Vol. 12, No. 1
5
REVIEW
The generation of spermatogonial stem cells
and spermatogonia in mammals
Agnieszka Kolasa1, Kamila Misiakiewicz, Mariola Marchlewicz,
Barbara Wiszniewska
Department of Histology and Embryology, Pomeranian Medical
University in Szczecin, Poland
Received: 24 June 2011; accepted: 16 November 2011
SUMMARY
Spermatogenesis is a complex series of cellular changes leading to the formation of haploid male gametes (spermatozoa) and includes mitotic, meiotic
and post-meiotic phases. Spermatogonial stem cells (SSCs) are essential
for the continuous lifelong production of spermatozoa. Spermatogenesis
is initiated when SSC is triggered to undergo mitosis that gives rise to
progenitors, which further differentiate into spermatogonia. In this review,
we describe the origin of SSCs and other spermatogonia populations
and summarize the knowledge concerning their markers. Reproductive
Biology 2012 12 1: 5-23.
Key words: spermatogonial stem cell, spermatogonia, markers, seminiferous epithelium
1
Corresponding author: Department of Histology and Embryology, Pomeranian Medical University
e-mail: [email protected]
Copyright © 2012 by the Society for Biology of Reproduction
6
SSCs and spermatogonia
INTRODUCTION
Mammalian spermatogenesis is initiated by the conversion of gonocytes
into spermatogonial stem cells (SSCs) that form the resident stem cells
in the seminiferous epithelium. Spermatogenesis begins 5-7 days after birth
in rodents and 10-13 years after birth in humans [21]. Spermatogonial stem
cells provide the foundation for the continual production of spermatozoa
throughout a male’s lifetime [63] with millions of spermatozoa produced
daily in adult testis.
ORIGIN OF THE SPERMATOGONIAL STEM CELL POOL
Progenitors of primordial germ cells (PGCs) are derived from the epiblast
of blastocyst. Shortly before the epiblast separates into three germ layers:
ectoderm, endoderm and mesoderm, the pluripotent cells of the epiblast
differentiate into PGCs !"#$%&'*+/%89&<<"9post coitum (dpc) in the proximal
part of the epiblast. After that, the PGCs start to move, and approximately
on 7.5-8.5 dpc they are observed at the base of allantois, which is located
in the extraembryonic mesoderm [1, 15, 44, 65].
].. Then, the PGCs are incorporated into the epithelium of hindgut, and on 9.5 dpc they start to migrate
into the dorsal mesentery which they reach on 10.5 dpc [15]. The mesoderm
contributes to the development of the future aorta-gonads-mesonephros
region (AGM region). Afterwards, PGCs migrate into the genital ridges lying on the dorsal body wall reaching them on 11.5 dpc [15, 44]. In humans,
the migration of PGCs occurs between 5 to 8 week of gestation [1, 47].
PGCs can be distinguished by the expression of molecular markers. These
early germ cells create small cluster of cells exhibiting high level of tissue
&?FKLOQ9!XX<# The process
of competence formation of these cells in murine epiblast depends on the expression of secreting bone morphogenetic proteins (BMPs: BMP4, BMP2
and BMP8b) that are released by the extraembryonic ectoderm [18, 35,
<<#Y9%Z8?$&%\O?^\ or
Kolasa et al
7
+/
!"<"#O&98%Z89
anti-proliferative function and may increase the length of the cell cycle
in embryonic germ cells, whereas Stella may affect development of pluripotency of these cells [1].
A member of the POU (Pit-Oct-Unc) family of transcription factors,
Oct4 (octamer-binding transcription factor 4; POU5F1) is strongly expressed
in migrating mouse and human PGCs and plays a role in PGCs survival.
The loss of Oct4 was shown to lead to cell apoptosis [1, 37, 38]. The migration of PGCs towards genital ridges is additionally guided by other
specific molecules: 1/ SDF-1 (stromal cell-derived factor 1) expressed
by cells of embryonic tissues through which the PGCs migrate including
gonad anlagen, and 2/ CXCR4 (receptor for SDF-1) expressed by the germ
cells [42, 44, 46]. It is anticipated that SDF-1/CXCR4 play a crucial role
in PGCs migration [55]. When PGCs start to migrate, additional markers
such as c-Kit receptor (c-Kit-R) begin to be expressed in the cells. The Steel
factor (Stem Cell Factor; SCF), ligand for c-Kit-R, is expressed in somatic
cells along the migratory path. The Steel factor/c-Kit-R signalling results
in the formation of a “travelling niche” [44, 65]. The expression of Steel
factor is necessary to regulate normal migration and proliferation as well
as to suppress apoptosis in PGCs [23, 24, 44].
