Gonocytes, the forgotten cells of the germ cell lineage

Gonocytes, The Forgotten Cells of the GermCell Lineage

Martine Culty*

Male germ cells, the repository cells of the genome, comprise severalsuccessive developmental stages starting in the embryo and ending upwith the spermatozoon. Gonocytes represent the fetal and neonatalstages preceding the formation of spermatogonial stem cells. Recentfindings shows that germline stem cells can be driven to pluripotencyand used as alternative for embryonic stem cells prompted more effortin identifying the processes regulating the development of their precur-sors, the testicular gonocytes. Also called pre- or pro-spermatogonia,gonocytes represent not one, but several successive developmentalstages between the time at which the germ cell becomes resident in theforming fetal testis to the time it migrates to the basement membraneof the seminiferous cord to adopt a spermatogonial phenotype. Thisreview summarizes the findings regarding the genetic identity of gono-cytes, providing a description of the ‘‘common’’ gene expression profilesof fetal and neonatal gonocytes, as well as information on the main re-gulatory factors of gonocyte functions. A better comprehension of gono-cyte development should help in the understanding of how germlinestem cells are formed, possibly providing valuable clues on the originsof germ cell tumors or infertility. Birth Defects Research (Part C)87:1–26, 2009. VC 2009 Wiley-Liss, Inc.

Key words: testis; germ cell development; gene profile

INTRODUCTIONTesticular gonocytes are the fetal/neonatal precursors of the undif-ferentiated spermatogonial stemcells (SSCs), the life-long reservoirof germline stem cells. The termgonocyte was originally proposedby Clermont and Perey (1957) todesignate the fetal germ cell afterit becomes resident in the devel-oping gonad. The term gonocytealso applies to the fetal femalegerm cell that resides in the devel-oping ovary, although very fewstudies, such as that of Morita andTilly (1999), appear to use this

terminology. Testicular gonocytesare also identified as prespermato-gonia, prospermatogonia or morebroadly as primitive germ cells indifferent articles (Gaskell et al.,2004), although the term gono-cyte appears in more studies thanthe other names.Germ cell development com-

prises two main phases leading tothe SSC and the spermatozoon,respectively. The first phaseencompasses the fetal and neona-tal periods leading to the forma-tion of the SSCs. This develop-mental period includes two main

types of germ cells, the primordialgerm cell (PGC), the first cell ofthe germline lineage in theembryo, and the gonocyte whichdifferentiates from PGC to formtype A spermatogonia, includingthe SSCs. Both PGCs and gono-cytes present the distinctive mor-phology of being large circularcells with a prominent nucleuscontaining one or two nucleoli(Baillie, 1964) surrounded by aring-like cytosol. They occupy thecenter of lumen-less seminiferouscords, easily distinguishable fromthe adjacent Sertoli cells (seeFig. 1). Although gonocytes arethe precursors of SSCs, recentstudies based on the differentialexpression of the basic helix-loop-helix transcription factor neuroge-nin 3 (Ngn3) in mouse gonocytesand spermatogonia have shownthat not all gonocytes (Ngn32)become SSCs (Ngn31), and that afraction of the gonocytes directlydifferentiate into Ngn32 differenti-ating spermatogonia that will sup-port the first wave of spermato-genesis (Yoshida et al., 2006).The second phase in germ cell de-velopment is the spermatogeniccycle, a highly regulated succes-sion of events including cell divi-sion by mitosis and meiosis, andcomplex differentiation processessuch as spermiogenesis, startingwith the formation of differentiat-ing spermatogonia and ending

REVIE

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VC 2009 Wiley-Liss, Inc.

Birth Defects Research (Part C) 87:1–26 (2009)

Martine Culty is from the Research Institute of the McGill University Health Centre, Montreal, Quebec, H3G 1A4, Canada andDepartment of Medicine, McGill University, Montreal, Quebec, H3G 1A4, Canada

Grant sponsor: Royal Victoria Hospital Foundation, Canada.

*Correspondence to: Martine Culty, The Research Institute of the McGill University Health Centre, Montreal General Hospital;1650 Cedar Avenue, Room C10.148.2. Montreal, Quebec H3G 1A4 Canada. E-mail: [email protected]

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.20142

with the production of spermato-zoa (Russell et al., 1990).Although several stages of sper-matogonia have been described inrodents, including the types Asingle,Apair, Aaligned, A1-4, A intermediate(In), and finally B spermatogonia,only three steps have beenobserved in human, the A pale, Adark, and B spermatogonia (Russellet al., 1990; de Rooij, 2001). DeRooij and Russell (2000) proposedthat only the SSCs, which retainpluripotency, the ability to self-renew or differentiate, and do notpresent intercellular bridges,should be called undifferentiated,whereas other spermatogonialstages should be referred to asdifferentiating, since they are allcommitted to progress to the nextstep without the possibility ofreverting to the previous stage (deRooij and Russel, 2000). However,spermatogonia are still classifiedas undifferentiated (Asingle toAaligned), differentiating (A1–A4),

and differentiated (In, B types) bymany investigators. Type B sper-matogonia and subsequent germcell stages are first visible at 4 to5 years old in human (Paniaguaand Nistal, 1984) and at postnatalday 10 in rodents (Russell et al.,1990), and they can be found sub-sequently throughout life. Thisreview will focus on the first phaseof germ cell development, morespecifically on the gonocyte.In contrast to studies on sper-

matogenesis that have over theyears generated many of publica-tions, articles dedicated to gono-cytes/pre- or pro-spermatogoniaare very few. Indeed, the neglectthat has affected gonocyteresearch over the years is easilyproven by the minimal number ofscientific articles that appear in asearch for this word in scientificjournals: only 100 to 200 articlesare found when including all possi-ble designations, against severalthousand studies related to more

mature types of germ cells fromspermatogonia to spermatid, andmore than 50,000 referenceswhen looking for spermatozoon orsperm. Even PGC, the direct pre-cursors of gonocytes, have beenstudied in more than 2,000articles. Thus, the scarcity of stud-ies published on gonocytes trulymakes them the ‘‘forgotten cell ofthe germ line.’’Nevertheless, the recent conver-

gence of studies in the fields ofstem cell research, environmentaltoxicology, and reproductive on-cology have led to a renewed in-terest in understanding how adultSSCs arise from their fetal/neona-tal precursors. Indeed, the recog-nition that SSCs and their precur-sor cells can be manipulated tobehave as pluripotent embryonicstem cells (ESCs)_revived thehope of developing a stem cellpool that could be used for thera-peutic purposes without insur-mountable ethical dilemma(Kanatsu-Shinohara et al., 2004;Guan et al., 2006; Izadyar et al.,2008). A series of studies identi-fied the fetal/neonatal testis asvery sensitive to environmentalexposure, raising the possibilitythat the precursor cells of SSCsmight be preferentially targetedby endocrine disruptors and posi-tion them at the origin of germ cellpathologies (Foster, 2006;Sharpe, 2006). In this regard,characterization of the gene andprotein expression profiles of car-cinoma in situ (CIS), also referredto as intratubular germ cell neo-plasia, unclassified (IGCNU)(Reuter, 2005), and testiculargerm cell tumors (TGCT), togetherwith their morphological appear-ance emphasize their similarityto the fetal/neonatal precursors ofthe SSCs, supporting the idea thatthe etiology of germ cell tumorsmight reside in the disruption ofSSC formation from their precur-sor cells, gonocytes, and PGCs(Rajpert-De Meyts and Hoei-Hansen, 2007). Deciphering thecellular and molecular mecha-nisms regulating gonocyte devel-opment must therefore be givenpriority, as it is the prerequisite tounderstanding SSC formation.

Figure 1. Morphological appearance of rat gonocytes from dpc 16 to dpp3. Paraffinsections of testes at different ages were dewaxed, rehydrated, and treated with anantigen retrieval solution before blocking treatments and immunostaining using theindicated primary antibodies and corresponding secondary antibodies followed by acolorimetric enzymatic reaction and hematoxylin counterstaining. One of the sectionswas incubated with secondary antibody only to determine background signal. Thescales represent either 10 or 100 lm. While at dpc16, the clustering of somatic andgerm cells outlines the general shape of the nascent seminiferous cord, it is only atdpc17 that a clear perimeter of peritubular myoid cells becomes visible. As illustrated,the size and morphological appearances of fetal to neonatal gonocytes are very simi-lar, despite critical differences in the gene expression and behaviors of the cells atdifferent ages. PY20: anti phosphotyrosine antibody.

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Birth Defects Research (Part C) 87:1–26, (2009)

DEVELOPMENTAL

TIME-LINE OF

GONOCYTE FORMATION

Before discussing in details aboutgonocytes, one must first definewhere these cells are positionedwithin the time-line of germ cell de-velopment. As mentioned above,gonocytes correspond to the fetal/neonatal stage between the PGCand the SSC. PGC precursors arefirst identified at around 5.5 daypost-coitum (dpc) in mouse, in theproximal epiblast located in the vi-cinity of the extra-embryonic ecto-derm, a position that has beenshown to be critical for the specifi-cation of epiblast cells to becomePGCs, under the influence of bonemorphogenetic proteins producedby cells of the extraembryonicectoderm (Lawson et al., 1999;Ying et al., 2000). Then, at 7.5 to 8dpc in the rodent and at 3 weeksgestation in human, PGC can beclearly identified by their stronggerm cell alkaline phosphatase(GCAP; also called placental-likealkaline phosphatase; PLAP) posi-tive staining (Cooke et al., 1993)as they migrate inside the extra-embryonic mesoderm at the baseof the allantois (Zhao and Garbers,2002). PGCs finally migrate towardthe genital ridge under the guid-ance of steel, the ligand for c-KIT,a tyrosine kinase receptor exp-ressed by PGCs (Sutton, 2000),where they become resident in theforming gonads at 4 to 5 weeksgestation in human and dpc 11 to13 in rodents (see Fig. 1). Interest-ingly, PGCs were recently shown toretain expression of pluripotencymarkers such as the stage specificembryonic antigen 1 (SSEA1), de-spite being restricted to the germcell fate in vivo, and to be able toform colonies of pluripotent embry-onic germ cells (EGC) in vitro(Guan et al., 2006; Kerr et al.,2008). Thus, the idea that all PGCsare unipotent has recently beenchallenged by studies suggestingthat a small portion of the PGCsmight remain pluripotent in utero,and that these cells might be theprecursors of EGCs in culture andembryonal carcinoma cells in vivo(Kerr et al., 2008). As the cell

becomes resident in the gonad, theexpression of the germ cell nuclearantigen (GCNA1) is turned on andremains as a germline marker insubsequent germ cell stages(Enders and May, 1994), whilealkaline phosphatase expression islost at around dpc 14.5 in mice(Richards et al., 1999). It is usuallyat the point that the germ cell namechanges from PGC to gonocyte.Although germ cell stages

between PGC and SSC are oftenindiscriminately called gonocytes,suggesting that these cells repre-sent a single developmental stage,morphometric and mitotic labelingstudies performed as early as inthe seventies indicated that rodentand human gonocytes comprisedsuccessive stages, described asmitotic (M) and transitional (T)prospermatogonia which wasfurther subdivided as T1 andT2 prospermatogonia (Fukudaet al., 1975; Wartenberg, 1976;Hilscher, 1991; Vergouwen et al.,1991). Comparison of the res-ponses of fetal and neonatal ratgonocytes to stimuli such as reti-noic acid (RA) (Boulogne et al.,1999b; Livera et al., 2000), andtransforming growth factor b1(TGFb1) (Olaso et al., 1998), fur-ther showed that fetal and neona-tal gonocytes do not behave in thesame ways, confirming that theterms gonocyte/prospermatogoniaencompass different developmen-tal stages. More recently, thesame conclusion was reachedregarding human prespermatogo-nia by several investigators whoobserved that M, T1, and T2 pre/prospermatogonia expressed dif-ferent levels and combinations ofgenes such as the Melanoma anti-gen-A4 (MAGE-A4), a gene of thecancer-testis family of antigensfound in spermatogonia, primaryspermatocytes and germ celltumors (De Plaen et al., 1999;Aubry et al., 2001), and GCAP(Franke et al., 2004; Gaskellet al., 2004; Pauls et al., 2006).Although Pauls et al. (2006) sug-gested that human fetal germ cellsincluded two major cell types, thegroups of Gaskell (Gaskell et al.,2004) and Franke (Franke et al.,2004) both identified three distinct

populations that were named gon-ocytes, intermediate cells and pre-spermatogonia in one case, andM, T1, and T2 prespermatogoniain the other. Hence, although thefetal and neonatal periods of germcell development can at first sightappear much more simple and ho-mogeneous than germ cell popula-tions found from prepuberty toadulthood, it is impossible todetermine precise time-framesthat would include homogeneouspopulations of germ cells repre-senting a single developmentalphase. Indeed, studies looking atmitotic index and migratorybehavior such as that of Naganoet al. (2000) and gene profilinghave shown that germ cells arenot synchronized within each de-velopmental period, but ratherthere is an overlap of subpopula-tions within each time-frame, cor-responding to the presence in thesame seminiferous cord section ofquiescent and mitotic germ cells,as well as cells located at both theperiphery and center of the cords,with the latter being in the processof differentiating to SSCs and theformer still dividing and pre-mi-gratory or destined to apoptosis.The schematic time-line pre-

sented in Figure 2 illustrates thesimilarities observed between thesuccessive mitotic and quiescentstages of rodent and human gono-cyte subpopulations, which extendfrom dpc 13 to postnatal day (daypostpartum, dpp) five in rodents,and from gestation week 7 topostnatal week 8 in human. Itshould be noted that the number-ing of the gestational and post-natal days can vary by 0.5 to 1day between studies, as either day0 or day 0.5 may be chosen forthe beginning of gestation,whereas day 0, 0.5, or 1 may beused to represent the day of birth.Moreover, an overview of the workpublished on this topic in rodentas well as human shows that theboundaries of each subpopulationin time can vary by 1 to 1.5 daysin rodents and several weeks inhuman, depending on the windowsof time examined in the studies.Thus, even when performed atspecific ages, studies will have the

TESTICULAR GONOCYTE DEVELOPMENT 3


tendency of presenting results cor-responding to the combination ofseveral sub-types of germ cells,and on occasion, literature rele-vant for the gonocytes might bepresented in studies dedicated toPGCs or SSCs.

