All You Wanted to Know About Spermatogonia but Were Afraid to Ask

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776
ReviewAll You Wanted to Know
About Spermatogonia but
Were Afraid to Ask
DIRK G. DE ROOIJ* AND LONNIE D. RUSSELL†
From the *Department of Cell Biology, University
Medical Center Utrecht, Utrecht, The Netherlands; and
the †Department of Physiology, Southern Illinois
University School of Medicine, Carbondale, Illinois.
Spermatogonia have entered the limelight in recent years,
given the intense interest in stem cells in general (Fuchs
and Segre, 2000) and specific interest in cloning (Wilmut,
1998; Wilmut et al, 1997, 1998), transgenesis (Erickson,
1999; Perry et al, 1999; Pintado and Gutie´rrez-Adan,
1999; Robl, 1999; Russell and Griswold, 1998), and reg-
ulation of spermatogonial numbers (de Rooij and Groot-
egoed, 1998). Spermatogonial terminology is confusing
to many and spermatogonial kinetics is perceived as com-
plex. The field is dominated by only a few individuals
whose work is highly specialized and whose techniques
are not generally used. Thus, there is a definite need to
simplify this little understood topic for others.
What follows is information about spermatogonia, in
particular the terminology applied to spermatogonia and
their function, a description of the kinetics and renewal
of spermatogonia, the origin of spermatogonia, the regu-
lation of spermatogonial numbers, and conditions that af-
fect spermatogonia. A question-and-answer format is used
in an attempt to simplify a complex area of research for
an audience that is generally not familiar with the topic
or that has in the past had difficulty with the topic. A
variety of other reviews on the topic are embedded in the
dialogue. Reviews are suggested for readers who have
mastered the basics and wish to examine the details of
spermatogonia and their properties. We limit our discus-
sion of spermatogonia to mammalian systems and, unless
otherwise specified, direct our comments primarily to ro-
dent systems, from which most of our information about
spermatogonia has been derived.
Role of Spermatogonia in Gamete Production
Question: What specifically do spermatogonia con-
tribute to the process of spermatogenesis?
Correspondence to: Lonnie D. Russell, Department of Physiology,
Southern Illinois University School of Medicine, Carbondale, IL 62901-
6512 (e-mail: lrussell@siumed.edu).
Received for publication May 1, 2000; accepted for publication June
26, 2000.
Answer: Spermatogonia have three roles. First, sper-
matogenesis is initiated via spermatogonia. Second, the
population of germ cells is greatly increased via the mi-
totic activity of spermatogonia. One spermatogonium on
average goes through 8 to 9 divisions before differenti-
ating into a spermatocyte (Lok and de Rooij, 1983b; Te-
gelenbosch and de Rooij, 1993). A spermatocyte carries
out only 2 meiotic divisions; therefore, on average, ulti-
mately 1 spermatogonium is capable of rendering 2048
or sometimes 4096 spermatozoa (Russell et al, 1990), de-
pending on the number of divisions. Third, regulation of
germ cell numbers is accomplished in the spermatogonial
population of cells; regulation ensures that the appropriate
ratio of germ cells to Sertoli cells is provided (de Rooij
and Janssen, 1987; de Rooij and Lok, 1987).
Spermatogonial Nomenclature
Question: How does one define a spermatogonium?
Answer: Spermatogonium is the term given to a rel-
atively unspecialized diploid germ cell present in the sem-
iniferous epithelium after the start of spermatogenesis that
is able to carry out mitotic divisions, ultimately giving
rise to primary spermatocytes.
Question: Is there more than 1 type of spermatogo-
nia?
Answer: Yes, there are a number of spermatogonial
types. Unfortunately, although the first assigned names
were logical to those who conceived of them, the detec-
tion of more spermatogonial cell types has made the no-
menclature rather confusing. The following questions
should help clarify this.
Question: How are spermatogonia named?
Answer: In the beginning only 2 types of spermato-
gonia were described. The first, type A spermatogonium,
did not display heterochromatin in the nucleus and the
second, type B spermatogonium, did display heterochro-
matin (Figure 1). Shortly thereafter, someone found a type
of spermatogonium that had an intermediate amount of
heterochromatin, which was termed intermediate (In)
spermatogonium. These names are historical, and they are
broad categories of cells. When more generations of sper-
matogonia were discovered, especially among the type A
category, new names were devised to categorize them.
Question: It was the amount of heterochromatin that
cells possessed that allowed them to be initially catego-
rized?
777de Rooij and Russell · Spermatogonia
Figure 1. Broad categories of spermatogonia.
Figure 2. A type A spermatogonium from a mouse testis.
Answer: Yes, it is a general rule in biology that the
more differentiated a cell is in a particular lineage, the
more heterochromatin one finds within the nucleus. We
can say that type A spermatogonia are the most primitive,
followed by type In spermatogonia, followed by type B
spermatogonia (Figure 1A and B).
Question: When species are compared, is the increase
in the amount of chromatin in the nucleus at the various
spermatogonial differentiation steps comparable?
Answer: Comparisons are only useful within a spe-
cies. One cannot necessarily expect the same amount of
heterochromatin in the same cell type of one species to
match that of the same cell type of another species.
Question: Which is the most primitive type A sper-
matogonium?
Answer: The most primitive is the type A-single (for-
merly called A-stem cell or A-isolated) or, simply, As or
the stem cell spermatogonium (Figure 2). The cell pic-
tured is a type A spermatogonium and, to the best of our
knowledge, it resembles a stem cell.
Question: Does a stem cell spermatogonium have all
the characteristics of other stem cells of the organism?
Answer: Yes, in that it initiates the development of
cells that are committed to form terminally differentiated
cells, and that it is able to give rise to more stem cells
(Figure 3).
Question: In what respect is it different?
Answer: For all other proliferating systems, the stem
cell is a functional definition, meaning it has the activities
described earlier (differentiation and self-renewal). In
mammalian testes, stem cell spermatogonia (As) are also
morphologically defined in that they are the only type of
spermatogonia without intercellular bridges.
Question: What is an intercellular bridge?
Answer: When a spermatogonium divides, telophase
is incomplete, leaving an open area of cytoplasmic con-
tinuity called a cytoplasmic bridge (ie, a ‘‘bridge’’; Faw-
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Figure 3. Two possible fates of a stem cell spermatogonium.
Figure 5. Symmetrical versus asymmetrical divisions of stem cells.
Figure 4. (A) The intercullar bridge connects two spermatogonia. (B) Two type B spermatogonia connected by intercellular bridges.
cett et al, 1959; Weber and Russell, 1987; Figure 4A and
B).
Question: What is the function of bridges?
Answer: Bridges allow sharing of gene products be-
tween cells of a clone. If gene products in adjacent cells
are shared, cellular activities become synchronized, re-
sulting in a synchronized development (Lee et al, 1995;
Braun et al, 1989). Because bridges are present in cells
that are committed to form sperm we can say that single
spermatogonia (without bridges) are stem cells.
Question: Do we know if the stem cell divisions are
symmetrical (dividing into similar cells) or asymmetrical
(dividing into dissimilar cells)?
Answer: We do not know that as yet. As depicted in
Figure 5, when divisions are symmetrical the fate of the
daughter cells could directly depend on a regulatorymechanism that decides whether or not a bridge is formed
in a subsequent cell division. A stem cell then either di-
vides to form 2 stem cells or to a pair (Apr), which is
destined to follow the differentiation path. When divi-
sions are asymmetrical, a stem cell divides into a stem
cell and a single cell that at its first division will give rise
to a pair. The other daughter cell produces 2 new stem
cells. A suggestion has been made that asymmetrical di-
visions may occur (Huckins, 1971b) but this has not been
confirmed (Lok et al, 1984). Until this is confirmed, we
assume that there are symmetrical divisions only and that
the only single type A spermatogonia are As cells.
Question: Now that we know the beginning cell in
the whole process is the type As spermatogonium, what
are the other types of spermatogonia in order of devel-
opmental progression?
Answer: The As in most species give rise to pairs and
779de Rooij and Russell · Spermatogonia
Figure 6. The so-called ‘‘undifferentiated spermatogonia.’’
Figure 7. Progression from As spermatogonia to B spermatogonia.
then chains from 4 to 16 cells, called Apr and Aal sper-
matogonia, respectively (Figure 6). The As, Apr, and Aal
spermatogonia together have been called undifferentiated
spermatogonia (Huckins, 1971b). Whereas the ‘‘undiffer-
entiated’’ label makes writing about spermatogonia easier
because they can be grouped under one label, the term
has been confusing.
Question: Why is the term ‘‘undifferentiated’’ con-
fusing?
