Seminars in Cell & Developmental Biology 17 (2006) 534–539

Review

Vertebrate reproductive stem cells:Recent insights and technological advances

Derek J. McLean ∗Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, WA 99164-6353, USA

Available online 13 July 2006

Abstract

How limited is the ability of stem cells to generate gametes or differentiated somatic cells? Recent outcomes of research with stem cells fromboth embryonic and adult origin will be discussed with particular attention to results that challenge conventional wisdom about the presence ofreproductive stem cells in adults and the plasticity of adult stem cell types. The ability of embryonic germ cells, primordial germ cells, oogonia,gonocytes and spermatogonial stem cells to differentiate or dedifferentiate into overlapping cell types is described as well as the implications ofgenerating differentiated somatic cells of multiple lineages from adult reproductive stem cells.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Embryonic germ cells; Primordial germ cells; Ovary; Testis; Spermatogonial stem cell

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5342. Reproductive stem cell origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5353. Germ cells from ES or somatic stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5354. Ovarian stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536

5. Spermatogonial stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537

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6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

The continual production of a vertebrate species is depen-dent upon successful reproduction. While the process oftenrequires complex behavioral interactions leading to copulationor gamete interaction, the production of the gametes, oogenesisor spermatogenesis, must occur for the successful production ofprogeny. Oogenesis and spermatogenesis are similar in that theend product is a haploid cell that is capable of forming a zygoteafter successful fertilization. However, the cellular differenti-

ation leading to the formation of an oocyte or spermatozoonis quite different. A central difference between these two pro-cesses is the presence of an adult population of stem cells, the

∗ Tel.: +1 509 335 8759; fax: +1 509 335 4246.E-mail address: dmclean@wsu.edu.

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1084-9521/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.semcdb.2006.07.003

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permatogonial stem cells, in the testis that provides a sourcef undifferentiated spermatogonia for the continual productionf sperm throughout the lifetime of the male [1]. In contrast,he conventional understanding of oogenesis in mammals is thatrimordial stem cells in the fetus differentiate into oogonia thatroliferate, enter into meiosis I and then arrest in prophase If meiosis before birth. Thus, a female animal is born with aomplete, finite population of oocytes that either ovulate or areost by follicular atresia.

The field of reproductive stem cells is characterized by find-ngs from researchers that challenge accepted models. For exam-le, recent research focusing on germline stem cells indicateshe origin and ability of these cells to differentiate into non-

ermline mature cells is not as limited as previously believed.imilarly, recent findings by Johnson et al. [2] suggest that therere germline stem cells present in adult females. These findingsighlight the stimulating and complex research environment of

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his field. This review will discuss research in the area of repro-uctive stem cells in vertebrates with a particular emphasis onecent findings challenging generally accepted knowledge ofhe origin and plasticity of these cell populations and the use ofeproductive stem cells for biotechnological applications.

. Reproductive stem cell origin

Embryonic stem (ES) cells represent the most undifferenti-ted cells in vertebrates with the potential to differentiate intoultiple lineages. The use of ES cells for developmental biology

nd cellular differentiation research as well as clinical therapyas been extensively reviewed [3,4]. The potential of ES cells toifferentiate into cells with germ cell morphology and markersuggests that the typical in vivo differentiation program requir-ng cells to migrate from a specific region of the embryo andnteract with somatic cells in a microenvironment or niche isot entirely required for the formation of a germ cell [5]. Theormal developmental program in mammals for the formationf adult germ cells in the gonad begins at 7.0–7.5 days of ges-ation as approximately 100 primordial germ cells (PGCs) areound in the embryonic ectoderm. These cells originate fromhe proximal epiblast adjacent to the extra-embryonic ectoderm.he PGCs migrate to the urogenital ridges and multiply to about5,000 cells by E13.0–13.5. The differentiation of the indiffer-nt gonad into a primitive ovary or testis also occurs during thisime to create the proper microenvironment necessary for sexetermination [6]. In the male, expression of the gene sry ini-iates a process leading to the formation of seminiferous cordshe encapsulate PGCs. During this process, the PGCs prolif-rate and differentiate into a primitive male germ cell calledonocytes or prespermatogonia. The male germ cells cease pro-iferation until just before birth in mice and shortly after birthn rats. Mitotic resumption of the gonocytes is associated withhe movement of these cells from the center to the peripheryf the seminiferous tubule. Subsequent to this event, gonocytesifferentiate into spermatogonial stem cells based on their abil-ty to colonize and initiate donor-derived spermatogenesis in theestes of recipient mice [7]. In addition, some gonocytes differ-ntiate directly into differentiated spermatogonia that initiate therst wave of spermatogenesis [1]. Although in mitotic arrest, a

