- Email: [email protected]
The generation of spermatogonial stem cells and spermatogonia in mammals
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 ! "#$% &' * +/% 89 & < <" 9post coitum (dpc) in the proximal part of the epiblast. After that, the PGCs start to move, and approximately on 7.5-8.5 dpc they are observed at the base of allantois, which is located in the extraembryonic mesoderm [1, 15, 44, 65]. ].. Then, the PGCs are incorporated into the epithelium of hindgut, and on 9.5 dpc they start to migrate into the dorsal mesentery which they reach on 10.5 dpc [15]. The mesoderm contributes to the development of the future aorta-gonads-mesonephros region (AGM region). Afterwards, PGCs migrate into the genital ridges lying on the dorsal body wall reaching them on 11.5 dpc [15, 44]. In humans, the migration of PGCs occurs between 5 to 8 week of gestation [1, 47]. PGCs can be distinguished by the expression of molecular markers. These early germ cells create small cluster of cells exhibiting high level of tissue & ?FKL OQ9!XX < # The process of competence formation of these cells in murine epiblast depends on the expression of secreting bone morphogenetic proteins (BMPs: BMP4, BMP2 and BMP8b) that are released by the extraembryonic ectoderm [18, 35, < < #Y9 %Z8?$&%\O ?^\ or
Kolasa et al
7
+/ ! " <"#O & 98%Z8 9 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. Z9 9% Q |% *%* ''|'?&8 O 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/ differen8L LX % ?$O % 8 ?&8 OFLs 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 '' 8 8% 9L % 8 &9
10
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&9 Q % 9!X "X#% 8 L LX $ '&8 *%* ''% 9!"X#F9% 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 *% 8 ?&8 O * * % 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 % 9 &% * ! <# 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 /%9% ' *&'9 88 '*&'9 * %'% Q
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
+ + +
+
+
+ + +
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
As Apr Aal
Table 1.+%% ? 8!"X# % & O
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ES
Continued on the next page
+
+
+
+
+
+
+
+
+
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&8 Q Q 8% 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 %9 9'% &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 L 9& Q
* * KZ * activation of anti-apoptotic genes B-cell CLL/lymphoma 6 member B protein +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Continued on the next page
+
+
Kolasa et al 13
+
+
+
+
+
+
+ +
+
+ + +
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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 % 8
8& 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 $'% '*%'< 8 F +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
+ + + + + + + – – ? ? ? ? ? + + + + +
+ + + + + + + + + + + + + + ? ? ? ? –
16
SSCs and spermatogonia
proliferative features. Similar to rodent SSCs, they co-express ITGA6, F +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 %* ''|' ! "#998' Q cell sorting and MACS, human SSCs were isolated from obstructive azoospermic (OA) and non-obstructive azoospermic (NOA) patients, and as ' %'+ZY 8< 8 F/| 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 |' *'9 89?&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 !\ X X #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 ?/^ O Q| '% |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 % *YF9 +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%%''%% 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.
REFERENCES 1. L
2006 Germ cells from mouse and human embryonic stem cells. Reproduction 132 699-707. 2. Albanesi C, Geremia R, Giorgio M, Dolci S, Sette C, Rossi P 1996 A cell- and deQ %8 & % Q *' protein during mouse spermatid elongation. Development 122 1291-1302. 3. Amann RP 2008 The cycle of the seminiferous epithelium in humans: a need to revisit? Journal of Andrology 29 469-487. 4. Aponte PM, van Bragt MPA, de Rooij DG, van Pelt AMM 2005 Spermatogonial stem cells: characteristics and experimental possibilities. Acta Pathologica, Microbiologica, et Immunologica Scandinavica 113 727-742. 5. Bandyopadhyay S, Banerjee S, Pal AK, Goswami SK, Chakravarty B, Kabir SN 2004 Primordial germ cell migration in the rat: preliminary evidence for a role of galactosyltransferase. Biology of Reproduction 71 1822-1827. 6. Caires K, Broady J, McLean D 2010 Maintaining the male germline: regulation of spermatogonial stem cells. Journal of Endocrinology 205 133-145.
