Homozygous Inactivating Mutation in NANOS3 in Two Sisters with Primary Ovarian Insufficiency

Research ArticleHomozygous Inactivating Mutation in NANOS3 in Two Sisterswith Primary Ovarian Insufficiency

Mariza G Santos1 Aline Z Machado1 Conceiccedilatildeo N Martins1 Sorahia Domenice1

Elaine M F Costa1 Mirian Y Nishi1 Bruno Ferraz-de-Souza2 Soraia A C Jorge3

Carlos A Pereira3 Fernanda C Soardi4 Maricilda P de Mello4 Andrea T Maciel-Guerra5

Gil Guerra-Junior6 and Berenice B Mendonca1

1 Unidade de Endocrinologia do Desenvolvimento Laboratorio de Hormonios e Genetica MolecularLIM-42Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo Avenida Dr Eneas de C Aguiar 155 2 andar Bloco 605403-900 Sao Paulo SP Brazil

2 Laboratorio de Carboidratos e RadioimunoensaiosLIM-18 Hospital das Clınicas da Faculdade de Medicina da Universidade deSao Paulo Avenida Dr Arnaldo 455 01246-903 Sao Paulo SP Brazil

3 Laboratorio de Imunologia Viral Instituto Butantan Avenida Vital Brasil 1500 05503-900 Sao Paulo SP Brazil4 Centro de Biologia Molecular e Engenharia GeneticaCBMEG Faculdade de Ciencias Medicas Universidade Estadual de Campinas13083-970 Campinas SP Brazil

5 Departamento de Genetica Medica Faculdade de Ciencias Medicas Universidade Estadual de Campinas Rua Tessalia Vieira deCamargo 126 13083-970 Campinas SP Brazil

6Departamento de Pediatria Faculdade de Ciencias Medicas Universidade Estadual de Campinas Rua Tessalia Vieira deCamargo 126 13083-970 Campinas SP Brazil

Correspondence should be addressed to Berenice B Mendonca beremenuspbr

Received 10 March 2014 Revised 30 May 2014 Accepted 3 June 2014 Published 26 June 2014

Academic Editor Svetlana Lajic

Copyright copy 2014 Mariza G Santos et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Despite the increasing understanding of female reproduction the molecular diagnosis of primary ovarian insufficiency (POI) isseldom obtained The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanosfamily have an evolutionarily conserved function in germ cell development andmaintenance by repressing apoptosisWeperformedmutational analysis of NANOS3 in a cohort of 85 Brazilian women with familial or isolated POI presenting with primary orsecondary amenorrhea and in ethnically-matched control womenAhomozygous pGlu120Lysmutation inNANOS3was identifiedin two sisters with primary amenorrheaThe substituted amino acid is located within the second C2HCmotif in the conserved zincfinger domain ofNANOS3 and in silicomolecularmodelling suggests destabilization of protein-RNA interaction In vitro analyses ofapoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by thismutation The identification of an inactivating missense mutation in NANOS3 suggests a mechanism for POI involving increasedprimordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins inhuman ovarian biology

1 Introduction

Primary ovarian insufficiency (POI) is characterized byovarian failure in women under the age of 40 years [1 2]POI may present as primary amenorrhea (PA) in severe cases

with prepubertal onset or postpubertally as secondary amen-orrhea (SA) associated with infertility hypoestrogenism andelevated gonadotropins (FSH gt 40UL) This complex spec-trum of progressive ovarian abnormality is largely relatedto the size of primordial germ cells (PGCs) pool where

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 787465 8 pageshttpdxdoiorg1011552014787465

2 BioMed Research International

prepubertal onset might reflect a complete lack of germcells since birth causing a failure in the maintenance ofovarian somatic structure and postpubertal onset wouldreflect a variably insufficient pool of oocytes This disorderis associated with female infertility and affects 1 to 2 of allwomen [3ndash5]