BUILDING THE ID CARD

OF GONOCYTES

Expression of Stem CellMarkers in Gonocytes

Because gonocytes correspondto one of the two intermediarystages between pluripotent ESCsand the adult germ-line stem cells,they were expected to retain theexpression of genes found in stemcells. The identification of specificsets of genes correlated withstemness in embryonic cellsemerged in the nineties, followingthe establishment of pluripotentembryonic cell lines from mouse

embryos, where the conditionsrequired to perpetuate and keepthese cells as undifferentiated celllines were determined (Evans andKaufman, 1981; Martin, 1981).This advance followed earlier stud-ies in the seventies where pluripo-tent embryonic cell lines were ei-ther derived from spontaneousteratocarcinoma containing bothstem cells and differentiated cells(Gachelin, 1976), or generated byinjecting cells of the Inner CellMass into ectopic sites, includingthe testis. The latter procedure ledto the formation of tumors consti-tuted of undifferentiated embry-onic stem cells called embryonalcarcinoma (EC), that could be keptas undifferentiated proliferatingcells or induced to differentiateinto cells of various lineages(Diwan and Stevens, 1976). Theavailability of ESCs and ECs andthe understanding that they bothrecapitulated aspects of early em-

bryonic development allowed forthe determination of key molecu-lar and cellular mechanisms regu-lating this process (Jakob, 1984;Rossant and Papaioannou, 1984).The subsequent comparison ofgene expression profiles betweenthese cell lines further led to theidentification of a panel of genesconsidered as embryonic stem cellmarkers, including the Stage Spe-cific Embryonic Antigens (SSEAs),Thy-1 (CD90), c-kit (CD117) (Lingand Neben, 1997), podocalyxin, asialomucin related to CD34 recog-nized by the monoclonal antibod-ies TRA-1-60 and TRA-1-81 at thesurface of both ESC and EC(Andrews et al., 1984; Schopperleand Dewolf, 2007) discovered inthe eighties, and the transcriptionfactors POU5F1 (OCT3/4),NANOG, SOX2, STELLAR, the TGFrelated ligand growth and differen-tiation factor 3 (GDF3), and theRNA binding protein DAZL, further

Figure 2. Schematic representation of the time-lines of gonocyte development in mouse, rat, and human, as summarized from theavailable literature. M, mitotic; T1, transitional 1; T2, transitional 2. The box on the human time line indicates the period betweengestation weeks 19 and 25, at which a progressive shift occurs between Oct4 and MAGE-A4 expression in the germ cell.

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identified in pluripotent stem cellsas well as in mammalian PGCsand/or human tumoral germ cells(Nichols et al., 1998; Yen, 2004;Ezeh et al., 2005; Clark, 2007;Kerr et al., 2008). Thus, in aneffort to better define rodent andhuman gonocytes, investigatorshave examined the expression ofthese genes, usually focusing on asingle gene or a panel of a fewselected genes.Among those genes, the tran-

scription factor Oct4 (Oct-3,POU5F1) that was initially consid-ered as an ESC marker because itis expressed in undifferentiatedtotipotent ESCs of the inner cellmass in blastocytes and in primi-tive endoderm (Nichols et al.,1998), was subsequently shown tobe restricted to the germline afterdpc7.5, using green fluorescentprotein (GFP) expression driven bythe Oct4 enhancer/promoter as asurrogate for Oct4 expression inmouse germ cells (Ohbo et al.,2003; Ohmura et al., 2004). Thus,Oct4 has become one of the majormarkers used to identify PGCs,gonocytes and spermatogonia(Zhao and Garbers, 2002). Sev-eral investigators have takenadvantage of the segregation ofOct4 in germ cells to producelacZ- or GFP-tagged germ cells bydriving lacZ or GFP expressionunder the control of Oct4enhancer/promoter in transgenicmice and rat (Ovitt and Scholer,1998; Cronkhite et al., 2005). Thisapproach can greatly simplify thecharacterization and isolation ofrodent PGCs and gonocytes, andhas been successfully used todemonstrate the plasticity andpluripotency potential of PGCs andSSCs in experiments where thesecells were induced to reverse to anESC phenotype, capable of differ-entiating into several somatic celllineages (Guan et al., 2006).Besides being one of the principalmarkers used to characterize earlygerm cells from PGCs to spermato-gonia, the finding of Oct4 expres-sion in CIS/seminoma has givencredit to the hypothesis that thesegerm cell neoplasms result from afailure of the PGCs or gonocytes toproperly differentiate, a hypothe-

sis originally based on morphologi-cal similarity between the fetal/neonatal and the tumoral germcells (Almstrup et al., 2004;Kristensen et al., 2008).Using Oct4/EGFP mice, Ohbo

et al. (2003) compared theexpression of Oct4 (as reflected byGFP protein expression) in dpp0.5gonocytes and dpp7.5 spermato-gonia (called prospermatogonia inthe study) to that of several genespresent in hematopoietic/progeni-tor cells, such as the cell-cell ad-hesion molecule Activated Leuko-cyte Cell Adhesion Molecule(ALCAM; CD166) (Bowen et al.,1997) and the tyrosine kinase re-ceptor c-kit (Ashman et al., 1991),or genes previously described inspermatogonia, including a6 andb1 integrins (Intg), the antigenrecognized by the EE-2 monoclo-nal antibody, and the stem cellantigen recognized by the anti-body TRA98. Intg a6 and b1 wereboth shown to be expressed at thesurface of adult mouse SSCs in ac-kit2 subpopulation of spermato-gonia, and to represent usefulmarkers for enrichment of adultSSCs using magnetic beads cellsorting (MACS) (Shinohara et al.,1999). In our own studies, wefound that dpp3 rat gonocytesexpress Intg a6 transcripts (Wangand Culty, 2007), whereas Tresand Kierszenbaum (2005)reported the expression of a6b1integrins in rat gonocytes duringthe time of migration. The EE2antigen has been shown to be acell surface 114 kDa glycoproteinwith affinity for lectins. It isexpressed on gonocytes, in peri-natal testis, in adult spermatogo-nia from A to B types, and in earlymeiotic germ cells, and is there-fore considered as a spermato-genic marker (Koshimizu et al.,1995). The antigen for TRA98 waspreviously shown to be 110kDanuclear protein expressed in PGCsand spermatogonia (Tanaka et al.,1997; Tanaka et al., 2000). Theproto-oncogene c-kit was firstdescribed as a hematopoietic stemand progenitor cell marker(Ashman et al., 1991). It was lateridentified as a PGC marker andshown to mediate PGC migration

toward the genital ridge(Koshimizu et al., 1992). c-kit wasalso found in spermatogonia fromdpp6 to adult and in preleptotene(Manova et al., 1990), but notin pachytene spermatocytesand post meiotic germ cells(Sorrentino et al., 1991). Sortingof dpp 0.5 gonocytes according tothe expression of these variousmarkers allowed Ohbo et al.(2003) to characterize the major-ity of gonocytes as Oct41/Tra981/a6b1 integrins1/EE-2 antigen1/ALCAM1/c-kit2. However, the factthat only 75% of a6 integrin 1

were also b11 highlighted the het-erogeneity of the gonocyte popu-lation. Nevertheless, the facts thatIntg a6 was found in dpp0.5 micegonocytes (Ohbo et al., 2003),dpp3 rat gonocytes (Wang andCulty, 2007) and SSCs (Ohboet al., 2003) suggest that thisintegrin is a hallmark of postnatalgonocytes and SSCs, whereas thesubsets of gonocytes not express-ing b1 intg might instead expressanother b integrin, such as b3integrin that was found in subsetsof gonocytes as discussed later(Ryu et al., 2004). Co-expressionof Oct4 and c-kit was observed indpc11.5 and 13.5 PGCs, but not infetal/neonatal quiescent gonocytesat dpp16.5 and dpp0.5 whereOct41 cells did not express c-kit.However, c-kit was re-expressedat later stages, with a distinctspermatogonial population atdpp7.5 clearly expressing bothOct4 and c-kit (Ohbo et al., 2003).The study also found that ALCAMwas not expressed in PGCs nor indpp7.5 spermatogonia, and that itwas transiently expressed on gon-ocytes between dpp0.5 and 3.5,suggesting a potential role in gon-ocytes migration toward the base-ment membrane. Moreover, thesorting of dpp7.5 spermatogoniaaccording to the expression ofthese various markers and theevaluation of their repopulationability by transplantation intobusulfan-treated mouse testisidentified two populations of sper-matogonia, including a group ofOct41/c-kit2 spermatogonia withhigh stem cell properties. Interest-ingly, the group of dpp7.5 Oct41/



c-kit1 spermatogonia still retainedsome repopulating activity, bycontrast with adult c-kit1 sperma-togonia that were devoid of anystem cell potential (Ohbo et al.,2003).The study of Ohmura et al.

(2004) extended this work byexamining also gonocytes fromdpp 1.5 and 3.5 mice. This studyconfirmed that Oct4 presence cor-relates with the stemness proper-ties of gonocytes by showing that98 to 51% of gonocytes from dpp0.5 to 3.5 mice, respectively,expressed Oct4 and showed stemcell repopulation activity. Thesecells were also positive for Tra98.The authors further reported aprogressive decrease in Oct4/Tra98 expression as the cellsadvanced toward more differenti-ated stages, with 30% of the sper-matogonia remaining Oct41/Tra981 at dpp7 to 7.5. In theirstudy, the expression of the Glialcell line-Derived Neurotrophic Fac-tor (GDNF) receptors was exam-ined. GDNF is a neurotrophic fac-tor related to TGFb that promotesthe differentiation and survival ofneurons and kidney morphogene-sis (Takahashi, 2001). GDNF re-ceptor comprises two distinct pro-teins, the tyrosine kinase RETwhich is the signaling sub-unit ofthe receptor complex, and the gly-cosyl phosphatidylinositol-anch-ored GFRa1 which represents theligand binding component of thecomplex. RET activates classicaldownstream signaling cascades,including several mitogen acti-vated protein kinase (MAPK) andphosphatidylinositol 3-kinasepathways in functions of the celltype considered. RET mutationshave been implicated in severalhuman diseases, including thyroidcarcinoma and multiple endocrineneoplasia (Takahashi, 2001). Thegeneration of Gdnf Knockout (KO)and overexpressing mice unveiledan additional role of GDNF in sper-matogenesis. Indeed, Sertoli cell-secreted GDNF plays a critical rolein the regulation of undifferenti-ated spermatogonia SSC fate,whereby the dosage of GDNF inthe testis determines whetherSSCs will self-renew (high GDNF

level) or differentiate (low GDNFlevel) (Meng et al., 2000). It wasalso reported that Ret and Gfra1were down-regulated by week twoafter birth in mice. Together witha study of Dettin et al. (2003)showing that Gfra1 is expressednot only in As spermatogonia butalso in Apr and Aal mouse sperma-togonia, these results imply thatGFRa1 is expressed from the stemcell stage to the type Aal sperma-togonia. Thus, the expression ofGDNF receptors is not sufficient initself to identify a germ cell as anSSC. However, they offer theadvantage of being among therare cell surface proteins that canbe used to enrich isolated germcells from juvenile to adult testisin undifferentiated spermatogonia,including the stem cell population(Hofmann et al., 2005).The work of Ohmura et al.

