Indian Journal of Experimenlal Biology Vol. 4 1. July 2003. pp. 724-739

Stem cell research: Its relevance to reproductive biology

Tarala Nandedkar* & Madhura arkar

31 ionaiinslilUIe for Researeh in Reproducti ve Health. ( ICM R). Jehangir M erwanji Street. Parel, Mumbai 400 012, India

Slem ce ll s provide an exce llenl model system 10 undersland the differentiation. developl'1elll and func tioning of gonads, and further use of these cell s in transplantation or ce ll -based Iherapi es. Embryonic germ ce ll s prcsenl as a beller ~ource of pluripotent slcm ce ll s. The germ ce ll s are spcc iali zed ce lls, which differentiale inlo sperm or oocytes. Spermalogonial stem cell s are the onl y stem ce l ls in Ihe adullmammali an bod y that can be recogni zed and slUdied at cellular level wilh respcct to proliferation and differellliution. In Ihe present slUdy. bas ic process of spermut genes is. tesli cular niche and molecular regulation of spe rmalOgenes is and density regulation has been disc sscd. Research 0, oogonial slem ce ll s ha. reccntl y been encouraged due to the demand for oocy tes for va rious rcsearch purposes. M echanism of regu lation of folli cle formal ion, oocy le ullrition and folli cle deve lopmenl and atresia arc onl y pania ll y understood . Hence, the ~lages of deve lopmenl , il s interaclion with the neighbouring somalic ce lls during each deve lopmental stage and the molecular regulat ion underl y ing it has been rev iewed. These siudies w ill result in es tab li shmen l of treatmelll f ovarian dison.icrs. and in identifying cure for infcrt i lity that occurs due lo ovari an palhophys iology. Indian scenario in terms of stem cell research and its benefits is also discussed.

Keywords: Alresia, Gonad. N iche. OocY le, Primordi al germ cell s

The las t five years have brought ex traordinary advances in biomedica l sc iences. Know ing the complete seq uence of the human genome was onl y the beginning of understand ing how the spec ific genes funct ion. With the advent of DNA microarray technology it became possible to define the gene expression profile of a large number of genes wi thin a cell typ I. But the most promising breakthrough came with the in venti on of human embryoni c stem cell s, which prov ided an attractive model system fo r the identification of biologica l roles of genes.

The stem cell s provided efficient means to in vestigate the ex pression, regulation and functions of genes involved in mam malian differenti ation and development. The studies have raised signi ficant interest in characteri zing various gene functions utili zing transgenic and gene targeting tcchniques" Employ ment of stem ce lls to determine the pharmacokinetics of drugs and tox ins has also been recognized~. However, what has rea lly attracted the attention of general public as well as scienti sts is the potential use of stem cells in transplantation and ce ll rep lacement therapy that could treat many debilitating diseases such as Parkinson's disease, coronary hean disease. di abetcs and cancer' .

Basics of stem cells Stem cell s are th ose master cell s that can self­

renovate and reproduce indefinitely to form the

* For correspondence: E-mail : cc llbioirr@holmail. com Fax: 9 1-22-24 1394 12

speciali zed ce lls of ti ssues and organs. Such stem cclls are present in many ti ssues of ad ult an imals and are important in tissue rep<.1ir and homeostas is. The stem cell s may ha ve vari ous differen ti ation potentials. They can be totipotent (able to produce enti re organ ism) e.g. zygote and first few cell s afte r divi sions; multipotenr (capable of producing various cell types) e.g. hematopoiet ic stem cell s that produce erythrocytes and all types f white blood cell s; or unipotent (can produce onl y single type of ti ssue) li ke the spermatogoni al stem cell s in tes tis that can produce on ly one type of cell i.e. spermatozoon. Current effort s are aimed towards produci ng a pure culture of stern cell s as well as direc ting the differenti ati on of stern ce ll into any des ired ce ll type (Fig. I).

Pluripotent stem cell s theoretica ll y can give ri se to every ce ll type in the body and are not derived from adult body but from embryor.ic tis s u e~. In human , two main sources of stem cell have bee n reported: embryonic stem ce ll s and embryoni c germ cell s. The human embryonic stem cell s (ESC) are deri ved from inner cell mass of ill vitro fert ili zed e bryos at the blastocyst stages. The embryoni c germ ce ll s (EGC) are obtai ned from the primordial germ cell s in ?enital ridge of 5 to 9 weeks old aborted fet ses. The pluripotent stem ce ll iso lated from any of these sources can be cul tivated on feeder laye r and purifi ed wi th specific molecul ar ma' kers, furth er processed and transp lanted into the patient' s bod/. However, the

NA DEDKAR & NARK AR : RELEVA CE OF STEM CELLS RESEARCH IN REPRODUCTI O 72S

Inner cell mass

H~mitopoodc st~m

j cells

1 Embryonic stem cells

Embryonic Germ cells

Adult Stem cells -. °6 °1 . --

Pluripotent stem cells

Ectoderm (c:xtertW !ayel) Mesoderm mWd.1c Jaycl ) Endoderm intcmallaycr) Germ cell

11111Jlln III DEJD B D ·· ~rnCf2]

cpl(k'nni euron Pigmem I: kin) (Brnjn) cell

(Skin)

Tubules (kidne )

mooth Pancreatic Th roid Lung ce ll Spenn Egg mu ' Ie cell ce ll (al eolar) (in gut)

Fig. 1- Di ffe rcnt iat ion or stCIn ce lls in culture

body being immunolog ical ly incompatible may rej ect these ce ll s. Hence, recently it has been proposed that human embryonic stem cell s could be utili zed 1'0 1'

ther:1peuti c cloningJ With thi s hypothesi s, damaged pan of the patient could be repaired with one's own cells. The nucleus o f any ce li oI' the body is inserted into an enuclea ted oocy te so th at nucleus gets reprogrammed to toti potency and forms an embryoS. The stem cell s deri ved from such embryo can be di f ferentiated into des ired ce ll type and tran splanted into the pati ent. However, thi s concept w ill ha ve to be investi gated in depth before it could be appli ed clinicall y.

A du lt stem cell s scattered in va ri ous body ti ssues are present as an alternati ve source. The adult stem ce ll is an undifferentiated (un. pec iali zed or parti all y speciali zed) cell that is found ill a differentiated ti sSLJ>. These ce ll s el well in vari ous ti ssues alld replace elamaged or dead ce iL w ith new ones . For example.

new layers or skin appear after shedding or dead ce ll s. The hematopoietic stem cells from bone marrow replace the blood cell s. Ce lls that line the gastrointes tinal tract are removed and replaeeel by new ones. The source of these new ce ll s th at repopulate the skin , bl ood and intestine is adult stem cells . The res idential stem cell s in these organs maintain a group o f self-renewing undifferentiated ce lls as we ll as prov ide a new population of elilTerentiated functional ce ll s th at replcni sh the dead ce ll s at regular interval s or in case of damage of the ti ssue. For example hematopoieti c stem ce ll s present in the bone marrow give ri se to all types of blood cell s. Hence hematopoietic stem cell s from a healthy indi vidual are utili zed in allogenic bone marrow tran splantation in order to reju venate a di seased pat ient 'S bl ood system. Thi s concept is kno. 11 as regenerati ve medicine. Thi s approach is superi or LO the drug therapy as it clues not just change Lhe ce ll

726 I DIAN .I EX P 1310L. JULY 2003

melabol ism but res tores th e normal functi on of the ce l l.