The PGCs are surrounded by Steel factor-expressing cells from the time
of their appearance in the allantois to the time they colonize the genital
8!X#+/%8&%|%Q%
and they establish contact with each other by extending processes. This
locomotion is controlled in part by the components of the extracellular matrix to which the migrating cells bind [5]. For instance, the adhesion protein
laminin may be involved in the regulation of integrin and/or proteoglycan
expression on the germ cell surface [44]. It was also reported that during
migration, a unique glycoconjugate is selectively and transiently expressed
on the surface of rat PGCs [5].
PGCs have the ability to undergo mitotic divisions during the migration
phase, and approximately three thousand PGCs colonize the genital ridges
[54]. Then, the PGCs are enclosed by differentiating Sertoli cells, and seminiferous cords are formed [4]. Once the germ cells arrive to the genital ridges,
8
A
SSCs and spermatogonia
B
C
Figure 1. Cross sections of immature testes of 6-day-old Wistar rats. Red arrows:
gonocytes located near the basement membrane (A, B) and in the middle portion
(A, C) of seminiferous tubules. Periodic Acid-Schiff (PAS)-staining; objective
%8&‚
some genes are up-regulated and some are down-regulated [44]. In the mouse
and rat fetal testes, PGCs start to proliferate (~13.5 dpc) and after a few days
they are arrested in the G0/G1 phase of the cell cycle [14]. PGCs no longer
proliferate when they cease to express the c-Kit-receptor [23].
At this time, the cells are called gonocytes or pre-spermatogonia [14].
These cells are the long-living primary round-shaped germ cells with promi'!\€#%8&*%niferous cords and shortly after birth some gonocytes resume proliferation.
Z99%Q|%*%*''|'?&8O
where they differentiate into spermatogonial stem cells [39].
Due to the degeneration process only a portion of gonocytes present
in immature testes is destined to become stem cells [51]. In neonatal (0-4
days postpartum) rat testes there are two populations of gonocytes [53].
One population of gonocytes develops cytoplasmic extensions, which
Kolasa et al
9
probably permit them to migrate to the seminiferous tubule basement membrane, a position important for establishing the germ line. The gonocytes
of the second population are round, fail to relocate and remain centrally
located in the seminiferous tubule where they probably degenerate [53].
The gonocytes that resume proliferation and reach the basement membrane
%*''|''8&***%&
wave of spermatogenesis and establish the initial pool of SSCs [54].
SPERMATOGONIA
Spermatogenesis is initiated in the mature testis when a spermatogonial stem
cell is triggered to undergo mitosis and form a differentiated type of spermatogonia. Thus, the spermatogenesis is maintained by the ability of SSCs
to provide a continual supply of differentiating spermatogonia [6]. In the rat
testis, six generations of differentiating spermatogonia are observed: A1, A2,
A3, A4, intermediate (In), and B spermatogonia [28]. Huckins [32, 33, 34],
in turn, divided all spermatogonia into three main categories: 1/ stem cells:
Asingle (As); 2/ proliferating cells: Apaired (Apr) and Aaligned (Aal); and 3/ differen8LLX%?$Oƒ%8?&8OFLs
spermatogonia are thought to be the SSCs, while Apr and Aal spermatogonia
are undifferentiated daughter progeny of SSCs [6, 17]. Spermatogonia A1A4, In and B are differentiated cells [28].