ow percentage of PGCs and embryonic gonocytes are capablef colonizing the seminiferous tubules of recipient adult mice8,9]. These data indicate primitive germ cells can differenti-te into spermatogonial stem cells if transplanted into a suitableicroenvironment.The female embryonic gonad is characterized by a lack of

eminiferous cord formation and germ cell mitotic arrest. Inact, as the embryonic ovary forms, oogonia initiate meiosis.mbryonic ovarian germ cells initiate meiosis at the same time

n development that male germ cells germ enter mitotic arrest.etinoic acid appears to be a key regulator of the initiation ofeiosis by inducing the expression of the gene stra8 [10]. In a

ell-designed set of experiments, Koubova et al. [10] demon-

trated that the initiation of meiosis in male germ cells at thisime of development is blocked by the metabolism of retinoiccid by the protein product of the cyp26b1 gene. These data

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rovide information on how the critical process of meiosis isnitiated. In the embryonic ovary, however, female germ cellso not complete meiosis I and at birth most oocytes are arrestedt the diplotene stage of prophase I. Stimulation of the ovaryy the LH surge prior to ovulation results in oocytes complet-ng meiosis I followed by arrest in meiosis II that is completedfter fertilization. Thus, the formation of the oocyte populationas been considered complete before or shortly after birth inammals and no true oogonial stem cells are present in adults.

. Germ cells from ES or somatic stem cells

Despite the precise and intricate series of events that leads toamete formation in both the testis and ovary, ES cells have beennduced to differentiate in vitro into germ cells if cultured underhe correct conditions. These remarkable results have been dis-ussed in previous reviews [11] so only information from thesetudies relevant to subsequent work outlined in this review wille noted. To characterize and follow the differentiation of ESells induced to differentiate toward the germ cell lineage; Hub-er et al. [12] used ES cells expressing GFP under control of theerm cell specific enhancer of pou5f1, previously called oct3/4.ulture conditions did not include a feeder layer and after differ-ntiation the cells expressed several PGC markers (pou5f1 and-kit) and eventually germ cell markers ddx4, also known as vasar mouse vasa homolog (mvh), and synaptonemal complex pro-ein 3 (scp3) as a marker for meiosis. Continued culture resultedn cells that were morphologically similar to oocytes and aggre-ates that produced estrogen. This remarkable finding indicateshe developing oocytes induced other cells to differentiate intoells with function similar to granulosa cells. Likewise, genesssociated with a zona pellucida were also detected [12]. Theifferentiation of ES cells into oocytes under conditions thatid not require cellular interactions normally occurring in thembryonic gonad provides a unique approach to the investiga-ion of the regulation of meiosis and factors that can manipulateogenesis.

Differentiation of ES into sperm required more complex cellssociations than reported for oocyte formation. Toyooka et al.13] used a similar strategy as Hubner et al. [12] to monitor germell formation using reporter genes whose expression mimickeddx4. Culture of ES cells to form embryoid bodies resulted inncreased reporter gene expression, indicating ddx4 promoterctivity, and other genes that are markers for PGCs and germells (e.g. GCNA). This process is enhanced if the ES cellsere exposed to Bmp4 or Bmp8b, two factors involved in PGC

unction [14]. Geijsen et al. [15] used retinoic acid treatmento generate PGCs from embryoid bodies. Some PGCs from thembryoid bodies differentiated into haploid round spermatidsnd upon injection into oocytes resulted in the formation oflastocysts. Lastly, human ES cells have also been induced toifferentiate and express germ cell markers following formationf embryoid bodies [16]. Interestingly, both XX and XY ES

ells expressed proteins that are specific for both oocytes andpermatids.