(2008) also showed that 55% ofthe Oct41/Tra981 gonocytes fromdpp0 mice were also positive forGDNF receptors, while by dpp7 to7.5, 25% were Ret1, a similar pro-portion of spermatogonia as thosefound to be Oct41/Tra981. Retexpression further decreased to5% Ret1 germ cells at dpp14 andless than 0.5% Ret1 germ cells inadult testis. The comparison of theproportions of germ cells present-ing Oct4, Tra98, and Ret positivesignals between dpp0 and 7.5showed a progressive decrease inthe three markers as cells pro-gressed from quiescent to mitoticgonocytes and finally to undiffer-entiated spermatogonia. The factthat only 55% of mouse gonocyteswere Oct41/Tra981 at dpp 3.5supports the idea of Yoshida(2006) that a subpopulation ofgonocytes might directly evolveinto differentiating spermatogoniawithout passing by the stem cellstage. Moreover, at dpp0, onlyhalf of the gonocytes were Ret1,whereas 98% were Oct41/Tra981,indicating that the population ofneonatal quiescent gonocytes isalso heterogeneous. To our knowl-edge, so far only one study hasexamined the expression of Gfra1and Ret in mouse fetal testis(Golden et al., 1999). In thisstudy, the mRNA expression of

both receptors was surveyedthroughout the whole embryo/fetus from dpc 10 to 18. BothGfra1 and Ret mRNAs were pres-ent in testis at dpc 14, where fetalgonocytes are mitotic, but absentat dpc16 and 18, corresponding tothe period of quiescence in mice.Moreover, a study using heterozy-gous mice expressing a dominantnegative Ret allele encoding for aprotein with diminished kinaseactivity (RetDN) found that thenumber of GCNA positive gono-cytes was comparable betweenwild type and RetDN/1 mice atdpp0, suggesting that Ret activity,and by deduction GDNF, is notinvolved in fetal gonocyte survivalor proliferation. By contrast, atdpp10, the number of spermato-gonia in RetDN/1 was decreased byhalf compared to wild-type testis,as well as the number of BrdU-la-beled proliferative spermatogonia,and by dpp17 the number of apo-ptotic germ cells nearly doubled(Jain et al., 2004). These dataare in agreement with GDNF play-ing a critical role in spermatogoniabut not at earlier stages. Thus,GDNF receptors appear to bedown-regulated in fetal gonocytesand to be re-expressed afterbirth, to finally remain only ina small sub-population of adultundifferentiated spermatogonia in-cluding SSCs.GDNF receptors and c-kit

expression in gonocytes and sper-matogonia were further exploredby Naughton et al. (2006) usingdpp 0, 3.5, 7, 14, and 28 testesfrom wild type as well as Gdnf-,Ret-, and Gfra1-KO mice. Thestudy examined the in vivo proteinexpression of Ret, Gfra1, Kit, andPlzf by immunohistochemistry andquantified the number of germcells positive for these markers atdifferent ages. Plzf is a transcrip-tional repressor of stem cell differ-entiation (Buaas et al., 2004) thatwas previously found in the nucleiof fetal dpc13.5 PGCs and dpc17.5 gonocytes in mice, as well asin spermatogonia nuclei of juvenilemice (Costoya et al., 2004). Theresults showed that more than80% of dpp3.5 gonocytes werepositive for Ret, Plzf, and c-kit,

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whereas in dpp7 spermatogonia,only 50% of Ret1 and 10% of Plzf1

remained Kit1, respectively. More-over, the study demonstratedunequivocally that GDNF-mediatedsignaling is required for SSCrenewal. The authors furtherconcluded that a subpopulationof RET1/PLZF1/KIT1 gonocyteswould be the precursors of RET1/PLZF1/KIT2 SSCs, giving rise toRET1/PLZF1/KIT1 differentiatingspermatogonia. Another studyconducted on mice reported thatgonocytes were c-kit2 at dpp2 butc-kit1 at dpp5 (Tajima et al.,1994). It should be noted thatgerm cell development in mice isadvanced by approximately 1 dayas compared with rat (see Fig. 2),and that mouse dpp2 gonocytesare already proliferative, whereasrat dpp2 gonocytes are still quies-cent (at least the majority ofthem). A study by Orth (1997)using gonocyte-Sertoli cell co-cul-tures from dpp1 to 5 rats reportedthat the proportion of c-kit1 gono-cytes gradually progressed fromless than 10% c-kit1 gonocytes atdpp1 to 30% c-kit1 cells at dpp3and 60% c-kit1 cells at dpp5, andthat 80% of dpp3-5 c-kit1 gono-cytes presented pseudopods indic-ative of migratory potential (Orthet al., 1997). Subsequently, the c-kit ligand stem cell factor (SCF;Steel) was shown to have noeffect on the colony formationproperties of neonatal mouse gon-ocytes, with c-kit mRNA beingpresent but the receptor non-func-tional (Hasthorpe et al., 1999).Ohta et al. (2000) showed usingSteel deficient recipient mice thatundifferentiated c-kit2 spermato-gonia were formed in the mutantmice, but that these cells couldnot further differentiate. Similarly,transplanting eGFP-labeled donordpp7 spermatogonia in Sl/Sld miceresulted in the proliferation ofc-kit2 undifferentiated spermato-gonia, but in the absence of SCFsignaling, the cells could not fur-ther differentiate. Thus, from thesediverse studies, c-kit emerges as amarker of PGCs and differentiatingspermatogonia, whereas it isabsent in SSCs, but it is expressedin a fraction of the gonocytes.

Another gene that has beenused as a marker of germline stemcells is Thy-1 (CD90), a glycopro-tein of the immunoglobulin familythat was originally found in hema-topoietic stem cells and furtheridentified in ESCs, and bone-mar-row derived multipotent mesen-chymal adult progenitor cells (Lingand Neben, 1997; Izadpanahet al., 2006). Using dpp3 rat gono-cytes and dpp8 to 14 spermatogo-nia, Ryu et al. (2004) showed thatThy-1 was expressed both in neo-natal gonocytes and in a mixedpopulation of spermatogonia.Interestingly, at dpp3, only Thy-11 (low and high expresser) gono-cytes were able to form colonies intransplantation assays, confirmingthe presence within the gonocytepopulation of a group of Thy-11

cells with stem cell potential. Thesituation was different in dpp12 to13 spermatogonia where Thy-12

or low were the cells with colonyforming potential, whereas highThy-1 expresser cells did not gen-erate colonies. These data illus-trate the fact that the germlinestem cells present in the neonatalrat are different from the adultSSCs. The same study used twoother parameters to characterizethe germ cell nature of the stemcell populations present at the twoperiods, Ep-CAM and b3 integrin.The expression of Ep-CAM wasoriginally shown by Andersonet al. (1999) to be restricted tothe germ line during gestation,and to occur only in type A and Bspermatogonia in adult mouse tes-tis. It was further used to isolatespermatogonia from dpp6 andadult mouse testis by immuno-magnetic cell sorting (Van derWee et al., 2001). Expression ofb3 integrin in gonocytes wasinvestigated by Ryu et al. (2004)because this cell adhesion mole-cule had been reported to beexpressed in PGCs together witha5 integrin (Anderson et al.,1999). The study of Ryu et al.(2004) revealed that there wasperfect co-expression betweenThy-1 and Ep-CAM, but that only75% of these cells were b3 integ-rin1 in dpp3 gonocytes. By dpp8to 14 though, only 6% of the Thy-

11, Ep-CAM1 spermatogoniaremained b3 integrin1. Thus, twoconclusions could be deductedfrom this work: (1) the subset ofcells with stem cell signaturewithin the neonatal gonocyte pop-ulation is Thy-11, Ep-CAM1, b3integrin1; (2) these cells differen-tiate in the pup into SSCs that areThy-1low, Ep-CAM1, b3 integrin2,further suggesting that the embry-onic stem cell marker Thy-1 is notretained in juvenile SSCs, andmost probably not in adult SSCseither.Nanog is a stem cell pluripo-

tency marker that has been exam-ined by investigators studyinggerm cell development. Nanog is adivergent homeodomain transcrip-tion factor that was originallyidentified by Chambers et al.(2003), who took inspiration fromthe name of the mythologicalCeltic land to name this geneNanog, because of its role in stemcell self-renewal function. Simulta-neously with the group of Mitsui,these investigators reported thatNanog is expressed in the foundercells of the preimplantation embryo,in post-implentation ESCs, whereit is essential for the maintenanceof pluripotency of the ESCs, forself-renewal and for the preven-tion of ESC differentiation, as wellas in PGCs (Chambers et al.,2003; Mitsui et al., 2003). Nanogwas shown to act in concert withOct4 (Yoshida et al., 1994). More-over, Oct 4 appears to be one ofthe several transcription factorsregulating Nanog expression (Panand Thomson, 2007). Nanogexpression in ESCs was also foundto have an additive effect on ESCself-renewal with leukemia inhibi-tory factor (LIF), a cytokine criticalin the maintenance of the self-renewal potential of ES cells in cul-ture (Yoshida et al., 1994). LIFacts via dimerization of Lif recep-tor and gp130 and the down-stream activation of the transcrip-tion factor Stat3 (Yoshida et al.,1994; Matsuda et al., 1999). How-ever, Lif/Stat3 pathway does notappear to be required for pluripo-tency in vivo, as shown by thefacts that Nanog overexpressionbypasses the requirement of Lif by



ESCs (Chambers et al., 2003;Mitsui et al., 2003); ICM formationoccurs in mutant embryos defi-cient in LIF or downstream signal-ing molecules (Stewart et al.,1992); and LIF is not involved inhuman blastocyst-derived stemcells self-renewal (Reubinoff et al.,2000).Most studies about testicular

Nanog have focused on its expres-sion and function in PGCs and onits potential use as a marker oftesticular germ cell neoplasms inhuman (Rajpert-De Meyts, 2006).Yamaguchi et al. (2005) exploredNanog expression through mousegerm cell development, reportingthat 89% of PGC are Nanog1 atdpc9.5, and that its expressiondecreases as PGCs become resi-dent in the forming testis, with13% of fetal gonocytes beingNanog1 at dpc15.5 and only 1%at dpc16.5, when gonocytesbecome quiescent. Finally, Nanogexpression was not detected inadult mouse testis. However, thisand other earlier studies in micedid not examine the germline de-velopmental stages spanningbetween dpc17 and dpp10, corre-sponding to the transition phasesfrom quiescent to mitotic gono-cytes and from gonocytes toSSCs. By contrast, several studiesexamined NANOG expression inhuman fetal testis (Kerr et al.,2008) in comparison with CIS andTGCT in search of genes thatmight provide indications on theorigins of these pathologies (Hoei-Hansen et al., 2005; Rajpert-DeMeyts, 2006). These studies indi-cated that NANOG is present inhuman PGCs and in fetal gono-cytes until gestation week 20, thelatter period corresponding to astage of mitotic gonocytes. Subse-quently, NANOG expression isretained only in a small group ofgonocytes until 3 months postna-tally, at which time most germcells were found to be quiescent,and it is absent in childhood andadult testis (Hoei-Hansen et al.,2005; Rajpert-De Meyts, 2006).However, a study by Ezeh et al.(2005) reported the detection ofpositive NANOG immunostainingin spermatogonia of a normal tes-

tis section, suggesting thatNANOG expression might beretained in a subset of adult sper-matogonia, probably correspond-ing to the SSCs. The discrepancyin these studies regarding NANOGexpression in spermatogoniamight be due to a difference in theantibodies used or to the limitednumber of samples used in thestudies (1 to 4 samples of normaladult human testis examined).Nevertheless, considering theclose homology in the sequence ofdevelopmental events betweenrodent and human germ cells, thedynamic aspect of gonocyte devel-opment, and the fact that NANOGis expressed in human gonocytesup to week 20 and in a subset ofneonatal germ cells, one cannotexclude that rodent gonocytesmight express Nanog perinatally.Recent studies dedicated to the

generation of mouse multipotentcell lines from SSCs (mGCs/maGSCs) reported expression ofNanog in the newly establishedcell lines, but this was attributedto the cells reversing to ESC phe-notype and no data were providedregarding Nanog levels in the par-ent neonatal gonocytes (dpp0 to3) or adult SSCs before mGCsgeneration (Kanatsu-Shinoharaet al., 2004; Guan et al., 2006;Izadyar et al., 2008). It is inter-esting to note that Nanog expres-sion was recently described in thenuclei of gonocytes (called primi-tive germ cells) from dpp2 to 4neonatal pig testis, where it wasprogressively lost with age (Goelet al., 2008). This study alsoshowed that Nanog1 neonatalgerm cells had the ability to formcolonies and proliferate in trans-plantation assays, suggesting thatin pigs, gonocytes with stem cellpotential express Nanog. Thus,the possibility that Nanog mightbe retained in a subpopulation ofgonocytes in rodent as it is thecase in human remains to beexamined.The Activator protein-2c (AP-2c)

(TFAP2C gene; originally namedAP-2.2) is a transcription factorbelonging to a family of proteinswith a helix-span-helix dimeriza-tion domain at the C terminal that

can either play transcription acti-vating or repressing roles in func-tions of their associated partners(Pellikainen and Kosma, 2007).AP-2c was initially described as anearly retinoic acid-induced gene inthe murine p19 embryonal carci-noma cell line, and shown to becritical in embryogenesis andexpressed in PGCs in the genitalridge (Chazaud et al., 1996). Thestudy of Pauls et al. (2005) furtherextended these observations bysurveying human fetal testes fromgestation week 12 to 37 for AP-2cexpression. This work showed thatAP-2c is expressed in fetal humangonocytes, with the highestexpression levels between w12and 20, including the periods ofmitotic and migratory gonocytes.AP-2c then progressivelydecreased and was absent atweek 36 (newborn). Thesechanges were paralleled by similarchanges in c-kit expression.Because c-kit had previously beenreported to be a target gene ofAP-2c (Yamamoto et al., 1993;Yasuda et al., 1993), the authorsproposed that AP-2c regulates c-kit expression in human fetal gon-ocytes. In a recent study compar-ing germ cell differentiationbetween marmoset and humans,Mitchell et al. (2008) reportedsimilar results as those of Paulset al. (2005, 2006) showing thatAP-2c was co-expressed with Oct4and Nanog in fetal gonocytes andprogressively decreased until nosignal was seen for these markersafter 6 months and 6 weeks of agein human and marmoset, respec-tively. However, this study alsohighlighted differences betweenfetal primate and rat gonocytes,with the former presenting amixed population of cells eitherexpressing exclusively Oct4 or thegerm cell-specific differentiationmarker VASA at fetal week 14,and other germ cells co-express-ing the two markers, while in therat at dpp15.5, the germ cell pop-ulation appeared to be more ho-mogeneous with all fetal gono-cytes co-expressing Oct4 andVASA. Most other recent studiesexamining AP-2c have beenrelated to its use as a tumor germ