Several genetic mechanisms may lead to POI includingchromosomal abnormalities of the X chromosome or auto-somes and autoimmune disorders [6] Despite the increas-ing understanding of female reproduction defined causesor mechanisms resulting in primary ovarian insufficiencyremain largely unknown [7] Persani et al have estimated theprevalence of known genetic alterations in POI patients orig-inally classified as idiopathic to be around 20 to 25 of cases[8] whereas others have found this prevalence to be loweraround 10 Rare mutations have been described in genesinvolved in ovarian development andor function such asFSHR (MIM 136435) LHCGR (MIM 152790) BMP15 (MIM300247) POF1B (MIM 300603) NOBOX (MIM 610934)INHA (MIM 147380) GDF9 (MIM 601918) NR5A1 (MIM184757) and FIGLA (MIM 608697) and in meiotic genes[9ndash23] Nevertheless mutations in these genes account fora minority of cases of ovarian dysfunction indicating thatadditional factors remain to be identified

Nanoswas first identified inDrosophila where it repressesthe translation of target mRNAs through binding to their 31015840UTR and has a conserved function in germ cell developmentacross species Members of the evolutionarily conservedNanos gene family are preferentially expressed in the ovariesand are known to play an important role in germ cell devel-opment maintenance and survival [24ndash30] In Drosophilathe single Nanos gene (Nos) is required for development ofthe abdomen as well as for germ line maintenance [31 32]Three Nanos homologues exist in mouse with Nanos2 andNanos3 functioning primarily inmale germ cell developmentandmaintaining PGCs viability respectively [33 34] InmiceNanos3 is expressed in the primordial germ cells (PGCs) fromtheir formation until shortly after their appearance in thegonads (E135 in female and E145 in male embryos) [24]Male and female mice deficient in Nanos3 are infertile andfemale 1198731198861198991199001199043minusminus mice have atrophic ovaries in which nogermcells are detectable due to loss ofmigrating PGCsduringembryogenesis [24] PGCs are lost by apoptosis in the absenceof Nanos3 establishing an essential function of Nanos3 as arepressor of apoptosis in the germ cell [34]

A similar conserved function for NANOS proteins hasbeen shown in humans NANOS1 and NANOS2 seem tobe mainly involved with male germ cell development andmaintenance [35] and indeed NANOS1 mutations have beenassociated with male infertility manifested as nonobstructiveazoospermia or oligozoospermia [36]NANOS3 on the otherhand is expressed in both male and female fetal and adultgonads and in vitro evidence suggests a pivotal function inhuman germ cell development [37]

Despite this compelling evidence for the importance ofNANOS3 in germ cell maintenance initial efforts to identifyNANOS3mutations in women with POI were not successfulin 2007 Qin et al analyzed 168 infertile Caucasian andChinese women and failed to identify pathogenic mutations

[38] More recently howeverWu et al performedmutationalanalysis of the coding regions of NANOS1 NANOS2 andNANOS3 in 100 Chinese POI patients identifying a het-erozygous pArg153Trp NANOS3 mutation in a 23-year-oldwoman [39]ThemutantNANOS3 proteinwas shown to havedecreased stability in vitro leading the authors to postulatethat reduced expression of NANOS3 in the ovaries wouldresult in decreased PGC population and POI [39] Herein wereport a homozygous missense mutation in Nanos3 found intwo sisters from our cohort of 85 Brazilian women with POIand present supporting in vitro functional data

2 Materials and Methods

21 Subjects This study was approved by the ethics commit-tee of Hospital das Clinicas University of Sao Paulo School ofMedicine Brazil (Protocol number 122607)

Eighty-five patients with POI were referred to the Devel-opmental Endocrinology Unit of Hospital das Clinicas SaoPaulo or to the Pediatric Department of University ofCampinas in Sao Paulo Brazil for evaluation of primaryamenorrhea (45 patients 10 familial cases) or secondaryamenorrhea (40 unrelated patients) The age at diagnosisof amenorrhea was 292 plusmn 59 years (18 to 39 yrs) Parentalconsanguinity was present in 80 of familial cases and noother relevant familial history was reported Each family had2 affected individuals and age at diagnosis of familial caseswas 194 plusmn 62 years (14 to 36 yrs) FSH levels were elevatedin all patients ranging from 27 to 150UL in patients withprimary amenorrhea and from 32 to 158UL in patients withsecondary amenorrhea Mutations in FSHR NR5A1 BMP15and GDF9 premutations in FMR1 and thyroid adrenalor ovarian autoimmune disorders had been excluded in allpatients Ethnically and age-matched (23 to 39 years old)women with normal menarche and menstrual cycles wereinvited to participate as controls Institutional review boardapproval and written informed consent were obtained fromall subjects before sample collection for DNA analysis