The ability to form oocytes in vitro does not appear to beimited to ES cells or even possibly pluripotent cells. Porcine

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kin stem cells from fetal pigs have been induced to differenti-te into neurons, astrocytes and adipocytes in vitro [17]. Dycet al. [18] followed this work by culturing stem cells from days5–45 gestation porcine fetuses in medium supplemented with% filtered porcine follicular fluid to determine if oocytes coulde derived from these cells. This approach resulted in cells in theulture expressing pou5f1 and several other germ cell specificarkers (gdf9b and dazl) after several days in culture. At day 20

f culture, cells expressed ddx4 and by days 30–40 of culture cellggregates resembled an oocyte surrounded by cumulus cells.ransfer of cell aggregates to oocyte-growth medium that con-

ained gonadotropins resulted in some aggregates extruding cellsrom 30 to 100 �m in diameter that expressed markers consistentith oocytes including zona pellucida proteins and scp3. Cell

ggregates also produced estradiol and progesterone indicatingteroidogenesis was stimulated in the cells of the aggregatesy gonadotropins present in the oocyte-growth medium. Theseesults appear to be consistent with those of Hubner et al. [12]hat the formation of an oocyte-like cell stimulate closely asso-iated cells to differentiate into follicle-like cells potentiallyupporting oocyte maturation. Lastly, Dyce et al. [18] reportedhat some oocyte-like cells spontaneously activated to becomearthenogenetic embryos. In contrast to the work described forS cells, the origin of the porcine oocyte-like cells was primaryells directly from a fetus. Perhaps the most striking finding ofhis study, in comparison with deriving germ cells from ES cells,s that fetal somatic stem cells were induced to dedifferentiatento germ cells. It is possible, as noted by the authors, that somendifferentiated or primordial germ cells did not migrate to therogenital ridge during development and reside in the skin andhis small population of cells were observed to differentiate intoocytes [18]. More works need to be done to fully characterizehe initial cell population but these results are intriguing in lightf recent evidence that germline stem cells in the bone marrownd peripheral blood can populate the adult ovary with oocytes2].

. Ovarian stem cells

The paradigm for oogonial proliferation since the 1950s ishat the formation of oocytes in mammals, with a few exceptions,nly takes place in the embryonic ovary [19,20]. This finding haseen questioned in mice by results presented by Johnson et al.2,21] that describe the regeneration of oocytes from putativeerm cells in bone marrow and peripheral blood. The abilityf ovaries to generate new follicles was tested by eliminatingndogenous oocytes with doxorubicin, a chemotherapy drug,nd then analyzing the ovaries for the formation of new folli-les. A rapid (24–36 h) regeneration of the follicular pool wasbserved and 2 months later the number of follicles in treatednd control animals was similar. Undifferentiated germ cellsere identified in the ovary but the number of cells in this popu-

ation was not large enough to explain the rapid regeneration of

he follicle pool. Thus, the researchers hypothesized that theres another source of germline stem cells in adult females. Bonearrow was investigated as a source of germline stem cells due

o the presence of hematopoietic stem cells in the bone marrow

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nd the common embryonic origin of hematopoietic stem andGCs. Bone marrow cells were positive for expression of sev-ral genes associated with germ cells including the germ cellpecific marker ddx4. Thus, the source of germline stem cellsopulating the ovary in adult female mice was hypothesized toe the bone marrow [2].

To determine if bone marrow cells could colonize and initi-te oogenesis in the ovary, bone marrow cells were transplantednto mice treated with cyclophosphamide and busulfan to elim-nate all existing germ cells [2]. Control mice had no or fewegenerating follicles in the ovary while mice injected withone marrow cells had several hundred follicles at all stagesf folliculogenesis that were present up to 11 months post-ransplantation. Lastly, these researchers also demonstrated theeripheral blood contained germline stem cells by transplant-ng peripheral blood from transgenic mice expressing GFP intoild-type mice treated with chemotherapy drugs to eliminate

ndogenous germ cells [2]. These results have stimulated a greateal of interest among reproductive biologists and specificallyesearchers investigating ovarian biology. Byskov et al. [22]aised several issues regarding the interpretation and validityf several of the results presented by Johnson et al. [21]. Oneoncern with these studies is the rapid regeneration of the ovaryith growing follicles, a process that occurs over the course of

everal days at a minimum. Likewise, concerns have been raisedbout the specificity of the genes used to verify the presence ofermline stem cells in the bone marrow.

The significant nature of germline stem cells in the bone mar-ow and peripheral blood that can repopulate the ovary withocytes has also lead to other scientists providing alternativeypotheses, some which had already been present in the liter-ture, for the origin of the oocytes observed by Johnson et al.2,21]. Specifically, Bukovsky [23] argues that Johnson et al. [2]id not consider data demonstrating germ cells are present in theurface epithelium of the ovary and that these cells have beenbserved to migrate away from the ovary through the peripheralloodstream [24]. These cells would then be responsible for theositive gene expression for germ cell markers observed in bonearrow cells [2]. Thus, according to this hypothesis, there is a