8 CULTY


cell marker, supporting the hy-pothesis of the gonocytal origin ofCIS (IGCNUs) and TGCT, similarlyto Nanog (Hoei-Hansen et al.,2004; Pauls et al., 2005;Biermann et al., 2006). Thus, theexpression of AP-2c has been con-firmed in primate gonocytes,encompassing stages including mi-totic, migratory, and quiescentgerm cells, but remains to be con-firmed in rodent gonocytes.Another pluripotent cell marker

that is often studied in concertwith Oct4 and/or Nanog is thetranscription factor, SOX2 that hasbeen shown to be required forESCs formation using Sox2 KOmice. Sox2 has also beendescribed in mouse PGCs (Perrettet al., 2008; Western et al.,2005). However, it appears to bedown regulated in human PGCs,because it was not expressed indifferent stages of human germcell development equivalent tomouse PGCs (Perrett et al., 2008).Thus, Sox2 represents an exampleof a gene expressed differently inrodent and human PGCs. Theseresults do not agree with the pro-posed model of Clark (2007),where PGCs and gonocytes wereassumed to express SOX2, whichmight have been partially basedon rodent studies. Therefore,more studies are needed to clarifySox2 expression in gonocytes.The murine gene Stella (PGC7;

Developmental Pluripotentcy Asso-ciated 3; DPPA3) encodes for atranscription factor that has beenfound in ESCs and PGCs (Saitouet al., 2002; Sato et al., 2002).Interestingly, the genomic posi-tioning of STELLAR, the humanhomolog of Stella, showed that itslocus is centrally positionedbetween NANOG and GDF3, onhuman chromosome 12, similarlyto its position between Nanog andGdf3 on murine chromosome 6(Clark et al., 2004). Indeed,STELLAR is one of several genes,together with NANOG, GDF3,Cyclin D2 (CCND2), and K-Ras,located on human chromosome12p, an area implicated in CIS andTGCT via genomic amplification(Rajpert-De Meyts, 2006). In astudy comparing the expression of

STELLAR, NANOG, GDF3, andOCT4 in normal human testis, twoseminoma samples and undiffer-entiated versus differentiatinghuman ES cell lines, Clark et al.(2004) reported that STELLARmRNA is expressed at much higherlevels in seminoma than in normaltestis, similarly to the other threegenes examined. The authors alsofound that STELLAR mRNA is pres-ent in the germ cell population offetal testis (gestation weeks 20–29), which corresponds to thestage of quiescent prespermatogo-nia. Moreover, the level of STEL-LAR transcripts were similar tothose of OCT4 in human ESCs,with NANOG being the most abun-dant transcript and GDF3 the leastabundant, and these genes wereall down-regulated upon differen-tiation of the cells in culture. Inmice, immunohistochemical analy-sis of normal embryonic and tes-ticular tissues (Sato et al., 2002)and the generation of Stella-GFPtransgenic mice (Payer et al.,2006) clearly showed that Stella isexpressed in mouse PGCs untiltheir relocation in the genitalridge, at which point its expressiondecrease to finally being absent indpp1 neonatal gonocytes andspermatogonia at dpp7 as well asin the adult. Moreover, the gener-ation of Dppa3-deficient mice byBortvin et al. (2004) demon-strated that this gene is notrequired for germ cell specifica-tion, as male Dppa3-deficient micepresented normal spermatogene-sis. By contrast, abnormality inthe development of zygotes issuedfrom female Dppa3-deficient miceindicated a role of this gene as amaternal factor required forproper cleavage between the 4and 8-cell stages. Taken together,these studies indicate that Stella/Stellar protein is expressed inPGCs but not in neonatal gono-cytes or spermatogonia in mice,whereas STELLAR mRNA is pres-ent both in PGCs and late gesta-tion prespermatogonia in human,suggesting the possibility that asmall subset of human fetal gono-cytes might express Stellar.The TGF related ligand, GDF3, is

another ESC pluripotency factor

that is mentioned as a marker ofgerm cells from the PGC to sper-matogonia stages in some reviewarticles. GDF3 acts by inhibitingthe bone morphogenetic protein(BMP) pathway both in human andmouse embryo. Because BMPsplay different roles in human andmouse embryogenesis, GDF3 in-hibitory action on BMPs resulted inthe inhibition of the differentiationof human ESCs, whereas itblocked the in vitro differentiationprocesses of ESCs grown withoutLIF (Levine and Brivanlou, 2006a,b). Studies have also shown thatGdf3 activates Nodal signaling inthe pre-gastrulation mouseembryo (Chen et al., 2006; Ander-sson et al., 2007), and Levineet al. (2008) recently proposed,using the frog embryo model, thatGDF3 corresponds to a bi-func-tional ligand capable of inhibitingBMPs or activating Nodal signalingin function of the conditions. Clarket al. (2004) showed that low lev-els of GDF3 transcripts were pres-ent in normal adult and fetal(week 20–29) human testis, andthat it is down-regulated in humanESCs induced to differentiate inculture. To our knowledge, thereis no available literature on GDF3expression or function in rodentPGCs, gonocytes, or spermatogo-nia. Thus, more studies areneeded to clarify the status ofGDF3 in these cells.

Germline-Specific Markers

Several genes have been identi-fied as germ cell markers, includ-ing DAZL and VASA (Mvh inmouse), two genes interactingwith RNA that are required formale germ cell development. Con-sidering the significant role playedby RNA processing during sperma-togenesis (Eddy, 2002), it is notsurprising that such RNA-interact-ing proteins will play critical rolesin germ cell development. DAZL isan autosomal member of theDeleted in Azoospermia (DAZ)family of RNA binding proteins,which also includes the Y chromo-some genes DAZ, a group of fourgenes expressed exclusively inhuman and primates, and the



autosomal gene BOULE (Xu et al.,2001). Studies of mouse KO mod-els of Dazl and Boule, togetherwith the existence of human infer-tility cases related to DAZ dele-tions, have established the require-ment of Dazl and Boule in germ celldevelopment, (Ruggiu et al., 2000;Saunders et al., 2003), whereasDAZ is involved but not absolutelyrequired for spermatogenesis tooccur because some patientsaffected by DAZ deletions presentoligospermia instead of azoosper-mia (Xu et al., 2001). Dazl hasbeen shown to be expressed fromPGC to spermatocytes stages bothin mouse and human by severalinvestigators. However, there is noconsensus on the expression ofDazl in more mature germ cellstages and on its subcellular local-ization. Indeed, some investigatorsobserved Dazl immunoreactivityfrom PGCs to round spermatidsand a predominant subcellularlocalization changing from nuclearin spermatogonia to cytosolic inmeiotic cells in human and mice(Reijo et al., 2000), whereas othersdescribed a predominantly cyto-solic location of Dazl in rat germcells, contrasting with DAZL signalbeing first nuclear in human fetalgonocytes but cytosolic in more dif-ferentiated human germ cells, andboth cytosolic and nuclear in mar-moset spermatogonia (Ruggiuet al., 1997, 2000). These discrep-ancies might be due to differencesin the antibodies used in thesestudies, and the limited numbers ofsamples examined in the case ofhuman studies. Nevertheless, onecan assume from these studiesthat DAZL is expressed in rodentand human gonocytes, whereasDAZ expression is initiated at laterstages but only in human, andBOULE expression starts at thespermatocyte stage. Because of itsgerm cell-restricted expression,DAZL might represent a usefulmarker in gonocyte studies.Another important germ cell-

specific gene is the ATP-dependentRNA helicase VASA (human)/Mvh(murine homolog) that is exp-ressed in both male and femalegonads. VASA has been shown inhuman to be a cytosolic protein

strongly expressed from the mi-gratory PGC stage to spermatocytestages, whereas it is only presentat low levels in spermatids(Castrillon et al., 2000). Using tar-geted disruption of exons 9 and 10of the Mvh gene, Tanaka et al.(2000) generated Mvh-deficientmale mice that were infertile, whilehomozygous female presentednormal fertility. Analysis of testicu-lar development of homozygousmice revealed that PGCs wereformed in Mvh-deficient fetuses.However, lower proportions of pro-liferative PGCs were observed inthe testes, but no change in apo-ptosis, indicating a role for Mvh ingerm cell proliferation. Spermato-genesis progressed until the lepto-tene spermatocyte stage in olderMvh-deficient mice, and higherlevels of apoptotic germ cells wereseen in deficient mice than normalmice at dpp15 and 35 mice. Thus,Mvh plays a critical role in the tran-sition from leptotene to zygotenestages. Moreover, abnormal extra-tubular germ cells could be foundin homozygous 1-year-old mice.Although VASA might not have afunctional role in gonocytes sinceMvh-deficient mice progressthrough this developmental step tothe next, VASA still represents aninteresting gene to include in thearray of genes that can be used asgonocytes markers.One advantage of using DAZL

and VASA as gonocyte markersthat emerged from these studiesis that both genes appear to behomogeneously expressed at eachgerm cell stage, by opposition ofmost of the stem cell markerslisted above that are expressed atdifferent levels in subsets of germcells within a specific developmen-tal stage.Miwi (mouse Piwi) and Mili (Miwi

like; also called Piwil2) representtwo members of the Piwi family, agroup of basic proteins that inter-act with small RNAs and havebeen implicated in stem cell self-renewal and in germ cell develop-ment (Kuramochi-Miyagawa et al.,2001). Miwi was absent from themale gonad until dpp14 in mice,and it was found in the cytoplasmof spermatocytes and round sper-

matids (Deng and Lin, 2002). Bycontrast, Mili presented a broaderrange of expression, spendingfrom the dpc 12.5 PGCs to adultspermatogonia and spermatocytes(Kuramochi-Miyagawa et al.,2001; Deng and Lin, 2002). Tar-geted mutations of Miwi (Dengand Lin, 2002) and Mili (Kura-mochi-Miyagawa et al., 2004)have been shown to result in ster-ile males, confirming the role ofthese proteins in germ cell devel-opment. Miwi and Mili are believedto maintain transposon silencing ingerm cells by interacting withsmall 29 to 30 nucleotides RNAs(piRNAs) that abound in testis(Girard et al., 2006; Xu et al.,2008). Another intriguing aspectof Mili/Piwil2 is that it has beenshown in an in vitro gain-of-func-tion cell culture model to modulatethe expression of stem cell-relatedgenes. These include severalgenes that are expressed in gono-cytes and/or participate in gono-cyte functions, such as plateletderived growth factor receptor b(PDGFRb), Thy-1, Itga6, andHsp90 (Lee et al., 2006). BecauseMili expression starts at the PGCstage and persists until spermato-cyte stages, one can predict thatgonocytes express this protein.Thus, Mili might be a usefulmarker for gonocytes.In summary, the survey of the

current literature reporting theexpression of stem cell and germcell related genes at various germcell developmental periods includ-ing gonocytes stages has allowed‘‘portrait robot’’ to be drawn of theaverage fetal and neonatal gono-cytes (Table 1). However, thesestudies also illustrate the hetero-geneity of these germline stages,precluding the determination of asingle ‘‘identity card’’ but ratherleading to the establishment ofsubsets of gonocytes presentingsubtle differences in their geneexpression signatures.

WHAT REGULATE

GONOCYTE FUNCTIONS?