20
SSCs and spermatogonia
7. Carlomagno G, van Bragt MP, Korver CM, Repping S, de Rooij DG, van Pelt AM 2010 BMP4-induced differentiation of a rat spermatogonial stem cell line causes changes in its cell adhesion properties. Biology of Reproduction 83 742-749. 8. Chiarini-Garcia H, Russell LD 2002 Characterization of mouse spermatogonia by transmission electron microscopy. Reproduction 123 567-577. 9. Clermont Y 1966 Renewal of spermatogonia in man. American Journal of Anatomy 118 509-524. 10. Clermont Y 1966 Spermatogenesis in man. A study of the spermatogonial population. Fertility and Sterility 17 705-721. 11. Clermont Y 1969 Two classes of spermatogonial stem cells in the monkey (Cercopithecus aethiops). The American Journal of Anatomy 126 57-71. 12. Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, Aicher W, Bühring HJ, Mattheus U, Mack A, Wagner HJ, Minger S, Matzkies M, Reppel M, Hescheler J, Sievert KD, Stenzl A, Skutella T 2008 Generation of pluripotent stem cells from adult human testis. Nature 456 344-349. 13. Dadoune J-P 2007 New insights into male gametogenesis: what about the spermatogonial stem cell niche? Folia Histochemica et Cytobiologica 45 141-147. 14. De Felici M 2009 Primordial germ cell biology at the beginning of the XXI Century. International Journal of Developmental Biology 53 891-894. 15. De Miguel MP, Arnalich Montiel F, Lopez Iglesias P, Blazquez Martinez A, Nistal M 2009 Epiblast-derived stem cells in embryonic and adult tissues. International Journal of Developmental Biology 53 1529-1540. 16. de Rooij DG 2009 The spermatogonial stem cell niche. Microscopy Research and Technique 72 580-585. 17. de Rooij DG, Russell LD 2000 All you wanted to know about spermatogonia but were afraid to ask. Journal of Andrology 21 776-798. 18. de Sousa Lopes SM, Roelen BA, Monteiro RM, Emmens R, Lin HY, Li E, Lawson KA, Mummery CL 2004 BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes & Development 18 1838-1849. 19. Dolci S, Pellegrini M, Di Agostino S, Geremia R, Rossi P 2001 Signaling through extracellular signal-regulated kinase is required for spermatogonial proliferative response to stem cell factor. Journal of Biological Chemistry 276 4022540233. 20. Dudley B, Palumbo C, Nalepka J, Molyneaux K 2010 BMP signaling controls formation of a primordial germ cell niche within the early genital rigdes. Developmental Biology 343 84-93. 21. Dym M, Kokkinaki M, He Z 2009 Spermatogonial stem cells: mouse and human comparisons. Birth Defects Research. Part C, Embryo Today: Reviews 87 2734. 22. Ehmcke J, Wistuba J, Schlatt S 2006 Spermatogonial stem cells: questions, models and perspectives. Human Reproduction Update 12 275-282.
Kolasa et al
21
23. Grimaldi P, Rossi P, Dolci S, Ripamonti CB, Geremia R 2002 Molecular genetics of male infertility: stem cell factor/c-kit system. American Journal of Reproductive Immunology 48 27-33. 24. Gu Y, Runyan C, Shoemaker A, Surani A, Wylie C 2009 Steel factor controls % 8%'QQ % 9* %% *& in the allantois, and provides a continuous niche throughout their migration. Development 136 1295-1303. 25. He Z, Kokkinaki M, Jiang J, Dobrinski I, Dym M 2010 Isolation, characterization, and culture of human spermatogonia. Biology of Reproduction 82 363-372. 26. Hermann BP, Sukhwani M, Simorangkir DR, Chu T, Plant TM, Orwig KE 2009 ' *%8%8 &'Q% 8 nial stem cells in rhesus macaques. Human Reproduction 24 1704-1716. 27. Hermann BP, Sukhwani M, Hansel MC, Orwig KE 2010 Spermatogonial stem cells in higher primates: are there differences from those in rodents? Reproduction 139 479-493. 28. Hermo L, Pelletier RM, Cyr D, Smith CA 2010'&8Q 9 *tory, and genes/proteins expresses by testicular germ cells. Part 1: Background to spermatogenesis, spermatogonia, and spermatocytes. Microscopy Research and Technique 73 243-278. 29. Hess RA, Cooke PS, Hofmann MC, Murphy KM 2006 Mechanistic insights into the regulation of spermatogonial stem cell niche. Cell Cycle 5 1164-1170. 30. Hofmann MC 2008 Gdnf signalling pathways within the mammalian spermatogonial stem cell niche. Molecular and Cellular Endocrinology 288 95-103. 31. Hofmann MC, Braydich-Stolle L, Dym M 2005 Isolation of male germ-line stem ' *+^KZDevelopmental Biology 279114-124. 32. Huckins C 1971 The spermatogonial stem cell population in adult rats. III. Evidence for a long-cycling population. Cell and Tissue Kinetics 4 335-349. 