Apa rt from hcmatopoieti c stem ce ll s the oth er :1dult stem cc ll s that ha ve been tested and put to clinical use include : myob lasts for cardiac and ske lda l musc le loss or degenerati on, cultured beta cc ll s for di ahetes mcl lilU :-, and cultured limhal epi thelial ce ll s for reconstructi on of ocular surface in limbal stem cel l s ~ . But there are cert ain disadvantages in usi ng adu lt stem ce ll s. The y icld of pure sLem cells ohtained in case of adul t stem ce ll s is vcry low. Stud ies on molccular markers that can separate adulL stem ce ll s from dilTerenti ated ce ll s of a ti ssue. arc lacking. Further. the adult stem ce ll s from Illouse bone marrow fuse with the cmbryoni c stem cell s formin!.! !.!ian t ce ll s with ahnormal chro moso ille number') . Tlli~ worri es the >.c icnti sts th at adult stem ce ll transp lantation may lead to cancerous growth . Furthcr. studi es on transd ilTerentiati on of musc le ce ll. to neura l ce ll s could not be con formed IU. Hence. these problems musl he overcome before adult stem cel ls arc used for cli nica l purpose.

Identifying stem cells A lthough basic characteri sti cs of Slem ce ll that is

self-renewal and differenti ation into vari ous ce ll types is known, a stem ce ll has not been defin ed completel y at molecular leve l. Thi s is essential to ensure the pur ity of stem cell cultureJ The pluripotent stem ce ll lines have many att ributes in cO lllmon. Researchers are in ves ti gating su itable means to identify stem cell s. Initial stud ies by Bongso el a/. on human embryon ic stem cells reli ed on typical stem ce ll - like morphology and pos iti ve staining for alkaline phosphatase ll . Thoms n' s group further showed lhat human ES ce ll lines ex press high tcloll1erase acti vit y. which is correlated with immortalit y in human ce ll lines. They also observed ex pression o f estab li shed ce ll surface markers of pluripotency such as SSEA-3. SSEA-4. TRA- I -60 and TRA-I-SI and alka line phosphatases.I". Stem cell cannot be identified onl y on the basis o f morphology and/or ex press ion of spec ific ant igenic prote ins . Heterogeneity in ce ll populati ons rema ins one of the biggest obstac les in identify ing a true stem ce ll. In a ce ll popu lat ion, iL is dilli cult to ensure that all the ce ll s can div ide indefiniLely and are true stem ce ll s'. Hence some researchers are looking for stem cell spcc ifie genes that would not be ex pres. ed in any other ce ll types . M olecular analyses of stem ce ll s has been carri ed out by Weissman and Lemschkal3.14 to generate markers for identi fica tion tag to differentiate

cach cell used. These in ve .. ti ga tors u,.,ed subtrac ti ve eDNA li braries and cD A from highl y homogenous eell popul at ions to gcnerate microarray and examine gene ex press i )n pro fil es of haelll<ltopo ·ti c (HSC) and ncu ral stcm ce ll ( SC) . Compari son of H C and NSC revealed non-ident ica l and overl app ing profil es . Thi s suggests presence of a comlllon genctic program that maintains an undillerent iated state of a ~tem cell and prov ide a unique combinati on o f genc expression th at gi ves identit y to stem ce ll i"'.

Directing the dillhcntiation or stem cells Oncc the stem ce ll s have een iso lated and purifi ed

th e nex t objec ti ve is to diffe renti ate thcm in to dcsi n::d ce ll type by prov id ing suitable growth fac tors. Scienti sts arc now look ing at the poss ibility of directed differentiati on o f embryoni c stem ce ll s. Pluripotelll . telll ce ll s are ab le to gi ve rise to a \v ide array of dilTerential cd dcri vati es. In the absence of fac tors th at inhi bit thei r di fferenti ati on . pluripotcnt stem cell differenti ati on has bcen typ ica ll y direc ted by manipul ating their environment. Thi s can be achieved by growing them on different types o f feeder ce ll s. by addit ion of growth factors or by grow th on crude or defined ex trace llu lar matrix substrates4

. The signaling pathways thai regulate the stem cell di fferen tiat ion have not been identified comp letely. S(~ h uld i n e r et a/. examined the potenti al of eight growth factors namcly basic fibroblast growth fact rs (bFGF), transforming grow th fac tor (TGF-~ I ), actl vln A, bone morphogenetic fac tor (BM P)-4, Hepatocyte growth fac tor (HGF), epidermal growth facto r (EGF), basic nerve grow th factor (bNGF) and retinoie ac id (RA)I (l . Human ES cell s initiall y dev lop as aggregates known as embryoid bodies which are composed of all three germ layers. These aggregat es possess receptors for all these factors. A nd th eir interac tion ca uses differenti ati on into cell s with different ep itheli al and mesenchymal moqJhologies. When idcnti fied by ce ll spec ific markers eleven different ti ssues that are from all three embryonic germ layers can be identi fi ed. Growth factors ac ti vin A and T(jF-~ 1 induce mesodermal cell s, RA, EG F. B M P-4 and bFG F ac t i vate ec todermal and mesoderma I markers and NGF and HGF induce diff rentiati on into all three germ layers. However, none of these growth factors uniquely di f ferentiated anyone celltyp 16. In ES ce ll s, several soluble factors, shown to direc t differentiation e.g. growth factors regu lating de elopment of haematopoietic l7 and neura l stem cell. IX have been studied ex tensi ve ly. None of these faetor_ exc lusively

NA DEDKAR & ARKAR: RELEVANCE OF STEM CELLS RESEA RCH IN REPRODUCTION 727

direct the differentiation to only one ce ll type, but rather give ri se to vari ed proportions of spec ific cell types. Hence. to obtain uniform populati on of di fferentiated ce ll s one needs to se lec t or sort it from a heterogeneous population. Another apprmch in volves DNA microarray technology. Dav id Kell y's group utili zed mouse cD A ex pression arrays to examine the express ion o f 588 known regulatory genes in OJ ES ce ll s and their RA- induced progeny. In thi s study they included a vari ety of molecular markers such as transcl"I ptJ on factors. growth factors and thei r receptors, cy toskeletal and ex tracellular mat ri x proteins, cell surface markers. intracellular signal transduct ion modulators and effectors I.

Niche: Microenvironmcnt for stem cell Apart from the know ledge of growth factors a

prec ise microen ironment known as niche is essential for directing different iati on of stem cell s. Prev ious ly it was thought that the stem cell functi on is dependent on gene ex press ion profi le of the ce ll. But now ev idence suggests that it is the loca tion of the stem cell th at determines the differentiation of the stem cell i!). Niche is composed of a subset of ti ssue ce ll s and ex trace llular substrates th at influence the behav ior of stem cell s. Schofo ld et al. was the first to introd uce thi s concept who studi ed the niche that

I h . . 11 ' 0 F . contro s ematopole[lc stem ce s-. or systematic identification o f niches. stem cell s are marked and th en their lineage is foll owed. Then the ce llular neighbors, ex press ion patterns of signal i ng molecules and local en ironmental factors such as ex trace llular matrices are characteri zed. The niches arc usuall y identifi ed by replacement assay. Niches are thought to operate in one of the two ways: lineage mechanism and populat ion mechani sm. I n lineage mechanism, onl y one daughter ce ll is maintained as a stem ce ll whereas the other ce ll differentiates . In popu lati on mechanism both the daughter ce ll either remain as stem ce ll s or both may differenti ate. Thus, understanding of the niche of the ti ssue is important fo r the study of the development and di fferenti ati on of a ti ssue llJ .

