects on
the piglets or on sow health or fertility
(Honaramooz et al., 2005). It was shown
previously in rodents that complete suppression
of spermatogenesis might not
be desirable in recipient animals as some
residual endogenous spermatogenesis is an
indication that the testicular environment
has not been permanently damaged by the
cytotoxic treatment (Brinster et al., 2003).
Donor Germ Cell Isolation
It has been estimated that there are only
about 2 × 10 4 stem cells in 10 8 cells of a
mouse testis (Meistrich and van Beek,
1993; Tegelenbosch and de Rooij, 1993). To
improve efficiency of donor-derived spermatogenesis
after transplantation and to
allow manipulation of donor cells prior
to transplantation, it is desirable to obtain
a cell population enriched in germ cells.
Isolation and enrichment of spermatogonia
from pre-pubertal animals where significant
testis growth has already occurred but germ
cell differentiation has not yet started should
yield the largest number of germ cells for subsequent
experimentation. Therefore, enrichment
of germ cells from immature testes is
more efficient than from adult testes. In the
mouse, selection of germ cells for expression
of a 6- and b 1-integrin, the absence of c-kit
receptor, as well as collection of cells from
experimentally induced cryptorchid testes,
resulted in a significant enrichment for
spermatogonial stem cells (Shinohara et al.,
1999, 2000a,b). More recently, expression of
Thy-1, CD9 or Egr3 was utilized as a marker
Germ Cell Transplantation 85
for enrichment of mouse germ line stem
cell populations (Kubota et al., 2003, 2004;
Kanatsu-Shinohara et al., 2004a; Hamra et al.,
2004). Fluorescence- or magnetic-activated
cell sorting (FACS or MACS) are frequently
employed to isolate spermatogonia in mice
(von Schonfeldt et al., 1999; Shinohara
et al., 2000a). Alternatively, testicular cells
subjected to velocity sedimentation and differential
adhesion to extracellular matrix
components like laminin or fibronectin were
shown to be enriched in spermatogonia in
mice and rats (Shinohara et al., 1999; Hamra
et al., 2004). In large animal models, it is
important that enrichment techniques yield
large enough cell numbers for transplantation
into a large animal recipient testis without
compromising cell viability (Honaramooz
et al., 2002b; 2003a,b). Because the number
of cells that can be isolated by FACS or
MACS is limited, these approaches are not
as suitable for enrichment of large numbers
of spermatogonia from large animal testes,
while differential plating and velocity sedimentation
are potentially more practical for
enriching spermatogonia from pre-pubertal
testes for transplantation into large animals.
A combination of differential plating and
a discontinuous Percoll density gradient
have been employed to enrich bovine type
A spermatogonia (Izadyar et al., 2002). We
recently reported a modified differential
plating approach to obtain enriched cell
populations from pre-pubertal porcine testes
that contained about 1 spermatogonial cell
in 6 total cells (Luo et al., 2006). While not
all spermatgonial cells are necessarily stem
cells, this degree of enrichment appears comparable
to those reported for surface markerbased
cell-sorting strategies for rodent germ
line stem cells (Ryu et al., 2005).
Germ Cell Culture
Nagano et al. (1998) showed first that
mouse germ line stem cells could be maintained
in culture for a long period of time.
Improving culture conditions for male germ
line stem cells still is under intense study
as evidenced by several reports of improved