33. Huckins C 1971 The spermatogonial stem cell population in adult rats. II. A radioautographic analysis of their cell cycle properties. Cell and Tissue Kinetics 4 313-334. 34. Huckins C 1971 The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. The Anatomical Record 169 533-557. 35. Itman C, Mendis S, Barakat B, Loveland KL 2006 All in the family: TGF-beta family action in testis development. Reproduction 132 233-246. 36. Kanatsu-Shinohara M, Toyokuni S, Shinohara T 2004 CD9 is a surface marker on mouse and rat male germline stem cells. Biology of Reproduction 70 70-75. 37. Kehler J, Tolkunova E, Koschorz B, Pesce M, Gentile L, Boiani M, Lomelí H, Nagy A, McLaughlin KJ, Schöler HR, Tomilin A 2004 Oct4 is required for primordial germ cell survival. European Molecular Biology Organization Reports 5 1078-1083. 38. Kellner S, Kikyo N 2010 Transcriptiona regulation of the Oct4 gene, a master gene for pluripotency. Histology and Histopathology 25 405-412.
22
SSCs and spermatogonia
39. Kim BG, Cho CM, Lee YA, Kim BJ, Kim KJ, Kim YH, Min KS, Kim CG, Ryu BY 2010% *'8 9 8% & % *'8 9 8% & '8 '8 viral transduction in pigs. Biology of Reproduction 82 1162-1169. 40. Kostereva N, Hofmann M-C 2008 Regulation of the spermatogonial stem cell niche. Reproduction in Domestic Animals 43 386-392. 41. Kubota H, Avarbock MR, Brinster RL 2004 Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proceedings of the National Academy of Sciences of the United States of America 101 16489-16494. 42. Kucia M, Machalinski B, Ratajczak M 2006 The developmental deposition of epiblast/germ cell-line derived cells in various organs as a hypothetical explanation of stem cell plasticity? Acta Neurobiologiae Experimentalis 66 331-341. 43. Lim JJ, Sung SY, Kim HJ, Song SH, Hong JY, Yoon TK, Kim JK, Kim KS, Lee DR 2010 Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions. Cell Proliferation 43 405-417. 44. McLaren A 2003 Primordial germ cells in the mouse. Developmental Biology 262 1-15. 45. Meng X, Lindahl M, Hyvönen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H 2000 Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287 1489-1493. 46. Molyneaux K, Wylie C 2004 Primordial germ cell migration. The International Journal of Developmental Biology 48 537-444. 47. Molyneaux K, Wylie C 2005 Primordial germ cell migration. The International Journal of Developmental Biology 48 537-543. 48. Morrow CM, Hostetler CE, Griswold MD, Hofmann MC, Murphy KM, Cooke PS, Hess RA 2007 ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood testes barrier function and testicular immune privilege. Annals of the New York Academy of Sciences 1120 144-151. 49. Nagano MC 2011 Techniques for culturing spermatogonial stem cells continue to improve. Biology of Reproduction 84 5-6. 50. Naughton CK, Jain S, Strickland AM, Gupta A, Milbrandt J 2006 Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biology of Reproduction 74 314-321. 51. Oatley JM, Brinster RL 2008 Regulation of spermatogonial stem cell self-renewal in mammals. Annual Review of Cell and Developmental Biology 24 263-286. 52. Oatley JM, Avarbock MR, Brinster RL 2007 Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. The Journal of Biological Chemistry 282 25842-25851. 53. Orwig KE, Ryu BY, Avarbock MR, Brinster RL 2002 Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes. Proceedings
Kolasa et al
23
of the National Academy of Sciences of the United States of America 99 1170611711. 54. Phillips BT, Gassei K, Orwig KE 2010 Spermatogonial stem cell regulation and spermatogenesis. Philosophical Transactions of the Royal Society of London. Series B, Biological sciences 365 1663-1678. 55. Rossi P, Sette C, Dolci S, Geremia R 2000 Role of c-kit in mammalian spermatogenesis. Journal of Endocrinological Investigation 23 609-615. 56. Scadden DT 2006 The stem-cell niche as an entity of action. Nature 441 1075-1079. 57. Schlatt S, Ehmcke J, Jahnukainen K 2009 Testicular stem cells for fertility preservation: preclinical studies on male germ cell transplantation and testicular grafting. Pediatric Blood and Cancer 53 274-280. 58. Schmidt JA, Avarbock MR, Tobias JW, Brinster RL 2009$ & *8 line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat. Biology of Reproduction 81 56-66. 59. Shetty G, Meistrich ML 2007 The missing niche for spermatogonial stem cells: do blood vessels point the way? Cell Stem Cell 1 361-363. 