Stem cells in reproductivc organs Stem cell s rep lace the dcgenerated tissue w ith new

ce ll s duri ng the normal t issue turnover. In reproductive sys tem, peri odic replenishment o f the ce ll s under the infl uence of hormones and growth factors happens to be a common phenomenon. For example, in the test is, sperms are being continuously

produced from the precursor germ cell s or spermatogoni a wh i Ie in the ovary, oocytes deve lop from the oogon ia. In the uterus. the endometri al li ni ng is sloughed of!" and regencrated by the precursor ce lls from the basa li s during each cycle. I--lence, the present study focuses on the relevance o f stem cell research in reproducti on. Stem cell s prov ide an exce llelll model sys tem to understand the lifferentiation. deve lopment and funct ioning of gonads. Such investi gati ons wi ll help in dev ising effecti ve contracepti ve approaches as well as in trea tin g inrertilit y and reproducti ve d isorders. It may also help in answering some or the basic questi ons such as how the interacti on between spermatogonia and Serto li cell s takes place and what are the factors invol ved in fo lli culogene. is and oogenes is. In this review, we di scuss embryonic germ cells. their deri vation rrom primordial germ ce lls as well as the ro le of stem cell s in male and female reproductive system. These in ve tigati ons are mainl y to ex plore the poss ible molecular markers and niche of these cells. Thi s wou ld assist in develop ing sui tab le culture conditions for the growth of stem cells, directing their differentiation in vitr(} and further use of the ce ll s in transplantation or ce ll -based therapie ·.

Embryonic germ cells The zygote and the few cell s produced during

initial di visions of the rertili zed egg. appear to be totipotent. As the embryo develops, the ce ll s dirferenti ate, acqu ire capacity to produce spec ific ce ll ­lineages losing their pluripotenti ality . However, totipotency is mainta ined in the cell s fo rming the germ line. Earl y in embryogenes i , few ccll s arc designated to become primordial germ ce ll s (PGC). The ancestors of PGC arc located in the epiblast close ly adjaL:ent to extraembryonic ectoderm from which a molecular signal (BMP-4 related) predisposes them to become germ cell s~l . These ce ll s are recognized through germ I ine speci fic transcri ptional acti v i ty, showing express ion fo r the Lranscri ption factor Oct-4 and the product of Drosophil a gene, vasa~~. Primordi al germ cell s are also recognized by their express ion of alkaline phosphatase6

. It should be noted that when ce ll s from other locati on of epiblast are transplanted to the same location they also develop as germ cell s. Thi s suggests that extern al signal IS in vo lved and the cell s are not cy top lasmica ll y programmed to a germ cell fa te. The primordial germ cell s undergo proliferati on and si mul taneous mi grati on into the undi fferentiated gonads. There these cell s differentiate in to male or

72H INDIAN J EX P BIOL. JULY 20m

fema le germ ce ll precursors according to the XX or XY sex chrc>nlosomes 21

. Studies in rodents have highlighted the importance of c-kit proto-oncogene receptor and its li gand, kit li gand (S tem cell factor or SCF) in the migrat ion of germ ce ll s and their subsequent survi val and deve lopment2.1. A ft er migrati on, all germ ce ll s whether XX or XY in constituti on. enter prophase of first meiotic di vision at abou t the same time unless an inhibiting factor from test is prevents development. Thus. aga in an ex terna l signa l and not the genotype of the ce ll determines the entry into the meiotic phase. Thi s underlin es the importance of the microenvironment or niche in the development o f germ ce ll s.

Germ ce ll s undergo a complex hi story during their formation. but by changing their environment in culture they can be revert ed all the way back to ce ll s with propcrti es similar to those of the epiblast from which they aroseJ John Gearhart 's group first reali zed the potential of primordial germ ce ll s as a source of pluripotent stem cell s. They deri ved embryonic germ cell s from the primordial germ ce lls present in the genital ridges and mesentri es of 5-9 weeks embryo postfertili zat ion. PGCs were cultured on mouse STO f ihrob las t feeder layers in the presence of human recombinant leukemia inhi bitory fac tor (L1 F), basic fibrob last growth factor (bFGF) and forsko lin . L1 F and bFGF in med ium resulted In indefini te proliferati on of PGCs now called as embryonic germ cell s. A long w ith being positi ve for alka line phosphatase, they were al so pos iti ve against a panel of five immunolog ica l markers (SSEA- I , SSEA-~, SSEA-4, TRA- I -60, and TRA- I -81) that are rou tinely used to charac teri ze pluripotent stem cell s6

. Oct-4 a marker for PGCs is ex pressed in EGCs but mouse vasa homologue (MVH ) express ion is not observed in PGCs unlike EGCs. Germ Cell Nuclear Antigen (GC A) is expressed in EG ce ll s but not in PGCs. The ability of EGCs to form chimeras is another charact ri sti c that is not observed in PGCs21

.

Identifi ca ti on of molecular and ce llular markers for PGCs and EGC. will not on ly help in distinguishing the two but also help in understanding the mechanism by which EGC deve lop qualities of pluri potent ialit y, sc i f-re newa l and pro l i ferati on. Li ke embryonic stem cell s, the cultured EGC showed normal and stable ka ryotype. in case of both XX and XY ce ll cultures. Immu nohistochemica l ana lys is of EGC deri ved embryoid bodi es showed ex pression of a vari ety of markers of var ious di fferentiated ce ll types that included all three germ layers('. Embryonic germ ce ll s

present as a beller source of pluripolent ce lls th an the embryoni c stem ce lls due to their abil ity to retain the genom ic imprints. They may serve as it model system for in "i'ro studi es on human embryogenes is. Further. in germ ce ll s genomic imprints ar erased anel reestab li shed in a stepwise manner. But the molecular mechanism underly ing thi s process is not known. Embryonic germ cell s may be used to understand the

. . h ' )4 7S F hi ' k I I ep igeneti c mec anlsms- '-' . ~ Ult er tll S now ec ge may be employed to reprogram somat ic nuclei to

pluripotency or to induc transd i ffe rentiation. Human EG ce ll s could be used for ce ll and ti ssue

therapy. Some peop le see fewer ethi ca l problems in uS1l1g aborted felLlses, rather than di .. carded preimplantati on embryos, to provide pluripotent germ cell s for therapeutic cause21

.

Thus, the germ ce ll s arc very speciali zed ce ll s. and become more so as they di rFerentiate into sperm and oocy tes. They usua ll y have to go th rough meios is to rea li ze their totipotency , and sperm achieve thi s on ly after fertili za ti on and reprogramming of the ir DNA by egg cy toplasm. I-Ience, th e potential of male and female germ ce ll s as pluripotent stem cell s has been fu rther inves ti gated.