Answer: The term ‘‘undifferentiated’’ was originally
used to describe the morphological appearance of such
cells. They show virtually no characteristics of any nu-
clear or cytoplasmic differentiation. Unfortunately, these
cells differ little from subsequent generations of A sper-
matogonia that we call ‘‘differentiating,’’ which also
show few differentiation characteristics. However, most
importantly, Apr and Aal spermatogonia in normal semi-
niferous epithelium are actually differentiating cells in the
sense that they are irreversibly committed to take further
developmental steps in the direction of spermatocytes. So,
although they are called ‘‘undifferentiated,’’ they are
functionally committed. Clearly, the terminology is con-
fusing.
Question: So should they be given a name that is less
confusing?
Answer: Let us agree to eliminate the word ‘‘undif-
ferentiated’’ and call spermatogonia by their names, As,
Apr, Aal, and so on.
Question: If the term ‘‘undifferentiated’’ was once
used, was the term ‘‘differentiated spermatogonia’’ also
used?
Answer: Yes, Aal spermatogonia are able to differ-
entiate into the first of 6 generations of so-called differ-
entiating spermatogonia in mouse and rat, which are sub-
sequently composed of A1, A2, A3, A4, In, and B sper-
matogonia. Again, the terminology, in this case differen-
tiating, should be dropped. The progression from As to B
is demonstrated in Figure 7.
Question: Why was the terminology inappropriate?
Answer: Differentiating should apply to all spermato-
gonia that are more advanced than stem cells. Because it
has been inappropriately named, we should discard the
term.
Question: What do advanced spermatogonia look
like?
Answer: Photographs of these cells taken from whole
mounts are provided in Figure 8A through L. When look-
ing at these photos, remember that only the nuclei stain,
so the bridges that link cells generally cannot be seen,
although we have one example of a whole mount in
which bridges are visible (Figure 8D).
Question: It seems like the A1 through A4 spermato-
gonial cells appear similar. How does one tell them apart?
Answer: You are correct, one cannot tell the individ-
ual cell types apart.
Question: Then if we can’t tell them apart, how do
we know which is a type A1, A2, A3, or A4 spermatogo-
nium?
Answer: A1-4s can be distinguished from each other
only by determining in which stage of the cycle of the
seminiferous epithelium they are present (Oakberg, 1956;
Clermont, 1962). For example, when the area of the rat
whole mount is in stage X, A2 spermatogonia will be
present. Although the great majority of A spermatogonia
in Stage X will be A2, some As, Apr, and Aal spermato-
gonia (which are present throughout the entire epithelial
cycle) will also be present. There should be more than 8–
16 cells in a clone for cells to be classified as A2 cells,
whereas a maximum of 16 cells would be present in Aal.
It has been shown that the bridges can be seen in specially
prepared whole mounts (Huckins, 1978b), but it is diffi-
cult to visualize bridges. In sectioned material, the various
types of A spermatogonia cannot be readily distinguished
from each other because the intercellular bridges are not
always in the plane of section and, as well, the cells that
comprise a chain will generally not all be in the same
section.
Question: Does a division take place from Aal to A1?
Answer: No! It is simply a transformation that takes
place in cells while they are in G0/G1 phase of the cell
cycle, which requires no division (Figure 9).
Question: According to a count in Figure 9, there are
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Figure 8. Photographs of the cells lying on the basal membrane of mouse
seminiferous tubules. As, Apr, and Aal spermatogonia can be distinguished
from A1-A4 spermatogonia because they do not go through the cell cycle
synchronously with the latter cells. Whole mounts of seminiferous tubules
are stained with hematoxylin and only the nuclei of the cells can be
discerned. (A) As spermatogonium presumably in G1 phase of the cell
cycle (arrow) among A1 spermatogonia (arrowheads) synchronously in
G2 phase just before division into A2 spermatogonia. Some Sertoli cells
(asterisks) and (leptotene) spermatocytes (small arrowheads) are also
indicated.
Figure 8C. A chain of 8 Aal spermatogonia, 7 of which are in the photo-
graph (indicated by in-between dashes) among telophasic A3 spermato-
gonia (some indicated by arrowheads). Magnification 660�. Bar � 10
�m.
Figure 8D. Apr spermatogonia, the focus of which is on the intercellular
bridge, which happens to be visible, among A1 spermatogonia in G2.
Magnification 660�. Bar � 10 �m.
Figure 8B. Apr spermatogonia (arrows) among A3 spermatogonia (arrow-
heads) in G2 phase. Magnification 660�. Bar � 10 �m.
9 or 10 spermatogonial divisions in the rat before sper-
matocytes are formed. Although there are usually 9 di-
visions, as many as 16 Aal cells have been seen at one
time (Huckins, 1978a), indicating that sometimes there
are 10 spermatogonial divisions.
Answer: Good, you are doing quite well!
Question: If we compare cell generations of sper-
matogonia, which is more constant in terms of what ac-
tually happens: that there are more or less 9 to 10 gen-
erations, or the uniqueness in the morphology of the nu-
cleus?
Answer: We rely on the number of generations and
the number of cells connected by bridges rather than the
morphology.
Question: How many stem cells are there in a rodent
testis?
Answer: A mouse testis contains about 35 000 As
spermatogonia (Tegelenbosch and de Rooij, 1993). In a
rat testis weighing about 10 times more, one can speculate
that there will be about 350 000 stem cells per rat testis.
Question: How does one determine the number of
stem cells?
Answer: In whole mounts (see later discussion), by
carrying out cell counts, we can determine the ratio be-
tween the numbers of As cells and Sertoli cells. In sec-
tions, one can determine the number of Sertoli cells per
testis using morphometric methods (eg, the disector meth-
od [Sterio, 1984; Russell et al, 1990; Tegelenbosch and
de Rooij, 1993]). From there we can use the ratio (Sertoli/
type As) to estimate the number of stem cells per testis.
781de Rooij and Russell · SpermatogoniaFigure 8E. A chain of 4 A1 spermatogonia in prophase of mitosis into a
chain of 8 (indicated by in-between dashes) among A4 spermatogonia in
G1 phase (some indicated by arrowheads). Magnification 660�. Bar �
10 �m.
Figure 8G. A1 spermatogonia (arrowheads). Magnification 1320�. Bar�
10 �m.
Figure 8H. A2 spermatogonia (arrowheads). Magnification 1320�. Bar�
10 �m.
Figure 8F. An apoptotic clone of A2 spermatogonia (asterisks) among
viable A2s (some indicated by arrowheads). Magnification 660�. Bar �
10 �m.
Spermatogonial Predecessors
Question: What cells give rise to spermatogonia?
Answer: The lineage leading to spermatogonia is rel-
atively straightforward (Figure 10) but may appear con-
fusing because there are alternative terminologies for pre-
cursor cells. The first cell in the lineage is the primordial
germ cell (PGC), which in turn, derives from epiblast
cells (embryonal ectoderm; Lawson and Pederson, 1992).
Later in development, PGCs migrate from the base of the
allantois along the hindgut to finally reach the genital
ridges at about embryonic day 11.5 in rats and embryonic
day 10.5 in mice. They proliferate during migration and
after migrating to the gonadal ridge, primordial germ cells
become gonocytes as they become enclosed within cords
formed by Sertoli precursor cells and are surrounded by
peritubular cells (Clermont and Perey, 1957a; Sapsford,
1962). They show a burst of mitotic activity followed by
an arrest in the G0 phase of the cell cycle, and then gon-
ocytes remain mitotically quiescent until after birth, when
they give rise to spermatogonia. The terms prosperma-
togonia (various types) (Hilscher et al, 1974) and pres-
permatogonia (Byskov, 1986) are other names given to
gonocytes.
Question: During pubertal development, do gono-
cytes give rise to As cells?
Answer: How gonocytes divide to form various sper-
matogonial cell types is still unclear. We know the fol-
lowing: 1) stem cells by definition must be present; 2)
bridges that connect some gonocytes have been described
(Zamboni and Merchant, 1973); and 3) as discussed later,
the kinetics of spermatogenesis at the start of spermato-
genesis suggest that gonocytes, aside from giving rise to
stem cells, also give rise to A2 spermatogonia (de Rooij,
1998). Thus, whereas at least some gonocytes give rise
to As spermatogonia at the start of spermatogenesis, other
gonocytes give rise to more differentiated types of sper-
matogonia, some behave like A1 cells and give rise to A2
spermatogonia (Figure 11). The latter observation implies
that just like Aal spermatogonia do in the course of normal
spermatogenesis, some gonocytes differentiate into A1
spermatogonia before division and, after their first divi-
sion, become A2 cells. They may not skip several divi-
sions because they are already in clones with intercellular
bridges, which suggests some differentiation has already
taken place.
Question: Are Apr and Aal spermatogonia present dur-
ing the first wave of spermatogenesis?
Answer: We do not know whether Apr and Aal sper-
matogonia are also formed directly at the start of sper-
matogenesis.
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Figure 8I. A3 spermatogonia (arrowheads). Magnification 1320�. Bar �
10 �m.
Figure 8K. In spermatogonia (arrowheads). Magnification 1320�. Bar �
10 �m.
Figure 8L. B spermatogonia (arrowheads). Magnification 1320�. Bar �
10 �m.