60. Tyagi G, Carnes K, Morrow C, Kostereva NV, Ekman GC, Meling DD, Hostetler C, Griswold M, Murphy KM, Hess RA, Hofmann MC, Cooke PS 2009 Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells '| %&Q *% 8Biology of Reproduction 81 258-266. 61. Western P 2009 Foetal germ cells: striking the balance between pluripotency and differentiation. International Journal of Developmental Biology 53 393-409. 62. Ying Y, Qi X, Zhao GQ 2001 Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways. Proceedings of the National Academy of Sciences of the United States of America 98 7858-7862. 63. Yoo JK, Lim JJ, Ko JJ, Lee DR, Kim JK 2010 & *8 & '%% 8 % '8' '|Q hybridization. Journal of Cellular Biochemistry 110 752-762. 64. Yoshida S, Sukeno M, Nabeshima Y 2007 A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 317 1722-1726. 65. Zwaka TP, Thomson JA 2005 A germ cell origin of embryonic stem cells? Development 132 227-233.
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 ! "#$% &' * +/% 89 & < <" 9post coitum (dpc) in the proximal part of the epiblast. After that, the PGCs start to move, and approximately on 7.5-8.5 dpc they are observed at the base of allantois, which is located in the extraembryonic mesoderm [1, 15, 44, 65]. ].. Then, the PGCs are incorporated into the epithelium of hindgut, and on 9.5 dpc they start to migrate into the dorsal mesentery which they reach on 10.5 dpc [15]. The mesoderm contributes to the development of the future aorta-gonads-mesonephros region (AGM region). Afterwards, PGCs migrate into the genital ridges lying on the dorsal body wall reaching them on 11.5 dpc [15, 44]. In humans, the migration of PGCs occurs between 5 to 8 week of gestation [1, 47]. PGCs can be distinguished by the expression of molecular markers. These early germ cells create small cluster of cells exhibiting high level of tissue & ?FKL OQ9!XX < # The process of competence formation of these cells in murine epiblast depends on the expression of secreting bone morphogenetic proteins (BMPs: BMP4, BMP2 and BMP8b) that are released by the extraembryonic ectoderm [18, 35, < < #Y9 %Z8?$&%\O ?^\ or
Kolasa et al
7
+/ ! " <"#O & 98%Z8 9 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. Z9 9% Q |% *%* ''|'?&8 O 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/ differen8L LX % ?$O % 8 ?&8 OFLs 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 '' 8 8% 9L % 8 &9
10
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&9 Q % 9!X "X#% 8 L LX $ '&8 *%* ''% 9!"X#F9% 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 *% 8 ?&8 O * * % 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 % 9 &% * ! <# 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 /%9% ' *&'9 88 '*&'9 * %'% Q
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
+ + +
+
+
+ + +
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
As Apr Aal
Table 1.+%% ? 8!"X# % & O
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ES
Continued on the next page
+
+
+
+
+
+
+
+
+
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&8 Q Q 8% 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 %9 9'% &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 L 9& Q
* * KZ * activation of anti-apoptotic genes B-cell CLL/lymphoma 6 member B protein +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Continued on the next page
+
+
Kolasa et al 13
+
+
+
+
+
+
+ +
+
+ + +
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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 % 8
8& 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 $'% '*%'< 8 F +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
+ + + + + + + – – ? ? ? ? ? + + + + +
+ + + + + + + + + + + + + + ? ? ? ? –
16
SSCs and spermatogonia
proliferative features. Similar to rodent SSCs, they co-express ITGA6, F +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 %* ''|' ! "#998' Q cell sorting and MACS, human SSCs were isolated from obstructive azoospermic (OA) and non-obstructive azoospermic (NOA) patients, and as ' %'+ZY 8< 8 F/| 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 |' *'9 89?&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 !\ X X #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 ?/^ O Q| '% |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 % *YF9 +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%%''%% 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.
