Abnormal development of the olfactory bulband reproductive system in mice lackingprokineticin receptor PKR2Shun-ichiro Matsumoto*†, Chihiro Yamazaki‡§, Koh-hei Masumoto‡¶, Mamoru Nagano‡, Masanori Naito*,Takatoshi Soga*, Hideki Hiyama*, Mitsuyuki Matsumoto*, Jun Takasaki*, Masazumi Kamohara*, Ayako Matsuo*,Hiroyuki Ishii*, Masato Kobori*, Masao Katoh*, Hitoshi Matsushime*, Kiyoshi Furuichi*, and Yasufumi Shigeyoshi†‡

*Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma, Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan;‡Department of Anatomy and Neurobiology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan; §Trans Genic, Inc.,Tokyo Office, Houkoku Building 7th Floor, 3-9-2 Kyobashi, Chuo-ku, Tokyo 104-0031, Japan; and ¶Department of Physics, Informatics,and Biology, Yamaguchi University, Yoshida, Yamaguchi 753-8512, Japan

Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved January 23, 2006 (received for review October 12, 2005)

Prokineticins, multifunctional secreted proteins, activate two en-dogenous G protein-coupled receptors PKR1 and PKR2. From in situanalysis of the mouse brain, we discovered that PKR2 is predom-inantly expressed in the olfactory bulb (OB). To examine the roleof PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice(Pkr1�/� and Pkr2�/�, respectively). Phenotypic analysis indicatedthat not Pkr1�/�but Pkr2�/�mice exhibited hypoplasia of the OB.This abnormality was observed in the early developmental stagesof fetal OB in the Pkr2�/� mice. In addition, the Pkr2�/� miceshowed severe atrophy of the reproductive system, including thetestis, ovary, uterus, vagina, and mammary gland. In the Pkr2�/�

mice, the plasma levels of testosterone and follicle-stimulatinghormone were decreased, and the mRNA transcription levels ofgonadotropin-releasing hormone in the hypothalamus and lutein-izing hormone and follicle-stimulating hormone in the pituitarywere also significantly reduced. Immunohistochemical analysisrevealed that gonadotropin-releasing hormone neurons were ab-sent in the hypothalamus in the Pkr2�/� mice. The phenotype ofthe Pkr2�/� mice showed similarity to the clinical features ofKallmann syndrome, a human disease characterized by associationof hypogonadotropic hypogonadism and anosmia. Our currentfindings demonstrated that physiological activation of PKR2 is es-sential for normal development of the OB and sexual maturation.

EG-VEGF � G protein-coupled receptor � Kallmann syndrome �knockout mouse � GnRH

Prokineticins (PKs), comprising PK1 (also called EG-VEGF)and PK2 (also called Bv8), are secreted bioactive proteins

possessing 10 conserved cysteines that form five disulfide bonds(1). The mature forms of PK1 and PK2 consist of 86 and 81amino acids, respectively, and share �40% amino acid identity.The N-terminal six-amino acid sequence (AVITGA) of maturePKs is completely conserved during molecular evolution and isessential for the bioactivities of PKs (2). Two endogenous PKreceptors (PKRs), termed PKR1 and PKR2, both of which areG protein-coupled receptors, mediate signal transduction of PKs(3–5). PKR1 and PKR2 show strong similarity (87% homology)in their primary structure. In humans, both PKRs are predom-inantly expressed in the testis. In addition, PKR1 shows prefer-ential distribution in the peripheral tissues, whereas PKR2 showsrelatively localized distribution in the CNS. Analysis usingreceptor-transfected mammalian cell lines showed that PK2binds PKRs with higher affinity than does PK1, suggesting thatPK2 is the stronger agonist for the PK�PKR system underphysiological conditions (3–5). As a result of binding with PKs,PKRs, which can couple to Gq protein, promote intracellularCa2� mobilization (3–5). Moreover, PKRs also couple to Gqiand Gs proteins, indicating that PKRs activate multiple intra-cellular signal-transduction pathways (6, 7). Activation of PKRs

