Male germ-line stem cell potential is predicted by
morphology of cells in neonatal rat testes
Kyle E. Orwig, Buom-Yong Ryu, Mary R. Avarbock, and Ralph L. Brinster*
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
Contributed by Ralph L. Brinster, July 11, 2002
Gonocytes are a transient population of male germ-line stem cells
that are derived from primordial germ cells in the embryo and give
rise to spermatogonial stem cells, which establish and maintain
spermatogenesis in the postnatal testis. In contrast to spermato-
gonial stem cells, gonocytes can be identified easily in neonatal rat
testis cell suspensions based on their large size and distinct mor-
transgenic rats demonstrated that gonocytes are the only cells that
express a lacZ reporter transgene. Two gonocyte subpopulations,
designated pseudopod and round, were identified and isolated
from neonatal (0–4 days postpartum) rat testis cell suspensions.
Male germ-line stem cells, identified by their ability to produce and
maintain colonies of spermatogenesis upon transplantation into
infertile recipient testes, were present almost exclusively in the
pseudopod gonocyte subpopulation. In contrast, annexin V stain-
ing indicated that the majority of round gonocytes undergo apo-
ptosis. These results indicate that a nearly pure population of male
germ-line stem cells can be prospectively identified in neonatal rat
testis cell suspensions by morphological criteria. Together, the
the establishment of spermatogenesis in the postnatal testis.
grate to the seminiferous tubule basement membrane, and give
rise to stem cell spermatogonia. Maintenance of spermatogen-
esis and other self-renewing systems in postnatal animals de-
pends on the activity of resident stem cells that have the capacity
to both self-renew and produce progenitors that give rise to the
specified cell lineage. Male germ-line stem cells are unique
among self-renewing systems of postnatal animals because they
can pass genes, through the germ line, to subsequent genera-
tions. Despite their critical position in mammalian physiology,
there is a paucity of information regarding the molecular and
biochemical characteristics of male germ-line stem cells. This
results, in part, from the fact that these cells are extremely rare,
comprising 1 in 3,333 cells of the adult mouse testis (1) and 1 in
500 cells of the adult rat testis (2, 3). Establishment of pure or
significantly enriched populations of male germ-line stem cells is
a critical first step that will facilitate biological and molecular
The development of a functional transplantation assay for
hematopoietic stem cells over 40 years ago (4, 5) enabled
investigators to identify biochemical markers (6), develop en-
richment strategies, and eventually purify hematopoietic stem
cells (7, 8). Spermatogenesis is the only other self-renewing
system for which a stem cell functional assay is available, and
male germ-line stem cells are defined by their ability to generate
and maintain colonies of spermatogenesis upon transplantation
into infertile recipient testes (9–11). Using this assay as a
functional endpoint, we have developed methods for enriching
spermatogonial stem cells from adult testis cell populations by
taking advantage of physical binding properties (3, 12, 13),
surgical manipulation (13), and immunoselection (14). In the
best case, a 166-fold enrichment of spermatogonial stem cells
was achieved by using fluorescence-activated cell sorting
permatogenesis is initiated in the testes of postnatal mam-
mals when quiescent gonocytes resume proliferation, mi-
(FACS) to isolate a subpopulation of cells from adult mouse
cryptorchid testes (14). This testis cell population, characterized
as cryptorchid?side scatterlo(SSClo)??6-integrinhi??v-inte-
grin(?), has a stem cell concentration of about 1 in 30 and
provides a valuable resource for further characterization of
spermatogonial stem cells. Continued development of enrich-
population of spermatogonial stem cells.