REFERENCES 1. L
2006 Germ cells from mouse and human embryonic stem cells. Reproduction 132 699-707. 2. Albanesi C, Geremia R, Giorgio M, Dolci S, Sette C, Rossi P 1996 A cell- and deQ %8 & % Q *' protein during mouse spermatid elongation. Development 122 1291-1302. 3. Amann RP 2008 The cycle of the seminiferous epithelium in humans: a need to revisit? Journal of Andrology 29 469-487. 4. Aponte PM, van Bragt MPA, de Rooij DG, van Pelt AMM 2005 Spermatogonial stem cells: characteristics and experimental possibilities. Acta Pathologica, Microbiologica, et Immunologica Scandinavica 113 727-742. 5. Bandyopadhyay S, Banerjee S, Pal AK, Goswami SK, Chakravarty B, Kabir SN 2004 Primordial germ cell migration in the rat: preliminary evidence for a role of galactosyltransferase. Biology of Reproduction 71 1822-1827. 6. Caires K, Broady J, McLean D 2010 Maintaining the male germline: regulation of spermatogonial stem cells. Journal of Endocrinology 205 133-145.
20
SSCs and spermatogonia
7. Carlomagno G, van Bragt MP, Korver CM, Repping S, de Rooij DG, van Pelt AM 2010 BMP4-induced differentiation of a rat spermatogonial stem cell line causes changes in its cell adhesion properties. Biology of Reproduction 83 742-749. 8. Chiarini-Garcia H, Russell LD 2002 Characterization of mouse spermatogonia by transmission electron microscopy. Reproduction 123 567-577. 9. Clermont Y 1966 Renewal of spermatogonia in man. American Journal of Anatomy 118 509-524. 10. Clermont Y 1966 Spermatogenesis in man. A study of the spermatogonial population. Fertility and Sterility 17 705-721. 11. Clermont Y 1969 Two classes of spermatogonial stem cells in the monkey (Cercopithecus aethiops). The American Journal of Anatomy 126 57-71. 12. Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, Aicher W, Bühring HJ, Mattheus U, Mack A, Wagner HJ, Minger S, Matzkies M, Reppel M, Hescheler J, Sievert KD, Stenzl A, Skutella T 2008 Generation of pluripotent stem cells from adult human testis. Nature 456 344-349. 13. Dadoune J-P 2007 New insights into male gametogenesis: what about the spermatogonial stem cell niche? Folia Histochemica et Cytobiologica 45 141-147. 14. De Felici M 2009 Primordial germ cell biology at the beginning of the XXI Century. International Journal of Developmental Biology 53 891-894. 15. De Miguel MP, Arnalich Montiel F, Lopez Iglesias P, Blazquez Martinez A, Nistal M 2009 Epiblast-derived stem cells in embryonic and adult tissues. International Journal of Developmental Biology 53 1529-1540. 16. de Rooij DG 2009 The spermatogonial stem cell niche. Microscopy Research and Technique 72 580-585. 17. de Rooij DG, Russell LD 2000 All you wanted to know about spermatogonia but were afraid to ask. Journal of Andrology 21 776-798. 18. de Sousa Lopes SM, Roelen BA, Monteiro RM, Emmens R, Lin HY, Li E, Lawson KA, Mummery CL 2004 BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes & Development 18 1838-1849. 19. Dolci S, Pellegrini M, Di Agostino S, Geremia R, Rossi P 2001 Signaling through extracellular signal-regulated kinase is required for spermatogonial proliferative response to stem cell factor. Journal of Biological Chemistry 276 4022540233. 20. Dudley B, Palumbo C, Nalepka J, Molyneaux K 2010 BMP signaling controls formation of a primordial germ cell niche within the early genital rigdes. Developmental Biology 343 84-93. 21. Dym M, Kokkinaki M, He Z 2009 Spermatogonial stem cells: mouse and human comparisons. Birth Defects Research. Part C, Embryo Today: Reviews 87 2734. 22. Ehmcke J, Wistuba J, Schlatt S 2006 Spermatogonial stem cells: questions, models and perspectives. Human Reproduction Update 12 275-282.