influences several physiological events in the CNS and periph-eral tissues (8), including intestinal contraction (9), hyperalgesia(10, 11), spermatogenesis (12), neuronal survival (13), circadianrhythm (14, 15), angiogenesis (16–18), ingestive behavior (19),and hematopoiesis (20). Recently, Ng et al (21) reported thegeneration of knockout mice lacking Pk2 (Pk2�/� mice). TheirPk2�/� mice showed marked reduction in the olfactory bulb(OB) size, loss of normal OB architecture, and accumulation ofneuronal progenitors in the rostral migratory stream. Based onthe abnormal phenotype observed in Pk2�/� mice, they dem-onstrated that PK2 plays critical roles in OB morphogenesis andOB neurogenesis. To date, however, which PKR activation isessential for normal OB development remains unknown.

In this study, we have generated two murine lines that aregene-disrupted for Pkr1 and Pkr2, respectively. Phenotypic anal-ysis of these two lines demonstrated that the Pkr2�/� miceshowed abnormal development of the OB and reproductivesystem, whereas Pkr1�/� mice did not.

ResultsIn Situ Hybridization Analysis for PKR2 Using Radiolabeled Probes.Based on our previous finding that PKR2 has strong expressionin the human fetal brain, we analyzed its mRNA expression inthe fetal mouse head region, upper cephalic portion of theembryo, or whole brain from embryonic day (E)9.5 to postnatalday (P)10 (see Fig. 5A, which is published as supporting infor-mation on the PNAS web site). Quantitative PCR study indi-cated that PKR2 was expressed strongly in both tissues duringthe embryonic stage from E9.5 to E18.5. Next, to study thedetailed expression pattern of PKR2 in the mouse brain, weperformed in situ hybridization analysis for PKR2 using radio-labeled probes of mouse Pkr2 cDNA. In accordance with theprevious findings of Cheng et al. (14), PKR2 was stronglyexpressed in the suprachiasmatic nucleus and moderately in theparaventricular thalamic nucleus (Fig. 5B). In addition, PKR2was predominantly expressed in the OB, especially in theependyma and subependymal layer of the olfactory ventricle(Fig. 5 C and D).

Generation of Pkr1�/� and Pkr2�/� Mice. To study the roles of PKRsin the OB, we generated gene-disrupted mice for Pkr1 (see Fig.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: En, embryonic day n; FSH, follicle-stimulating hormone; GnRH, gonado-tropin-releasing hormone; HE, hematoxylin and eosin; KS, Kallmann syndrome; LH, lutein-izing hormone; OB, olfactory bulb; Pn, postnatal day n; PK, prokineticin; PKR, PK receptor.

†To whom correspondence may be addressed. E-mail: shunichiro.matsumoto@jp.astellas.com or shigey@med.kindai.ac.jp.

© 2006 by The National Academy of Sciences of the USA

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6 A–C, which is published as supporting information on thePNAS web site) and Pkr2 (Fig. 6 D–F), respectively. For design-ing the targeting vectors for Pkr1 and Pkr2, an exon containingthe initiating ATG codon corresponding to the first Met of eachORF was deleted, and a reverse-oriented Neo-cassette wasinserted, respectively (Fig. 6 A–D). Generation of heterozygousand homozygous mice for Pkr1 (Fig. 6B) and Pkr2 (Fig. 6E) wasconfirmed by Southern blot analysis. Pkr1�/� mice were obtainedat the expected Mendelian rate, whereas �50% of Pkr2�/�micedied at an early neonatal stage from respective heterozygousmouse mating. By performing quantitative PCR analysis, we alsoconfirmed that the mRNA expression levels of PKR1 (Fig. 6C)and PKR2 (Fig. 6F) were decreased by �50% and 100% in theheterozygous and homozygous mice, respectively.