Figure 8J. A4 spermatogonia (arrowheads). Magnification 1320�. Bar �
10 �m.
Question: It would seem that if gonocytes have the
dual responsibility of producing As, and possibly also Apr
and Aal spermatogonia and dividing to form A2 cells, that
the yield of spermatogenesis is low during pubertal de-
velopment.
Answer: The first wave is less efficient, possibly for
this reason, but surely also because many cells degenerate
early in spermatogenic development (Kluin et al, 1982;
Russell et al, 1987).
Question: When do the first A2 spermatogonia appear
in various rodent species? It appears that this would be
important to know because the division of gonocytes to
form the first A2 spermatogonia marks the beginning of
spermatogenesis, right?
Answer: Yes, the appearance of A2 spermatogonia is
the signal that spermatogenesis has started. The data for
the beginning of spermatogenesis in several species/
strains is summarized in Table 1.
Question: There is something strange with these data.
In the rat, spermatogenesis starts at day 5 and spermato-
genesis is complete at day 43. That means that 4 epithelial
cycles (from A2 to spermiation) took place within 38
days, while 1 epithelial cycle takes 12.8 to 12.9 days in
the rat. One would expect spermatogenesis not to be com-
plete before day 56. Apparently, in the developing rat,
each epithelial cycle lasted only 9.5 days on average in-
stead of 12.8 to 12.9 days.
Answer: The epithelial cycle in young animals is
much shorter than in adults (Kluin et al, 1982; van Haas-
ter and de Rooij, 1993b). In rats, the epithelial cycle takes
about 5 days during the first weeks of life (van Haaster
and de Rooij, 1993b) versus 12.8 in adults (Hilscher et
al, 1969); in Chinese hamsters, the comparable figures are
9 days (van Haaster and de Rooij, 1993b) versus 17 days
in adults (Oud and de Rooij, 1977). Spermatogonial cell
cycle times and spermatocyte development are faster in
prepubertal animals. It is not known what slows down the
process to the adult rate. The switch to a slower sper-
matogenic process correlates with testicular descent (and
a lower testicular temperature), the appearance of stage
VII pachytene spermatocytes, and the start of the for-
mation of a tubular lumen (van Haaster and de Rooij,
1993b).
Question: A correlation between testicular tempera-
ture and the rate of spermatogenesis seems perhaps the
most plausible cause for differing rates of spermatogen-
esis. Is there any evidence for such a correlation?
Answer: Yes, one paper has indicated a role for testis
temperature in modifying the rate of adult mouse sper-
matogenesis (Meistrich et al, 1973); however, more stud-
ies will be necessary to extend this to the prepubertal
situation.
Question: Do some PGCs fail to become incorporated
into cords and, if so, what happens to PGCs that do not
become incorporated into cords?
Answer: This has been studied by Byskov (1978;
1986) and it was found that such cells, when they occur,
begin the meiotic process. One of us (L.D.R.) has found
a condition in a desert hedgehog mouse knockout in
which gonocytes residing outside of cords do not enter
meiosis (unpublished).
Question: If gonocytes do enter meiosis, wouldn’t
that be like the normal situation in the formation of the
ovary in which gonocytes normally never enter cords?
Answer: Yes (Byskov, 1986).
783de Rooij and Russell · Spermatogonia
Figure 9. Progress of spermatogonia from As to B.
Figure 10. Predecessors of As spermatogonia.
Spermatogonial Kinetics
Question: The preceding section described the types
of spermatogonia. Can we presume the arrows in Figure
9 imply the pattern of kinetics?
Answer: Yes, most investigators in the field now
agree on a single stem cell (As) theory, which was orig-
inally put forward by Huckins (1971b) and Oakberg
(1971).
Question: Can this theory be simplified?
Answer: Yes, As cells divide to either renew As cells
or to become Apr cells. Apr cells divide to form Aal cells
that reach clonal sizes of 4, 8, or 16 cells. As, Apr, and
Aal spermatogonia divide at random during the cycle of
the seminiferous epithelium, but most actively during rat
stages X–II and only occasionally in stages III–IX. Dur-
ing the proliferative activity of these cells the numbers of
As and Apr remain about constant but many Aal are
formed. At about stages VII–VIII nearly all of the Aal that
were formed transform intoA1 cells (no mitosis), which
resume the cell cycle. A1 cells divide to form A2 cells (in
stage IX); these divide to form A3 cells (in stage XI in
mouse, stage XII in rat) and finally, a division occurs to
form A4 cells (in stage I). A4 cells divide to form In type
spermatogonia (in Stage II) and, in turn, In spermatogonia
divide to form type B spermatogonia (in Stage IV). The
last spermatogonial division forms preleptotene spermato-
cytes (in Stage VI).
Question: I thought you were trying to make this a
simple explanation?
Answer: The diagram in Figure 12 simplifies what
has been proposed for rats and mice (Huckins, 1971b;
Oakberg, 1971).
Question: If divisions that lead to Aal cells are random
during the cycle of the seminiferous epithelium, there
must be a temporary arrest for some of them prior to their
stage-related commitment to form sperm. Right?
Answer: Right, although the relation between cell cy-
cle arrest of Aal spermatogonia and their transformation
into A1 spermatogonia is unclear. On the one hand, the
arrest of Aal spermatogonia can be prolonged in vitamin
A-deficient rodents when vitamin A deficiency is induced
(van Pelt and de Rooij, 1990a; van Pelt et al, 1995). Cells
are arrested in G1/G0 phase of the cell cycle in all tubules
and do not progress beyond the Aal to A1 transition; thus,
all tubules will contain large numbers of Aal cells. Then,
when retinoic acid (vitamin A) is administered, all sper-
matogonia in all tubules go forward at the same time,
which leads to a more or less synchronized testis in terms
of germ cell development (Morales and Griswold, 1987;
van Pelt and de Rooij, 1990b). On the other hand, the
arrest can be shortened or may not occur when no In or
B spermatogonia are present. A kind of a feedback reg-
ulatory mechanism exists due to which the proliferation
of As, Apr, and Aal spermatogonia is prolonged when in-
sufficient numbers of A1 spermatogonia are locally pro-
duced during the preceding cycle (de Rooij et al, 1985).
Question: What is the cell cycle time of spermato-
gonial generations?
Answer: The cell cycle duration of A2 through B
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Figure 11. Gonocytes apparently have two possible division patterns.
Figure 12. Some divisions occur anytime during the spermatogenic cycle
(random) and others occur at specific stages.
Figure 13. The A0/A1 scheme of spermatogonial renewal.
Table 1. The age at which spermatogenesis starts and at which it
is complete, in various species and strains of rodents (day of birth
was taken to be day 1)
Species Age of Start
Age of
Completion References
Cpb-N mice
CBA
Mouse strain not
specified
Wistar rat
Djungarian
hamster
Chinese hamster
Day of birth
Day of birth
Day 3
Day 5
Day 5
Day 9
Day 32
Day 35
Unknown
Day 43
Day 32
Day 63
Kluin et al, 1982
Vergouwen et
al, 1991, 1993
Sapsford, 1962
van Haaster and
de Rooij, 1993
van Haaster et al,
1993
van Haaster and
de Rooij, 1993
spermatogonia has been estimated to be 28.5 hours (Mo-
nesi, 1962) in mice, 42 hours in rats (Hilscher et al, 1969;
Huckins, 1971a), and 60 hours in Chinese hamsters (Lok
and de Rooij, 1983a). For As, Apr, and Aal spermatogonia,
cell cycle times have been found to be 56 hours in rats
(Huckins, 1971c) and about 90 hours in Chinese hamsters
(Lok et al, 1983).
Question: Most people believe the scheme originally
proposed by Huckins (1971b) and Oakberg (1971); are
there other schemes?
Answer: Yes, another major stem cell renewal
scheme has been advanced by Clermont and Bustos-Ob-
regon (1968), Dym and Clermont (1970), Clermont and
Hermo (1975), and Bartmanska and Clermont (1983). In
fact, this was the first theory advanced.
Question: Can we examine and compare both
schemes?
Answer: Certainly, it is only through doing so that
readers can make up their minds about which scheme is
correct and only through knowing both schemes can one
or the other scheme be modified or confirmed.
Question: What is the name of the other stem cell
renewal scheme?
Answer: It is the A0/A1 theory. It has also been called
the ‘‘reserve stem cell’’ theory or ‘‘A0 theory.’’
Question: What is a reserve stem cell?
Answer: It is proposed to be a reserve stem cell,
which is normally a quiescent cell, and only divides when
needed; thus, it has an indefinite or very slow cell cycle.
This has been proposed by Clermont and Bustos-Obregon
(1968), Dym and Clermont (1970), Clermont and Hermo
(1975), and Bartmanska and Clermont (1983).
Question: If A0 cells are normally nonproliferative,
then how do spermatogonia renew according to the A0/
A1 scheme?