As spermatogonia divide cyclically, but little is known if mammalian
spermatogonial stem cells divide symmetrically and/or asymmetrically
[17]. Two daughter cells derived from one A s spermatogonium can form
a pair of spermatogonia (Apr). Due to incomplete cytokinesis the Apr spermatogonia are connected by intercellular cytoplasmic bridges. Furtherurthermore, the Apr spermatogonia are forced to differentiate or they separate
and enlarge the As stem cells pool [4, 28]. The mitotic division of Apr
spermatogonia produces a chain of four Aal spermatogonia, and further cell
divisions create chains of 8, 16 or even 32 Aal spermatogonia [54]. In an
adult testis under normal conditions, the Aal spermatogonia differentiate
''88%9L%8&9
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SSCs and spermatogonia
Figure 2. Schematized spermatogonial multiplication and stem cell renewal in ro'%?8!X#%&O
of differentiated cells [17]. The A1 cells divide by mitosis and form A2
cells which, in turn, divide and create A3, a division of which generates
A4 spermatogonia. Next, two mitotic divisions form In and B spermatogoF&9Q%9!X„"X#%8
LLX$ƒ'&8*%*''%
9!"X#F9ƒ%8&%
preleptotene spermatocytes. In the rat testis, spermatogonial differentiation takes about (strain-dependent) thirteen days – the equivalent of one
cycle of the seminiferous epithelium [28]. A1 spermatogonia undergo six
more divisions before entering the meiotic prophase, and in theory, from
one stem cell division arise 4096 spermatids [57].
Kolasa et al
11
For non-human primates, two classes of A spermatogonia are present:
Adark%8&Q%Lpale spermatogonia
which proliferate continuously during each spermatogenic cycle producing
B spermatogonia [11]. It was established that the Apale spermatogonium can
give rise either to two new Apale or two new B1 spermatogonia. It seems,
however, that the Apale spermatogonium is unable to produce one Apale and one
B1 spermatogonium after mitosis. If Apale spermatogonium division results
in two B1 spermatogonia, their further divisions form four B2, eight B3
spermatogonia, and sixteen B4 spermatogonia, 32 spermatocytes and 128
spermatids [22, 27].
In human testis, three types of spermatogonia are present: Adark, Apale
8*ƒ%8?&8O**%8tor cell during the spermatogenesis arise maximally 16 spermatids [22].
According to the Clermont model [9, 10], the Apale spermatogonium divides
once every a seminiferous epithelium cycle (once every 16 days).
SPERMATOGONIA-SPECIFIC MARKERS
As mentioned above, in the rodent testis, Asingle spermatogonia are classified as SSCs [28]. Phillips et al. [54] summarized germ cell markers
?|O& Until now little was known
on the characteristics of SSCs and spermatogonia in non-human primates
(see previous paragraphs). However, the expression of proteins which are
considered to be markers of SSCs in rodents (GFRa1, PLZF, NGN3) was
investigated recently in the rhesus testis [26]. It was shown that the rhesus
%9ˆ&%*!<#
Similar to primates, both Adark and Apale spermatogonia are present
in the human testis. Spermatogonia Adark are believed to be the reserve
pool of stem cells, whereas the proliferation of active Apale spermatogonia
maintains spermatogenesis by balancing the production of differentiating
B spermatogonia and renewing Apale pool [3, 21, 27]. Studies on phenotype
*'%%8/ˆ%9%'*&'9
88'*&‰'9*%'%ŠQ
GFR-1
6-integrin
(CD49f)
1-integrin
(CD29)
Ep-CAM
Oct4 (POU5F1)
Stra8
DAZL
EE2 antigen
VASA (MvH)
GCNA1
c-Kit-R
Marker
Epithelial cell adhesion molecule
Octamer-binding transcription factor 4
Receptor for glial cell line-derived neurotrophic
factor (GDNF)
A cell surface receptor which mediates cell-cell
and cell-extracellular matrix attachments
The transmembrane tyrosine kinase receptor;
receptor for stem cell factor (SCF)
Germ Cell Nuclear Antigen 1
ATP-dependent RNA helicase (from DEADbox family)
GTP-binding protein; required for cell
transformation and interaction with the putative
effector protein GAP
Deleted in azoospermia-like; an RNA-binding
protein, one of the members of the DAZ family
Stimulated by retinoic acid gene 8; required for
premeiotic DNA replication
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As Apr Aal
Table 1.+%%?8!"X#%&O
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A1-4 B Spc RS
12
SSCs and spermatogonia
Bcl6b
TAF4B
Sox-3
RBM
PLZF
NGN3
EGR3
CD9
Nanos3
Thy1 (CD90)
CD24
A glycophosphatidylinositol (GPI)-linked
membrane sialoglycoprotein
Thymus cell antigen 1, a GPI linked cell surface
glycoprotein member of the immunoglobulin
superfamily