Malformation of the OB in Pkr2�/� Mice. Anatomical analysis of thebrain demonstrated that all the Pkr2�/� mice (n � 13) displayeda decreased OB size compared with the control wild-typelittermates (n � 10) (Fig. 1A Right). No significant abnormalityof the OB size was observed in Pkr2�/� heterozygous mice (datanot shown). There was a wide-open space between the right andleft OB in Pkr2�/� mice. In contrast, no remarkable morpho-logical differences in the OB were observed between Pkr1�/�

and wild-type mice (n � 4 each) (Fig. 1 A Left). Histopatholog-ically, the typical layered structure of the OB was absent andmalformed in Pkr2�/� mice (Fig. 1B). The glomerular layer wasindiscernible in the OB of Pkr2�/� mice. We next examined themorphogenesis of the developmental OB. During the embryonicstage at E14.5 (Fig. 1C), which is the developmental stage afterestablishment of the first olfactory sensory connections from theolfactory epithelium to the OB (22), no remarkable macroscopicdifferences could be observed between the Pkr2�/� mice andtheir wild-type littermates (n � 19). However, at E16.5 andE18.5 as well as at P0, projection of the OB from the telenceph-alon was evident in all of the wild-type mice, whereas thisprojection was very slight in all of the Pkr2�/� mice (n � 5, 7, and5 at E16.5, E18.5, and P0, respectively).

Abnormal Development of the Reproductive System in Pkr2�/� Mice.Anatomical analysis of the non-CNS tissues of Pkr1�/� andPkr2�/� mice indicated that both the male and female repro-ductive organ weights were reduced in Pkr2�/� mice, but not inPkr1�/� mice (mean weight of testis, 114.9 � 11.8 mg in wild-typemice and 2.8 � 0.5 mg in mutant mice, P � 0.001; mean weightof ovary, 3.4 � 0.7 mg in wild-type mice and �0.1 mg in mutantmice, P � 0.001) (Fig. 2A; and see Table 1, which is publishedas supporting information on the PNAS web site). However, nosignificant differences were observed in the reproductive systemsof the male and female Pkr2�/� heterozygous mice comparedwith the wild-type mice (data not shown). Histopathologicalanalysis revealed overt atrophy of the testis of all Pkr2�/� miceexamined (n � 5) (Fig. 2B). The seminiferous tubules werereduced in diameter. No sperm were present in the lumen of thetubules in the mutant testis. Spermatogenic cells and spermato-cytes were present, but spermatids were not observed. Theinterstitium was sparse, and the few Leydig cells observed were

Fig. 1. Macro- and microscopic analyses of OB from Pkr2�/� mice. (A)Macroscopic view of the male brain at 8 weeks of age from the controlwild-type littermates and Pkr1�/� mice (Left), and control wild-type litter-mates and Pkr2�/� mice (Right). The OB size is small in the Pkr2�/� mice. (Scalebars, 5 mm.) (B) Nissl-stained sagittal sections of the male OB at 8 weeks of agefrom the wild-type littermates (Upper) and Pkr2�/� mice (Lower). Highermagnifications of the OB (boxes, Left) from each genotypic mouse are shown(Right), respectively. The arrow (Upper Right) indicates the glomerular layer(GL) of the OB from wild-type littermates. Note that no glomerular layer isdiscernible in the OB from the Pkr2�/� mice (Lower Right). [Scale bars, 0.5 mm(Left) and 0.1 mm (Right).] (C) OB morphogenesis during the embryonic stageat E14.5 (Upper Left), E16.5 (Upper Right), E18.5 (Lower Left), and P0 (LowerRight). (Scale bars, 1 mm.)

Fig. 2. Hypoplasia of the reproductive organs in Pkr2�/� mice. (A) Macro-scopic view of the testes at 8 weeks of age from the control wild-typelittermates and Pkr1�/� mice (Left), control wild-type littermates and Pkr2�/�

mice (Center), and the ovary and uterus (Right) at 12 weeks of age from thewild-type, Pkr1�/�, and Pkr2�/� mice. The male and female reproductiveorgans are small in the Pkr2�/� mice. (Scale bars, 5 mm.) (B) HE-stained sectionsof wild-type and Pkr2�/� testis at 20 weeks of age. Higher magnification(rectangle) shows normal Leydig cells in wild-type testis (Lower Left), whereasthe mutant testis shows small Leydig cells and no spermatid (Lower Right).SPG, spermatogenic cell; SPC, spermatocytes; SPT, spermatid; LC, Leydig cells.[Scale bars, 100 �m (Upper) and 10 �m (Lower).] (C) HE-stained sections ofwild-type and Pkr2�/� ovary at 20 weeks of age. A magnified region (rectan-gle) shows normal antral follicle (arrow) in wild-type ovary (Lower Left). Amagnified region (rectangle) shows undeveloped follicles (arrows) in Pkr2�/�

ovary (Lower Right). [Scale bars, 200 �m (Upper) and 50 �m (Lower).]