22 Mutational Analysis of NANOS3 Two protein-codingtranscripts of Nanos3 are described in the Ensembl databasea 581 bp transcript (ENST00000339133) resulting in a 192-residue protein and a 776 bp transcript (ENST00000397555)resulting in a 173-residue protein only transcriptENST00000339133 is part of the human consensus codingsequence set (CCDS)

Following genomic DNA extraction from peripheralblood leukocytes the entire exonic region and at least 15 bpof exonintron boundaries of transcript ENST00000339133(RefSeq NM 0010986222) were PCR-amplified and submit-ted to direct automated sequencing in an ABI PRISM 310(Applied Biosystems Foster City CA)

23 Apoptosis Detection by Flow Cytometry A pCMV6-AC-GFP-NANOS3 expression vector (OriGene Technolo-gies Rockville MD) was used as a template to generate ac358GgtAmutant using the QuikChange site-directed muta-genesis kit (Stratagene La Jolla CA) and specific primers

BioMed Research International 3

Wild type

c358GgtA

(a)

1 192

2nd C2HC motif

112 128

Zinc finger

pGlu120Lys (pE120K)

D melanogasterMacaca mulattaHomo sapiensPan troglodytesBos taurusRattus norvegicusMus musculus

(b)

Figure 1 AMutational analysis ofNANOS3 revealed a homozygous c358GgtA substitution in exon 1 in two sisters with primary amenorrheaThe GAG to AAG change in codon 120 results in a substitution from glutamic acid to lysine in the codified protein (pGlu120Lys) B Cartoonrepresentation of the NANOS3 protein (192 amino acids) with the zinc finger domain (amino acid 76 to 130) depicted in yellow showing thelocation of the pGlu120Lys mutation Glutamic acid 120 (shown as E120) is localized within one of two highly conserved Cys-Cys-His-Cys(C2HC) motifs (shaded red and yellow boxes) Conservation among species of the second C2HCmotif sequence (human residues 112 to 128)is shown in detail glutamic acid 120 (E) is highly conserved Alignment was performed in Clustal Omega using protein sequences obtainedfrom UniProt

(F 51015840 GGC GCC ACA CGT AAG CGC GCC CAC AC31015840 R 51015840 GTG TGG GCG CGC TTA CGT GTG GCGCC 31015840) African green monkey kidney COS-1 cells weretransiently transfected using Lipofectamine 2000 (InvitrogenCarlsbad CA) and analyzed 48 hours later with or withouttreatment with 5mM sodium butyrate (Calbiochem SanDiego CA) Cells were stained with Annexin-V conjugates(Molecular Probes Invitrogen Carlsbad CA) according tomanufacturerrsquos protocol GFP and Annexin-V fluorescencewere excited at 488 nm and 650 nm and emission measuredusing 53030 nm and 66030 band pass filters respectively

24 Confocal Microscopy Confocal microscopymicrographswere obtained using a 488 nm laser line and light emittedbetween 500 and 600 nm for GFP detection and a 650 nmlaser line and light emitted between 660 and 700 nm forred Annexin-V detection Images were collected on a METALSM 510 laser scanning confocal microscope equipped with a630 times 12Wobjective (Carl Zeiss Jena Germany)The samesettings for image acquisition and processing were applied forall samples to allow comparison of the fluorescence intensitiesamong different samples