Spermatogonial stem cells Spermatogonial stem cells are the only stem cdl s

in the adult mammal ian body th at c;l n be recognized and studi ed at ce llular level with respec t to proliferati on and eliffercntiat ion lv. They are potentiall y totipotent ce ll s res ieling wi thin the basal layer or the seminiferous tubul es of the tes ti s. These ce ll s arc known as spematogoni a in the prepubertal and adult peri od and can be di vided into three primary stages: Stem cell spermatogon ia!. pro l i fera ti e spermatogoni a and di fferenti ating spermatogonia26

.

S permatogon ial stem cell s are un iq ue among aelu I t stem cell s because they pa~;s genetic in fo rmati on to the nex t genera ti o n ~7 The abi lity to recover :;pematogonial stem cell s and transfer them to another testi s would provide a vulnerable techn ique to study spermatogenesis. Hence in the presen t study, bas ic process of spermatogenesi s, testi cular niche and molecular regu lation of spermatogenes is and density regulation has been discussed.

Sf'e nlla/ogoll ial di[lerelll ia' iOIl In the tes tis, germ ce ll developmen t is main ta im:d

by germline stem cell (GSC). These are undifferentiated A spermatogonia which according to thei r topographical arrangement on the basa l membrane arc subdi vided

NA DEDKAR & NA RK AR : RELEVANCE OF STEM CELLS RESEARCH IN REPRODUCTI ON 729

into A single (As), A paired, (Apr) and A aligned (A"I) spermatogon ia (Fig 2). The As spermatogonia are considered to be the stem cell s of spermatozoa. Di vision of A, gives ri se to daughter ce ll s that migrate away from each other and become either two new stem cell s or stay connected by an intracell ular bridge to become Apr spermatogoni a that will ultimately become spermatozoa. A bnormal rati o of stem cell renewal and Apr formati on would lead either to lLimor formation or to exhausti on of the stem cell poo l. The Apr spermatogonia di vide further to form chains of 4,8 or 16 A"I spermatogonia. During each cyc le, in the seminiferous epithelium at about stage V II , most Aal spermatogoni a di ffe renti ate into A I sperm atogoni a

Spennatogonia A ~, } .~ Self renewal

'-------'t--~---'

AI • + A2 , A3 • + Differentiation A4 • .. In 8

+ B '. ~

Spennatocytes "

Fig. 2 - Earl y stages in spe rma togenes is: Diffe re ntia tio n of spe rJlla toge!lCs is

Testicular Niche

Sperm-Sertoli cell

Spennatids ~

Secondary spennatocyte

that are f irst generation of differenti ating type spermatogonia. These di fferentiating spermatogoni a go through a seri es of six di visions to gi ve ri se to A2 . A 3, A4, Intermediate and B and finall y pri mary

?X 1<) ' spermatocy te- .- .

The spermatocytes carry out the meiot ic di vi sions and develop into spermatids. III vivo , in the normal tes ti s all these steps in germ ce ll deve lopment take pace at the same time and therefore are di f fi cu lt to stud y. To in ves tigate each of these steps. in I' ilm ex periments using iso lated germ ce ll s have been performed. However, only a limited number of ce ll s can be iso lated, germ cell s have a l imited viability in cul ture, and it is diff icul t to disti nguish spermatogonial stem ce ll from more di f fe rentiated A spermatogonia in vitro, due to the lack o f spec i f ic stem cell markers. The es tabli shment of spermatogonial ce ll l ines would overcome most of

' 0 these problems" .

TesliclIlar niche Maintenance of the stem cell compartment invo lves

complex interacti ons w ith other stem cell s, di fferentiated germ cell s, somati c ce ll s and ex trace llular matri x components of testi s. Together these interac tions create a microenvironment or niche for the spermatogonial stem cellol (Fig. 3).

N iche-based regulati on has been stud ied extensive ly in mammal ian test is. Init ially,. A, lie in contact w ith the basement membrane of the semi ni ferous tubule. The spermatogonia can move

Ovarian Niche

Oocyte­granulosa cell

Oocyte

cells Sertoli Type A2 Type B

cells spennatogonia spennatogonia

Fi g. ~-Gerlll cell and sO llla lic ce ll int e raction: Nie he in both sexes

730 INDI AN J EXP UIOL. JUL Y 2003

laterall y along the basement membrane and are enca:ed by Serto li ce ll s that med iate Illany aspects of male germ cell development. As germ ce ll s enter meiosis and begi n to differentiate into sperm they lose contact with the basement membrane and move towards the lumen of the seminiferous tubule, while maintaining their assoc iation with the Sertoli cell s.

T he niches in the tes tis were demonstrated by transplantation or earl y germ cells into the semini ­ferous tubul es of hos t males whose GSCs had been dep leted. Int roduced ce ll s co lonize at the site on the walloI' tubu les and developed clones o f sperms. This restored the fertilit y of the host male. These germ ce ll s initially fill ed all the empty niches with the undifferentiated stelll ce ll s. Only when the co lony had expanded fully , differentiation of sperm resumed. This reveals the presence o f th ousands or gennline ce ll niches along the \ all of seminiferous tu bu les and also provides informati on on their deve lopmental

. IlJ 'h l' R I' I I I properti es .- .. -. ecent stut les laV C S l own t lat immature testes provide a more fa vorabl e environ­ment than adult tes ti s but adu lt testes in contras t are a ri cher source of stcm ce lls in compari son to immature tes tis.13

Mo/ecu/or reg I t/{{fiOi I OJ"sIJI!nllologOll io/ difTerell1 iOI if J/I

Each stage in spermatogonial development involves a specific se t of genes and proteins. Some are produced by the spermatogoni a while the testi cular niche contributes some. Studi es with knockout mice helped in identify ing many o f the regulatory factors of spermatogenes is.

Self-renewal and differentiati on o f stem cell s normall y seems to play an important ro le in regulation of Glial ce ll line deri ved neuro troph ic factor (G ONF).

RA and GFRcx I receptor system. As Sertoli ce ll s produce GONF, they apparen tl y regulate this aspect of spermatogonial stem cell behavior. The complex ity of the seminiferous ep ithelium makes it difficult to study the spe rmatogonial multiplication and stem cell renewal at the molecular leve l.

C-kit receptor li gand system plays an important role in spermatogonial pro liferat ion and dillcren­ti ati on. Testes from W-mice lack normal c- Kit functi on and arc defic ient in endogenous spermatogenesis . c­Kit i~ invo lved in the differenti ati on step from /I. , to AI. It i~ predom inantl y expressed in germ ce ll s of tesli s and also ex pressed in Leydig ce ll s. T he presence of functiona l c-K it receptor has been impli cated in sl1Lrm:llogonial proliferation . survi va l and adhesioll to Sertoli ce ll s. Vincent ('I 0/. showed th e a press ion of

c-Kit by pachytene spermatocy te and proposed thJt kit and kit-ligand is essential for meiosis34

.

cx6 and ~ I integri n present on the spermatogonial stem ce ll is enriched in the basa l layer and may help to attach spermatogonial stem cell s to laminin ill the basement membrane"'). Cons istent wi th thi s idea, transp lant assays show that crude cell population can be enriched for GSCs by binding to laminin , the target of cx6~ I integrin but not to collagen I V or fi bronectin I ~ . Bone morphogenetic protei Il (B M P)-4 produced by ex tra embryoni c.: ectoderm stimulates the growth alld development of early germ ce ll s. BMP-8a and BMP-8b are also needed for germ ce ll developmcnt. But an ongoing functi on o/" BMP has not yet been establi shed.16 . .l7 Sertoli ce ll s produce

another GONF thaI affects proliferati on of pre­meioti c germ line ce ll s including perhaps stem cell s.lx.