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small. These findings suggest that spermatogenesis was arrestedin the pachytene spermatocyte stage. Apparent atrophy was alsoobserved in the ovary, uterus, vagina, and mammary gland of allfemale Pkr2�/� mice (n � 5). The ovary contained mainlyundeveloped follicles, and growth seemed to be arrested in thepreantral phase of follicle development (Fig. 2C). Only a fewfollicles showed antral formation, but it was incomplete. Nocorpora lutea were seen, and the interstitium was severelyatrophic. These findings suggest abnormalities in the mainte-nance and growth of the follicles. A high level of atrophy was alsoobserved in the endometrium, the muscle layer of the uterus, andin the epithelium of the vagina.

Analysis of plasma hormone concentrations revealed thattestosterone (see Fig. 7A, which is published as supportinginformation on the PNAS web site) and follicle-stimulatinghormone (FSH) (Fig. 7B) were lower in male Pkr2�/� mice thanin the age-matched wild-type mice (P � 0.001 for testosteronecompared in male mice, and P � 0.001 for FSH compared inmale mice and P � 0.078 in female mice, respectively). On theother hand, no statistically significant difference was found for

the plasma luteinizing hormone (LH) concentration (Fig. 7C). Inaddition to the plasma levels of those sex hormones, the mRNAtranscriptional levels for pituitary FSH (P � 0.004 and P � 0.01for male and female mice, respectively) (Fig. 7D) and LH (P �0.001 and P � 0.009 for male and female mice, respectively) (Fig.7E) were lower in the Pkr2�/� mice.

Disappearance of Gonadotropin-Releasing Hormone (GnRH) Neuronsin Pkr2�/� Mice. To detect GnRH neurons in the brain, weperformed GnRH immunohistochemistry on both the Pkr2�/�

mice and their control wild-type littermates. In contrast to thefinding that GnRH-immunoreactive neurons were present in thepreoptic region and median eminence of the hypothalamus ofwild-type mice [n � 4 (male, 2; female, 2)] (Fig. 3 A Left and B),no such reactivity was found in the corresponding regions of thePkr2�/� mice [n � 4 (male, 2, female, 2)] (Fig. 3A Right).GnRH-immunoreactive neurons were also observed in the hy-pothalamus of the Pkr2�/� heterozygous mice (data not shown).In parallel with this observation, the mRNA transcriptional levelfor hypothalamic GnRH was significantly decreased in thePkr2�/� mice (P � 0.001 and P � 0.001 for males and females,respectively) (Fig. 3C).

To study the mechanism of abnormal GnRH neuron and OBdevelopment observed in the Pkr2�/� mice, histopathologicalanalysis was performed in the upper nasal region in the mutantand wild-type fetal mice at E12.5 and E13.5. In the Pkr2�/� miceat E12.5 (Fig. 4 B, F, and J) and E13.5 (Fig. 4 D, H, and L), theolfactory�vomeronasal axons in mutant mice failed to reach theforebrain, and the terminals of the axons formed a large tangledsphere-shaped structure. In contrast, the wild-type littermates atE12.5 (Fig. 4 A, E, and I), E13.5 (Fig. 4 C, G, and K) showed nosuch tangled fibers, and the olfactory�vomeronasal axonsreached the forebrain.

DiscussionHere, we report that Pkr2�/� mice exhibited hypoplasia in both theOB and the reproductive system, whereas Pkr1�/� mice did not.