25 Molecular Modeling of NANOS3 A model for mutatedhuman NANOS3 was built using the resolved structureof L3MBTL1 (PDB-ID 2RI7) as template since NANOS3structure has not yet been resolved In order to find thebest model for NANOS3 a Blast alignment with proteinsavailable in PDB was performed Highest homology wasfound with the crystal structure of Nanos from zebrafish(PDB-ID 3ALR) with 58 identity However this structure

did not include the region where the pGlu120Lys mutationis located Therefore we looked for another protein bearingsimilarities with NANOS3 Such protein was L3MBTL1Its X-ray resolved structure presented the second highesthomology with NANOS3 (33 of identity) and includedthe pGlu120Lys mutant region Molecular modeling wasperformed with the SwissModel web-served program andimages examined and edited using the web-based BlueStarSTING (CNPTIA-Embrapa Brazil) and PyMOL softwares(freely available at httpwwwpymolorg)

3 Results

31 Identification of NANOS3 Mutation Mutational analysisof NANOS3 revealed that two sisters carried a homozygousc358GgtA pGlu120Lys mutation (Figure 1) which was notdetected in 113 ethnically matched control women and hasnot been reported in the 1000 Genome Project or dbSNPdatabases Their mother was heterozygous and the fatherrsquosDNA was not available for study In the remaining 83 womencomposing our POI cohort no new variants were identifiedin NANOS3 Two previously described SNPs were identifiedin our cohort rs897790 and rs2016163 (Table 1)

The patients carrying the homozygous c358GgtA muta-tion were from the northeast region of Brazil (Piauı State)and born to nonconsanguineous parents At 12 and 16 yearsold they were evaluated for breast underdevelopment Bothgirls had not reached menarche and physical examinationrevealed Tanner stage I breast development and Tanner stageIII pubic hair Basal gonadotropin levels were elevated withpredominantly high FSH levels (LH = 63 and 21UL

4 BioMed Research International

Table 1 Polymorphic variants in NANOS3 identified in POI patients and controls

Genotype frequency ()Variant dbSNP id Patients with POI Controls

Wild-type Heterozygote Homozygote Wild type Heterozygote Homozygotec-23CgtT (51015840 UTR) rs897790 60 341 59 725 257 18c353AgtG (exon 1 synonymous) rs2016163 506 40 94 70 274 26

FSH = 150 and 61UL resp) Estradiol levels were withinthe prepubertal range in both girls Measured heights were145 and 156 cm and weights were 38 and 46Kg respectivelyPelvic ultrasonography showed no masses and ovaries werenot visualized Treatment with conjugated estrogens followedby progesterone replacement resulted in complete breastdevelopment and normal cycling

Their heterozygous mother has a history of menarche at16 years old and difficulties to conceive Her first pregnancywas at 27 years old after 4 years of attempts to conceive andher second and last gestation occurred at 30 Menopauseoccurred at 51 years of age

32 Increased Apoptosis In Vitro with pGlu120Lys MutantNANOS3 Considering that Nanos3 has been shown tomaintain germ cell development inmodel organisms throughrepression of apoptosis [34] in vitro studies were performedto assess the effects of the pGlu120Lys substitution on theapoptotic rate of cultured cells COS-1 cells were transfectedwith GFP-tagged wild-type or mutant NANOS3 and viabilityassessed by Annexin-V staining Flow cytometry analysisshowed that the percentage of Annexin-V stained GFP-positive cells was significantly higher in cells transfected withmutant NANOS3 in comparison to wild type in untreated orsodium butyrate-treated cells (119875 lt 005) (Figure 2) indicat-ing that the pGlu120Lys substitution profoundly impairs cellviability

Since NANOS3 has been shown to act in the cell nucleus[37] studies were performed to investigate whether thepGlu120Lys mutation would affect intracellular localizationTransfected COS-1 cells were analyzed by confocal laser scan-ning microscopy for cellular localization of GFP-tagged wildtype or mutant NANOS3 and Annexin-V staining (Figure 3)Nuclear localization was observed in cells transfected withboth wild type and mutant cDNA However Annexin-Vstaining in the plasma membrane indicating apoptosis wasonly detected in cells expressing the pGlu120Lys mutantNANOS3