The RNA heli case vasa is al so expressed in develop ing germ cc lls of many species. In the absence of vaso , male germ cell proliferati on is reduced

. . I 19 severely In spermatogonIa stages . Oazl (deleted in azoospermi a - like) RNA binding

protein , cyc lin 02 and retin oic acid (RA) are among the ot her fac tors th at regulate spermatogenes is. RA and Retinoid X receptor is invo lved in di f ferentiation of spermatogonia. In an imal with vitamin A defi ciency, A"I lose their ab ilit y to diffe rentiate. Since both spermatogonia and Sertoli ce lls possess nuclear receptors for RA it is not clear whether the differentiation occurs indirec tl y v ia ertoli ce ll or

. ,9 ·111 SCF K ' . . I d' A dIrec tly'. - c- It sys tem IS Invo ve 111 I spermatogonia dillerenti ati or!. Mutations in c-kit and SCF gene have a variabl e effect on spermatogonia at various steps. One mutant, T he SII7 1-1 /S1171-1 mouse showed an arrest spec i f ically at the differenti ation step of A"I to AI spermatogonia indica ting SCF-c-Kit sys tem is essential at thi s step. In accOI"dance w ith thi s. from about stage V I onwards, the A "I

. I I' I . · ' ~ ·11 I . spermatogoil ia s lOW ex press Ion 0 c-..;W · . n mice deficient in RNA binding protein encoded by On l gene. the differentiati on of A .II spermatogonia into A I does not take place. The Oaz! protein is thus essential for spermatogonial differentiati on. Finally, a study of express ion of va ri ous cyc li n 0 su,;ges t that only Cyc lin 0 2 is expressed arou nd epi thel ial stage V II I A .II different iates into A I . Cyclin 0 2 is not expresscd

.. . .p in A" Aill and A"I spennatogon l{\ In testi s -.

Dcnsity rcgulaltion Germ ce ll d '~gene ral ion has been ful ly es tabl ished

as a factor that limits effici ency of spermatogenesis.

NANDEDKAR & NARKAR : RELEVANCE OF STEM CELLS RESEARCH IN REPRODUCTION 731

The prevemion of apoptotic wave in three-week-old mice leads 10 drastic changes in spermatogenesis and fertility in adults. Apoptosi s plays a key role in maintaining equilibrium between the number of spermatogon ia and the Sertoli cells. The excess spermatogonia in testi s enter apoptosis. It has been suggested that adjusting the number of proliferating germ cell s to match the number of supporting Sertoli ce lls mighl ensure the quality of the gametes produced. M olecular mechani sm of apoptosis in spermatogonia is al. 0 important. Bcl -2 famil y is clearly in vo lved in thi s apoptot ic wave. Bcl-21 Bax rati o has b~en known to regulate this process. Overexpression of Bcl-2 and Bcl-x l and deficiency of Bax protein has been known to cause an accumulat ion of spermatogonia in tes ti s leading to apoptosis in all ce ll s soon after the meioti c prophase. Kit! SCF has been reported to influence the apoptot ic process. It is not yet clear how a surplus germ cell is sensed and what tri ggers the apoptoti c mechanism in some of the A2-A4 spermatogonia in a parti cular area. The role of Sertoli ce lls in density regulat ion of spermatogon ia is not well u nderstood41 .

In conclusion, few molecules such as GD F, SCF­C- Kit system, cyc lin D2, Dazl protein, RA, Bcl-2 fam il y have been identified to playa rol e in regulati on of spermatogonial stem cell di fferentiation and apoptosis. A lthough studi es w ith these molecules prov ide clues to the understanding of spermatogonial stem cell behavior in its niche, experiments in transgenic mice and development o f advanced culture systems should lead to complete understanding of spermatogenesi s.

These studies w ill be important in development of better male contracepti ves and treatment for male infertility. The establi shment o f effecti ve culture systems is prerequisite for better understanding of the developlllenLal mechani sm by which male germ cell s de clop into matu re sperm ce ll. . Brinster-Zimmerman reported that stem cell s iso lated from tes tes of the donor male Illi ce repopulate steril e test es when injec ted in to sem iniferou s tubul es. Donor ce ll spermatogenes is in rec i pienl tes tes showed normal morpholog ica l characteri sti cs and produced mature spermatozoa. These results are encouraging for the treatment of male infertility"6 Culturing the self­renewable spermatogonial stem cell s coupled with the recent breakthrough in our ability to tran sp lant these ce ll s into testes or infertil e Illice wou ld establi sh a test system for assessment o f the developmental potential or cultured spermatogenic ce ll s. These studi es w ill

also ·enable to genetica lly alter the male germ cell s such that the alterati on can be passed on to the nex t generation through mature sperm and fertili zation. Thi s will help in allev iati on of certa in forms of male infertility and the discovery of novel contraceptive approaches as well as a wide array of other potential benefits4J.

The focus of the spermatogonial stem ce ll ex peri ments is to improve isolati on, identi fication. characterization and culture of spermatogonial tem cell s. Such data wi ll result In successful transp lantation of spermatogonial stem cell s into the seminiferous tubules.J.J. It will allow the development of nove l routes for the generation of transgenic lifes tock, treatment for infertility . targeting male for contraception and alternate strateg ies for fertili ty preservati on.J3. A lso such research w i II prov ide means to correct defec ti ve genes in spermatogoni al stem cell lines that can be used therapeuti cally to eli minate devastating geneti c diseases .

Oogonial stem cells Research on oogon ial stem cells has recently been

encouraged due to the demand for oocytes for various research purposes. M echan ism of regulation of follicle formation, oocyte attrition and foil icle development and atresia are only partially understood. By culturing follicle from the oogon ial stem cells in vill'O at the earliest stages of growth may provide us with better understanding of these processes. To culture the oocytes one must closely investigate the stage of development. its interact ion with the neighboring somatic ce lls during each developmental stage and the molecular regulation underl yi ng it. This will result in creating a precise microenvironmcnt for the growth of oocyte in cul ture

.J5 system .