Answer: The idea is that a small number of A4 cells
divide to form A1 cells, whereas most of the A4 cells go
forward to form Intermediate-type spermatogonia.
Question: A0 cells are around to respond to emergen-
cy situations when there is a problem with the A4 to A1
transition; for example, after irradiation? On a routine ba-
sis, A1 cells are produced from A4 cells?
Answer: Yes, that is correct (Figure 13).
Question: What is the major evidence for each of the
schemes? Can we start by reviewing the Huckins-Oak-
berg scheme (As)?
Answer: In whole mounts of seminiferous tubules
(see later discussion of techniques), thanks to the orga-
nization provided by the spermatogenic cycle, one can
follow the subsequent germ cell stages of the cycle along
the length of the tubule. Consequently, one can, for ex-
ample, see A1 spermatogonia in G2 phase; somewhat fur-
ther on they can be seen in mitosis and, after that, A2
spermatogonia are present but are small because they are
in G1 phase. Then these A2s become larger as they enter
S phase, G2 phase, and then they divide into A3, etc—
sort of a logical progression. Spermatogenesis is a highly
synchronized process and therefore all spermatogonia in
785de Rooij and Russell · Spermatogonia
a particular clone will synchronously enter mitosis and
even adjacent clones are fairly synchronous. Huckins
(1971b) observed other A spermatogonia topographically
arranged as singles, pairs, and chains of up to 16 cells
that did not follow the synchrony of the A1-B spermato-
gonia, whose chain size is much larger. Furthermore, in
stage IX (rat) these stage-asynchronized spermatogonia
were few in number while many more were present in
stage II. Cell kinetic analysis of the spermatogonial cell
types revealed that the asynchronous A spermatogonia
have clearly different cell cycle times from the synchro-
nous ones (see earlier; Huckins, 1971a, 1971c). In mouse
testis sections, scrutiny of small morphological differenc-
es brought Oakberg to the conclusion that mice also pos-
sess a population of A spermatogonia that do not follow
the general synchronous developmental pattern of A1-B
spermatogonia (Oakberg, 1971). Huckins and Oakberg
then jointly proposed the scheme of spermatogonial mul-
tiplication and stem cell renewal, which was outlined ear-
lier (Huckins, 1971b; Oakberg, 1971). Later on this hy-
pothesis was supported and extended by tubular whole
mount studies in mice (de Rooij, 1973) and Chinese ham-
sters (Lok et al, 1982, 1983; Lok and de Rooij, 1983a,
1983b). In essence, the Huckins-Oakberg theory says that
the spermatogonia that give rise to A1 divide irregularly
during the epithelial cycle and are not population-derived
from the A1 or their progeny, the latter dividing in a stage-
related manner.
Question: What is the major evidence for the Cler-
mont scheme (A0/A1 theory)?
Answer: Clermont and Leblond were pioneers in es-
tablishing the cycle of the seminiferous epithelium and
its stages using spermatid development as a guide to iden-
tify stages (Leblond and Clermont, 1952; Clermont and
Perey, 1957b). Spermatogonial multiplication and stem
cell renewal were studiedby counting cell numbers and
mitotic figures in the various epithelial stages. A scheme
evolved in which A1-A4, In, and B spermatogonia were
distinguished and in which the last generation of type A
spermatogonia (A4) gave rise to both A1 and In sper-
matogonia (Clermont, 1962), based on the idea that there
are no principle differences between the various genera-
tions of A spermatogonia. Clermont and Bustos-Obregon
(1968) introduced the seminiferous tubular whole-mount
technique to study spermatogonial kinetics. Additional A
spermatogonia were detected, topographically arranged as
singles or pairs. These were present throughout the cycle
of the seminiferous epithelium, did not change in numbers
much, and were only rarely seen to divide (Clermont and
Bustos-Obregon, 1968; Clermont and Hermo, 1975). Ac-
cordingly, and also because results of studies after irra-
diation suggested that these cells were involved in the
repopulation of the seminiferous epithelium (Dym and
Clermont, 1970), the single or paired cells were termed
reserve stem cells or A0 spermatogonia. The finding that
the numbers of the singles and pairs are relatively con-
stant during the epithelial cycle was confirmed in rats
(Huckins, 1971b), mice (de Rooij, 1973), and Chinese
hamsters (Lok et al, 1982).
Question: Can you summarize the main differences
between the 2 schemes?
Answer: The difference of opinion about the scheme
of spermatogonial proliferation and stem cell renewal
originates from different results with respect to the pro-
liferative activity of the As and Apr spermatogonia (A0 in
Clermont’s scheme) and a different idea about the nature
of the asynchronous chains of A spermatogonia (Aal ac-
cording to Huckins and A1-A4 according to Clermont).
Question: So where do you have a problem with the
Clermont scheme?
Answer: Although Clermont and coworkers did not
find much proliferative activity of the singles and pairs
of A spermatogonia, Huckins (1971c) and Lok and De
Rooij (1983b) did find appreciable 3H-thymidine incor-
poration by these cells, indicating active proliferation. It
is difficult to reconcile this difference in results with re-
spect to the proliferative activity of As and Apr spermato-
gonia. Furthermore, the Clermont group did not distin-
guish chains of A spermatogonia that we call Aal sper-
matogonia. Chains of asynchronous A spermatogonia
were observed but were considered to be delayed or quick
A1-A4 spermatogonia, depending on the epithelial stage
in which they were encountered. However, in cell kinetic
studies in which the labeled mitoses technique was used
to study cell cycle times of spermatogonial cell types, it
was found that the asynchronous chains of A spermato-
gonia have a cell cycle time similar to that of As and Apr
spermatogonia (Huckins, 1971c; Lok et al, 1983) and not
similar to A1 to A4 spermatogonia. Furthermore, because
there was a clear second peak in the labeled mitoses curve
of the chains of asynchronous A spermatogonia, it could
be concluded that these cells consistently have a cell cycle
time that is different from that of A1-A4 spermatogonia.
Thus, the widely different cell kinetic properties of Aal
spermatogonia versus A1-A4 clearly indicate that these
are 2 different cell populations.
Question: This is a huge amount of information to
digest. Would you help by simplifying this more?
Answer: OK, here is a summary for simplicity.
Essential Features of the Two Major Spermatogonial
Renewal Schemes
A0/A1 Theory—A4 spermatogonia normally give rise to
A1 spermatogonia as well as In spermatogonia. A0 sper-
786 Journal of Andrology · November/December 2000
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matogonia divide to form A1 spermatogonia when sper-
matogenesis is in need of more cells.
As Stem Cell Theory—Only As cells act as stem cells,
they give rise to committed cells that divide irregularly
(Apr and Aal) during the spermatogenic cycle and these,
in turn, give rise to A1 spermatogonia.
Major Historical Difference in the As and the A0/A1
Theories—In the As theory, As, Apr, and Aal cells are a
separate population of cells that are different from A1 to
A4 cells. In the A0/A1 theory, As and Apr cells are consid-
ered reserve stem cells and Aal are considered A1-A4 cells,
and are no different from each other.
Question: There are other actively proliferating tis-
sues in the body such as the hemopoetic system, the gas-
trointestinal tract, and skin. What kind of stem cell re-
newal do these tissues possess? Are their stem cell re-
newal schemes more like the As model or the A0 model?
Answer: The Huckins and Oakberg scheme (As the-
ory) is similar to other cell-renewing systems in that stem
cells divide the least frequently (Lajtha, 1979). It can be
argued that this is advantageous because there is only a
small chance that stem cells will be damaged by inac-
curate DNA duplication. In the Clermont scheme stem
cells theoretically divide 4 times each epithelial cycle; in
the Huckins-Oakberg scheme, it has been estimated that
in mice and Chinese hamsters As divide 2 to 3 times (Lok
and de Rooij, 1983b; Tegelenbosch and de Rooij, 1993).
Question: Is there a proposed dedifferentiation sys-
tem from A4 to A1 spermatogonia in the Clermont sys-
tem?
Answer: The Clermont scheme supposes that A1-A4
spermatogonia are at a similar phase of differentiation and
that no differentiation takes place; thus, no dedifferenti-
ation is required. At present, no molecular markers have
been described that indicate differentiation in A1 to A4
spermatogonia.
Question: Although no molecular markers have been
found, are not intercellular bridges an indication of dif-
ferentiation?
Answer: Some would say so because As cells do not
have bridges. Someday we will know the answer to this
question.
Question: According to Clermont’s theory, A4 sper-
matogonia would have to break their bridges to form A1
cells. By counting the number of cells in bridges, are there
examples when this might have occurred?
Answer: The point is not easy to explain within the
Clermont scheme. It could be established in Chinese ham-
sters that the A1 spermatogonia in stage VII are composed
of chains of 4, 8, and 16 cells only (Lok et al, 1982) and
that chains virtually never posses odd numbers of cells.