33 In SilicoMutant NANOS3Modelling Glutamic acid120 isa negatively charged amino acid locatedwithin the zinc fingerdomain This residue is found at the surface of NANOS3within the second Cys-Cys-His-Cys (C2HC) motif whichis important for its ability to bind RNA [40] In generalsubstitution of amino acids that share similar hydrogenbinding capabilities and tridimensional requirements such asglutamic acid and lysine has limited structural consequencesHowever lysine has a positive charge on the aliphatic sidechain that can affect DNA binding activity when inserted

0

1

2

3

4

5

Flu

ores

cent

cells

(Ann

exin

-V) (

)

NTWTMUT

Apoptosis detection

NaBuminus NaBu+

Figure 2 Detection of apoptosis using flow cytometry andAnnexin-V staining in COS-1 cells transfected with wild type (WT)or pGlu120Lys mutant (MUT) NANOS3-GFP before and afterinduction of apoptosis with sodium butyrate (NaBu) Expressionof mutant NANOS3 results in significantly higher apoptosis incomparison to wild type (119875 lt 005) Error bars represent SEM fornine replicates NT nontransfected control

into the zinc finger domain In silico analysis shows thatlysine 120 establishes a new contact with Lys96 and abolishesthe original contact with His123 (Figure 4) Consequentlythese changes could lead to internal contact conformationalmodifications resulting in impaired protein function Inaddition 3D structure indicates that the negatively chargedglutamic acid 120 residue is surrounded by negatively chargedresidues (Figure 5) Therefore the presence of the positivelycharged lysine 120 residue in the protein surface might causestrong electrostatic repulsion among the side chains and leadto destabilization upon protein-RNA interaction

4 Discussion

The present study aimed to investigate the contribution ofvariants in NANOS3 to human POI We have identifieda nonsynonymous homozygous NANOS3 mutation in twosisters with primary amenorrhea

During development once germ cells populate thegonads they are exposed to several apoptotic wavesNANOS3 plays an important role in the maintenance andsurvival of primordial germ cells and therefore constitutesan interesting candidate gene for nonsyndromic POI IndeedNANOS3 has been shown to function in germ cell devel-opment in a breadth of organisms from flies and wormsthrough frogs and mammals In mice loss of Nanos3 results

BioMed Research International 5

Nanos3-GFP Annexin-V Merge

WT

5120583m 5120583m 5120583m

(a)

Mut

5120583m 5120583m 5120583m

(b)

Figure 3 Confocal microscopy analysis of apoptosis in COS-1 cells following 48 h induction of apoptosis with sodium butyrate In cellstransfected with wild type (WT upper row) or pGlu120Lys mutant (Mut lower row) GFP-tagged NANOS3 expression of green NANOS3-GFP is seen predominantly in the nucleus Red Annexin-V staining in the cytoplasm and plasma membrane indicating apoptosis is seen incells expressing pGlu120Lys mutant NANOS3 but not in those expressing WT NANOS3

Wild type Mutant

T124

E120

K96

G116

T124

H123

K120

G116

Figure 4 In silico analysis of amino acid interactionmodification shows the different conformation conferred by the pGlu120Lys substitutionGlutamic acid 120 (shown as E120) makes a hydrophobic interaction with lysine 96 (K96) and hydrogen bonds with both glycine 116 (G116)and threonine 124 (T124) The substitution to lysine 120 (K120) modifies structural points of contact establishing a new interaction withhistidine 123 (His123) through aromatic stacking and abolishes the hydrophobic interaction with K96

6 BioMed Research International

H123

H90

R119

K93

R121R126

E120

H123

H90

R119

K93R121

R126

K120

Figure 5 Bulk representation of wild-type NANOS3 with pGlu120Lys mutant shown in details box The dashed circle highlights a proteinsurface region rich in basic residues shown in blue At the center of this region lies the acidic residue glutamic acid 120 (E120) shown in redIn the details substitution by lysine 120 (K120) disturbs the electrostatic interactions among adjacent residues

in infertility due to apoptosis of migrating PGCs during fetallife resulting in atrophic gonads where germ cells are absent[24 34] Furthermore the contribution of Nanos3 to PGCmaintenance and survival seems to be dosage-dependent inmice as a model with attenuated Nanos3 transcript levelsdisplayed markedly reduced numbers of PGCs [39]