In situ hybridization analysis showed that PKR2 was predom-inantly expressed in the ependyma and subependymal cell layerof the adult OB. It is well known that development of the OBrequires generation and differentiation of several lines of cells in

Fig. 3. Analysis of GnRH neurons in Pkr2�/� mice. (A) Immunohistochemicalstudy of GnRH neurons in the wild-type (Left) and Pkr2�/� mice (Right).Coronal sections of the preoptic region (Top and Middle) and median emi-nence (Bottom). (Scale bars, 0.1 mm.) (B) Higher magnification of the preopticregion [box (A Middle Left)] from a wild-type mouse. The arrow indicatesGnRH-positive neuronal cell bodies. (Scale bar, 0.2 mm.) (C) Mean � SD oftranscriptional mRNA level of GnRH in the hypothalamus of Pkr2�/� mice.Quantitative PCR was performed to compare the mRNA level between thewild-type littermates (male, n � 6; female, n � 6) and Pkr2�/� mice (male, n �5; female, n � 6) at 20 weeks of age. Data shown are representative of twoexperiments.

Fig. 4. Abnormal morphology in the upper nasal region in Pkr2�/� mice atE12.5 and E13.5. HE-stained parasagittal sections of the upper nasal region inwild-type littermates at E12.5 (A, E, and I), Pkr2�/� mice at E12.5 (B, F, and J),wild-type littermates at E13.5 (C, G, and K) and Pkr2�/� mice at E13.5 (D, H, andL). Note that a sphere-shaped structure was observed at the region betweenthe olfactory pit and forebrain in Pkr2�/� mice at both E12.5 and E13.5(arrowhead in B and D). This structure is particularly evident when viewed athigher magnification (arrowhead in F, H, J, and L). OP, olfactory pit, F,forebrain. [Scale bars, 200 �m (A–D), 100 �m (E–H), and 50 �m (I–L).]

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the early developmental stage (22). Together with our currentfinding that PKR2 was strongly expressed in the mouse fetalbrain as well as in the human fetal brain (5), we deduce that thePK�PKR2 system plays important roles in OB morphogenesis.The discovery of OB malformation, which originated in theembryonic stage, only in the Pkr2�/� mice suggests that notPKR1 but PKR2 activation is essential to drive the normaldevelopment of the OB during embryogenesis.

In addition to the abnormal development of the OB, thePkr2�/� mice exhibited hypoplasia of the reproductive system.Our assays of the plasma hormones revealed that the testoster-one and FSH levels were considerably decreased in the Pkr2�/�

mice. We also found that the mRNA transcription levels of FSHand LH were significantly lower in the Pkr2�/� mice in thepituitary, which is the source of circulating FSH and LH. Inaddition to these circulating hormones, it is well known thatsexual maturation is tightly regulated by hypothalamic GnRH(23). Moreover, the pathological findings for the testis and ovaryof the Pkr2�/� mice were highly consistent with those seen inGnRH-deficient mice (24). Accordingly, the defective sexualdevelopment in the Pkr2�/� mice is considered to be the resultof possible defects in GnRH secretion. In the present compar-ison of the plasma FSH level, a statistically significant differencewas not found between the female Pkr2�/� mice and their controlwild-type littermates. We surmise that, because, in rodents, theplasma FSH level is generally lower in females than in males (25,26), individual variability in the plasma FSH level masked thetrue difference between the mutant and wild-type mice. Asimilar explanation could be applied to our result that the plasmaLH level showed no statistically significant difference (25). Inview of the fact that PKR2 is also expressed in the gonads (4, 5,18), we cannot exclude the possibility that the hypoplasia of thereproductive systems arose from the loss of a direct role of PKR2in the development of these tissues. Further investigation isneeded to confirm such an additional activity for PKR2 ongonadal development.

In contrast to the report of Ng et al. (21), indicating that �50%of Pk2�/� mice displayed asymmetric OB formation, all of ourPkr2�/� mice exhibited symmetrical OB malformation. More-over, in their article, there was no description of any abnormal-ities of the reproductive system in their Pk2�/� mice. It may beargued that PK1, another PKR2 ligand, compensated for theabsence of PK2 bioactivity and played roles in OB morphogen-esis and pubertal maturation in the Pk2�/� mice. It is not clear,at this moment, what is the main cause of the frequent neonatallethality observed in Pkr2�/� mice. One possibility is, as de-scribed previously in regard to gene-disrupted mice for FGFreceptor 1 (27) and lysophosphatidic acid receptor (28), thatdysfunction of the olfactory system of Pkr2�/� mice might leadto inability to suckle milk because of smelling disability. Addi-tional studies would be required to elucidate this issue directly.