OocYle del'elopll1elli Gnd/olliclI logell esis On arri va l in the genital ridge the primordial germ

cell s in the female vertebrate are named as oogonia. Thc medullary cords in the pn1111tl ve gonad degenerate and is replaced by highly vascu larised ovarian stroma. The cord ce ll s proliferate and the mesenchymal ce ll s encase the oogonia giving ri se to pri mordial 1'011 icles . Foil icle formation inhuman begin ' between week 16- 18 of the fetal life. The mesenchymal cell s secrete a basement membrane around the fo lli cle to form primordial folli cle. and thc same cell s give ri se to granulosa cell s in the folli cle. Simultaneously during the envciopment the oogonial germ cell s enter meios is and arrest at the diplotene stage of the lirst meiotic phase. Oogonial gcnn ce lls

732 INDIAN J EXP BIOL, JULY 2003

entering meiosis are known as primary oocytes. The formation of primordial follicle to primary follicle is completed around the time of birth or immediately after that. The excess of oogonia that are not enveloped, are expelled by the ovary. The primordial follicle thus has a primary oocyte surrounded by flattened granulosa cells. When a few flattened granulosa cells become cuboidal, the follicle is known as primary follicle which contains a growing primary oocyte and proliferating granulosa cells. Preantral follicle is a secondary follicle containing two layers of granulosa cells. The spaces between granulosa cells get filled with follicular fluid along with the growth of the follicle in diameter to about 200 /-lm. This is the early antral stage follicle. The antral follicle then becomes 'preovulatory' or 'atretic' follicle. The later stages of follicular development are dependent on pituitary gonadotropins45. Thus, every stage of folliculogenesis occurs in an organized manner due to the appearance of growth factors (Fig. 4). These factors are either secreted by the oocyte or the neighboring somatic cells. However, regulation of early folliculogenesis is not well understood.

Ovarian niche Ovarian niche is composed of the oocyte,

surrounding somatic cells and the stroma (Fig 3). For

Primordial

Secondary (Preantral)

Antral follicle

Graffian follicle

understanding ovarian niche, study of three aspects is important: factors regulating follicular development, oocyte-granulosa cell interaction and apoptotic mechanism involved in the regulation of ovarian function. The molecular signaling between oocyte and granulosa cells plays an important role in growth of the oocyte as well as the development of follicle. The decision to become an ovulatory follicle is also mediated by specific signals. Finally surplus cells at some of the stages, for example, excess of unenveloped primary oocytes after completion of primordial follicle formation or cells in the atretic follicles are removed by apoptosis .

Molecular regulation of folliculogenesis The factors regulating folliculogenesis can be

divided into two phases: gonadotropin independent i.e. primordial to secondary or preantral stage and gonadotropin dependent i.e. antral phase onwards. Interestingly, at birth only primordial follicles are b d · . 46 47 h d . o serve In mIce marmosets as t ~t reporte In

human and rhesus monkeys48. Further, follicular development in these species takes place postnatally. On the other hand, in sheep follicular development upto Type Sa i.e. antral stage occurs in the ovary during fetal life in uter049. The molecular mechanisms

GDF-9 c-kit-kitL

Gonadotropin independent

BMP-15, T1MP-I

Steroid hormones

& Inlm(W~ri':!O

Gonadotro:pin dependent

fig. 4 - Factors regulating carly and late stages of folli culogenesis

-

.,

- f

NANDEDKAR & NARKAR: RELEVANCE OF STEM CELLS RESEARCH IN REPRODUCTION 733

that regulate the selection and initiation of growth of the primordial follicles is not well understood. Expression of oncogenes c-myc and erb-A is observed in oocytes of the primordial follicle and pregranulosa cells, hence these genes may be involved in autonomous growth 50. During preantral period the growth of the primary oocyte is arrested. Interaction between c-Kit in the oocyte and kit ligand in granulosa cell is the key regulator of initiation of growth from the resting pool of primordial follicles51

.

53. The tissues surrounding the primordial follicle coordinate the initiation of growth. This niche is evident since removal of stromal compartment in vitro led to marked increase in granulosa cell proliferation54

.

Anti-Mullerian Hormone (AMH) has been shown to inhibit recruitment of primordial follicles in knock out

mice and in follicle culture system. The TGF-~ superfamily includes TGF-~, actlvlIl, inhibin, Mullerian inhibiting substance (MIS/ AMH), growth and differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMP). These factors play important role in wide range of biological effects such as cell growth, morphogenesis, cell differentiation and apoptosis. Smad-2 and Smad-3 have recently been reported to be present in stage specific manner in ovarian follicles55

. BMP-15 and its close homologue GDF-9 are exclusively expressed by oocyte throughout folliculogenesis. GDF-9 is involved in the regulation of proliferation, cytodifferentiation and clonal expansion of granulosa cells ill vitro49

. BMP-15 knock out female mice exhibit reduced fertility . Mutation in bmpl5 gene (FecX') caused increased ovulation rates in ewes in heterozygotes while those with homozygous mutation showed arrest in follicle development at primary stage leading to infertility. BMP-15 is involved in granulosa cell proliferation during early follicular growth.

Thy-l a differentiation protein of vascular origin can signal the oocyte to remove a hypothetical arresting factor originating in the oocyte and acting upon the pregranulosa cellss6. Localization of erb-A, myc50 and Wilm's tumour (WT) gene on oocytes suggests that they might be involved in initiation of early follicular growth. WT-l expression decreases from the oocytes at primordial stage upto the antral stage follicles455 7

. Basic fibroblast growth factor (bFGF) in the primordial oocyte may interact with the granulosa cells from early preantral stage. FGF-2 and FGF receptor have been localized in the human fetal ovarian tissue5R

. Neurotropins such as NGF, BDNF, neurotropin 3 and 4 have been reported to regulate

ovarian follicular growth at early stages59. From this

stage onwards, LH and FSH control follicular growth along with the steroid hormones and intraovarian factors . At the formation of secondary follicles , granulosa cells develop FSH receptors and theca cells are formed. Theca cells express LH receptors. In gonadotropin dependent stage alongwith the local factors60 FSH and LH play an important role in follicular development.

Thus in early stages of follicular development the expression of several genes and proteins from various gene families have been reported . Yet the mecha·nism of the initiation of follicular growth and maturation is unknown.

Oocyte-granulosa cell interaction Throughout follicular development the

communication between the oocyte and its companion somatic cells, the granulosa cells is essential for oocyte development. The granulosa cell development and function are regulated by signals from oocytes.

The ligand-receptor system is also reported in the ovary with expression of both the AMH ligand and its receptor61 and kit-kit ligand62 postnatally in preantral follicles; receptors being located on granulosa cells while ligand expressed in the oocyte. Thus, these reports proposed a close proximity between oocyte and granulosa cells during early folliculogenesis which continues until fertilization of the egg with sperm. It is suggested that the presence of the receptor-ligand pair is essential to prevent apoptosis.

Oocyte promotes granulosa cell proliferation and differentiation63

. In turn, the oocyte depends on somatic cells to support its growth and development64

,

regulate meiosis65, and modulate transcriptional

activity in the oocyte genome66. A developmental

program, intrinsic to the oocyte, controls the rate of follicular development in neonatal mice suggesting a crucial role of the oocyte in regulating progression of foil icular development. Knock out mice lacking Factor In the Germline a (FIG a) an oocyte specific helix-loop-helix transcription factor, fail to develop

primordial follicles67. GDF-9 - a TGF ~ superfamily

member has been reported to be secreted by oocytes throughout folliculogenesis68

. Defects in granulosa cell proliferation and differentiation in the GDF9 knock out mouse ovary demonstrate arresl in primary stage of folliculogenesis indicating importance of the oocyte in follicular cell functions69

. In the absence of GDF-9, there is increased granulosa cell kit L expression and other granulosa cell anomalies that

734 INDIAN J EXP BIOL, JULY 2003

cause precocious oocyte growth and defects, eventually leading to oocyte demise7o. Kit ligand (Kit L) is derived from granulosa ceIls6H

• Mice homozygous for a hypomorphic kit L allele exhibit infertility as a result of block in follicular development preceding formation of secondary follicles 71. Recently , a negative feedback system between oocyte; bone morphogenetic protein IS, BMP-lS, and granulosa cell kit L has been reported by Otsuka and Shimasaki72 by co-culturing rat oocyte and granulosa cells. Yet, factors that trigger the development of primary follicles and oocyte-granulosa cell interaction are not well understood although somatic cell-derived anti­Mullerian hormone (AMH)73.74 and activin75 are implicated in the regulation of this process.