Gonocytes are cells in transitionfrom a fetal phenotype presentingaspects common with embryonic

10 CULTY


TABLE 1. Summary of Stem Cell and Germline-Specific Marker Expression in Gonocytes Compared to PGCs

and Spermatogonia

Germ cell stage References

PGC Gonocyte

Mitotic fetal Quiescent Mitotic postnatal

Spermatogonia

dpc13.5-16/18 (m/r)

dpc16/18-dpp0.5/2(m/r)

dpp1-4/dpp3-4(m/r)

Stem cell markersOct41/c-kit1ALCAM2

Oct41/c-kit-a6 Intg1b11

Intg (75% ofa61) EE-2

Ag1; ALCAM1

Oct41/c-kit2ALCAM1

Oct41/c-kit1Oct41/c-kit-

ALCAM2

Ohbo et al., 2003

Oct4/Tra98(98%) Ret1

(55%)

Oct4/Tra98(54%)

Oct4/Tra98(30%) Ret1

(25%)

Ohmura et al.,2004

GFRa11/RET1 GFRa1- RET2 Golden et al., 1999PLZF1 PLZF1 Costoya et al.,

2004RET1/PLZF1/c-

kit1(80%)50% RET1 areKIT1; 10%

PLZF1 are KIT1

Naughton et al.,2006

c-kit2 c-kit1 Tajima et al., 1994c-kit1 (10%) c-kit1 (30%) c-kit1 (60%) Orth et al., 1997

Thy-11/Ep-CAM175% are

b3 Intg 1

Ryu et al.,2004

Nanog1 (89%) Nanog1

(13%)Nanog1 (1%) ? Nanog2 Yamaguchi et al.,

2005Human PGCNANOG1

NANOG1 in human fetal gonocytes until w20 and infew cells until postnatal month 3

Nanog2 Hoei-Hansen et al.,2005; Rajpert-De

Myets, 2006Few Nanog1

goniaEzeh et al., 2005

AP-2c1 Chazaud et al.,1996

AP-2c1 AP-2c1 in human fetal gonocytes w12–20 AP-2c2 Pauls et al., 2005AP-2c1/OCT41/NANOG1 in human fetal gonocytes Mitchell et al., 2008

Sox21 mouse Western et al.,2005; Perrettet al., 2008

Human SOX22 Perrett et al., 2008Stella1 Sato et al., 2002

Saitou et al., 2002STELLA in human fetal germ cell at w20–29 Clark et al., 2004

Stella1 Stella2 Stella2 Payer et al., 2006Low GDF3 in fetal (week 20–29) human testis Low GDF3 Clark et al., 2004

Germline-specific markersDazl1 Dazl1 Dazl1 Dazl1 Dazl1 Reijo et al., 2000

Human VASA1 Human VASA1 Human VASA1 Castrillon et al.,2000; Tanaka et

al., 2000Mvh1 Mvh1 Mvh1 Mvh1 Mvh1

Mili1 Mili1 Mili1 Mili1 Mili1 Kuramochi-Miyagawa et al.,2001; Deng and

Lin, 2002

The data presented refer to either mouse (m) or rat (r) models, except when applying to human as indicated. 1: pres-ent; –: absent; (%): percent of cells expressing the gene in question.



stem cells, yet committed to theunique fate of germline lineage, toan adult undifferentiated pheno-type that includes a subset ofstem cells. Although this processtakes several months in human, ithappens in less than 2 weeks inrodent, where a cell can adopt 2very different phenotypes in amatter of a day. How this progres-sion occurs is still a mystery. Forexample, once a gene is turned onat a specific time of development,does the product it encodes needto reach a certain threshold beforeits effects become visible, and ifso, is there simultaneously in thetestis several subsets of germ cellsexpressing different levels of thesame gene product, potentiallycorresponding to different pheno-types and behaviors? In otherwords, does the progression fromone stage to the other occur grad-ually or as a succession of plus orminus events? Studies such asthat of Ryu et al. (2004), whoidentified groups of gonocytes andspermatogonia with low to highlevels of Thy-1 that could berelated to the ability of the cells torepopulate a recipient testis, sug-gest that changes might occurprogressively. Moreover, germcells do not appear to besynchronized in their changes,because investigators have shownthat, at a given time, one can findsubsets of cells positive and nega-tive for a specific gene product, asdescribed in the previous section.The same observation is also truefor the study of gonocyte func-tions, where not all cells appear tobehave in the same way at thesame time. Since the early nine-ties, several investigators havestudied the regulation of gonocytefunctions, including proliferation,survival, apoptosis, differentiation,and migration toward the base-ment membrane, using the mouseand rat models. A review by Olasoand Habert (2000) presented athorough description of variousmutations and gene KO models,as well as several culture modelsused to study both PGCs and gon-ocytes and a comprehensive list ofthe factors identified up to thattime as regulating PGCs and gono-

cytes functions. Thus, the presentreview will only mention brieflysome of the factors described indetail by Olaso and Habert, focus-ing more on subsequently pub-lished findings regarding hor-mones, growth factors, cytokines,and signaling molecules expressedor acting on gonocyte functions.

Factors Regulating GonocyteProliferation

Gonocyte proliferation occurs intwo phases in rodents, a fetalphase that takes place when thegerm cell becomes resident in theforming testis (between dpc13.5and 16 in the mouse and dpc13–18 in the rat), and a postnatalphase taking place between dpp1and 4 in the mouse and dpp3 to 4in the rat, separated by severaldays of quiescence. In human, theavailable literature suggest thatthere is only one mitotic phaseoccurring between the third andsixth months of gestation, fol-lowed by a quiescent phasestretching until 2 to 3 months af-ter birth. Only a few studies haveexamined simultaneously theresponses to stimuli of fetal andneonatal gonocytes or comparedtheir gene profiles. From those,two studies by the team of Habert(Boulogne et al., 1999b; Liveraet al., 2000) highlighted differen-ces in the response of dpc14.5 anddpp3 rat gonocytes to RA,whereas recent work from Ohmuraet al. (2008) reported differencesin expression levels of the nucleo-lar GTP binding protein nucleoste-min between dpc14.5 and dpp0.5mouse gonocytes, emphasizingphenotypic differences betweenthe two stages. Thus, it is likelythat proliferation in fetal and neo-natal gonocytes is differently regu-lated and studies reporting dataon these two developmentalphases are presented separatelybelow.

Fetal gonocytes

In contrast to PGCs where theregulation of proliferation hasbeen well studied in the nineties,leading to the identification of LIF

as a major regulator of PGC prolif-eration and survival and the find-ing that SCF is involved in PGCsurvival (see review of Olaso andHabert, 2000 for details), very fewstudies have examined the regula-tion of fetal gonocyte functions.Thus, information on this topic isstill very sparse. A molecule thathas been shown to have someeffect on fetal gonocyte prolifera-tion is RA, the bioactive metaboliteof Vitamin A. RA is the ligand ofthe nuclear receptors RA receptors(RAR a, b, and c) and retinoidreceptors (RXR a, b, and c). RA,which is a critical factor in embry-onic development and organogen-esis (Duester, 2008), was recentlyshown to be instrumental in deter-mining fetal germ cell fate (Bowleset al., 2006). Vitamin A has beenknown for years to be required formale fertility. Indeed, the testes ofVitamin A-deficient rodents pres-ent altered spermatogenesis witheither arrest at the type A sperma-togonia stage in mice (van Peltand de Rooij, 1990; Gaemerset al., 1996) or preleptotene stagein rats (van Pelt, 1991; van Peltet al., 1995). Moreover, RA wasfound to induce the proliferation ofgrowth-arrested A spermatogoniain Vitamin A deficient mice(Gaemers et al., 1996), suggest-ing that RA might be involved inphysiological germ cell differentia-tion processes. A study from thelaboratory of Habert (Livera et al.,2000) reported that RA exerts adual effect on dpc14.5 rat gono-cytes in organ cultures by inducinga slight increase in proliferation,simultaneously to a broad increasein apoptosis, resulting in an overalldecrease in fetal gonocyte num-ber. However, in dpc16.5 rat gon-ocytes co-cultured with Sertolicells, RA induced a decrease in thenumber of mitotic gonocytes after6 days of treatment, as measuredby BrdU incorporation (Boulogneet al., 2003). Similarly, the phor-bol ester PMA, activator of proteinkinase C, decreased fetal gonocytemitosis, suggesting an anti-prolif-erative role of PKC in fetal gono-cyte proliferation. More recently,Trautmann et al. (2008) reportedthat RA reduced or prevented the

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time-dependent decrease indpc11.5, 12.5, and 13.5 gonocytemitosis in organ cultures, but notthat of dpc14.5 gonocytes whichwere entering mitotic arrest. Inthis system, RA induced a largeincrease in the number of BrdU-positive dpc12.5 and 13.5 gono-cytes that was dependent on PI3-Kinase activity. Moreover, RA pre-vented the mitotic arrest of fetalgonocytes, thus impeding theirdifferentiation, while also inducinggonocyte apoptosis. The authorsproposed that the dual action ofRA in stimulating proliferation andapoptosis of fetal gonocytes mightcorrespond to a physiological proc-ess that would allow for the elimi-nation of improperly differentiatinggerm cells. Taken together, thesestudies suggest that RA does exerta positive effect on fetal gonocyteproliferation that is age-depend-ent, both in rat and mouse,although it simultaneously inducesapoptosis.

Neonatal gonocyte

From the few studies that havequestioned the nature of the sig-nal(s) regulating postnatal gono-cyte proliferation, several factorshave emerged as important,including platelet derived growthfactor (PDGF)-BB, 17b-estradiol(E2), LIF, and RA. In search of thefactor(s) regulating neonatal ratgonocyte proliferation, we foundthat both PDGF-BB and E2 stimu-lated dpp3 rat gonocyte prolifera-tion in vitro, in a dose-dependentand non-additive manner (Liet al., 1997). Moreover, E2 effectwas abolished by an estrogen re-ceptor (ER) antagonist, indicatingthat the effect required E2 bindingon its receptor. By contrast, nei-ther NGF nor EGF had an effect ongonocyte proliferation, whereasbasic FGF (FGF-2) induced a smallbut not significant increase. Inthese studies, proliferation wasmeasured by BrdU incorporation inisolated gonocytes maintained inculture for 1 day in the presenceof a low level (2.5%) of fetal bo-vine serum, found to maintaingood cell viability over a period of3 days, while inducing only low

basal levels of proliferation.Although this model system wasdevoid of the natural somatic cell-germ cell interactions, thisapproach allowed us to demon-strate a direct effect of PDGF andE2 on gonocytes proliferation.Both PDGF and E2 have beenshown to be produced by Sertolicells, and thus, keeping these cellsin culture with the gonocyteswould have probably prevented usfrom observing the effects of ex-ogenously added compounds,since the same molecules wereproduced locally in the tissue. Wesubsequently reported that gono-cytes express both full lengthPDGFR a and b (Thuillier et al.,2003), as well as a variant trun-cated PDGFRb, V1-PDGFRb, that ispreferentially expressed in devel-oping gonads (Wang and Culty,2007). Because V1-PDGFRb islacking the N-terminal domaincontaining PDGF binding site, itcannot be involved directly inPDGF regulation of proliferation.Moreover, we showed that dpp3gonocytes express ERb and theER-associated proteins, Hsp90,p23, and Cyp40 (Wang et al.,2004). Using inhibitors of PDGFRpathway and ER antagonists, wefound that PDGF and E2 are bothrequired simultaneously to acti-vate dpp3 gonocyte proliferation(Thuillier et al., 2004). Althoughthese studies were performed inan artificial environment for gono-cytes, they nevertheless show thatgonocytes proliferation is activatedby a crosstalk mechanism betweenPDGF and E2 and the activation oftheir respective receptors, sug-gesting that an interactionbetween ER and PDGFR pathwaysmight regulate in vivo rat gono-cytes proliferation.Although our studies did not

indicate whether PDGF-BB wasexerting its effect by binding tothe PDGFRb homodimers orPDGFRa/b heterodimers, a recentstudy by Basciani et al. (2008)reported that PDGFRb is mediatingin vivo neonatal gonocyte prolifer-ation in mice. These investigatorstook advantage of the predomi-nance of PDGFRs over c-kit in gon-ocytes during the first 5 postnatal

days to inhibit PDGFRs by treatingmice with imatinib (gleevex), aninhibitor of both PDGFRs and c-kittyrosine kinase activities. Theyexamined the expression ofPDGFRs and c-kit in testes fromdpp3 mice, as well as the effect ofimatinib treatment on the expres-sion and phosphorylation of theseproteins and on gonocytes prolif-eration and apoptosis. This studyshowed that dpp3 mouse gono-cytes expressed PDGFRb and V1-PDGFRb, but no detectable levelsof PDGFRa or c-kit. These resultsdiffer to some extent from thoseobtained by other investigators onmouse gonocytes where a subsetof cells was found to express c-kit(Naughton et al., 2006), and withour data on rat gonocytes whereboth PDGFRa and b are expressed,although not homogeneously in allcells (Thuillier et al., 2003). How-ever, these discrepancies mayreflect differences in the antibod-ies and sensitivities of the meth-ods used, rather than a totalabsence of PDGFRa and c-kit in alldpp3 gonocytes. The authorsreported that imatinib treatmentresulted in a reduced proportion ofproliferating gonocytes at dpp2and 3, together with the inhibitionof PDGFRb and V1-PDGFRb phos-phorylation in gonocytes at dpp3,suggesting a link between theactivation of PDGFRbs and in vivogonocyte proliferation.The positive effect of E2 on dpp3

gonocyte numbers was also shownin vivo by the study of Vigueras-Villasenor et al. (2006) describingthat daily subcutaneous injectionsof E2 in rat pups starting at dpp1resulted in a two-fold higher num-ber of gonocytes at dpp3 in E2-treated than in control pups.Quantification of the number ofproliferating gonocytes by immu-noreactivity with an anti-phospho-histone H3 showed a small but notsignificant increase in positivegonocytes in E2-treated rats,while apoptosis levels were notchanged by E2 treatment at thisage. However, continuation of E2treatment until dpp8 to 16 had theopposite effect, resulting in asignificant decrease in proliferat-ing spermatogonia as well as



an increase in spermatogonialapoptosis.Another indication of the com-

plex effect of E2 in germ cell pro-liferation comes from the studiesof Habert et al. (Delbes et al.,2004). Using a mice model carry-ing an ERb inactivation due toexon 3 disruption, these authorsshowed that the absence of ERbcorresponded to a 2-fold decreasein apoptotic gonocytes and a smallincrease in the number of BrdU-la-beled gonocytes at dpp2, as com-pared to wild-type mice. Theseresults suggested that estrogenexerts a dual anti-mitotic and pro-apoptotic effect of neonatal mousegonocytes. However, a recentstudy by the same authors usingdpp3 rat testes organ cultures(Delbes et al., 2007) did not findany decrease in gonocyte numbersin the presence of E2 and foundno change in the numbers of BrdUlabeled gonocytes. It should benoticed that these studies wereperformed on organotypic culturesof testes in which the aromatase-expressing Sertoli cells areexpected to locally produce E2,which could potentially mask aneffect of exogenously added E2.The fact that E2 did not exert del-eterious or positive effects on neo-natal rat gonocytes in organ cul-tures, whereas disruption of its re-ceptor ERb had a protective effecton gonocytes in neonatal micesuggests that the effects of estro-gen on germ cells might differbetween species and in function ofthe developmental stage of thecells exposed to estrogens. Fur-thermore, as will be discussedlater, E2 has also been found toexert pro-apoptotic effects in fetalgerm cells, whereas it has beenshown to act as a survival factorfor adult human germ cells (Penti-kainen et al., 2000). Similarly, soyisoflavones, among which the phy-toestrogen genistein is a majorcomponent, were shown to par-tially rescue spermatogenesis inaromatase KO mice (Robertsonet al., 2002). Lastly, Wahlgrenet al. (2008) recently reportedthat spermatogonial DNA synthe-sis was stimulated by the ERbselective ligand 5a-androstane-