The pathology observed in Pkr2�/� mice bears a strikingresemblance to the clinical manifestations of Kallmann syn-drome (KS), which is a human developmental disease withcombined features of hypogonadotrophic hypogonadism andanosmia (29–32). KS is categorized into three types, KAL1,KAL2, and KAL3, which are an X-linked form (Online Men-delian Inheritance in Man (OMIM) entry no. 308700), anautosomal dominant form (OMIM entry no. 147950), and anautosomal recessive form (OMIM entry no. 244200), respec-tively (29). The gene responsible for KAL1 encodes an extra-cellular matrix protein, anosmin-1 (33, 34), whereas KAL2 iscaused by loss-of-function mutations in the gene encoding FGFreceptor 1 (35). On the other hand, the gene responsible forKAL3 remains unknown. In KS patients, OB development iscompletely or partially absent, and failed puberty is often thefirst manifestation of the disease in both sexes (31). Based onhistopathological examinations, the pathogenesis of KS has been

proposed to be that malformation of the OB causes abnormallocalization of GnRH neurons, leading to the hypogonadotropichypogonadism through loss of GnRH activity in the hypothal-amus (29–32, 36, 37). The remarkable phenotypic similarities toKS, i.e., simultaneous hypoplasia in the OB and the reproductivesystem, observed in all of our Pkr2�/� mice, inspired us tohypothesize that the hypothalamic GnRH neurons have a de-velopmental abnormality in these mice. Our immunohistochem-ical study confirmed this hypothesis that GnRH neurons are,indeed, absent in the hypothalamus of Pkr2�/� mice. Theabsence of GnRH neurons in the hypothalamus could be attrib-uted to impaired migration of the GnRH neurons from theolfactory pit to the brain. We observed that the olfactory�vomeronasal axon runs into a sphere-shaped structure in theupper nasal region in Pkr2�/� fetal mice at E12 and E13 andfailed to reach the OB. The structure seemed like a fibrocellularmass (FCM) that has been observed in mutant extratoes mice(38) and Arx homeobox-gene-deficient mice (39). In these mice,most of the migrating olfactory axons failed to reach the OB andterminated in an axon-tangled FCM to cause failure of OBmorphogenesis. It is well documented that the prior establish-ment of a migrating pathway by the olfactory�vomeronasal axonsis essential for developing GnRH neurons to move properly fromthe olfactory pit to the rostral forebrain (31, 40). Therefore, inPkr2�/� mice, it is possible that the defect in the olfactory�vomeronasal axonal route in the nasal region blocked GnRHneurons from migrating into the forebrain. In line with thisconsideration, it is tempting to speculate that olfactory neuronsexpress PKR2 and that their axons would be guided by thePK1�PK2 to establish the axonal route during embryogenesis.

The current results suggested that, in Pkr2�/� mice, the failure ofdevelopment of GnRH neurons took place in conjunction withdevelopmental agenesis of the OB during the embryonic period andeventually led to the defects in sexual maturation. In the geneticallyengineered mice, however, the OB malformation does not neces-sarily cause abnormal GnRH localization. No pathological featuresof a hypoplastic reproductive system were observed in all hitherto-reported mouse lines possessing a pathological OB (31). OurPkr2�/� mouse is a genetically engineered murine line that showshypoplasia in both the OB and the reproductive system.

Recent reports demonstrated that knockout mice lacking Gpr54(Gpr54�/� mice) showed a striking phenotype of isolated hypotha-lamic hypogonadism (25, 41). Similar to PKRs, GPR54 is also amember of the G protein-coupled receptor family. GPR54 activa-tion leads to direct release of GnRH from hypothalamic GnRHneurons and is essential for mammalian puberty, including in man(26, 42–44). In Gpr54�/� mice, which possess intact hypothalamicGnRH neurons (45), decreased pituitary LH�FSH secretion causesthe characteristic hypogonadism due to the loss of GPR54 activa-tion (25, 43). In view of the fact that neither agenesis of the OB norabnormality of the hypothalamic GnRH neurons was observed inthe Gpr54�/� mice, we surmise that the physiological roles ofGPR54 are different from those of PKR2.

