RESEARCH ARTICLE

Molecular Reproduction & Development 76:151–159 (2009)

Identification of Oocyte-Selective NLRP Genes in RhesusMacaque Monkeys (Macaca mulatta)

PATRICK McDANIEL AND XUEMEI WU*

Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University,

Beaverton, Oregon

SUMMARY

Oocyte-selective genes control multiple aspects of female gamete development andpreimplantation embryogenesis. Several key oocyte-selective factors have beenidentified in mice recently; however, these factors are not well documented in moreadvanced species such as nonhuman primates. One of such oocyte-selective factorsis NLRP5 (NLR family, Pyrin domain containing 5), also known as Maternal AntigenThat Embryos Require (MATER), which is required for preimplantation embryodevelopment beyond the 2-cell stage in mice. Human NLRP family contains 14members. We identified 14 NLRP gene homologues and examined their spatial andtemporal expression in rhesus macaque monkeys (Macaca mulatta). While all 14NLRP genes are detectable in the macaque gonad, eight of them (NLRP2, 4, 5, 8, 9,11, 13, and 14) are specifically or preferentially expressed in the ovary. In situhybridization elucidated a specific oocyte expression pattern of the eight NLRP geneswithin the ovary. During the oocyte-to-embryo transition, seven of these oocyte-selective NLRP transcripts (excluding NLPR2) are enriched in maturing oocytes andearly preimplantation embryos but diminish upon embryo genome activation,indicating an exclusive maternal origin of these transcripts. Though functionallyunknown, the spatial and temporal distribution of these oocyte-selective NLRP genesimplies important roles of the NLRP family in oogenesis and early embryodevelopment in nonhuman primates.

Mol. Reprod. Dev. 76: 151–159, 2009. � 2008 Wiley-Liss, Inc.

Received 13 January 2008; Accepted 17 April 2008

* Corresponding author:Assistant ScientistDivision of Reproductive SciencesOregon National Primate

Research CenterOregon Health & Science UniversityWest Campus, 505 NW 185th AvenueBeaverton, OR 97006.E-mail: wux@ohsu.edu

Published online 28 May 2008 in Wiley InterScience(www.interscience.wiley.com).

DOI 10.1002/mrd.20937

INTRODUCTION

Mammalian folliculogenesis requires precisely coordinat-ed expression of intra-ovarian factors. While the contributionof granulosa cells to oocyte development has been welldocumented (Erickson and Danforth, 1995), it has onlybecome apparent recently that the oocyte plays a centralrole in directing its own fate as well as the growth anddifferentiation of its surrounding somatic cells (Dean,2002; Hutt and Albertini, 2007). Several oocyte-selectivefactors have been demonstrated to play critical roles atvarious stages of oogenesis and follicular development inmice. Factor In the GermLine, Alpha (FIGLA) is an oocyte-specific basic helix–loop–helix (bHLH) transcription factor.

Female mice lacking FIGLA are unable to form primordialfollicles and are thus sterile (Soyal et al., 2000). Nobox(Newborn Ovary homeoBOX)-null females are born withnormal number of primordial follicles; however, the transitionfrom primordial to primary follicles is blocked and the oocytesare depleted rapidly within 2 weeks after birth (Rajkovicet al., 2004). GDF9 (Growth Differentiation Factor 9), amember of TGFb superfamily, is preferentially expressedin mouse oocytes and required by the transition from primary

This article contains supplementary material, which may be viewed at theMolecular Reproduction and Development website at http://www.interscience.wiley.com/jpages/1040-452x/suppmat/index.html.

� 2008 WILEY-LISS, INC.

to secondary follicles (Dong et al., 1996). Three oocyte-specific extracellular zona pellucida glycoproteins (ZP1,ZP2, and ZP3) play important roles in maintaining theintegrity of zona matrix and are critical for the oocyte-spermrecognition (Rankin et al., 1996, 1999, 2000, 2001). MouseNlrp5 (NLR family, Pyrin domain containing 5) gene, previ-ously known as Maternal Antigen That Embryos Require(MATER), was originally identified during an autoimmuneantigen search in the ovary and is exclusively expressed inthe oocyte (Tong et al., 2000b). Female mice lacking NLRP5are infertile due to a developmental arrest at the 2-cellembryo stage (Tong et al., 2000a). Thus, Nlrp5 belongs toa small group of identified maternal effect genes (i.e., genesthat are derived from the oocyte and function at embryodevelopment).