A 173 amino acid protein that contains one
9&8QQ8%
proliferation
A transmembrane glycoprotein that plays a role
in cell-cell adhesion
Early growth response transcription factor 3
Neurogenin 3; a transcriptional regulator that
determine cell fate
%99'%&8
RNA binding motif protein, Y-linked;
is involved in spermatogenesis
Comprised a family of genes that are related to
the mammalian sex determining gene SRY; Sox
genes encode putative transcriptional regulators
implicated in the decision of cell fate
L9&Q
**KZƒ*
activation of anti-apoptotic genes
B-cell CLL/lymphoma 6 member B protein
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Kolasa et al
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As: Asingle spermatogonia; Apr: Apaired spermatogonia; Aal: Aaligned spermatogonia; A1-A4: differentiating type A1 to A4 spermatogonia; B: type B spermatogonia; Spc: spermatocytes; RS: round spermatids; ES: elongated spermatids
Lin28 (Tex17)
UTF1
Nucleostemin
CDH1 (CD324)
GPR125
Sohlh2
Ret
Lrp4
Numb
Neuronal cell fate decisions, a signaling adapter
protein plays a role in the determination of cell
fate during development
Low-density lipoprotein receptor-related
protein 4, also known as multiple epidermal
growth factor-like domains 7: MEGF7
Proto-oncogene, structurally related to the
growing family of tyrosine kinase transmembrane
receptor; involved in GDNF signaling
%88&
basic helix-loop-helix 2; a nuclear protein
that functions as a transcription factor during
oogenesis and spermatogenesis
E-cadherin, Ca2+-dependent adhesion molecules
G protein-coupled receptor 125
GTP-binding protein 3 that maintains
the proliferative capacity of stem cells
Undifferentiated embryonic cell transcription
factor 1
RNA-binding, cytoplasmic protein that
controls the timing of events during embryonic
development
14
SSCs and spermatogonia
Kolasa et al
15
it has been shown that markers for spermatogonia and their progenitors
in humans share many markers with rodents (tab. 2).
F%'*'*'&* spermato8$'%'*%'‘<8FŠ’+ZY‘
and CD133 were used to select spermatogonia by magnetic-activated cell
separation (MACS; [12, 21]). He et al. [25] used GPR125 to effectively
isolate and purify both Adark and Apale subpopulations of human spermatogonia by MACS. The freshly isolated GPR125-positive spermatogonia are
phenotypically putative human SSCs and under in vitro conditions possess
Table 2. A comparison of markers for human and rodent spermatogonia (accord8!#%&O
Marker
Oct4 (POU5F1)
6-integrin (CD49f)
GPR125
PLZF
GFR-1
Thy1 (CD90)
ITGA6
c-kit-R
1-integrin (CD29)
CD9
NGN3
RET
CDH1 (CD324)
Stra8
CD133
MAGE-A4 (melanoma antigen family A4)
CHEK2 (CHEK2 point homolog)
K“?'&O
F’?&’O
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–
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SSCs and spermatogonia
proliferative features. Similar to rodent SSCs, they co-express ITGA6,
FŠ’+ZY‘F'%%8%'88
be conservative between rodents and humans. The frequency of GPR125positive cells in human testes is estimated on one or two spermatogonia per
%*''|'!"#ƒ998”'Q
cell sorting and MACS, human SSCs were isolated from obstructive azoospermic (OA) and non-obstructive azoospermic (NOA) patients, and as
'ˆ%'+ZY‘
8‘<8•F/ˆ|8'QQ
apoptosis rates in vitro, and there were no differences in these parameters
between the two types of azoospermic patients [43]. Therefore, the culture
of SSCs is expected to become an important tool in the study of SSC survival, self-renewal, proliferation and differentiation [49].
SPERMATOGONIAL STEM CELLS NICHE
Spermatogonial stem cells reside and are maintained throughout life
in the basal compartment of the seminiferous epithelium with a specialized
%Q%–—F8'&
of the stem cells including pluripotency, self-renewal, quiescence and single
or multiple lineage differentiation [16, 28, 40]. Somatic cells play an important role in the formation and functioning of SSCs niche. The niche is formed
by Sertoli cells, peritubular cells and interstitial Leydig cells, and is regarded
|'*'989?&8\!\#O
Sertoli cells, the only somatic cells within the seminiferous epithelium,
secrete many growth factors which include glial cell line-derived neutro*?+^KZO|&||8*?|Z+ZO%
growth factor (EGF) which are needed for SSCs growth and self-renewal
!\XX#L*%”'**%
outside of the seminiferous epithelium i.e. the peritubular myoid cells,
Leydig cells as well as blood-derived factors control the generation of SSCs
and spermatogonia [16, 28, 40]. All these external signals have a crucial role
in controlling the fate of stem cells [30].