In humans NANOS3 is expressed in fetal and adultovaries during multiple stages of oogenesis and in vitro stud-ies of human embryonic stem cell-derived germ cells haveshown coexpression of NANOS3 in nuclei with importantgerm cell factors such as BLIMP1 VASA and STELLA inthe cytoplasm [37] In these cells reduction of NANOS3expression resulted in altered gene expression patterns andreduced number of cells in active division suggesting thatin humans too NANOS3 has an important function ingerm cell maintenance and survival Recently a heterozygouspArg153Trp NANOS3 mutation was reported in a 23-year-old Chinese woman out of a cohort of 100 women withPOI [39] Familial segregation was not defined but themutation was not found in 200 ethnically-matched controlsIn vitro studies suggested decreased protein stability due tothe pArg153gtTrp mutation leading the authors to postulateamechanismof reduced dosage ofNANOS3 expression in theovaries leading to decreased PGC population and resulting inPOI [39]

The pGlu120Lys mutation identified in our patients lieswithin the NANOS3 zinc finger domain which is composedof two C2HCmotifs located between amino acids 77 and 131The differences in chemical properties between amino acidsmight lead to impaired binding of NANOS3 to its mRNAtargets as suggested by protein modeling In vitro experi-ments show that the mutant NANOS3 identified in the twosisters with POI was associated with increased apoptosis in

transfected cells suggesting that loss of NANOS3-mediatedprotective effect against apoptosis in primordial germ cellsmay be the mechanism underlying POI in these patients

Primordial germ cell pool size in the developing ovaryand maintenance of these cells throughout life are at the cen-ter of the pathogenesis of POI It is remarkable that our twopatients with homozygousNANOS3mutation have presentedwith primary amenorrhea perhaps indicating more severelyPGC-depleted ovaries while their heterozygous mother haddifficulty to conceive which could represent a manifestationof insufficient PGC pool size Indeed the patient describedby Wu et al carried a heterozygous missense change andpresented with secondary rather than primary amenorrhea[39] supporting the concept of a dosage effect for NANOS3-related POI

In conclusion we report a rare homozygous missensemutation in NANOS3 in two Brazilian sisters with primaryamenorrhea from a cohort of 85 Brazilian women withPOI In vitro and in silico functional studies support thatthis inactivating mutation abolishes NANOS3 ability to pre-vent apoptosis suggesting a mechanism for POI involvingincreased PGC apoptosis during embryonic cell migration

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank patients and their families for partici-pating in this study They are grateful to Jorge Mario C F

BioMed Research International 7

Junior (Flow Cytometry Laboratory Instituto Butantan) andToshie Kawano andAlexsander Seixas de Souza (ParasitologyLaboratory Instituto Butantan) for technical assistance Thiswork was supported by Conselho Nacional de Desenvolvi-mento Cientıfico e Tecnologico (CNPq Grant 3013392008-9) and the Sao Paulo Research Foundation (FAPESP Grants0504726-0 07512156 and 1051102-0)

References

[1] C K Welt ldquoPrimary ovarian insufficiency a more accurateterm for premature ovarian failurerdquo Clinical Endocrinology vol68 no 4 pp 499ndash509 2008

[2] L M Nelson ldquoClinical practice Primary ovarian insufficiencyrdquoThe New England Journal of Medicine vol 360 no 6 pp 606ndash614 2009

[3] D Goswami and G S Conway ldquoPremature ovarian failurerdquoHuman Reproduction Update vol 11 no 4 pp 391ndash410 2005

[4] L M Nelson S N Covington and R W Rebar ldquoAnupdate spontaneous premature ovarian failure is not an earlymenopauserdquo Fertility and Sterility vol 83 no 5 pp 1327ndash13322005

[5] P Beck-Peccoz and L Persani ldquoPremature ovarian failurerdquoOrphanet Journal of Rare Diseases vol 1 no 1 article 9 2006

[6] D Toniolo ldquoX-linked premature ovarian failure a complexdiseaserdquo Current Opinion in Genetics and Development vol 16no 3 pp 293ndash300 2006