It remains unclear whether PKR2 activation regulates OBdevelopment directly or indirectly. Based on the results of Ng etal. (21), demonstrating that PK2 promoted postnatal and adultOB neurogenesis, the current result infers that PKR2 activationis also necessary for embryonic OB morphogenesis, including thedevelopment of OB projection neurons. Moreover, the fact thatPK2 has binding affinity for heparan sulfate proteoglycans(HSPG) (18) has something in common with the character ofanosmin-1 (KAL1 product), and FGFs (endogenous ligands forthe KAL2 product FGF receptor 1 (FGFR1) also interact withHSPG (30). It might be possible that PK–PKR complexes mightinfluence the physiological roles of these KS-responsible prod-ucts, including that there might be some interactions betweenFGFR1 and PKR2 signaling pathways.

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In conclusion, we report, in this study, that activation of PKR2and not PKR1 is critical for OB morphogenesis and localizationof GnRH neurons, which is vital for maturation of the repro-ductive organs. It is also noteworthy that this is an animal modelfor KS that manifests simultaneous hypoplasia for both the OBand reproductive organs. The Pkr2�/� mouse can be a usefulmodel for studying this human disorder.

Materials and MethodsIn Situ Hybridization Using Radiolabeled Probes. Radiolabeledprobes for 670 bp of mouse Pkr2 ORF-cDNA were made by using[35S]UTP (PerkinElmer) with a standard protocol for cRNAsynthesis. Mice were deeply anesthetized with ether and intra-cardially perfused with 10 ml of saline and 20 ml of a fixativecontaining 4% paraformaldehyde in 0.1 M phosphate buffer(PB), pH 7.4. Brains were postfixed in the same fixative for 24 hat 4°C, soaked in 0.1 M PB, pH 7.4, containing 20% sucrose for48 h, and, finally, stored frozen at �70°C. Stored tissues were cutto a thickness of 40 �m on the coronal plane by using a cryostat.The in situ hybridization of mRNA was performed as describedin detail in refs. 46 and 47.

Gene Targeting of Pkr1 or Pkr2. To construct a Pkr1 targetingvector, the short arm of the 2.1-kb region, which includes exon1, containing initiating methionine and an intron, and the longarm of the 7.8-kb region, which includes an intron, exon 2, and3� UTR, of the mouse Pkr1 gene, and, for the Pkr2 targetingvector, the long arm of the 6.6-kb region, which is 5� UTR, andthe short arm of the 2.0-kb region, which includes an intron ofthe Pkr2 gene, were ligated into the targeting vector pPNT(48). The targeting vector was transfected into TT2 cells (49)by electroporation, and clones resistant to G418 and ganci-clovir were selected and further screened by Southern hybrid-ization. Chimeric male mice were mated with C57BL�6 Jfemales (CLEA Japan, Tokyo) to obtain F1 heterozygotes.Genotyping using the tails for detecting the genes of Pkr1,Pkr2, and neomycin was performed by PCR with the primersets of (5�-ATGGAGACCACTGTCGGGGCTCTGGGTG-3�and 5�-CCTGTCAATGGCAATGGCCAGTAGGGCG-3�);(5�-CATGGGACCCCAGAACAGAAACACTAGC-3� and5�-CCTGTCAATAGCGATGGCCAGCAGAGCG-3�); and(5�-TATGGGATCGGCCATTGAAC-3� and 5�-CCTCA-GAAGAACTCGTCAAG-3�), respectively. F1 heterozygousmice were crossed with C57BL�6 mice to produce a largenumber of F2 heterozygous mice, which were then intercrossedto produce homozygous mice for analysis. In this study, weused mice of the F2 generations, and, in all experiments,comparisons were made with littermate wild-type mice. Allexperiments were performed in compliance with the regula-tions of the Animal Ethics Committee of Astellas Pharma, Inc.

