download report - Istituto Pasteur

P a r t i c i p a n t s :
Carla Boitani, professor; Elena Vicini, researcher; Barbara
Muciaccia, Rossella Pugliesi, post-doc fellows; Margherita
Grasso, Laura Grisanti, PhD students; Stefania Fera, Tiziana
Menna, technicians.
Report of activity
The highly efficient process of sperm production is
dependent on proper hormone balance, local cellular
interactions and, more importantly, it relies on the
biological activity of spermatogonial stem cells
(SSCs), the stem cells of the germline. In the last
years the activity of our group has focused on the
isolation and characterization of SSCs. Our previous
work has led us to the identification of SSCs in the
testis “side population” (T-SP). Further characterization
of T-SP SSCs by double vital staining with
Hoechst and rhodamine, indicated that T-SP SSCs
represent only a subset of SSCs. Of note, other
groups were able to derive pluripotent stem cells
from mouse and human germ cells in culture.
Therefore, SSC subsets may be endowed with different
functional properties and developmental potency.
The aim of this project was to further characterize
and exploit functional differences among SSC subsets.
A complementary project carried out in our
group was aimed to develop experimental strategies
to achieve conditional gene inactivation in the
germline.
Identify spermatogonial stem cells
heterogeneity in vivo
Evidence for phenotypic and functional heterogeneity
have been obtained after prospective isolation of
SSC and their biological assay, namely germ cell
transplantation. However there is no evidence for the
presence of such stem subsets in vivo. In the accepted
model, the SSCs are type Asingle spermatogonia
(As). The As either self-renew by forming single
Principal investigator: Mario Stefanini
Professor of Histology and Embryology
Dipartimento di Istologia ed Embriologia Medica
Tel: (+39) 06 49766570; Fax:(+39) 06 4462854
mario.stefanini@uniroma1.it
67
Molecular genetics of eukaryotes - AREA 3
Biological characterization and in vitro culture of spermatogonial
stem cells
cells or generate pairs of cells, named type Apaired
spermatogonia (Apr), connected by an intercellular
bridge, which are committed to differentiate. To test
the hypothesis that, in adult mouse testis, As spermatogonia
are phenotypically heterogeneous, we
have analyzed GFRA1 expression in undifferentiated
spermatogonia. GFRA1 is the co-receptor for GDNF
a niche-derived factor essential for SSC self-renewal
and differentiation. As spermatogonia can be easily
identified in whole mounted seminiferous tubules
after immunostaining for the nuclear repressor
PLZF. By immunofluorescence, confocal microscopy
and morphometric analysis, we were able to demonstrate
that 10% of the As cell population did not
express this receptor. A common mechanism to generate
cell diversity is the asymmetrical inheritance of
proteins that specify daughter cell fate. We therefore
analyzed by morphometric analysis the distribution
of GFRA1 in daughter cells. We found doublet of
daughter cells asymmetric for GFRA1 expression
(5% of clones). This suggest that generation of SSC
subsets may hold on asymmetric germ stem cell division.
We next analyzed cell cycle kinetics of the two
subsets by in vivo bromodeoxyuridine (BrdU) administration.
Our results indicated that both subsets are
actively engaged in cell cycle. A surrogate markers
largely employed in different tissues for stem cells
identification has been their preferential BrdU label
retention, labeling-retaining-cells (LRCs). In fact,
stem cells are expected to divide more slowly than
many of their cell progeny. Time-course analysis
after BrdU pulse labeling showed some LRCs at one
month of chase. However, at two months no LRCs
were identified. All together this indicate that germ
stem cells are actively engaged in cell cycle and no
quiescent stem cells are present in the adult testis,
regardless of their GFRA1 expression pattern.
Complementary observation in human testis samples
also revealed that the most primitive germ cells are
also heterogeneous for the expression of GFRA1.
This observation lend further support to germ stem