Kolasa et al
21
23. Grimaldi P, Rossi P, Dolci S, Ripamonti CB, Geremia R 2002 Molecular genetics of male infertility: stem cell factor/c-kit system. American Journal of Reproductive Immunology 48 27-33. 24. Gu Y, Runyan C, Shoemaker A, Surani A, Wylie C 2009 Steel factor controls % 8%'QQ % 9* %% *& in the allantois, and provides a continuous niche throughout their migration. Development 136 1295-1303. 25. He Z, Kokkinaki M, Jiang J, Dobrinski I, Dym M 2010 Isolation, characterization, and culture of human spermatogonia. Biology of Reproduction 82 363-372. 26. Hermann BP, Sukhwani M, Simorangkir DR, Chu T, Plant TM, Orwig KE 2009 ' *%8%8 &'Q% 8 nial stem cells in rhesus macaques. Human Reproduction 24 1704-1716. 27. Hermann BP, Sukhwani M, Hansel MC, Orwig KE 2010 Spermatogonial stem cells in higher primates: are there differences from those in rodents? Reproduction 139 479-493. 28. Hermo L, Pelletier RM, Cyr D, Smith CA 2010'&8Q 9 *tory, and genes/proteins expresses by testicular germ cells. Part 1: Background to spermatogenesis, spermatogonia, and spermatocytes. Microscopy Research and Technique 73 243-278. 29. Hess RA, Cooke PS, Hofmann MC, Murphy KM 2006 Mechanistic insights into the regulation of spermatogonial stem cell niche. Cell Cycle 5 1164-1170. 30. Hofmann MC 2008 Gdnf signalling pathways within the mammalian spermatogonial stem cell niche. Molecular and Cellular Endocrinology 288 95-103. 31. Hofmann MC, Braydich-Stolle L, Dym M 2005 Isolation of male germ-line stem ' *+^KZDevelopmental Biology 279114-124. 32. Huckins C 1971 The spermatogonial stem cell population in adult rats. III. Evidence for a long-cycling population. Cell and Tissue Kinetics 4 335-349. 33. Huckins C 1971 The spermatogonial stem cell population in adult rats. II. A radioautographic analysis of their cell cycle properties. Cell and Tissue Kinetics 4 313-334. 34. Huckins C 1971 The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. The Anatomical Record 169 533-557. 35. Itman C, Mendis S, Barakat B, Loveland KL 2006 All in the family: TGF-beta family action in testis development. Reproduction 132 233-246. 36. Kanatsu-Shinohara M, Toyokuni S, Shinohara T 2004 CD9 is a surface marker on mouse and rat male germline stem cells. Biology of Reproduction 70 70-75. 37. Kehler J, Tolkunova E, Koschorz B, Pesce M, Gentile L, Boiani M, Lomelí H, Nagy A, McLaughlin KJ, Schöler HR, Tomilin A 2004 Oct4 is required for primordial germ cell survival. European Molecular Biology Organization Reports 5 1078-1083. 38. Kellner S, Kikyo N 2010 Transcriptiona regulation of the Oct4 gene, a master gene for pluripotency. Histology and Histopathology 25 405-412.