After division this would render clones of 8–32 A2 sper-
matogonia and ultimately 32–128 A4 spermatogonia. If
the A0/A1 theory is correct, clones of 2, 4, and 8 cells
would then have to be pinched off from clones of A4
spermatogonia to divide into new clones of 4–16 A1 sper-
matogonia. One cannot say that this does not occur or is
impossible, but it does not seem to be a very likely course
of events.
Question: Why is there not universal acceptance of
the Huckins-Oakberg theory?
Answer: It is difficult for most people to critically
examine the literature because it requires a detailed
knowledge of staging as well as an examination of nu-
merous tables of cell counts at various stages. Most peo-
ple are unwilling to put in the effort. Moreover, as long
as a few people keep advocating the A0/A1 theory, there
will always be controversy.
Question: I will ask more about a theory of ‘‘density
dependent regulation’’ later, but there is an important
point to be brought up now. In this theory, which indi-
cates that the density of advanced germ cells is regulated
by spermatogonial apoptosis, you say that the numbers of
cells entering meiosis is controlled by apoptotic events,
primarily in A2 through A4 spermatogonia. If Clermont’s
scheme is correct would it make good ‘‘biological sense’’
to eliminate cells prior to stem cell renewal rather than
after renewal?
Answer: As discussed earlier, cells in every stem-cell
system divide as little as possible (Lajtha, 1979). Density
regulation among potential stem cells would mean that
spermatogonial stem cells divide more thanstrictly nec-
essary. In this way one would enhance the chance of mu-
tations in the kind of cells one should protect the most.
Logically, stem cell renewal should come before elimi-
nation of cells as it does in other systems of the body as
proposed in the As theory.
Question: I understand that intercellular bridges are
not ‘‘sacred’’ in terms of designating stem cells because
they have been described in gonocytes, right?
Answer: All gonocytes may not be alike; some may
not be stem cells. As discussed earlier, at least some gon-
ocytes give rise to A1 cells and thus gonocytes may be
comparable to the population of As, Apr, and Aal sper-
matogonia, as a whole, with respect to the occurrence of
intercellular bridges. It may well be that only single gon-
ocytes after the start of spermatogenesis will give rise to
As spermatogonia. Do not give up as yet about the sa-
credness of intercellular bridges in this respect. Future
research will surely enlighten us.
Techniques to Study Spermatogonia
Question: Because spermatogonia are the rarest cell
type within the epithelium and are the most difficult to
787de Rooij and Russell · Spermatogonia
Figure 14. Spermatogonia as seen using conventionally sectioned tu-
bules and using whole mounted tubules. In the latter the microscope
focuses on a plane just under the coverslip.
identify in the testis, must it take a special technique to
identify them?
Answer: Yes, whole mounts of seminiferous tubules
have traditionally been the most useful techniques (Cler-
mont and Bustos-Obregon, 1968).
Question: How is a whole mount prepared?
Answer: Tubule segments of up to several centime-
ters in length are isolated by teasing apart rodent testis
tissue in a Petri dish. The tubules are stained with he-
matoxylin (a nuclear stain), partially flattened by the pres-
sure of a cover slip, and examined by a microscope that
is focused only on the plane of cells on the basal lamina
(Clermont and Bustos-Obregon, 1968; Meistrich and van
Beek, 1993; Figure 14).
Question: How does one identify the types of sper-
matogonia?
Answer: The criteria are as follows:
As: No other similar A spermatogonia are within 25
�m of these cells.
Apr: Only 2 spermatogonia of the same nuclear mor-
phology are closer than 25 �m to each other.
Aal: More than 2 A spermatogonia can be construed as
branched or straight chains of the same morphology with
no intervening space of more than 25 �m between mem-
ber nuclei. The chains are generally no greater than 16
cells.
Question: What if an As cell is near, for example, a
chain of A3 spermatogonia? Does that mean that an As
cell could be misidentified?
Answer: One could misidentify the As cell. The eas-
iest way to identify As, Apr, and Aal spermatogonia is to
look for them in areas in which A1-A4 spermatogonia are
in late G2 phase or in mitosis. As-Aal spermatogonia then
stand out because they are not synchronized in the cell
cycle with A1-A4 spermatogonia and their nuclei will ap-
pear different. In areas in which A1-A4 spermatogonia are
in G1 or S phase, the differences between these cells and
As-Aal are too small to allow identification in each in-
stance and there will be too many doubtful cases to allow
reliable counting. Examples appear in Figure 8A through
E.
Question: With your staining technique, you don’t see
the intercellular bridges?
Answer: No, we generally don’t. Figure 8D is an ex-
ception; we presume intercellular bridges are present be-
cause the cells have the same morphology and, presum-
ably, are all in the same phase of the cell cycle. We can
imagine the chain of cells by the pattern of cells and the
expected distance between cells.
Question: What other techniques do you use to study
spermatogonia?
Answer: We utilize germ cell-depletion techniques,
irradiation, or vitamin A deficiency.
Question: How does irradiation affect the testis?
Answer: Irradiation kills the proliferating cells (ie,
spermatogonia). A1 through A4 spermatogonia are the
most radiosensitive, followed by Apr and Aal spermato-
gonia. As are the most radioresistant (van der Meer et al,
1992a, 1992b). Enough irradiation will deplete the entire
population of spermatogonia. Spermatocytes and sper-
matids are much more radioresistant and, after irradiation,
develop in an apparently normal way and ultimately leave
the testis as spermatozoa. If stem cells are able to survive,
they will usually repopulate the seminiferous epithelium
(Dym and Clermont, 1970; van den Aardweg et al, 1982),
but are found to first replenish their own numbers before
again producing differentiating cells (van Beek et al,
1990).
Question: What use can be made of vitamin A-defi-
cient testes?
Answer: As described earlier, the seminiferous epi-
thelium in a vitamin A-deficient testis contains only As-
Aal spermatogonia. Because of this, these testes can be
used to purify these cells (van Pelt et al, 1996). Further-
more, because after replacement of vitamin A, virtually
all Aal spermatogonia throughout the testis synchronously
differentiate into A1 spermatogonia and then start their
series of divisions, spermatogenesis in these animals be-
comes synchronized. Hence, in the testes of vitamin A-
deficient rats (Morales and Griswold, 1987; Griswold et
al, 1989; van Pelt and de Rooij, 1990a; van Beek and
Meistrich, 1991) and mice (van Pelt and de Rooij, 1990b),
after replacement of vitamin A, only a few epithelial stag-
es can be seen. Which stages are present depends on the
duration of vitamin A replacement and can be calculated
from the duration of the epithelial cycle and its stages.
These testes can be used to study epithelial stage-depen-
dent processes (Klaij et al 1994).
Question: How does one determine if particular sper-
matogonia have divided or are going to divide?
Answer: There are two ways to determine this. One
is to examine the epithelial stage and look for mitotic
figures. We have also relied heavily on the use of 3H
788 Journal of Andrology · November/December 2000
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Figure 15. Type A spermatogonia isolated and cultured from 9-day-old
rat testes using the STAPUT procedure (Dym et al, 1995). The cells
display large, spherical nuclei with a thin rim of cytoplasm and perinu-
clear organelles. The morphology of the cultured spermatogonia is very
similar to that seen in vivo.
thymidine or bromodeoxyuridine labeling. Both substanc-
es become incorporated into DNA during the S-phase of
the cell cycle and may be given as a pulse (Lok and de
Rooij, 1983a, 1983b; Lok et al, 1983; van de Kant et al,
1988, 1990; van Pelt et al, 1995), as a series of injections,
or continuously (Lok et al, 1984).
Question: Has anyone cultured spermatogonia?
Answer: Various culture systems for spermatogonia
of various species have been described. In a Sertoli cell–
germ cell coculture from neonatal mouse germ cells, no
apparent loss of viability was found after 3 days of culture
(Maekawa and Nishimune, 1991). Nagano et al (1998)
were able to keep germ cells, isolated from adult mice,
alive for more than 4 months in serum containing medi-
um. No attempt was made to quantify the number of vi-
able cells. It is interesting that cells that had been cultured
on feeder layers were able to colonize busulphan-emptied
recipient testes after spermatogonial stem cell transplan-
tation. Because this was not the case for germ cells cul-
tured without a feeder layer, this suggests that a feeder
layer is required for the survival of spermatogonial stem
cells. Recently, 50% of cultured, purified spermatogonia
from 80-day-old boars were shown to be viable when the
cells were cultured in a potassium-rich medium, called
KSOM, after 3 days (Dirami et al, 1999). However, in
general, a system for the long-term culture of purified
spermatogonia is still lacking. Long-term survival of
spermatogonialstem cells on a feeder layer has been
shown, but this system is still poorly defined in terms of
efficiency.
Question: Has anyone frozen spermatogonia?
Answer: Yes. Brinster’s laboratory has now reported
spermatogonial stem cell freezing for up to 6 months
(Avarbock et al, 1996; Clouthier et al, 1996; Ogawa et
al, 1999) and now has done so for 2 years (personal com-
munication). However, no estimate was made of the ef-
ficiency of storing spermatogonial stem cells by way of
cryopreservation.