[7] M M Matzuk and D J Lamb ldquoThe biology of infertilityresearch advances and clinical challengesrdquoNatureMedicine vol14 no 11 pp 1197ndash1213 2008

[8] L Persani R Rossetti and C Cacciatore ldquoGenes involvedin human premature ovarian failurerdquo Journal of MolecularEndocrinology vol 45 no 5 pp 257ndash279 2010

[9] P Laissue G Vinci R A Veitia and M Fellous ldquoRecentadvances in the study of genes involved in non-syndromic pre-mature ovarian failurerdquo Molecular and Cellular Endocrinologyvol 282 no 1-2 pp 101ndash111 2008

[10] R Rossetti E D Pasquale A Marozzi et al ldquoBMP15 mutationsassociated with primary ovarian insufficiency cause a defectiveproduction of bioactive proteinrdquo Human Mutation vol 30 no5 pp 804ndash810 2009

[11] Y Qin Y Choi H Zhao J L Simpson Z Chen and ARajkovic ldquoNOBOX homeobox mutation causes prematureovarian failurerdquo The American Journal of Human Genetics vol81 no 3 pp 576ndash581 2007

[12] D Toniolo and F Rizzolio ldquoX chromosome and ovarian failurerdquoSeminars in Reproductive Medicine vol 25 no 4 pp 264ndash2712007

[13] A LacombeH Lee L Zahed et al ldquoDisruption of POF1B bind-ing to nonmuscle actin filaments is associated with prematureovarian failurerdquo American Journal of Human Genetics vol 79no 1 pp 113ndash119 2006

[14] A N Shelling K A Burton A L Chand et al ldquoInhibin acandidate gene for premature ovarian failurerdquo Human Repro-duction vol 15 no 12 pp 2644ndash2649 2000

[15] A Marozzi C Porta W Vegetti et al ldquoMutation analysis ofthe inhibin alpha gene in a cohort of Italian women affected byovarian failurerdquo Human Reproduction vol 17 no 7 pp 1741ndash1745 2002

[16] H Dixit L K Rao V V Padmalatha et al ldquoMissensemutationsin the BMP15 gene are associated with ovarian failurerdquo HumanGenetics vol 119 no 4 pp 408ndash415 2006

[17] P Laissue S Christin-Maitre P Touraine et al ldquoMutationsand sequence variants in GDF9 and BMP15 in patients withpremature ovarian failurerdquo European Journal of Endocrinologyvol 154 no 5 pp 739ndash744 2006

[18] E Kovanci J L Simpson P Amato et al ldquoOocyte-specific G-protein-coupled receptor 3 (GPR3) no perturbations found in82womenwith premature ovarian failure (first report)rdquo Fertilityand Sterility vol 90 no 4 pp 1269ndash1271 2008

[19] X X Zhao N Suzumori M Yamaguchi and K SuzumorildquoMutational analysis of the homeobox region of the humanNOBOX gene in Japanese women who exhibit prematureovarian failurerdquo Fertility and Sterility vol 83 no 6 pp 1843ndash1844 2005

[20] D Loureno R Brauner L Lin et al ldquoMutations in NR5A1associated with ovarian insufficiencyrdquoTheNew England Journalof Medicine vol 360 no 12 pp 1200ndash1210 2009

[21] B Mandon-Pepin P Touraine F Kuttenn et al ldquoGeneticinvestigation of four meiotic genes in women with prematureovarian failurerdquo European Journal of Endocrinology vol 158 no1 pp 107ndash115 2008

[22] H Zhao Z Chen Y Qin et al ldquoTranscription factor FIGLA ismutated in patients with premature ovarian failurerdquo AmericanJournal of Human Genetics vol 82 no 6 pp 1342ndash1348 2008

[23] Z Z Zhao J N Painter J S Palmer et al ldquoVariation in bonemorphogenetic protein 15 is not associated with spontaneoushuman dizygotic twinningrdquo Human Reproduction vol 23 no10 pp 2372ndash2379 2008