Signaling between the developing oocyte and the surrounding granulosa cells is facilitated by transzonal projections from granulosa cells that contact the oocyte surface and it also occurs via gap junctions. Gap junctions are collections of intercellular membrane channels that allow adjacent cells to share small molecules of less than I kDa. Gap junction channels are composed of connexins, a homologous family of more than 20 proteins. In developing follicles , gap junctions couple the growing oocyte and its surrounding follicle cells into a functional syncytium. Many connexins such as Cx26, Cx30.3, Cx32, Cx37, Cx40, Cx43, Cx4S and CxS7 are expressed within the oocyte-granulosa cell complex depending on the species, but role of each of these has not yet been identified. Gap junctions between cumulus cells contain predominantly connexin-43.The importance of connexin 43 for granulosa cell function is demonstrated by the fact that follicles lacking thi s connexins are arrested in early preantral stages and produce incompetent oocytes76. Connexin 37 appears to be the only connexin contributed by oocytes to the gap junctions coupling these with granulosa cells, and loss of this conncxin interferes with the development of antral follicles 77

. Thus, connexins may have multiple functions regulatin~ gap junctional communications in folliculogcnesis7 .It is well documented that ZP2 and ZP3 null mice are inferti Ie and ZP l null mice are only subfertile and ZP antigens play an important role in sperm egg interaction. Activin receptors and Smad proteins are present in the oocyte as well as granulosa cells in postnatal ovary suggesting that they playa functional role in early folliculogencsis79.

Granulosa cell proliferation is regulated by cell cycle genes such as cyclin 02 which may have

control over early folliculogenesis. It is therefore essential to study the bottle neck genes and proteins in the key pathway of recruitment of primordial follicles . Key questions in follicular development are which signals control the exit of follicles from the resting pool; and how are the dominant follicles selected49 . The oocyte specific factor GDF-9 suppresses the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA. Hence, the cumulus cells in large preovulatory follicle do not express LHCGR mRNA but mural granulosa cell do express it. The oocyte also regulates the expression of kit ligand (kitl) gene in mural granulosa cells but not in cumulus granulosa cells of the preovulatory follicles . Kit ligand or SCF from granulosa cells is known to be regulator of oocyte growth. Fully grown oocytes on the other hand suppress the kit ligand expression in cumulus cells but not the mural granulosa cells. Thus complex interactions occur between the oocytes and granulosa cells that are essential for their normal growth and function which needs to be explored.

Hom eostasis of the ovarian cells Study of proliferation and apoptotic mechanisms is

important in ovarian cells. Excess of non-viable germ and granulosa cells are eliminated early in ontogeny (often beginning before birth) and thereafter continuously throughout reproductive life. Developmentally regulated loss of female germ cells at various stages of pre and postnatal development is considered a normal physiological phenomenon ensuring the ovulation of best possible oocytes. However, abnormally high rates of attrition often result in subclinical or clinical infertility or the premature termination of fertility for example reproductive senescence or human menopause. Ovarian follicle atresia in vertebrate is mediated via apoptosis a process that can be initiated from within ovarian germ or follicle somatic cellsHO

.X1 or in

response to physiological (cytokines) s ignals~2 Survival and apoptosis of ovarian cells is mediated

by several intracellular factors . These include bcl-2 family of genes/proteins, members of Fas-Fas ligand pathway, inhibitor apoptosis proteins, Caspases and other cell signaling molecules such as adenylyl cyclase-cAMP, mitogen activated protein kinase and phosphoinositol-3-kinase-AKTx3

. Lee et al. M4 studied expression of apoptosis related genes and described the sequence of expression of genes known to induce cell death by apoptosis in earliest stages of follicular development. According to these studies Bcl-2 or Bax

NANDEDKAR & NARKAR: RELEVANCE OF STEM CELLS RESEARCH IN REPRODUCTION 735

express ion was absent in primordi al folli clesx4 . Fas and Fas ligand were present onl y in oocytes from primordi al follicles but not the surrounding granulosa cell s. In preantral follicl es again BcI-2 and Bax were abo ent but a weak staining for Fas and Fas Ligand was observed in oocyte as well as granulosa ce ll s. In atreti c follicl es Bax was hi ghly ex pressed in granulosa and theca ce ll sR5

. Fas and Fas li gand express ion was observed moderately in atreti c fo lli clesg6

.g7

. T he role of apotosis in ovari an tissue is important for understandi ng the process of atres ia and also to maintain oocytes in culture avoiding ce ll death .

Knowl edge regarding mechani sm of ovarian apoptos is wi II hel pin the development of strateg ies to treat ovarian cancers. Recentl y, role of caspase­mediated apoptosis w ith respect to ovarian cell type has been reported. Apoptos is initiated w ith oogonia and oocytes from primordial to preantral foll icles is caspase-2 dependent and can be attenuated by cell survi va l fact ors such as i nterleukin- I a ( I L-I a) and

III ~ retinoi c acid, LI F and insu lin like grow th factor (IGF)-l and IGF II and stem cell factor (SCF) via its receptor, Kit. The death of the oocyte leads ultimately to granul osa cell death and fo lli cul ar atres ia. Caspase-3 dependent apoptosis within the granulosa cell layer appears to be in volved in fo lli cle atres ia during an tral to preovulatory stages o f development. Factors such as LH, FSH. IGF-I , EGF and TGF-a are known to promote gran ulosa ce ll survivalx, .

While deve lop ing a culture of ovarian ce ll s whether oocytes, fo llicles or granulosa cell . , one has to combine knowledge o f all the growth factors and mol cular markers. Thi s w ill help in crea ting precise environment for the ovari an cell s ill I'il ro. Develop ing fo llicle culture systems and culturing immalUre oocytes has many benefits which includes fertility preservati on fo r humans. conserva ti on of rare animals and development of oocyte banks for research purpose. Cancer pat ients lose their fertility due to rad iation therapy. Therefore it is advantageous to cryoprcscrvc large number of gametes pri or to init iati ng cancer treatment. I f culture techniques are avai lab Ie, afterwards they can be grown and differentiated ill vi l ro.

In certain reproducti ve pathologica l condi tions such as Premature Ovarian Fai lure there is a drop in the number of oocytes in th e ovary, thus reducing chances of conception. These conditions also lead to drasti c hormonal imbalances that affect the reproductive and overall hea lth of the woman. In such cases stem cell therapy may help to regain functioning

of the ovary , allev iate hormonal insufficiency and overcome infertility .