3b,17b-diol (3bAdiol) in rat semi-niferous epithelium, further illus-trating the stage-dependent differ-ential effects of ER ligands in germcells. Taken together, these stud-ies and our results suggest thatestrogen has different and some-times opposite effects on germ cellproliferation in function of thegerm cell stage at the time of ex-posure and that, at least in neona-tal rat gonocytes, the effect ofestrogen on proliferation involvesinteraction with another signalingpathway.Concerning the role of LIF in

neonatal gonocytes, it was shownby De Miguel (1996) to increasethe proportion of proliferatingdpp1 rat gonocytes after 3 days inco-culture with Sertoli cells,whereas it had no significant effecton the proportion of proliferatingdpp3 gonocytes after 3 or 6 daysin culture. Considering that ratdpp1 gonocytes are quiescent andthat the observed changes wereseen after 3 days in culture withSertoli cells, it is possible that theeffects of Lif were indirect via aprimary effect on Sertoli cells, orthat the role of Lif was to allowgonocytes to exit the Go/G1 phaseand become responsive to factorsproduced by Sertoli cells duringthe 3 days in culture. This possibil-ity is supported by the fact thatLIF did not change the proportionof proliferating gonocytes whenadded to co-cultures from dpp3rat testes, at an age when gono-cytes are responsive to proliferat-ing signals produced by Sertolicells. Alternatively, the lack ofeffect of LIF in dpp3 gonocytesmight be due to the fact that max-imal proliferation was alreadyachieved in response to factorsproduced by the Sertoli cells,including PDGFs B and D (Love-land et al., 1995; Basciani et al.,2008), E2 (Papadopoulos et al.,1986), and LIF (De Miguel, 1996).Thymulin was reported in two

studies by Prepin et al. (Prepinet al., 1994; Prepin and Le Vigour-oux, 1997) to stimulate [3H]thy-midine incorporation in gonocytesfrom dpc13.5 rat fetuses and indpp2 postnatal rat gonocytes, butnot at other early postnatal ages.

The observation that the numberof mitotic gonocytes remainedlow, despite the increase in[3H]thymidine labeling, led theauthors to propose that thymulinmay affect DNA duplication, ratherthan the whole mitotic processleading to the formation of daugh-ter cells. This would fit with thekinetics of events whereby themajority of rat gonocytes at dpp2are still non-mitotic, althoughsome cells might have re-enteredthe cell cycle and initiated DNAduplication before mitosis can beobserved at dpp3.Similar to its role on early fetal

gonocyte proliferation, RA hasbeen shown to exert a small butsignificant positive effect on dpp3rat gonocyte proliferation in organculture (Livera et al., 2000). Simi-larly, Zhou et al. (2008a) recentlyreported that RA induced a signifi-cant increase in the numbers ofBrdU-positive mouse dpp2 gono-cytes after one day in organ cul-ture, simultaneously to the induc-tion of gonocyte differentiation. RAwas also found to increase dpp3gonocyte proliferation after 3 daysof treatment in gonocyte-Sertolicell co-cultures (Boulogne et al.,2003). Moreover, RA was reportedto induce a small but significantincrease in the proportion of Ki67-labeled human fetal gonocytesfrom 6 to 10-week old fetuses af-ter 4 days in organ culture, indi-cating an increase in gonocyteproliferation, simultaneously withan increase in apoptotic germ cells(Lambrot et al., 2006). All to-gether, these data indicate thatRA is capable of stimulating neo-natal gonocyte proliferation,although it is not its only functionin germ cells.Finally, a recent study by

Braydich-Stolle et al. (2007) usingGFRa11 germ cells isolated fromdpp 4 to 5 mice reported a prolif-erative effect of GDNF on thesecells, identifying Src kinase andPI3K/Akt activation as part of thedownstream cascade of GDNF inthis process. Although the authorsreferred to the cells as spermato-gonia, the fact that these experi-ments were performed on germcells isolated from pooled dpp4

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and dpp5 mouse testes, andknowing that postnatal gonocytesexpress GFRa11, one cannotexclude that the results observedreflect the combined effects ofGDNF on gonocytes and nascentspermatogonia.

Factors Regulating GonocyteDifferentiation

Differentiation was, untilrecently, the least studied ofgonocyte functions. However, therecent understanding of SSC pluri-potency and their potential fortherapeutic use generated arenewed interest in studying theprocesses regulating SSC forma-tion. The main challenge faced inthis endeavor was to get first aclear profile of the starting andending stages, to identify genes/proteins that would be uniquelyexpressed in either gonocyte orundifferentiated spermatogonia.Indeed, until recently, the onlyclear differences between a neo-natal gonocyte and a spermatogo-nium at transitional days, such asdpp4 to 5 where both cell typescould co-exist, were their morpho-logical appearances and their dif-ferent location within the seminif-erous cord, gonocytes being largespherical cells at the center of thecords, while spermatogonia aresmaller half-moon shaped cellspositioned along the basementmembrane. Indeed, location byitself is not a sufficient criterion,because germ cells presenting thephenotype of gonocytes can beobserved adjacent to the base-ment membrane during the transi-tional days between gonocyte andspermatogonia stages. Then, whatdifferentiates a proliferating neo-natal gonocyte from a proliferatingundifferentiated type A spermato-gonia? As illustrated in the previ-ous section depicting proteinsexpressed in gonocytes, many ofthem are also present in sperma-togonia, and more specificallyin SSCs. However, recent advan-ces in the characterization ofSSCs have uncovered new genemarkers, allowing for revisitationof the question of gonocyte iden-tity and to identify several genes

that might help discriminatingbetween the two stages.One of these genes is Stimulated

by retinoic acid gene 8 (Stra8),originally identified by the team ofChambon (Bouillet et al., 1995)among a series of genes activatedby all-trans RA in pluripotentmouse P19 embryonal carcinomacells. These authors subsequentlycloned Stra8 in P19 cells andshowed that all-trans RA alsoinduced its expression in mouseF9 embryonal carcinoma cells andthe 129/Sv mouse blastocyte-derived D3 ES cell line, whereas inP19 cells both all-trans RA and 9-cis RA induced Stra8 expression(Oulad-Abdelghani et al., 1996).Stra8 was found to be a glutamicacid-rich cytosolic protein differen-tially phosphorylated by all-transRA and 9-cis RA. The study ofStra8 expression in adult mouserevealed that Stra8 was present inadult premeiotic germ cells. Usinga Stra8-luciferase construct, Giuiliet al. (2002) confirmed that Stra8is expressed in spermatogonia andpremeiotic cells, but not in post-meiotic or Sertoli cells. A 400-bpfragment of the Stra8 promoterwas then used to drive the expres-sion of CD4, utilized as a surfaceprotein tag, in transgenic mice,allowing for the enrichment of asubset of cells expressing stemcell markers and capable of repo-pulating germ cell-depleted testes,demonstrating that Stra8 is alsoexpressed in SSCs. Griswold et al.(Zhou et al., 2008b) recently per-formed a detailed analysis of thein vivo expression of Stra8 proteinin postnatal mouse testis, thatshowed a progressive increase ofStra8 in the germ cells of the firstspermatogonial wave, startingwith a few positive spermatogoniaat dpp5, followed by an increasingnumber of positive germ cells overtime, identifyied as preleptotenesand early leptotene spermato-cytes. In adult testis, Stra8expression was also observedfrom spermatogonia to early lep-totenes, whereas it was absent inmore differentiated germ cells.Other studies showed that RA-induced Stra8 acts as a meiosisinitiator in fetal ovary and in post-

natal testis (Menke et al., 2003;Koubova et al., 2006), whereas itsexpression is repressed in fetaltestis via localized degradation ofRA by cytochrome p450 26b1(cyp26b1) (Bowles and Koopman,2007).Using Stra8 as an indicator of

gonocyte to spermatogonia transi-tion, several studies, includingours, recently identified RA as akey regulator of gonocyte differen-tiation in rat (Wang and Culty,2007) and in mouse (Zhou et al.,2008a). Addition of RA onto iso-lated rat dpp3 gonocyte culturesfor 1 day induced a large increasein Stra8 transcripts (Wang andCulty, 2007). Simultaneously,there was an increase in themRNA expression of c-kit, a genepreferentially expressed in differ-entiating type A spermatogonia,and a decrease in GFRa1 tran-script, a gene expressed mainly inthe SSC sub-population, whereasthe levels of Intg a6 mRNAremained constant. These resultssuggested that RA induced in vitrogonocyte differentiation, resultingin a reduction in the proportion ofSSCs that was paralleled by anincrease in the proportion of moreadvanced type A spermatogonia.Despite the reports by severalinvestigators that Intg a6 isretained only in a subset of sper-matogonia corresponding to theSSC in dpp7 to 10 mice (Ohboet al., 2003; Buageaw et al.,2005; Ohmura et al., 2008), Intga6 transcripts did not decreaseupon RA treatment of isolatedgonocytes (Wang and Culty,2007). The discrepancy betweenthe lack of effect of RA on Intg a6expression and its induction of alarge decrease in GFRa1 expres-sion in the total gonocyte popula-tion suggests that this in vitromodel recapitulates only a portionof the differentiation process ofgonocyte in the absence of sup-port cells, probably due in part tothe lack of basement membranethat would have provided anchorssuch as laminin for the integrinsexpressed at the surface of thegonocytes. In these experiments,rat gonocyte treatment with RAwas also found to increase the



mRNA expression of V1-PDGFRb,suggesting a potential role of thetruncated form of PDGFRb in gon-ocyte differentiation (Wang andCulty, 2007).Using isolated Thy1-positive

mouse dpp2 gonocytes and dpp2testis organ cultures, Zhou et al.(2008a) showed that RA treat-ment induced similar increases inStra8 transcripts to thoseobserved in the rat (Zhou et al.,2008a). Moreover, retinol and 4-oxo-RA were found to have similarbut smaller effects on Stra8expression than RA, and c-kittranscripts were also found toincrease simultaneously to Stra8.In addition, this study confirmedthat Stra8 expression wasincreased by RA treatment notonly at the mRNA but also at theprotein level, changing from aweak to a strong signal in 24hours. Taken together, these stud-ies suggest that RA induces gono-cyte differentiation both in rat andmouse gonocytes, and that theup-regulation of Stra8 and c-kitexpression represent two keysteps in the progression from gon-ocyte to spermatogonia. Thus, it istempting to extrapolate to humanand postulate that the samemechanisms might be at play inhuman gonocyte differentiation.