Question: Has anyone purified spermatogonia?
Answer: Yes, several techniques have been pub-
lished. First, to isolate germ cells, a testis has to be de-
capsulated, mechanically minced into small pieces, and
then enzymatically dissociated (Meistrich et al, 1973;
Barcellona and Meistrich, 1977; van Pelt et al, 1996).
Various procedures have been developed to further purify
spermatogonia from human and rodent testis cell suspen-
sions, which involve sedimentation velocity (Meistrich
and Eng, 1972; Bellve´ et al, 1977), equilibrium density
centrifugation in Percoll gradients (Meistrich and Trostle,
1975), and centrifugal elutriation (Grabske et al, 1975).
However, the efficiency of the purification of spermato-
gonia not only depends on the isolation technique but also
on the relative abundance of these cells in the testis and,
consequently, in the testis cell suspension. Starting from
young mice, Bellve´ et al (1977) succeeded in obtaining
greater than 90% pure A spermatogonia and 76% pure B
spermatogonia. About 85% pure A spermatogonia could
be prepared from young rat testes (Morena et al, 1996).
Recently, Dirami et al (1999) described the purification
of 95%–98% pure A spermatogonia from 80-day-old pig
testes using sedimentation velocity and differential ad-
hesion to eliminate contaminating Sertoli cells, which ad-
here to culture flasks. Figure 15 shows spermatogonia af-
ter they have been isolated.
Question: Can the genetic information be modified in
stem cell spermatogonia?
Answer: There have been numerous claims that the
genetics of a small number of spermatogonia can be mod-
ified through various manipulations such as electric shock
of the testis after introduction of a vector. In a very recent
paper from Brinster’s laboratory, spermatogonia were iso-
lated, cultured, and transfected with a retrovirus and then
transplanted with resulting stable integration of the viral
vector (Nagano et al, 2000).
Regulation of Spermatogonial Number
Question: Can one simply determine what the even-
tual yield of sperm will be on the basis of the kinetics of
spermatogonia, spermatocytes, and spermatids?
Answer: It is not that simple. As it turns out, the germ
cell population can be regulated. To determine yield of
sperm, one must also consider cell degeneration during
spermatogenesis. Although there are several sites for pos-
sible regulation during spermatogenesis, the regulation at
the spermatogonial level appears to have the greatest im-
pact on the eventual yield of cell numbers.
789de Rooij and Russell · Spermatogonia
Question: Where does the regulation of spermatogo-
nia take place?
Answer: Studies have been performed to examine the
cell density of A1 and other types of spermatogonia and
preleptotene spermatocytes in Chinese hamsters (de Rooij
and Janssen, 1987; de Rooij and Lok, 1987). Large dif-
ferences were found in the density of A1 spermatogonia
in different areas of the seminiferous tubular basal mem-
brane. Apparently, there is no mechanism that regulates
the proliferation of As-Aal spermatogonia in such a way
that A1 spermatogonia are evenly distributed. However,
the density of preleptotene spermatocytes is the same
everywhere. Hence, density regulation must take place
somewhere in the process from A2 to B spermatogonia.
Question: So, where is the site of regulation under
normal conditions?
Answer: Regulation under normal conditions takes
place among A2, A3, and A4 spermatogonia.
Question: What appears to be the raison de eˆtre for
density-dependent regulation?
Answer: The basis is the density of cells; thus, the
theory is called ‘‘density-dependent regulation.’’ The
greater the density of A2 though A4 spermatogonia in a
particular region of the tubule, the greater the amount of
cell degeneration is necessary to down-regulate the den-
sity to a ‘‘normal’’ level.
Question: Could you tell me more?
Answer: It works like other systems of the body (Pot-
ten et al, 1997; Conton and Raff, 1999). At first, certain
cells are overproduced and are later reduced to physio-
logical numbers by the apoptotic process (Figure 8F). If
all the cells produced as spermatogonia were to become
sperm, there would be a twofold to fivefold increase in
sperm production in the mouse (de Rooij and Lok, 1987).
Increasing sperm production sounds nice, but the sup-
porting cells of the testis could not handle such a load.
Indeed, this has been found to be the case in some trans-
genic mice in which spermatogonial apoptosis was inhib-
ited (Knudson et al, 1995; Furuchi et al, 1996; Rodriguez
et al, 1997). There was an accumulation in these mice of
spermatogonia and early spermatocytes that eventually all
entered apoptosis, apparently by way of a backup apo-
ptotic pathway.
Question: What would be the mechanism for density-
dependent regulation?
Answer: We must assume that a mechanism exists
that senses too many spermatogonia of more advanced
types in a particular area, and which triggers the apoptotic
mechanism. Such a mechanism could reside in the Sertoli
cells but also in spermatogonia themselves. Possibly,
when the clones consisting of at least 16 A2 to up to 128
A4 get in each other’s way trying to carry out divisions,
apoptosis may be induced. This area will need further
study.
Question: Do cells apoptose individually or as a
clone?
Answer: Huckins (1978a) found that the entire clone
is always lost during density-dependent regulation. In
normal epithelium, clones of even-numbered cells are
found exclusively. Odd-numbered clones are found under
experimental or adverse conditions (van Beek et al, 1984;
de Rooij et al, 1999), but this does not necessarily imply
‘‘single-cell death’’ because there may be forces under
abnormal conditions that cause a single cell to separate
from a chain.
Question: When all is said and done, how finely
tuned is the spermatogenic process?
Answer: Reviewing the ratio of elongated spermatids
per Sertoli cell reveals that for many of the species stud-
ied, there is about 20%–40% difference in the number of
germ cells each Sertoli cell supports, and the number of
spermatids that can be supported by a single Sertoli cell
is characteristic of the species (Russell and Peterson,
1984).
Question: Why would a biological system be so
wasteful?
Answer: Stem cells are rare and it may be difficult to
regulate their proliferative activity in such a way that they
constantly produce a particular number of differentiating
cells in all areas. When stem cell renewal and differen-
tiation depends on stochastic events that gives them a
50% chance of self-renewal or differentiation in normal
epithelium, there will always be areas in which by chance
too many stem cells carried out self-renewal or differen-
tiation, and lead to the production of too few differenti-
ating cells in the same or the next epithelial cycle, re-
spectively. Therefore, it may be better to keep on the safe
side and produce so many cells that in all cases, sufficient
differentiating cells are produced. In addition, such a sys-
tem will be better able to cope with situations in which
cells are lost by damage or insult to the testis. For ex-
ample, a toxic insult may result in increased cell loss, but
because apoptosis can be cut back, sufficient cells may
still be produced to ensure normal fertility. Enough tele-
ological reasoning, let’s get back tofacts.
Question: What factors have been proposed to regu-
late spermatogonial divisions/apoptosis?
Answer: The literature on this topic is voluminous
and in keeping with the idea that we should keep this
review simple, we present a table (Table 2).
Question: Of all the proposed regulators of spermato-
gonia, which have the strongest physiological evidence to
support their proposed role?
790 Journal of Andrology · November/December 2000
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File # 21em
Table 2. Factors or receptors shown to be expressed in spermatogonia and that may play a direct role in the regulation of proliferation,
differentiation, and apoptosis in spermatogonia
Factor/Receptor in
Spermatogonia Growth Factor Role References
c-ret, GFRalpha 1
c-kit
Retinoid receptors RXR�,
RXR�, RXR�
Dazl
Estradiol receptor � (ER�)
p53 (after induction of DNA
damage)
Bax
Bcl-xL
TGF�-RII
FGFR-4
Prolactin receptor
NGF
Glial cell line–derived neurotroph-
ic factor (GDNF)
Stem cell factor (SCF)
All trans retinoic acid, 4-oxo-reti-
noic acid, 9-cis-retinoic acid
Estradiol
TGF�
FGFs
Prolactin
NGF receptor
Regulation of stem cell renewal
and differentiation
Aa1-A1 transition; development of
A1–A4 survival factor in culture
Aa1-A1 transition
Aa1-A1 transition
Promotes proliferation at start of
spermatogenesis
Role in spermatogonial response
to DNA damage
Role in the regulation of sper-
matogonial density
Role in the regulation of sper-
matogonial density
Meng et al, 2000
de Rooij et al, 1999;
Koshimizu et al, 1991;
Schrans-Stassen et
al,1999;
Yoshinaga et al, 1991;
Yan et al, 2000
Gaemers et al, 1996;
Gaemers et al, 1998a;
Gaemers et al, 1998b
Schrans-Stassen et al,
personal communi-
cation
Li et al, 1997;
Saunders et al, 1998;
van Pelt et al, 1999
Beumer et al, 1998;
Hasegawa et al, 1998;
Odorisio et al, 1998
Knudson et al, 1995
Rodriguez et al, 1997;
Beumer et al, 2000
Olaso et al, 1998
Cancilla and Risbridger,
1998
Hondo et al, 1995
Wrobel et al, 1996
Answer: The most interesting regulators are the Glial
cell line–derived neurotrophic factor (GDNF)/c-ret-
GFRalpha1 system, which seems to be necessary for reg-
ulation of spermatogonial stem cell renewal and differ-
entiation (Meng et al, 2000) and the SCF/c-kit system (de
Rooij et al, 1999) and the Dazla RNA-binding protein
(Schrans-Stassen et al, in preparation), both of which
seem to be indispensable in the Aal-A1 transition. Retinoic
acid may also be directly involved in the Aal-A1 transition
(van Pelt et al, 1995; Gaemers et al, 1998b), but an in-
direct effect via Sertoli cells can still not be excluded.