Endometrium: Potential research area on stem cells Endometrium is another reproducti ve organ where

stem cell research may be carri ed out. Such stud ies w ill help in better understanding of embryo implantati on. The functi onali s wh ich occupies two third of the endometrial lining present immediately near the lumen is shed at the end of the menstrual cyc le in the absence of implantat ion. The basal i layer takes over in the new cyc le and repleni . hes th functi onali s layer in the fo lli cul ar phase8x

. Hence. th presence of adult stem cell s in the basa li s layer is evident. These are the same stem cell s, wh ich differentiate and prov ide a niche fo r the developing embryo at the time of implantat ion. Investi gation on the endometri al stem cell s is essenti al for under­standing on embryo implantat ion and development. Stem cell s w ill also prov ide an in vitro model for study of endometri al disorder such as uterine fibroids, hyperpl asia and adenocarcinoma. It may answer some of the basic questi on such as why implantation fai ls in some of the women, how menstrual irregulariti es occur and so on. Recently. it has been reported that the molecules involved in implantation and those preventing immune reject ion have been identified to be the sameR'i. Hence studies in thi s area will also be usefu l in transplantation of the tissues.

However, to our know ledge identi fi cati on and culture of stem cells from endometrium has not been reported and thi s is an important area, wh ich can be explored further.

Ethical considerations of stem cell research The employment of hu man IVF embryos for

production of stem cell s has raised a debate about ethics. Hence many countri es have restri cted their use. Since the primit ive streak starts forming around day 14 of gestati on, it has been argued th at Ii fe begi ns onl y then. So the countri es w ith la\ovs that allow human embryo research , set a limit for usuall y of 14 day-embryos. The cultu re o f embryonic ce ll s after the 141h day should face ethica l prob lems since the organization of the embryo is lost in the process of cell culture and monolayer of ES- like ce ll s with no potenti al of developing into a human embryo is formeLi. However. as these cell s possess the capabil ity to produce clones utmost caut ion should be taken to mili imize the ri sk. In European countri es such as

736 INDIAN J EXP RIOL. JULY 2003

Denmark , Hungary, Spain , Sweden and Un ited Kingdom embryo research is regulated by l aw~o Recentl y French law on bioethi cs has been proposed to be rev ised. A llowance o f studi es on human c loning is mentioned in the proposa l'!l. Some cou ntri es such as Australia and German y all ow work ing w ith imported stem ce ll lines. Japan and Israel permit s work w ith 'spare' I VF embryos . The United States Con!!rcss prohibits federal fund ing for human cmbryo research but pri vately funded proj ec ts are al lowed to

kIlO C 'd' I I" (. wor · . onsl erlng tl l! app Ica tl on 0 stem ce ll research in elucidating the mechanism of dillerent iat ion and as a source o f ce ll s for drug discovery and tran splantat ion therapi es , th e studi es in thi s area need to be strongl y promoted.

Indian scenario Ev idence from ancien t references such as

M ahabharat sugges ts that stem ce ll research is not new to India. Ad iparva o f the Mahabharat mentions the birth of one hundred KaliN/ IIOS deri ved from a pilld (a ball of fl esh) o f a si ngle aborted fetus. The method sage Dwa ipayana describes for the treatment of the feta l ti ss ue is comparable to that used for embryonic stem cell s in modern times. A group o f sc ienti . ts beli eve it to be one or the los t sc iences of Ind i a<)~.

In rece nt times too India is one of the major countri es contri buting to stem cell research. Reli ance Life Sciences is working w ith ten of the sixty - four stem ce ll lines approved by the US at ional Institute o f Health. Dr. Panicker's group from ati onal Centre for Biological Sc iences al so has been working in thi s area. In India there are few ethi ca l concerns so far in the use of embryonic stem cell s although scienti sts agree th at the donor's consent is essential. The Department of Biotechno logy has framed guidelines for such research. Just recently, Indian Council for Medica l Research has released a draft of guidelines. which recommends ban on export o f human embryonic ti ssue93

. This is parti cularl y to prevent misuse of human embryos ava ilable in several fertility clini cs that have sprung up throughout the country. It also prohibits selling o f embryos by the couple although donati on of spare embryos is al lowed. But Indian sc ienti sts are afra id that it may affec t the research work on embryonic stem cell s that is In co ll aborat ion wi th the foreign laboratories.

Studies in adult stem ce ll. ' are also in progress In many inst itutes in India. L.V.Pra. ad Eye Institute in Hyderabad has success fully used limbal stem cells ror

transplantation. It is creditabl e that apart from India such trea tment is ava ilable on ly in two other countri es that are the US and Taiwan. Dr Matarurkar and hi s co lleagues from Maulana Azad M edica l Co llege, New Delhi have obtained US patent for a technique which enables the regenerati on of damaged organs and ti ss ues')4 A mong the other studi es in India on stem ce l ls are: studies on bone marrow stem cell s for the trea tment of thalassaemia and sick Ie cell anaemia by Institut e or Il11munohaematology. MUl11ba i anel neural stem ce ll studi es for the treatment or A lzheimer's disease by -.!a tional Brain Research Center. Delhi. However the stem cell research in th e fi eld o f reproducti on appe, rs to be neglec ted in the India. Studies in thi s area should be promoted. as it w ill prov ide l' l"fec tive contracepti ve measu res that are essential for population contro l in Indi a.

Su 1111113 ry Looking at the past and future areas o f stem cell

research in reproducti on there is a vas t scope for studies in thi s f ield . E luc idating the molecular markers of stem cell ~ and d irecting their differentiati on ill lI il m by provid ing accur' te combinati on o f growth factors are two major chall enges for stem cell researchers. I n reproducti on stem ce ll s are important in various ways. Embryonic germ ce ll s deri ved from the genital ridge of aborted fe tu s se rve as the use ful source of pluripotent stem cell s. They serve as good model fo r genomic imprinting and embryogene:' is studies. The germ cell s are also useful for the stud ies on male and rema le germ cell development. Us ing thi s knowledge it w ill be possible to dev ice new contracepti ve measures and improve treatment ror infertility. T es ticul ar and ovari an niche are being in ves ti gated by va ri ous scienti sts aiming at their culture ill vitro. The cultured germ cell s may be utili zed for cell based therapi es for reproductive disorders and transpl antati on in reproducti ve organs. The future perspect i ves of ·tem cell research are preserv ation of germ ce lls in onco logical pati en ts, generation of tran. genic animals and preservati on of enda.ngered animals. Ethical issues about embryonic ti ssue have led many countri es including India to restri ct their use. But since it holds potential to develop contracepti ve measures it should be encouraged in India.

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regu laled during the di lTerenlialion or embryonic Slem ce ll s. Mil l I?eprod Del'. 56 (2000) I 13.

NANDEDKAR & NARKAR : RELEVANCE OF STEM CELLS RESEA RCH IN REPROD UCTION 737

2 Vemuganti G S & Balasubramanian D. Heralding the dawn of cuilured adult stem cell transplantation. II/dial/ J Biolechl/ol. I (2002) 39.

3 Love ll -Badge R, The futu re for SlCm ce ll research, Nil/lire, 414 (200 1) l.'R.

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6 Shalllb ioll M J, Axelm<l n J & Wang S. el al . Deri vati on of pluripotent stem ce ll s from cullllred germ ce ll s. PNAS. 95 ( 1998) 13726.

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9 Ezzell C, The child within. Sci Alii. (2002) 16. 10 D'A mour K A & Gage F.H. A re somati c stem ce ll s

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