Factors Regulating GonocyteMigration to the Periphery of theSeminiferous Cord

Several studies have questionedthe identity of the factor(s) con-trolling gonocyte relocation to thebasement membrane, because themigration of gonocytes from thecenter of the seminiferous cords tothe basement membrane appearsnecessary for their differentiation,while germ cells remaining at thecenter of the cords after dpp5 areeventually targeted to apoptosis(Tres and Kierszenbaum, 2005).Orth and et al. (1997) proposed c-kit as a mediator of neonatal gon-ocyte migration in view of datausing rat gonocyte-Sertoli cell co-cultures. Their studies showedthat the majority of gonocytespresenting pseudopods were c-kit1, although this represented

only a small fraction of the gono-cytes. Furthermore, blocking c-kitwith an antibody decreased theproportion of gonocytes with pseu-dopods, while activating c-kit byaddition of its ligand in the me-dium had the opposite effect onpseudopod formation. Moreover,the comparison of c-kit expressionwithin various studies showed thatc-kit presents a pattern of expres-sion correlated with the migratorybehavior of the cells, being absentin non-migratory quiescent gono-cytes, becoming expressed in sub-sets of gonocytes at the timewhen gonocytes resume mitosisand migration, and then down-regulated in SSCs, which reside atthe basement membrane of thecords and do not migrate. Thus, itis possible that c-kit is transientlyexpressed in subsets of gonocytesto allow their migration and that itis subsequently down-regulated inSSCs. Its subsequent re-expres-sion in type Apair to differentiated-spermatogonia might not berelated to cell movement butrather to cell survival.A group of proteins that have

been proposed as potentiallymediating gonocyte migration arethe ADAM-Integrin-Tetraspanincomplexes, previously identified inpostnatal germ cells (presperma-togonia/gonocytes) (Tres andKierszenbaum, 2005). These com-plexes comprise ADAM (A Disinte-grin and A Metalloprotease do-main) 1 and 2 proteins, the integ-rins a3b1 and a6b1, and thetrespanins (transmembrane-4superfamily proteins), CD9 andCD81. ADAM 1 and ADAM 2 pro-teins (also called fertilin a and b)are multifunctional proteins con-taining a metalloprotease domainand a disintegrin domain thatbinds integrins. ADAM 1 andADAM 2 were originally found atthe surface of sperm and believedto play a role in sperm-egg inter-action (Chen and Sampson,1999), although the generation ofADAM2 and b1 KO mice subse-quently showed that these pro-teins were not required for gametefusion (Cho et al., 2000; He et al.,2003). ADAM 1 and ADAM 2 arenot expressed in PGCs and appear

at dpp1 (ADAM1) and dpp2(ADAM2) mouse gonocytes(Rosselot, 2003). Integrins a3b1and a6b1 bind laminin, a majorcomponent of the seminiferoustubule basement membrane, andthey play a critical role in Sertoli-germ cell interactions as well asgerm cell survival (Siu and Cheng,2004a, b). CD9 is a transmem-brane protein binding a3b1 anda6b1 integrins, which is expressedin mouse and rat SSCs (Kanatsu-Shinohara et al., 2004), as well asin human testis at a location cor-responding to the spermatogonia(Nakamura et al., 2001). Becauseof the expression of these proteinsin gonocytes at the time of theirmigration from the center to theperiphery of the seminiferouscords, and because of their knowninteractions, Tres and Kierszen-baum (2005) proposed thatADAM-Integrin-Tetraspanin inter-actions are mediating the reloca-tion of the gonocytes.More recently, Basciani et al.

(2008) proposed that PDGFRbs,including a V1-PDGFRb truncatedform that lacks a PDGF binding do-main but has retained tyrosine ki-nase activity (Wang and Culty,2007), play a role in the regulationof gonocyte migration. We havepreviously reported that dpp3 ratgonocytes express several formsof PDGFRa and b, which appear tobe down-regulated by dpp6 to 7(Li et al., 1997; Thuillier et al.,2003). Interestingly, PDGFRb andc-kit belong to the type III familyof receptor kinases, share thesame signaling cascades and existboth as truncated forms and fulllength receptors in testicular germcells at specific stages (Rossiet al., 1992; Sette et al., 2000;Thuillier et al., 2003; Wang andCulty, 2007). The study of Bas-ciani et al. reported that in vivotreatment of neonatal mice withthe PDGFR and c-kit inhibitor,imatinib, disrupted gonocytemigration to the basement mem-brane and led to an increasednumber of centrally located apo-ptotic gonocytes. Moreover, PDGF-B and D, two ligands of full lengthPDGFRb produced by Sertoli cellsduring the same developmental

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period, exerted chemotacticeffects on gonocytes in vitro.Interestingly, the authorsobserved that a subset of the gon-ocytes migrated to the basementmembrane regardless of the treat-ment, but these cells did not cor-respond to the gonocytes that givedirectly rise to the first spermato-genic wave without passing by theSSC stage described by Yoshidaet al. (2006), since the first roundof spermatogenesis was missing inimatinib-treated mice. Thus, it istempting to think that these gono-cytes represent a group of cellsthat depend on other moleculessuch as the ADAM-Integrin-Tetra-spanin complexes for their migra-tion. Furthermore, the work ofBasciani et al. (2008) showed thatthe numbers of spermatozoa weregreatly reduced at dpp35 and 70in imatinib-treated mice, but thatthe numbers of spermatozoareturned to normal levels bydpp90 and dpp120. These datasuggest that the few gonocytesunaffected by imatinib and inde-pendent of PDGFRs and c-kit fortheir migration toward the base-ment membrane were capable ofproliferating and differentiatingsufficiently to compensate for theearly loss of germ cells and torecover normal spermatogenesiswith time. Thus, it would be inter-esting to examine whether thisspecific pool of gonocytesexpresses Ngn3 or not, which wasdescribed by Yoshida et al. (2004)as a marker of SSCs. These gono-cytes could correspond to thesmall pool of gonocytes that willdifferentiate into SSCs. Taken to-gether, these studies support theidea that at least two populationsof neonatal gonocytes co-exist,including a small fraction of gono-cytes destined to become SSCsthat will populate the SSC niche(Shinohara et al., 2002; Yoshidaet al., 2007; Seandel et al.,2008), and a group of gonocytesthat will progress directly to theApair stage, committed to differen-tiation. Since it takes 38.7 daysfor a mouse germ cell to progressfrom type A1 spermatogonia tospermatozoa (Franca et al., 2005)and in imatinib-treated (dpp1–

dpp5) mice germ cells at all stageswere still depleted at dpp70, theobservation of Basciani et al.(2008) that animals recoverednormal levels of spermatozoa atdpp90 suggests that the gono-cytes altered by imatinib treat-ment were supporting more thanthe first spermatogenic wave.Alternatively, it is possible that thedelay in recovery reflects the factthat the few remaining imatinib-resistant gonocytes could notprogress through differentiationuntil specific conditions werereached in their environment,such as a somatic feedbackresponse to germ cell depletionthat would induce the replenish-ment of the SSC niche beforefurther differentiation.

Factors Regulating GonocyteSurvival

Two major factors have beenshown to regulate gonocyte sur-vival, LIF and Fibroblast growthfactor-2 (FGF-2/basic FGF). VanDissell Emiliani et al. (1996)reported in 1996 that FGF-2induced a greater than 50%increase in the number of dpp1and 3 gonocytes after 6 days inco-culture with Sertoli cells, butnot after 3 days in culture, whilethe number of proliferating gono-cytes as assessed by [3H]thymi-dine incorporation, increased byapproximately 50% after 3 days inculture, but only by 22% after 6days. Because of the delay effectsobserved in gonocytes, theseauthors suggested that the FGF-2effect on gonocytes was indirect,via a primary effect on Sertoli cellsor on myoid cells present in theco-cultures. Moreover, the differ-ence between the amplitudes ofthe increase in germ cell numbersand the proportion of mitotic cellsfurther suggests that FGF-2 mightplay more of a role in gonocytesurvival than proliferation. Usingthe same co-culture system, DeMiguel et al. (1996) reported thatboth LIF and Ciliary NeurotrophicFactor (CNTF) acted as survivalfactors on dpp3 rat gonocytesover a period of 6 days in culture,and that LIF, but not CNTF,

increased the proportion of prolif-erative dpp1 gonocytes after 3days in culture, whereas it had nosignificant effect on the proportionof proliferative dpp3 gonocytes af-ter 3 or 6 days in culture. Thesedata suggest that the main func-tion of LIF in dpp3 gonocytes is tomaintain their survival, while itmight play an additional role atdpp1 in the re-entry of quiescentgonocytes into the mitotic cycle.Because LIF has been shown invitro but not in vivo, to maintainESCs in a stemness phenotype, itwould be interesting to examinewhether treating gonocytes withLIF will block or delay their differ-entiation beyond the SSC stage,while sustaining their survival.Because GDNF receptors are

present in postnatal mouse gono-cytes (Naughton et al., 2006) anddpp3 rat gonocytes (our unpub-lished data), and GDNF is criticalfor SSC survival and proliferation(Meng et al., 2000; Nagano et al.,2003; Hofmann, 2008), GDNF isexpected to act on gonocyte sur-vival. However, to the best of ourknowledge, no study has beenpublished yet on the effect ofGDNF on gonocytes. GDNF wasshown by Hu et al. (1999) to stim-ulate the proliferation of rat dpp6Sertoli cells after 3 days in organcultures. In this study, the authorsalso reported that the BrdU indexof the germ cells (referred to as‘‘gonocytes’’) in the testicularfragments were increased by acombination of FSH and GDNF,while FSH alone had no effect.However, they did not report thepotential effects of GDNF alone.Moreover, dpp6 rat germ cells rep-resent spermatogonia and notgonocytes, and thus, this studywas in fact describing the effectsof GDNF on spermatogonia, at anage when the testis contains ahigher percentage of SSCs than inlater ages when the differentiatingtype of spermatogonia appear.Thus, the results of this study can-not be extrapolated to gonocytes.Dazl is another factor that has

been shown to be required for qui-escent gonocyte survival byexperiments using Dazl KO mice inwhich germ cells survived until



dpc19 only (Ruggiu et al., 1997).In contrast with Dazl that repre-sents an endogenous regulatoryfactor for survival, the thyroid hor-mone triiodothyronine (T3) wasreported by Jannini (1993) to indi-rectly increase the number ofdpp3 rat gonocytes and decreasetheir apoptosis via a primaryaction on Sertoli cells, both in vivoand in organ culture, while T3 hadno effect in adult testis. Janninifurther identified the thyroid hor-mone receptor a in immature Ser-toli cells, showing that the recep-tor was strongly expressed duringthe perinatal period until dpp5,greatly decreasing at dpp15, anddisappearing in the adult (Jannini,1994). Although they did not iden-tify the Sertoli cell factor secretedin response to T3 that acted ongonocytes, they reported in a sub-sequent study that T3 inhibitedboth basal and FSH-stimulatedaromatase activity in prepubertalSertoli cells (Ulisse et al., 1994),suggesting a potential linkbetween the effects of T3 on theability of Sertoli cells to produceestrogen and gonocyte survival.However, this possibility was notexamined further. Moreover, areview reporting human thyroidpathologies with regard to malereproduction, together with stud-ies exploring T3 and T4 effects onanimal reproduction, suggestedthat thyroid hormones act on so-matic cells, but did not identify themechanisms by which the somaticeffects translated into changes ingerm cells (Krassas and Ponti-kides, 2004). Another potentialcandidate in the regulation of gon-ocyte survival is V1-PDGFRb, atruncated variant of PDGFRbexpressed in dpp3 gonocytes,recently reported to increase thesurvival of the embryonal terato-carcinoma F9 cell line (Wang andCulty, 2007). F9 cells represent aclassical cell model to study em-bryonic cell differentiation (Strick-land et al., 1980). We found sev-eral analogies between F9 cell sig-naling pathways and those ofgonocytes, including the expres-sion of V1-PDGFRb and full lengthPDGFRa (Vu et al., 1989; Wangand Culty, 2007), RA receptors

(Martin et al., 1990; Clifford et al.,1996) and ER, although theyexpress ERa and not ERb as ingonocytes (Culty M. unpublisheddata), PDGF-induced proliferation,and RA-regulated differentiation(Strickland and Mahdavi, 1978;Wang and Culty, 2007). Thus, it ispossible that V1-PDGFRb wouldexert a similar role in gonocytesurvival as it does in F9 cells, butthis hypothesis remains to betested.Gonocyte survival appears to

depend not only on paracrine andautocrine factors, but also on theintratubular position of the cells atspecific developmental periods.Indeed, the failure of gonocytes tomigrate toward the basementmembrane in a timely manner hasbeen associated with the inductionof their apoptosis (Sinha Hikim andSwerdloff, 1999; Tres and Kiers-zenbaum, 2005). Thus, pro-sur-vival factors are important but notsufficient to ensure gonocyte sur-vival. It is possible that Sertoli cellsare programmed through their owndevelopmental sequence to locallysecrete pro-apoptotic factors afterdpp5, and that the gonocytes thatsucceeded in relocating at thebasement membrane escape thesesignals, or alternatively, duringtheir differentiation at the base-ment membrane, gonocytes mightevolve to become unresponsive tothe apoptotic signals.