[24] M Tsuda Y Sasaoka M Kiso et al ldquoConserved role of nanosproteins in germ cell developmentrdquo Science vol 301 no 5637pp 1239ndash1241 2003

[25] L Mosquera C Forristall Y Zhou and M L King ldquoA mRNAlocalized to the vegetal cortex of Xenopus oocytes encodes aproteinwith a nanos-like zinc finger domainrdquoDevelopment vol117 no 1 pp 377ndash386 1993

[26] M Pilon and D A Weisblat ldquoA nanos homolog in leechrdquoDevelopment vol 124 no 9 pp 1771ndash1780 1997

[27] K Subramaniam and G Seydoux ldquonos-1 and nos-2 two genesrelated toDrosophila nanos regulate primordial germ cell devel-opment and survival in Caenorhabditis elegansrdquo Developmentvol 126 no 21 pp 4861ndash4871 1999

[28] A Forbes and R Lehmann ldquoNanos and Pumilio have criticalroles in the development and function of Drosophila germlinestem cellsrdquo Development vol 125 no 4 pp 679ndash690 1998

[29] M Koprunner C Thisse B Thisse and E Raz ldquoA zebrafishnanos-related gene is essential for the development of primor-dial germ cellsrdquo Genes amp Development vol 15 no 21 pp 2877ndash2885 2001

[30] H Kurokawa Y Aoki S Nakamura Y Ebe D Kobayashiand M Tanaka ldquoTime-lapse analysis reveals different modes ofprimordial germ cell migration in the medaka Oryzias latipesrdquoDevelopment Growth and Differentiation vol 48 no 3 pp 209ndash221 2006

[31] S Kobayashi M Yamada M Asaoka and T KitamuraldquoEssential role of the posterior morphogen nanos for germlinedevelopment inDrosophilardquoNature vol 380 no 6576 pp 708ndash711 1996

[32] H Sano M Mukai and S Kobayashi ldquoMaternal Nanos andPumilio regulate zygotic vasa expression autonomously in the

8 BioMed Research International

germ-line progenitors of Drosophila melanogaster embryosrdquoDevelopment Growth amp Differentiation vol 43 no 5 pp 545ndash552 2001

[33] A Suzuki and Y Saga ldquoNanos2 suppresses meiosis and pro-motes male germ cell differentiationrdquo Genes amp Developmentvol 22 no 4 pp 430ndash435 2008

[34] H Suzuki M Tsuda M Kiso and Y Saga ldquoNanos3 maintainsthe germ cell lineage in the mouse by suppressing both Bax-dependent and -independent apoptotic pathwaysrdquo Develop-mental Biology vol 318 no 1 pp 133ndash142 2008

[35] K M Kusz L Tomczyk M Sajek et al ldquoThe highly conservedNANOS2 protein Testis-specific expression and significancefor the humanmale reproductionrdquoMolecularHumanReproduc-tion vol 15 no 3 pp 165ndash171 2009

[36] K Kusz-Zamelczyk M Sajek A Spik et al ldquoMutations ofNANOS1 a human homologue of the Drosophila morphogenare associated with a lack of germ cells in testes or severe oligo-astheno-teratozoospermiardquo Journal of Medical Genetics vol 50no 3 pp 187ndash193 2013

[37] V T Julaton and R A Reijo Pera ldquoNANOS3function in human

germ cell developmentrdquoHumanMolecular Genetics vol 20 no11 pp 2238ndash2250 2011

[38] Y Qin H Zhao E Kovanci J L Simpson Z Chen and ARajkovic ldquoMutation analysis of NANOS3 in 80 Chinese and 88Caucasian women with premature ovarian failurerdquo Fertility ampSterility vol 88 no 5 pp 1465ndash1467 2007

[39] X Wu B Wang Z Dong et al ldquoA NANOS3 mutation linkedto protein degradation causes premature ovarian insufficiencyrdquoCell Death and Disease vol 4 article e825 2013

[40] H Hashimoto K Hara A Hishiki et al ldquoCrystal structure ofzinc-finger domain of Nanos and its functional implicationsrdquoEMBO Reports vol 11 no 11 pp 848ndash853 2010