During development, germ-line stem cells first can be iden-
tified as a distinct population of primordial germ cells (PGCs)
that arises from the embryonic ectoderm (15). PGCs proliferate
and migrate to the genital ridge where they associate with
somatic cells of the presumptive gonad (16). In females, PGCs
form oocytes, which stop dividing and enter meiosis. In males,
the germ cells are enclosed in the sex cords, become gonocytes,
and cease dividing until after birth (17). The differentiation of
PGCs into gonocytes marks the transition from a cell with
multiple potentials, because pluripotent embryonic germ cells
can be derived from PGCs (18, 19), to one that has the restricted
potential to develop the male germ cell lineage (17, 20). There-
fore, gonocytes are the first stem cells committed to male
germ-line development and the only germ cells in the neonatal
testis. Shortly after birth, some gonocytes resume proliferation,
migrate to the basement membrane of seminiferous tubules
(21–25), presumably differentiate into spermatogonial stem
cells, and initiate spermatogenesis (21, 22, 26, 27). However,
gonocytes of the immature testis are a complex population from
which only a portion are destined to become stem cells (17). A
significant number of gonocytes (30–75%) degenerate (22, 23,
28, 29), whereas some appear to differentiate directly into type
A1 spermatogonia (17). The molecular and biochemical char-
cell, differentiate, or die are not known. Histological and in vitro
studies demonstrate that gonocytes can be readily identified in
immature testes and testis cell cultures based on distinct mor-
phological characteristics (27), they can be isolated to homoge-
neity by micromanipulation (30, 31), and they can be maintained
in culture (24, 25, 30–34).
We took advantage of the morphological characteristics of
gonocytes, their purity as the only germ cells in the neonatal
testis, and their unique expression of a transgene to identify two
subpopulations of gonocytes (pseudopod and round) from dis-
persed neonatal rat testis cell suspensions. These two popula-
tions have distinct developmental potential, which is already
established at the time of birth; pseudopod cells become stem
pure subpopulations of gonocytes with specific destinies has
enormous potential for identification of their biological and
Donor Rats and Cell Collection. Donor testis cells were obtained
from neonatal Sprague–Dawley rats of 0–4 days postpartum
Abbreviations: 6-CFDA, 6-carboxyfluorescein diacetate; dpp, days postpartum; MT, metal-
lothionein I; PGC, primordial germ cell; X-Gal, 5-bromo-4-chloro-3-indolyl ?-D-galactoside.
*To whom reprint requests should be addressed.
September 3, 2002 ?
vol. 99 ?
staining provided a rapid assay to evaluate strategies for gono-
cyte selection, and (iii) the spermatogonial transplantation
system allowed definitive identification?confirmation of sper-
matogonial stem cell activity in selected gonocyte populations.
Although gonocyte migration and degeneration are not ob-
served until after postnatal day 2 in the rat (23, 25, 28, 50), the
present investigation suggests that developmental fate is already
determined at the time of birth. This conclusion follows from the
consistent relationship between the number and biological ac-
tivity of pseudopod and round gonocytes from 0 to 4 dpp. It
seems unlikely that significant numbers of cells changed mor-
phology?function from pseudopod to round, or the reverse,
because we rarely observed a round cell develop a pseudopod
(?1%), round cells almost never made colonies, and the fraction
of pseudopod cells that produced colonies (5%) remained
constant. The disparate developmental destinies (stem cell vs.
death) are undoubtedly orchestrated by divergent genetic pro-
grams. Thus, the pseudopod and round gonocyte cell popula-
tions will provide valuable tools for identifying molecular mech-
anisms controlling cell fate and the establishment of
spermatogenesis in the postnatal testis. In addition, examination
of these cell populations may provide general insight into stem
cell biology and apoptotic processes that are critical for the
development and maintenance of a variety of self-renewing
We thank Drs. R. Behringer, H. Kubota, and E. Sandgren for critical
evaluation of the manuscript and helpful comments. We appreciate the
assistance of C. Freeman and R. Naroznowski with animal maintenance
and experimentation, C. Brensinger for statistical assistance, and J.
Hayden for help with photography. The MT-lacZ transgenic rat line was
a gift from R. Hammer. Microscopic sections were produced in the
Institute for Human Gene Therapy, Cellular Morphology Core, Uni-
versity of Pennsylvania (5-P30-DK-47747-07). Financial support for the
research was from the National Institutes of Health Institute of Child
Health and Human Development Grant 36504; the Commonwealth and
General Assembly of Pennsylvania; and the Robert J. Kleberg, Jr., and
Helen C. Kleberg Foundation.
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