Question: Do the GDNF/c-ret-GFRalpha1 results im-
ply that we now actually have the potential to influence
spermatogonial stem cell behavior?
Answer: Yes, in principle we have. GDNF is pro-
duced by Sertoli cells. Overexpression of GDNF in mouse
testis inhibits differentiation, and tubules in 3-week-old
GDNF-overexpressing mice contain clusters that primar-
ily consist of single A spermatogonia. Later on, the clus-
ters disappear and the tubules become lined with stem
cells. In heterozygotes, in which 1 copy of the gdnf gene
is knocked out, the result is an early depletion of sper-
matogonial stem cells, which is evidenced by the appear-
ance of more and more tubules that lack spermatogenic
generations or that completely lack germ cells (Meng et
al, 2000).
Question: Would it be possible to manipulate the ex-
pression of GDNF and its receptors in the testis?
Answer: Yes, potentially! That is exactly the next
question to work on. Human patients presenting with hy-
pospermatogenesis could possibly be helped in this man-
ner.
Question: There also are W/W and Sl/Sl mutant mice
that show few germ cells in the testis due to a lack of c-
kit receptors and its ligand, stem cell factor (SCF), re-
spectively. Is there a block to spermatogonial develop-
ment in the SCF/c-kit system?
Answer: There is a wide variety in the severity of the
various W/W and Sl/Sl mutant mice. Indeed, there are no
germ cells at all with some alleles, whereas with others,
mice are fertile (Koshimizu et al, 1992). Anyhow, in
Sl17H/Sl17H mutant mice the Aal-A1 transition is inhib-
ited (de Rooij et al, 1999), indicating that besides a role
in primordial germ cell migration and proliferation
(Mintz, 1957; Mintz and Russell, 1957; Russell, 1979;
791de Rooij and Russell · Spermatogonia
Figure 16. Spermatogonial proliferation under normal conditions and un-
der conditions of germ cell depletion.
Kitamura et al, 1985), SCF/c-kit also has an indispensable
role in the Aal-A1 transition in the spermatogenic process.
Furthermore, SCF/c-kit has an important role in the reg-
ulation of the development of A1 to A4 spermatogonia, as
these cells are killed upon administration of a c-kit anti-
body in vivo (Yoshinaga et al, 1991) and are protected
by addition of SCF to cultured seminiferous tubules (Yan
et al, 2000). A recent experiment (Ogawa et al, 2000) was
successful in transplanting spermatogonia from Sl mu-
tants into W mutants to achieve spermatogenesis and fer-
tility. In essence, the experiment showed that placing
spermatogonia with an intact c-kit receptor from a Steel-
mutant mouse into an animal whose endogenous sper-
matogonia had no receptor allowed spermatogonia to pro-
gress to fertile sperm.
Question: Does regulation of spermatogonial num-
bers take place under adverse conditions other than what
has been described as density-dependent regulation?
Answer: One important site of regulation is at the
level of the As cell. As mentioned earlier, the stem cell
makes a choice when it divides to either become 2 new
stem cells (As) or to become an Apr. The choice is appar-
ently not random. Under normal circumstances it provides
sufficient cells to renew the stem cell population and also
divides to form cells that are committed to the spermat-
ogenetic process (Figure 16). However, if all spermato-
gonia, including many As cells, are destroyed by irradia-
tion the remaining stem cells devote their early efforts to
forming more stem cells (As) before they go forward to
form committed Apr cells (van Beek et al, 1990).
Question: Do spermatogonia possess receptors for
any of the major hormones stimulating the testis?
Answer: No one has reported testosterone receptors
in spermatogonia, but there is a controversy whether or
not follicle-stimulating hormone receptors are present on
spermatogonia (Orth and Christensen, 1978a, 1978b;
Wahlstrom et al, 1983; Baccetti et al, 1998). Furthermore,
receptors for estradiol have been found in spermatogonia
(Saunders et al, 1998; van Pelt et al, 1999).
Comparative Data
Question: Have we based all of our conclusions on
rat and mouse data?
Answer: No, but everyone has to start somewhere.
We just started with the species that have been most thor-
oughly studied.
Question: So what about spermatogonial types in oth-
er species?
Answer: Table 3 presents some representative species
and their spermatogonial types and kinetics.
Question: I see by the table that primates are very
different from other mammals. Can you tell me more?
Answer: In principle, on the basis of morphology, in
primates, too, there are 2 kinds of spermatogonia, types
A and B. The nuclei of A spermatogonia do not show
heterochromatin, but some nuclei stain heavily with he-
matoxylin and are called Adark spermatogonia and others
stain less heavily and are termed Apale (Clermont, 1966b).
Generally, there are equal numbers of pale and dark-stain-
ing A spermatogonia.
Question: What is their pattern of cell division?
Answer: Apale spermatogonia divide once every epi-
thelial cycle, which in humans, means once every 16 days
(Heller and Clermont, 1963; Clermont, 1966a). Adark sper-
matogonia normally do not divide andare apparently qui-
escent for a very long time. However, when the number
of Apale spermatogonia is diminished, for example, after
irradiation, Adark spermatogonia again become active (van
Alphen and de Rooij, 1986). In the process of their ac-
tivation, Adark spermatogonia transform into Apale sper-
matogonia and after this transition they start to proliferate.
Question: It seems that primates, including humans,
have a very different spermatogonial renewal scheme. In
fact, Adark spermatogonial act like what Clermont calls the
A0, or reserve stem cells, correct?
Answer: Yes, they do.
Question: Then would the presence of reserve stem
cells in the primate suggest, phylogenetically, their pres-
ence in the rodent and thus tend to support Clermont’s
A0/A1 scheme of spermatogonial self-renewal?
Answer: Adark spermatogonia in primates do not seem
to be a separate class of spermatogonia that renew them-
selves and give rise to differentiating-type spermatogonia.
In monkeys, it was found that after irradiation, Adark sper-
matogonia transform into Apale spermatogonia and then
divide further (van Alphen and de Rooij, 1986). Further-
more, while in normal epithelium the density of Adark and
Apale spermatogonia is such that in whole mounts, it is not
792 Journal of Andrology · November/December 2000
andr 21_621 Mp_792
File # 21em
Table 3. Spermatogonial cell types and number of generations in those nonprimate mammals in which the occurrence of As, Apr and Aa1
spermatogonia was studied in primates
Species
No. Spermatogonial
Generations Names References
Mouse, rat
Hamster
(Chinese and Djungarian)
Ram
Pig
Human
Monkey
(Macaca mulatta, Cercopithe-
cus aethiops, Macaca arcto-
ides)
9–11
9–11
9–11
9–1
Unknown number of divisions of A
spermatogonia, 1 generation of
B spermatogonia
Unknown number of divisions of A
spermatogonia, 4 generations
of B spermatogonia
As, Apr, Aa1 (4, 8, 16),
A1–A4, In, B
As, Apr, Aa1 (4, 8, 16), A1-A3, In,
B1, B2
As, Apr, Aa1 (4, 8, 16), A1–A3, In,
B1, B2
As, Apr, Aa1 (4, 8?, 16?), A1–A4, In,
B
Apale, Adark, B
Apale, Adark, B1–B4
Huckins, 1971b; Oak-
berg, 1971; Tegelen-
bosch and de Rooij,
1993
Oud and de Rooij,
1977; Lok et al, 1982;
van Haaster and De
Rooij, 1993a
Lok et al, 1982
Frankenhuis et al, 1982
Clermont, 1966a
Clermont and LeBlond
1959; Clermont,
1969; Clermont and
Antar, 1973
possible to distinguish separate clones of these cells. After
irradiation, when density is lower, it can be seen that both
these types of cells consist of single cells, pairs, and
chains (van Alphen et al, 1988). Finally, during repopu-
lation, after irradiation new Adark spermatogonia are
formed by Apale spermatogonia (van Alphen et al, 1988).
So, Adark spermatogonia differ from Clermont’s A0 sper-
matogonia in that they also consist of chains of cells in
addition to singles and pairs and that they do not divide
as such. Before proliferation, they become Apale first and
they are also renewed by Apale. Adark spermatogonia seem
to be set aside by Apale spermatogonia, which can be re-
cruited to become Apale again when needed. Hence, Adark
are reserve As, Apr, and Aal spermatogonia, but do not
differ from active As, Apr, and Aal spermatogonia except
that their proliferative activity has been down-regulated.