onstant flow of germ cells from the ovary to the blood ratherhan from the bone marrow into the ovary. In humans, theseells arise from a population of mesenchymal cells that reside inhe ovarian tunica albuginea. Primitive germ cells combine withrimitive granulosa cells to form follicles in the ovarian cortex.his suggests that the pool of follicles is changing more thanriginally thought and this mechanism could represent a wayo maintain or even extend reproductive performance past thembryonic or neonatal period characterized with a high degreef follicular atresia. It is possible that mechanisms regulating fol-iculogenesis and ovarian biology present in humans and otherammals are different such that separate hypotheses for the

resence or origin of female germline stem cells must be testedn multiple species. It is important that such significant and

aradigm shifting results are independently repeated and newechniques are developed and applied to further refine the spe-ific nature of germline stem cells associated with the ovary ofdult mammals.

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. Spermatogonial stem cells

In contrast to the mammalian female ovary, an adult popu-ation of stem cells is present in the adult testis. As describedbove, this population of cells, called spermatogonial stem cells,orms in mice during early neonatal development. The numberf spermatogonial stem cells increases in the testis until adult-ood although the population in rodents is small (3000–25,000n mouse; 830,000 in rats) in comparison to the total number ofesticular cells [25–27]. During development, the spermatogo-ial stem cells establish niches within the seminiferous tubulehat are thought to regulate cell activity [28]. A firm physicalescription of a spermatogonial stem cell niche is not possi-le because no specific spermatogonial stem cell marker haseen identified. Therefore, the precise location of the spermato-onial stem cells has not been described. The niche conceptncorporates information from other adult stem cell systemsn which stem cells interact with somatic cells and with thextracellular matrix components [29]. All elements of the nichere thought to regulate stem cell activity including the fate ofhe stem cell at division to either self-renew or differentiaten the case of spermatogonial stem cells into undifferentiatedpermatogonia.

The opportunity to investigate spermatogonial stem cell biol-gy has been enhanced by the ability to identify these cells withhe use of the spermatogonial stem cell transplantation assay30]. Cells can be transplanted from one mouse to the testis of aecipient mouse and the presence of spermatogonial stem cellss determined by the ability of cells in the donor cell populationo colonize the seminiferous tubules of the recipient mouse andstablish donor-derived spermatogenesis. This powerful assayrovides researchers the ability to determine the number andiological activity of spermatogonial stem cells in a cell pop-lation. A second approach that has facilitated research withpermatogonial stem cells is the ability to culture these cells inerum free conditions to investigate how certain factors regulateell activity [31–33]. Culture of spermatogonial stem cells alsorovides mechanism to genetically manipulate the cells prioro transplantation. Colonization and donor-derived spermato-enesis in the testes of recipient mice from genetically manipu-ated spermatogonial stem cells provides an alternative approacho produce transgenic offspring through the male germline34,35].

Culture of spermatogonial stem cells also provides a meanso evaluate the gene expression profile of these cells in cul-ure. Although spermatogonial stem cells are cultured on feederayers, these cells can be isolated from cultures in a popula-ion that is greater than 90% pure for gene expression analysisith the use of microarrays. The embryonic and postnatal tes-

icular transcriptome has been characterized with the use offfymetrix microarrays [36,37]. However, the complexity of

he different germ and somatic cell types present in the develop-ng testis and the changing proportions of these cells presents a

hallenge when attempting to identify factors critical for thectivity of a specific cell type, especially a cell type in lowbundance. Oatley et al. [38] cultured enriched spermatogonialtem cells from day 6 mouse pups on feeder layers using con-

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itions that have been shown to maintain spermatogonial stemells in a proliferative, self-renewing state that included glialell line derived neurotrophic factor (GDNF) in the medium32]. GDNF regulates spermatogonial stem cells to self-renewalather than differentiate. To identify genes regulated by GDNF,hese researchers removed it from the medium for 18 h andampled cells for gene expression at 2, 4 and 8 h after GDNFeplacement. Using this approach, the expression of six genesramatically declined upon GDNF removal and then increasedpon GDNF replacement. The biological significance of onef these genes, bcl6b, was investigated in vitro with the use ofiRNA to knockdown protein production. Maintenance of sper-atogonial stem cells in vitro was reduced by siRNA directed to

uppress bcl6b and in mice with targeted deletion of bcl6b degen-ration of the seminiferous tubules was observed [38]. Theseata demonstrate the utility of a genomic approach to investi-ate factors important for spermatogonial stem cell biologicalctivity.

