Spermatogenesis begins in the neonatal mouse testis with the segregation of prospermatogonia into
distinct undifferentiated and differentiating populations. A proportion of undifferentiated spermatogonia
retain stem cell potential (as foundational spermatogonial stem cells, or SSCs), and the remainder
becomes progenitor spermatogonia that proliferate and differentiate in response to retinoic acid (RA).
This initial fate decision is critical, as imbalances cause spermatogenic defects that can lead to human
testicular cancer or infertility. It is currently unknown how spermatogonial fate decisions are regulated.
We have recently made the exciting discovery that neonatal male germ cells differ in their
responsiveness to RA both in vivo and in vitro (in the absence of the niche environment). As early as
postnatal day (P) 1, we can identify two distinct subpopulations of prospermatogonia: 1 – those that
cannot respond to RA (‘RA-non-responsive’) either in vitro or in vivo, and 2 – those that can respond
but do not yet in vivo (‘RA-responsive’). The objective of this proposal is to determine whether the
foundational SSC pool forms from prospermatogonia that are intrinsically preprogrammed
(predetermination) or from those that happen to occupy a limited number of stem cell niches (selection).
Our data strongly suggest that some prospermatogonia are predetermined to remain undifferentiated
and become SSCs, and that this fate is manifest by an inability to respond to the proliferation and
differentiation signal provided by RA. In the proposed aims, we will explore whether this RA insensitivity
is intrinsic (germ cell autonomous) or determined by somatic cells (germ cell non-autonomous), define
when during postnatal development differential RA responsiveness appears, and determine whether
the foundational SSC pool is formed solely from these RA-insensitive germ cells. In Aim 1, we will
assess gene expression heterogeneity associated with neonatal germ cell RA responsiveness.
Heterogeneous expression of genes involving RA responsiveness by either the germline or soma could
explain which compartment dictates the fate of SSC precursors. In Aim 2, we will define the roles of RA
reception and catabolism in RA responsiveness. We predict that RA insensitivity is intrinsically
preprogrammed, long lasting, and defines the foundational SSC population. In Aim 3, we will determine
whether RA-insensitive prospermatogonia preferentially give rise to foundational SSCs. We will
modulate RA levels in vivo and examine the subsequent effect on formation of the foundational SSC
pool. Together, the results of these aims will define how RA responsiveness contributes to formation of
the foundational SSC pool in the neonatal testis.