Ironically, with this interpretation we can say that pri-
mates are similar to rodents and the As theory is still alive.
Question: Is there density-dependent regulation of
spermatogonial numbers in primates?
Answer: This topic has not studied as yet in primates.
Question: How often do spermatogonia in primates
divide?
Answer: Apale spermatogonia in primates divide only
once (Clermont and Leblond, 1959; Clermont, 1969) or
twice each cycle of the seminiferous epithelium (Cler-
mont and Antar, 1973) and then B spermatogonia are
formed, of which there are 4 generations in monkeys (B1-
B4; Clermont and Leblond, 1959; Clermont, 1969; Cler-
mont and Antar, 1973) and probably only 1 in humans
(Clermont, 1966a). It has been suggested that Apale and
Adark spermatogonia in primates are comparable to As, Apr,
and Aal spermatogonia in rodents, the single Apale and Adark
spermatogonia being the stem cells (de Rooij, 1983). Apale
spermatogonia directly produce B spermatogonia without
in-between generations of A1-A4 spermatogonia.
Question: What insight have we gained about sper-
matogonial renewal from studying species other than ro-
dents?
Answer: Several species have been studied and, un-
fortunately, the same controversy arose about the nature
of the spermatogonial stem cell. An A0/A1-type scheme
of spermatogonial multiplication and stem cell renewal
has been described for bulls (Hochereau, 1967; Hocher-
eau-de Reviers, 1976; Hochereau-de Reviers et al, 1976)
but Wrobel et al (1995) concluded that an As-type scheme
would be more appropriate. Studies in whole mounts of
seminiferous tubules (Frankenhuis et al, 1982) also re-
vealed the presence of As, Apr, and Aal spermatogonia in
boars, and cell counts in rams indicated that spermato-
gonial multiplication and stem cell renewal in rams is
principally similar to that in Chinese hamsters (Lok et al,
1982).
Question: Has anyone shown a functional assay for
stem cells such as in the hemopoietic system, in which
stem cells form colonies in vivo or in culture?
Answer: Yes, the current assay for stem cells is to
transplant them to determine if spermatogenesis, or at
least spermatogonial proliferation, is initiated. This has
been performed in mouse recipients from mouse (Brinster
and Avarbock, 1994; Brinster and Zimmermann, 1994),
rat (Clouthier et al, 1996; Russell and Brinster, 1996),
793de Rooij and Russell · Spermatogonia
hamster (Nagano et al, 1999), and rabbit and dog (Dob-
rinski et al, 1999) donors and in rat recipients from rat
and mouse donors (Ogawa et al, 1999).
Question: So, if the dogma holds true, then only sper-
matogonial stem cells would initiate spermatogenesis and
be able to continue spermatogenesis in transplanted ani-
mals?
Answer: Yes, that is correct; other spermatogonia and
germ cell types can go through limited differentiation
within the seminiferous epithelium of germ-cell–depleted
recipients (Parreira et al, 1998), but only stem cells can
provide continuous spermatogenesis.
Toxicants, Physical Agents, and Conditions Affecting
Spermatogonia
Question: Are spermatogonia particularly vulnerable
to toxicants or physical agents such as irradiation?
Answer: Yes, in particular because of their mitotic
activity, they will be susceptible to cytostatic and che-
motherapeutic agents that block mitosis or kill cells in S
phase. Also, DNA-damaging agents such as irradiation
and particular chemotherapeutic drugs kill spermatogonia
(van der Meer et al, 1992a, 1992b; Meistrich, 1993). As
DNA damage kills the cells when they try to divide, sper-
matogonia are more vulnerable than Sertoli and Leydig
cells and spermatids. Also, spermatocytes that carry out
meiotic divisions are less vulnerable than spermatogonia.
Question: There are many conditions that adversely
affect the testis. Are there any particular points in the
progression of spermatogonial kinetics that are vulnerable
to many insults?
Answer: Yes, it has been recently shown that the tran-
sition from Aal to A1 is particularly susceptible in a variety
of conditions that affect the testis (de Rooij et al, 1999,
2000; Shuttlesworth et al, 2000).
Question: Which cells are the most vulnerable to cy-
tostatic/chemotherapeutic agents?
Answer: It varies with the agent used. Generally, A1
to A4 spermatogonia are the most vulnerable but, for in-
stance, in the case of busulphan, stem cells are the most
vulnerable (de Rooij and Kramer, 1970).
Question: Can you give enough irradiation, for ex-
ample, to kill all the As spermatogonia?Answer: Yes in principle, high doses and especially
fractionated irradiation for which stem cells are more sen-
sitive will result in the death of virtually all spermato-
gonia.
Question: What if you don’t kill them all, but leave
a few. Will they repopulate a small area of the tubule or
the whole tubule?
Answer: Good question! They first repopulate a focal
area. In time, new spermatogenesis spreads along the
length of the tubule. That means that spermatogonia at
either end of the growing zone must be using their energy
to preferentially perform self-renewing divisions and also
must be moving down the tubule. It has been calculated
that after irradiation, repopulating spermatogenic colonies
expand approximately 33 �m along the tubule in both
dimensions per day (van den Aardweg et al, 1982). After
transplantation of spermatogenesis they can extend about
50–60�m/day (Nagano et al, 1999). Eventually, the entire
tubule will become repopulated.
Question: Can the hormonal environment influence
the regeneration of spermatogenesis from spermatogonia
after irradiation or chemotherapy or transplantation?
Answer: Yes, in recent years various conditions have
been described in which the Aal-A1 transition (likely) has
become inhibited, including irradiation (Kangasniemi et
al, 1996), administration of cytotoxic drugs (Meistrich et
al, 1995), administration of a Sertoli cell toxicant to rats
(Boekelheide and Hall, 1991), and the juvenile spermato-
gonial depletion (jsd) genotype in mice (de Rooij et al,
1999). In all these situations, compounds that affect pi-
tuitary secretions (gonadotropin-releasing hormone
[GnRH] antagonists and GnRH agonists) were found to
increase spermatogonial differentiation again and to par-
tially restore spermatogenesis (Blanchard et al, 1998;
Meistrich, 1998; Matsumiya et al, 1999; Shuttlesworth et
al, 2000). They are believed to work by lowering lutein-
izing hormone and, consequently, testosterone levels.
Transplantation of spermatogonia is favored in an envi-
ronment of low testosterone (Ogawa et al, 1998). In some
cases of xenotransplantation between species that are not
so phylogenetically related, and where success is com-
promised (Dobrinski et al, 1999), there may be other
causes.
Question: You mean the hormone that is well-known
to enhance spermatogenesis is detrimental to spermato-
gonial differentiation?
Answer: It appears to be the case. The idea is that
testosterone levels in small testes are relatively high be-
cause normal amounts of testosterone are produced within
a much smaller testicular volume. Hence, high testoster-
one levels may negatively affect spermatogonial differ-
entiation. In fact, during early pubertal development,
when spermatogonial divisions are very active, testoster-
one levels are relatively low.
Question: So what can we expect from future work
on spermatogonia?
Answer: Because we are now able to purify sper-
matogonia and the upcoming DNA chip technology, we
can expect new findings on the genes that regulate sper-
matogonial proliferation and differentiation. Furthermore,
794 Journal of Andrology · November/December 2000
andr 21_621 Mp_794
File # 21em
many new transgenic mice are constantly being made, a
number of which show interesting problems at the sper-
matogonial level (see earlier discussion). All this will lead
to fast-growing knowledge on the molecular regulation of
spermatogenesis as a whole and certainly also of sper-
matogonial multiplication and differentiation.
Comment: I think I have all the answers I want for
now. I’ll wait a few years to ask more questions.
Answer: Good, we will know more then.
Note: Very recently, a method has been developed to
obtain an enriched fraction of spermatogonial stem cells
(Shinohara et al, 2000). This method starts from crypt-
orchid mouse testis cells that are fractionated by fluores-
cence-activated cell sorting analysis based on light-scat-
tering properties and expression of the cell surface mol-
ecules alpha6-integrin, alphav-integrin, and the c-kit re-
ceptor. The most effective enrichment strategy, in this
study, selected cells with low side scatter light-scattering
properties, positive staining for alpha6-integrin, and neg-
ative or low alphav-integrin expression, and resulted in
a 166-fold enrichment of spermatogonial stem cells. C-
kit was determined not to be present in stem cells.
Acknowledgments
Support to the authors for aspects of this work was provided by National
Institutes of Health grants HD36476-03 and HD35494-01A2. Helio
Chiarini-Garcia and Rene´ Scriwanek graciously helped us with photog-
raphy. The authors also thank Martin Dym for providing the beautiful
figure of isolated spermatogonia (Figure 15).
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