Quantitative PCR. DNase-treated total RNA was isolated from thehead of mice from E9.5 to E13.5, the whole brain of mice fromE14.5 to P10 (male, n � 2; female, n � 2), and the hypothalamusand pituitary of mice at 20 weeks of age (wild-type littermates(male, n � 6; female, n � 6) and Pkr2�/�mice (male, n � 5;female, n � 6). Tissue expression of PKR2 in the head or brain,GnRH in the hypothalamus, and FSH and LH in the pituitarywere quantitatively analyzed by using a Prism 7700 SequenceDetector (Applied Biosystems) as described in ref. 5. To confirmthe disappearance of the mRNA corresponding to the target

genes, total RNA was isolated from the hypothalamus of malemice at 5 weeks of age (wild-type littermates, heterozygous andhomozygous mice (n � 2 for both Pkr1- and Pkr2-mutant mice).The oligonucleotide primer sets used for PCR were as follows:5�-AGCACTGGTCCTATGGGTTGC-3� and 5�-AGTGTT-CAGTGTTTCTCTTTCCCC-3� for GnRH; 5�-GTAGCCACT-GAATGTCACTGTGG-3� and 5�-GCAGTCAGTGCT-GTCGCTGT-3� for FSH; 5�-CGGCTCAGTAGCTCTG-ACTGTG-3� and 5�-ACAGGCCATTGGTTGAGTCC-3� forLH; 5�-GCCCCTGGATGAAGAGGAAG-3� and 5�-GCAG-CAAAGAAAGTCCGAGAA-3� for Pkr1; and 5�-ACCAAC-CTCCTCATTGCTAACC-3� and 5�-GATCGCCACCAG-GAAGTCAG-3� for Pkr2. The relative abundance of transcriptswas normalized to the constitutive expression of G3PDHmRNA.

Histopathological Examination. The brain, testis, ovary, uterus, va-gina, and mammary gland were dissected, fixed, and preserved in10% neutral buffered formalin (the testis was fixed in Bouin’ssolution). Sections of these tissues were stained with hematoxylinand eosin (HE) and observed microscopically. For the fetal brainpreparation, the time of performance of in vitro fertilization andembryo transfer was considered as E0, and E14.5–E18.5 embryoswere collected from pregnant mice by Cesarean section. For theanalysis of the upper nasal region in fetal mice, embryos from the12.5–13.5 days of gestation were collected from pregnant mice byCesarean section. E12.5–13.5 embryos were immersion-fixed in 4%paraformaldehyde in 0.1M PB overnight at 4° C. Specimens weredehydrated through graded alcohols and xylene and embedded inparaffin. Serial sections (8-�m thick) were cut in the sagittal planesand were mounted on silane-coated slides. These sections werestained with HE and observed microscopically.

Hormone Assays. Plasma testosterone, FSH, and LH were mea-sured with commercially available kits: DPC Total Testosteronekit (Diagnostic Products), rat FSH [125I] RIA System (Amer-sham Pharmacia Biosciences), and rat LH EIA System (Amer-sham Pharmacia Biosciences), respectively.

Immunohistochemistry. Immunohistochemical analysis was per-formed as described in ref. 50. Briefly, adult mice were anes-thetized with diethyl ether and intracardially perfused with PBcontaining 4% paraformaldehyde. Brains were removed, frozenin dry ice, and coronally sectioned with a cryostat at a thicknessof 20 �m. The sections were incubated for 2 days at 4°C inprimary anti-GnRH (Chemicon) diluted 1:60,000 in PBS con-taining 0.3% Triton X-100. The sections were sequentiallyincubated with biotinylated anti-rabbit goat IgG (Vector Lab-oratories) diluted 1:1,000 and avidin-conjugated horseradishperoxidase (Vector Laboratories) diluted 1:1,000. They werethen treated with 0.035% diaminobenzidine�0.05 M Tris�HClbuffer (pH 7.4), dehydrated with a graded series of ethanolrinses, immersed in xylene, and embedded in Entellan (Merck).

Statistical Analysis. Results are expressed as the mean � SD.Differences between groups were examined for statistical sig-nificance by using Student’s two-tailed unpaired t test.

We thank A. Adachi, H. Miyamoto, M. Isshiki, K. Honda, K. Arai, andJ. Kawano for their skillful technical assistance and M. Sato and J.Tanaka for helpful discussion.

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