NLRP family is a subfamily of the newly describedCATERPILLER (CAspase-recruitment domain (CARD)Transcription Enhancer, R (purine)-binding, Pyrin, Lots ofLEucine Repeats) family which is comprised of proteinswith a nucleotide-binding domain and a leucine-richregion (Ting and Davis, 2005). The CATERPILLER familyhas gained rapid prominence because of its demonstratedand anticipated roles in immunity, cell death and growth, anddiseases (Ting and Davis, 2005; Ting and Williams, 2005).The NLRP gene family constitutes of 14 members in humans(Tschopp et al., 2003). As these genes are only discoveredrecently, information about their expression and functions isvery limited. Interestingly, besides Nlrp5, several other Nlrpgenes are also found exclusively expressed in the oocyte inmice, and the expression of which declines with oocyte aging(Dade et al., 2004; Hamatani et al., 2004). By in vitro RNAinterference (RNAi), mouse NLRP14 appears to function asa maternal effect protein as well (Hamatani et al., 2004).Mutations of human NLRP7 gene are associated with intra-uterine embryo death, therefore NLRP7 is considered thefirst identified maternal effect gene in humans (Murdochet al., 2006; Qian et al., 2007).

Our group is interested in identification and characteriza-tion of novel oocyte-selective factors that may play importantroles in oogenesis and preimplantation embryo develop-ment. We chose rhesus macaque as our modeling systemdue to their close resemblance to humans in reproductivephysiology and genetics. Combining database miningand reverse transcription (RT) polymerase chain reaction(PCR) methodologies, we identified 14 NLRP gene homo-logues in rhesus macaques. Proteins encoded by theseNLRP genes share nearly 90% amino acid identity with theirhuman counterparts. Surprisingly, all 14 macaque NLRPtranscripts are detectable in the gonad by RT-PCR, andseveral NLRP family members show a very similar oocyte-specific expression pattern to mouse Nlrp5. Although theNLRP gene family is conserved on the whole, it has beenthrough some major changes during evolution. As a result,some primate NLRP homologues may not exist in lowermammalian species. Though the majority of NLRP familymembers are functionally unknown in primates, the spatialand temporal distribution of these NLRP genes impliesthat NLRP family members may be key players inoogenesis, folliculogenesis, and preimplantation embryodevelopment.

RESULTS

Identification of NLRP Homologues inRhesus Macaques

Macaque cDNA sequences showing homology to humanNLRP genes were downloaded from databases and ana-lyzed by ORF deduction and protein alignment with theirhuman counterparts. We discovered that the predictedmacaque NLRP4, 10, 13, and 14 (GenBank accessionnos. XM_001090206, XM_001098361, XM_001090549,and XM_001107454, respectively) were full-length codingcDNAs whereas the rest of the NLRP cDNA sequences inthe database were incomplete. For example, the predictedmacaque NLPR5 (GenBank accession no. XR_011260)cDNA is a chimeras: while approximately 2 kb (kilobase)-long sequence at its 50-end corresponds to human NLPR8,the remaining 3 kb-long sequence at its 30-end is homolo-gous to human NLRP5. Combining database mining andRT-PCR methodologies, we deduced full-length codingcDNAs for the rest of macaque NLRP genes and depositedthe new sequences in GenBank (http://www. ncbi.nlm.nih.gov/sites/entrez?db¼nucleotide). The accession num-bers for newly deduced full-length macaque NLRP cDNAsequences were listed in Table S1, and the evolutionaryrelationships between the mouse and primate (macaqueand human) NLRP proteins were shown in Figure 1.