Kolasa et al
17
Figure 3. F%8%?8!"X#%&O
In the basal compartment of the seminiferous tubules, SSCs are connected with elements of the basement membrane via adhesion molecules
[28, 30, 40, 54]. Adhesion of the SSCs to laminin of the basal lamina is possible because of the glycoprotein receptors (integrins) present on the surface
*/F|%8‘<?/^X€*O
8•?/^€OQ|'%|9*liferation, differentiation, survival and migration of the cells [4, 28, 36, 54].
REGULATION OF SSCS AND SPERMATOGONIA FUNCTION
SSCs properties are maintained by the microenvironment of their niches.
The niches play an important role in SSCs’ fate, such as self-renewal or
differentiation, and involve complex interactions among the SSCs, their
differentiating daughters, neighbouring cells and the extracellular matrix
[13, 30, 56]. The regulation of SSCs and spermatogonia functions is mul-
18
SSCs and spermatogonia
tidirectional and multifactorial, and not fully understood. The in vivo as
well as in vitro studies have shown that the proliferation and differentiation
of SSCs and uncommitted spermatogonia is under GDNF control. GDNF
is produced by Sertoli cells after FSH stimulation [16, 28, 29]. The in vitro
and in vivo'&%/*9
+^KZ88'8%ˆ*Y“F9˜+YZ‘!\
30, 45, 50-52, 58]. It is suggested that the fate of SSCs during the perinatal
period is regulated by GDNF [50], whereas in the pubertal and adult testes,
it is dependent on the Ets-related molecule (ERM) secreted by Sertoli cells
[29, 60]. Probably, ERM is needed for the maintenance of the blood-testis
barrier function and testicular immune privilege [48].
Fibroblast growth factor 2 (FGF2) acting via FGF2 receptor (FGFR2)
is another growth factor produced by Sertoli cells. FGF2 is involved
in the regulation of the balance between self-renewal and differentiation
of SSCs, and, indirectly, in the maintenance of the cells by the regulation
of GDNF production [16]. Another factor implicated in SSCs differentiation is BMP4. Earlier in the development, during organogenesis, BMP4
controls the survival of primordial germ cells and their colonization within
the genital ridges [20] probably through changes in the adhesion properties
of the cells during cell migration [7]. The in vitro studies also shown that
the BMP4-induced differentiation was accompanied by changes in the adhesion properties. It also appears that BMP4 regulates the expression of cKit-R, an important factor for cell differentiation [7]. SSCs do not express
c-Kit-R, but it is re-expressed in subsequent spermatogonia populations
[7]. Stem cell factor (SCF) is also produced by Sertoli cells of the adult testis. The production of both soluble and transmembrane SCF occurs under
FSH stimulation [28, 55]. It is hypothesized that the c-Kit-R/SCF system
in the adult testis plays a role in the proliferation and survival of mitotic
germ cells [23]. SCF stimulates the progression of A1-A4 spermatogonia into
the mitotic cycle and reduces the apoptosis of the cells [19, 55]. Moreover,
c-Kit-R is expressed also in premeiotic and meiotic spermatocytes, as well
as in postmeiotic germ cells [2, 19, 28, 55].
The in vivo study showed that the localization of SSCs and undifferentiated spermatogonia in the seminiferous epithelium is connected with the dis-
Kolasa et al
19
tribution of blood vessels in the interstitial tissue [6, 16, 59, 64]. Yoshida et al.
[64] postulated that the transfer of factors transported in the blood or from
the interstitial cells is critical for SSC maintenance and/or differentiation.
Therefore, the close association of the niche to the interstitial blood vessels,
Leydig cells and other cells is necessary.
CONCLUDING REMARKS
The seminiferous epithelium has a high regenerative potential because
of the present spermatogonial stem cells that are responsible for the transmission of genetic information from an individual to the next generation.
SSCs represent only a small portion of spermatogonia, as the latter term
&8%%'Š'%%8
some phenotypes with rodents and monkeys’ SSCs and their progenitors
and express some similar markers. A detailed analysis of both phenotype
&%*%8'Q a better understanding
of stem cell regulation in the testis.
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