Factors Regulating GonocyteApoptosis

Apoptosis has been clearlyshown to be part of the develop-mental program of germ cells(Russell et al., 1990; Furuchiet al., 1996; Rodriguez et al.,1997; Sinha Hikim and Swerdloff,1999). Indeed, mice deficient inthe proapoptotic factor Bax pres-ent decreased germ cell apoptosisat dpp5 and 15, that correlateswith an initial accumulation oftype A spermatogonia at dpp12,together with intermediate typespermatogonia at dpp15 and pre-leptotenes at dpp25, followed byan extensive germ cell degenera-tion and testicular atrophy (Russellet al., 2002). On the other hand,

mice overexpressing the anti-apo-ptotic factors BclxL or Bcl2 havebeen reported to present abnor-mal spermatogenesis reminiscentof the Bax KO phenotype, sug-gesting that apoptosis is neces-sary for normal spermatogenesis,possibly by maintaining a specificratio between the numbers ofgerm cells and the Sertoli cellsthat support them (Rodriguezet al., 1997). The work of Jahnu-kainen et al. (2004) clearly illus-trated that in rat testis, Bcl2 is stillstrongly expressed in germ cells atddp8 (which, according to theauthors, still included cells with agonocyte phenotype, togetherwith spermatogonia), while apo-ptotic factors including Bax, cas-pase 3, and the poly-(ADP-ribose)-polymerase (PARP), areonly increasing later, with a strongexpression at dpp18 to 26. More-over, in this study, germ cell apo-ptosis was found to be stage-spe-cific and to rarely affect dpp8germ cells while apoptosis wasprominent in midpachytene sper-matocytes, suggesting thatpostnatal gonocyte apoptosisrepresents a minor factor physio-logically as compared to apoptosisin more differentiated germ cellstages.Among the main pro-apoptotic

factors in germ cells are theTGFbs, which belong to the TGFbcytokine superfamily, whichincludes also bone morphogenicproteins, activins, inhibins, MIS,and GDNF (Loveland et al., 2007).These factors act by binding to theserine/threonine kinase receptorTbRII that phosphorylates TbRI,leading to the activation of Smadproteins (for details, see reviewfrom Shi and Massague, 2003).TGFb1 and TGFb2 were found toincrease dpc13.5 fetal gonocyteapoptosis in organ cultures of rattestes (Olaso et al., 1998) and toincrease pubertal germ cell apo-ptosis (Konrad et al., 2006). Bycontrast, TGFbs were found tohave only a minimal effect ondpp3 gonocyte numbers (Olasoet al., 1998), further stressing thedifferences in the physiologicalresponses of fetal and postnatalgonocytes.

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Despite its small positive effecton gonocyte proliferation, RA hasalso been shown to act as a pro-apoptotic factor in germ cells.Although rat fetal gonocytes(Livera et al., 2000), and humanfetal gonocytes (Lambrot et al.,2006), were reported to undergoapoptosis under the control of RA,this effect was not observed inneonatal gonocytes (Livera et al.,2000). Indeed, RA had no effecton the apoptosis of either late fetalgonocytes (dpc18.5) or neonatal(dpp3) gonocytes in organ cultureof rat testes (Livera et al., 2000),but it had a negative effect ongonocyte numbers in dpp3 gono-cyte-Sertoli co-cultures (Boulogneet al., 2003). Using specific ago-nists and antagonists, the sameinvestigators subsequently showedthat the negative effects of RA ondpc14.5 fetal gonocytes weremediated by the RA receptor a(RARa) (Livera et al., 2001),although this receptor could notbe detected in fetal gonocytes atthis age (Boulogne et al., 1999a;Vernet et al., 2006), suggestingan indirect effect of RA on germcells.Estrogens represent another

class of compounds with pro-apo-ptotic effects in fetal gonocytes, assuggested by the work of Lassur-guere et al. (2003). Using organcultures of dpc14.5 rat testes,these investigators found thatincubation with 4 3 1026 M of thepotent estrogenic compounddiethylstilbestrol (DES) for 1 to 3days resulted in significantdecreases in fetal gonocyte num-bers, associated with increasedapoptosis. However, incubationwith 4 3 1028 M DES had only asmall effect on germ cell numbersafter 3 days in culture, whereas 43 1026 M E2 induced a small butsignificant decrease in fetal gono-cyte numbers but no change in therate of apoptosis, suggesting thatthe changes in gonocyte numberswere unrelated to apoptosis.Rather, the reduction of gonocytenumbers appeared to correlatewith a small decrease in gonocyteproliferation rate observed uponE2 treatment. Considering that E2and DES exhibit similar ranges of

binding affinities and reportergene assay potencies for ERb(Barkhem et al., 1998; Gutendorfand Westendorf, 2001), which isthe ER form expressed in gono-cytes, the broad differenceobserved by Lassuguere et al.(2003) in the effects of E2 andDES on fetal gonocytes apoptosissuggests that the effects of estro-gens on apoptosis are ligand-spe-cific and involve more than a sim-ple interaction of the ligands totheir receptor. The potential roleof estrogen as a regulator of gono-cyte apoptosis was also suggestedby a study of Delbes et al. (2004),where transgenic mice expressingan inactive ERb were found tohave a two-fold lower level of apo-ptotic gonocytes concomitant withan increased proliferation rate atdpp2 in comparison to wild typemice. Interestingly, the positiveeffect of ERb disruption on germcell numbers started in the fetus,because there was already a smallbut insignificant increase in thenumber of fetal gonocytes atdpc15.5 and 17.5, which appearedto broaden during the phase ofgonocytes quiescence, leading to50% more germ cells at dpp2 anddpp6 in ERb deficient mice than inwild type mice. Thus, these resultssuggested that endogenous estro-gen stimulates apoptosis andinhibits germ cell growth in fetal/neonatal males via ERb activation.However, the recent finding by thesame authors that E2 treatmentdid not decrease gonocyte num-bers in dpp3 rat testes organ cul-tures (Delbes et al., 2007) sug-gests that the pro-apoptotic effectof E2 observed in mice might notapply to all species. Alternatively,it is possible that dpp2 mousegonocytes do not correspond tothe exact same stage as dpp3 ratgonocytes. Similarly, Lassuguereet al. (2003) reported no effect ofE2 on dpc14.5 rat gonocyte apo-ptosis, whereas Delbes et al.(2004) found a small increase infetal gonocytes in mice carryinginactive ERb, suggesting that fetalmouse and rat gonocytes mightnot be identical in their responsesto estrogen. In this regard, itshould be noted that both dpp2

mouse and dpp3 rat gonocytesrepresent heterogeneous germ cellpopulations, as demonstrated bythe gene expression studies dis-cussed earlier in this review. Thisin turn evokes the possibility thattwo sub-populations of gonocytesmight co-exist at these ages, cor-responding to two developmentalstages that are differentially re-sponsive to estrogens, and thatthe proportion of each sub-popula-tion might be different betweenmice and rats.In view of the studies described

above, it appears that fetal gono-cytes undergo apoptosis under thecontrol of TGFbs, RA, and possiblyestrogens, whereas the postnatalwave of germ cell apoptosis doesnot physiologically take place inthe gonocyte stage, but rather atthe next developmental phaseonce the cells have become sper-matogonia.

Regulation of GonocyteNumbers by Androgens

Androgens have been known tobe critical for male sex determina-tion, development of male repro-ductive tissues, and spermatogen-esis for many years (Nieschlaget al., 2001; Nieschlag et al.,2004). Because testicular somaticcells, but not germ cells, havebeen shown to express androgenreceptors (ARs), the effects ofandrogens on germ cells werebelieved to occur via a primaryeffect on their support cells, theSertoli cells (Dohle et al., 2003).This explanation does not apply atperinatal ages, when ARs arefound in the peritubular myoidcells but not in Sertoli cells, sug-gesting that at early ages theeffects of androgens on germ cellsmight be mediated by the myoidcells. However, a recent study byMerlet et al. (2007) questionedthe notion that fetal and neonatalgerm cells do not express ARs byreporting the presence of AR tran-scripts as well as proteins inmouse fetal gonocytes, openingthe possibility that androgens pro-duced by fetal and neonatal Leydigcells might exert direct effects onfetal gonocytes. To further explore



the role of androgens in fetal gon-ocytes, these authors used thetesticular feminized mice (tfm),which carry a natural inactivatingmutation of the AR. These studiesshowed that the testes of dpc17.5tfm fetuses presented increasednumbers of gonocytes as com-pared to wild-type mice, due to ahigher rate of proliferation. More-over, addition of 1026 M dihydro-testosterone (DHT) in organotypiccultures of testes from dpc13.5mice resulted in decreased prolif-eration, whereas DHT addition tocultures of enriched dpc15.5 gono-cytes transfected with an Andro-gen Responsive Element-drivenGFP expression vector led to theappearance of GFP-expressinggonocytes in the cultures, demon-strating the presence of transcrip-tionally active AR in these cells.However, this study did not exam-ine whether the rates of gonocyteapoptosis in tfm mice were differ-ent from those of wild type mice.Nevertheless, these studies sug-gest that androgens might be im-portant physiological regulators offetal gonocyte numbers.

Effects of Endocrine Disruptorson Gonocyte Functions

The deleterious effects of endo-crine disruptors on the male repro-ductive system of wild life havebeen known for years (Colbornet al., 1993). Among those, xen-oestetrogens such as DES, Bisphe-nol A (BPA) and the phytoestrogengenistein, have generated a largeamount of studies in rodents and afew in primates, identifying effectson Leydig and Sertoli cells(O’Donnell et al., 2001; Sharpe,2006). Prepubertal germ cellswere also shown to be affected byneonatal estrogen exposure, withDES increasing germ cell apoptosisand decreasing germ cell numbersat high doses, whereas low DESdoses and BPA had the oppositeeffect on germ cells (Atanassovaet al., 2000). On the other hand,molecules with anti-androgenicproperties such as the phthalateesters have also been reported toexert deleterious effects on testisdevelopment and male reproduc-

tion (Fisher et al., 2003; Foster,2006). Moreover, studies by thelaboratory of Sharpe (Atanassovaet al., 2005) have shown the im-portance of the androgen-estro-gen balance in the development ofmale reproductive tract and pre-sented results demonstrating thattestosterone administration couldprevent some of the deleteriouseffects of DES in male rat repro-ductive tract, indicating that atleast some of the effects of xen-oestrogens were due to their neg-ative effect on testosterone pro-duction. More recently, perinatalphytoestrogen exposure wasshown to lead to decreased testos-terone production at puberty anduntil adulthood in rat (Akingbemiet al., 2007). However, very fewstudies have examined gonocytefate in perinatal rodent exposed toestrogenic or anti-androgeniccompounds. Among those, ourown studies described the effectsof in utero exposure to BPA, genis-tein, DES and the phytoestrogencoumestrol on the expression ofPDGFRs and ER-associated pro-teins in dpp3 testis. Our studiesshowed that estrogen exposureled to the up-regulation of PDGFRsin testis, more specifically ofPDGFRb in gonocytes (Thuillieret al., 2003), whereas it inducedincreased expression of heat-shock protein 90 (Hsp90), a chap-erone molecule that regulates thestability and function of many pro-teins, including ERs and c-Raf(Wang et al., 2004). Althoughthese in vivo studies did not deter-mine whether gonocytes wereindirect or direct targets of estro-gens, the presence of ERb onthese cells suggests this possibil-ity. More recently, Ferrara et al.(2006) reported that in utero ex-posure to di(n-butyl) phthalate(DBP) delayed fetal as well aspostnatal germ cell development,inducing germ cell apoptosis in thefetus, followed by a decrease indpp6 germ cell proliferative index,and a subsequent increase in thesame parameter at dpp25, despitea decrease in the numbers ofgerm cells in this condition. Otherinteresting observations includedthe transient apparition of multi-

nucleated gonocytes, and the sur-prising return to normal numbers ofgerm cells in dpp90 rats. Hereagain, the in vivo nature of thesestudies did not allow the authors todetermine whether DBP exerted itseffects directly or indirectly on germcells. In summary, endocrine dis-ruptors have been reported to exertdeleterious effects on gonocytes,but it is not clear at present whetherthese effects are secondary to ini-tial effects on somatic cells.

CONCLUSION

Despite the limited number ofstudies focusing exclusively onperinatal germ cell development,the existence of articles related toPGCs or spermatogonia thatextended their observation to agescorresponding to gonocytes, haspermitted the identification ofgenes or combinations of genesexpressed in gonocytes. Fromthese studies, partial ‘‘identitycards’’ of the fetal and neonatalgonocytes have emerged, empha-sizing differences between thegene expression profiles of fetaland neonatal gonocytes. More-over, the comparison of theirbehaviors in response to variouseffectors clearly indicates that fe-tal and neonatal gonocytes indeedrepresent distinct developmentalstages. In view of these facts, sev-eral authors have proposed to dif-ferentiate fetal and neonatal germcells by retaining the term ‘‘gon-ocyte’’ for the fetal period andusing the term ‘‘prespermato-gonia’’ to identify neonatal gono-cytes. However, this distinction isnot entirely accurate either,because both fetal and neonatalstages can be further subdividedin quiescent and proliferative peri-ods, which comprise germ cellswith distinct and sometimes oppo-site behaviors, most probably phe-notypically different. Indeed,changes from one gonocyte stageto the next seem to occur progres-sively, or at least in anunsynchronized manner that ren-ders difficult the characterizationof each individual sub-group.Thus, any measurement or profil-ing performed on gonocytes at a

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specific age will represent the av-erage for the biological function/gene expression found within thedifferent subsets of gonocytespresent at this time. As moretransgenic mice models becomeavailable, new key molecules ingonocyte functions will be identi-fied, holding the promise thatmarkers, or rather sets ofmarkers, specific for different sub-sets of gonocytes will be discov-ered, allowing for better cell sort-ing and stage specific studies.Similarly, more studies on Sertolicell development will provide newclues on gonocyte regulation, asSertoli cells are the main source ofgonocyte effectors.

ACKNOWLEDGMENTS

We thank Jane Ebreo for her as-sistance in the preparation of thearticle.

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Gonocytes, the forgotten cells of the germ cell lineage