Characteristics and biological information about spermato-onial stem cells from a variety of species have been recently andxtensively reviewed [39–41]. Likewise, research and potentialpplications of spermatogonial stem cells in livestock speciesas also been recently reviewed [42]. Readers are referred tohese publications for more details on this cell type. However,he results from recent research suggesting spermatogonial stemells have a surprising capacity to dedifferentiate into ES-likeells that are pluripotent will be discussed in detail.

Two publications have reported that cells from the testes canstablish ES-like cells after culture [43,44]. Interestingly, theource and selection of cells for the initial cultures in thesexperiments was quite different. Kanatsu-Shinohara et al. [43]sed neonatal testes (0–2 days after birth) and negative selec-ion with gelatin-coated plates for 24 h to select cells for furtherulture and dedifferentiation into ES-like cells. After severalassages, the cells were transferred to cultures including mitoti-ally arrested mouse embryonic fibroblasts. ES-like cell coloniesere detected by appearance and expanded when transferred to

ultures using standard ES-cell culture conditions. Derivation ofS-like cells occurred in 19% of the experiments. Interestingly,

he original testis cells used to initiate cultures had been cul-ured using conditions that support male germline stem cells foreveral weeks prior to switching to conditions that support ESells. To determine if adult testis cells also had the ability to formS-like cells in culture, adult germ cells were positively selectedsing an antibody to CD9 followed by culture of these cells usingimilar conditions as the neonatal testis cells. This approach didot result in the formation of ES-like cells. However, culture ofD9 selected germ cells from newborn and 3–8-week-old p53nockout mice did result in the formation of ES-like cells in mul-iple experiments. The factor GDNF was required to establishermline stem cell cultures prior to ES-like cells in all experi-ents [43]. Although the ES-like cells formed teratomas when

njected into the seminiferous tubules of mice, when injected

nto blastocysts contributed to chimeras in 25% of embryos and5% of newborn animals. The ES-like cells contributed to mul-iple somatic cell lineages and the germline in chimeras [43].he age of the donor testis cells raises interesting questions as

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o the cell population in the neonatal testis such as what exactell type was selected for and propagated in this system. Theumber of spermatogonial stem cells in the testis in the first feways of life is low and the majority of the germ cells are con-idered gonocytes. In addition, the possibility that non-germlineells were selected and expanded using these conditions shouldlso be considered. The presence of germline stem cells in theeripheral blood suggested by Johnson et al. [2] could explainhe low percentage of ES-like cells derived from testes from 0o 2 days old mice.

Cells present in the testis of adult mice also appear to maintainhe potency to generate somatic cells in addition to contributingo the germline. Guan et al. [44] used transgenic mice that havehe stra8 promoter directing the expression of GFP. Testis cellsn culture positive for GFP were selected by cell sorting andultured in varying conditions to determine if these cells wouldevelop into ES-like cells. Inclusion of LIF in the medium orulture with mitotically inactive mouse embryonic fibroblastsesulted in the formation of ES-like cell colonies. These cellsxpressed germ cell markers in culture and gene markers asso-iated with different somatic cell lineages after embryoid bodyormation. Similar to Kanatsu-Shinohara et al. [43], ES-likeells from of testis origin contributed to organs and the germlinehen injected into blastocysts [44]. The impact of these find-

ngs for human and animal health could be significant. Theseesults suggest testis cells could generate cells that could besed to regenerate a damaged tissue or organ. It is interestingo note this possibility is only available for males at present,nless a similar approach could be developed from femaleissues.

. Conclusion

The importance of reproductive stem cells for the propa-ation of vertebrates cannot be argued. The progeny of theseells, oocytes and sperm, are responsible for passing geneticnformation to the next generation and the process of meio-is significantly increases genetic diversity. There is much wenow about the complex process of gamete formation and like-ise the requirements of the stem cells that initiate this process.owever, information about the specific microenvironments andey factors that regulate the processes leading to stem cell dif-erentiation are still relatively obscure. Indeed, recent resultsemonstrating that ES cells can differentiate into germ cells initro and previously unknown germline stem cell populationsxist in locations in the body external to the gonad representaradigm shifting discoveries forcing reproductive biologists toeconsider long held dogmas about germline stem cells. Theseesults also provide novel tools to investigate factors that regulateametogenesis and the efficiency of the process. The possibilityhat adult germline stem cells have pluripotent potential couldave a significant impact on human and animal health by pro-iding cells for tissue repair, once thought to only be possible

hrough ES cell manipulation. The pace of discoveries in theeld of reproductive stem cells promises to continue to chal-

enge scientists and spark the interest of the public in the nearnd distant future.

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