22
SSCs and spermatogonia
39. Kim BG, Cho CM, Lee YA, Kim BJ, Kim KJ, Kim YH, Min KS, Kim CG, Ryu BY 2010% *'8 9 8% & % *'8 9 8% & '8 '8 viral transduction in pigs. Biology of Reproduction 82 1162-1169. 40. Kostereva N, Hofmann M-C 2008 Regulation of the spermatogonial stem cell niche. Reproduction in Domestic Animals 43 386-392. 41. Kubota H, Avarbock MR, Brinster RL 2004 Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proceedings of the National Academy of Sciences of the United States of America 101 16489-16494. 42. Kucia M, Machalinski B, Ratajczak M 2006 The developmental deposition of epiblast/germ cell-line derived cells in various organs as a hypothetical explanation of stem cell plasticity? Acta Neurobiologiae Experimentalis 66 331-341. 43. Lim JJ, Sung SY, Kim HJ, Song SH, Hong JY, Yoon TK, Kim JK, Kim KS, Lee DR 2010 Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions. Cell Proliferation 43 405-417. 44. McLaren A 2003 Primordial germ cells in the mouse. Developmental Biology 262 1-15. 45. Meng X, Lindahl M, Hyvönen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H 2000 Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287 1489-1493. 46. Molyneaux K, Wylie C 2004 Primordial germ cell migration. The International Journal of Developmental Biology 48 537-444. 47. Molyneaux K, Wylie C 2005 Primordial germ cell migration. The International Journal of Developmental Biology 48 537-543. 48. Morrow CM, Hostetler CE, Griswold MD, Hofmann MC, Murphy KM, Cooke PS, Hess RA 2007 ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood testes barrier function and testicular immune privilege. Annals of the New York Academy of Sciences 1120 144-151. 49. Nagano MC 2011 Techniques for culturing spermatogonial stem cells continue to improve. Biology of Reproduction 84 5-6. 50. Naughton CK, Jain S, Strickland AM, Gupta A, Milbrandt J 2006 Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biology of Reproduction 74 314-321. 51. Oatley JM, Brinster RL 2008 Regulation of spermatogonial stem cell self-renewal in mammals. Annual Review of Cell and Developmental Biology 24 263-286. 52. Oatley JM, Avarbock MR, Brinster RL 2007 Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. The Journal of Biological Chemistry 282 25842-25851. 53. Orwig KE, Ryu BY, Avarbock MR, Brinster RL 2002 Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes. Proceedings
Kolasa et al
23
of the National Academy of Sciences of the United States of America 99 1170611711. 54. Phillips BT, Gassei K, Orwig KE 2010 Spermatogonial stem cell regulation and spermatogenesis. Philosophical Transactions of the Royal Society of London. Series B, Biological sciences 365 1663-1678. 55. Rossi P, Sette C, Dolci S, Geremia R 2000 Role of c-kit in mammalian spermatogenesis. Journal of Endocrinological Investigation 23 609-615. 56. Scadden DT 2006 The stem-cell niche as an entity of action. Nature 441 1075-1079. 57. Schlatt S, Ehmcke J, Jahnukainen K 2009 Testicular stem cells for fertility preservation: preclinical studies on male germ cell transplantation and testicular grafting. Pediatric Blood and Cancer 53 274-280. 58. Schmidt JA, Avarbock MR, Tobias JW, Brinster RL 2009$ & *8 line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat. Biology of Reproduction 81 56-66. 59. Shetty G, Meistrich ML 2007 The missing niche for spermatogonial stem cells: do blood vessels point the way? Cell Stem Cell 1 361-363. 60. Tyagi G, Carnes K, Morrow C, Kostereva NV, Ekman GC, Meling DD, Hostetler C, Griswold M, Murphy KM, Hess RA, Hofmann MC, Cooke PS 2009 Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells '| %&Q *% 8Biology of Reproduction 81 258-266. 61. Western P 2009 Foetal germ cells: striking the balance between pluripotency and differentiation. International Journal of Developmental Biology 53 393-409. 62. Ying Y, Qi X, Zhao GQ 2001 Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways. Proceedings of the National Academy of Sciences of the United States of America 98 7858-7862. 63. Yoo JK, Lim JJ, Ko JJ, Lee DR, Kim JK 2010 & *8 & '%% 8 % '8' '|Q hybridization. Journal of Cellular Biochemistry 110 752-762. 64. Yoshida S, Sukeno M, Nabeshima Y 2007 A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 317 1722-1726. 65. Zwaka TP, Thomson JA 2005 A germ cell origin of embryonic stem cells? Development 132 227-233.