Tissue Distribution of Macaque NLRP GenesTo elucidate the tissue distribution of NLRP genes, spe-

cific primers were designed according to the confirmed full-length or partial macaque NLRP cDNAs (Table S1) and usedfor RT-PCR amplification of NLRP1–14 in 13 macaquetissues. As shown in Figure 2, the expression of macaqueNLRP1 is almost ubiquitous, and this expression pattern isconsistent with previous report on human NLRP1 gene(Hlaing et al., 2001). NLRP3, which was thought to beimmune cell-specific in humans previously (Manji et al.,2002), is highly expressed in the testis, spleen, thymus andkidney in rhesus macaques. Interestingly, all NLRP genetranscripts are detectable in either the ovary or the testis, orboth. While many of these NLRP genes are also expressedin somatic tissues, NLRP4, 5, 8, and 9 are only transcribed inthe ovary and testis. Moreover, NLRP10, 11, and 13 arepreferentially expressed in the gonad, only barely detectablein the kidney (NLRP10), spleen (NLRP11), or oviduct(NLRP13), respectively. NLRP2 is mostly enriched in theovary and testis, although it can also be detected in thethymus, spleen, and pituitary.

Cellular Localization of NLRP Transcripts in theRhesus Macaque Ovary

To demonstrate the cellular localization of macaqueNLRP genes that are specifically or preferentially expressedin the ovary, we conducted in situ hybridization with probesthat are specific to NLRP2, 4, 5, 8, 9, 11, 13, and 14 (Fig. 3).All eight NLRP transcripts examined are exclusively local-ized in the oocyte in the macaque ovary, although there isslight difference in the earliest stage when each NLRP

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mRNA becomes detectable. For example, while NLRP2, 4,and 14 can be detected in the oocyte in as early as primordialfollicle stage, NLRP5, 9, 11, and 13 are expressed in theoocyte in primary follicles and beyond. Transcription ofNLRP8 begins in the oocyte surrounded by at least two-layer of granulose cells (i.e., secondary follicles). One com-mon expression feature shared by all eight NLRP genes isthat their transcripts seem to accumulate during oocytegrowth and reach the highest expression level in late antralfollicles.

Temporal Expression of Oocyte-Selective NLRPsDuring the Oocyte-to-Embryo Transition

Using individual NLRP gene-specific primers listed inTable S1, we also examined the temporal expression ofoocyte-expressed NLRP genes in fully grown, maturing

oocytes and preimplantation embryos by RT-PCR(Fig. 4). In the primate, the developmental control isswitched from the oocyte to the embryo between the 4-celland the 8-cell stage embryos, upon which time the maternaltranscripts decay rapidly and the embryo genome activationoccurs (Fig. 4A) (Braude et al., 1988; Zheng et al., 2004).The expression level of NLRP2 is consistent throughoutoocyte maturation and preimplantation embryogenesis, in-dicating that NLRP2 is transcribed from both maternal andembryonic genomes. The other seven oocyte-specific NLRPgenes examined (i.e., NLRP4, 5, 8, 9, 11, 13, and 14) arehighly expressed in fully grown GV-intact, maturing (MI) andmature (MII) oocytes. While the degradation of NLRP11 and14 mRNA begins at MII stage oocyte, the relatively high-levelexpression of NLRP4, 5, 8, 9, and 13 persists until the 4-cellstage and diminishes thereafter (Fig. 4B). Q-PCR dataconfirmed the RT-PCR results with NLRP4, 5, 8, 9, 11,13, and 14 (Fig. 4C). Furthermore, among the seven mater-nally expressed NLRP genes, NLRP4 and 5 appear to be themost enriched in fully grown (GV intact) oocytes, andNLRP13 transcript is the least abundant.

Figure 1. Phylogenetic tree of NLRP proteins in mice, macaques andhumans. [See color version online at www.interscience.wiley.com.]

Figure 2. Expression of NLRP1-14 in macaque tissues. Total RNAsextracted from the brain cortex (Br), hypothalamus (Hyp), pituitary(Pit), heart (He), lung (Lu), liver (Li), kidney (Ki), uterus (Ut), oviduct(Ovi), thymus (Thy), spleen (Sp), ovary (Ov), and testis (Te) weresubjected to RT-PCR as described in Materials and Methods Section.Cyclophilin A (PPIA) was used as positive internal control.

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Subcellular Localization of NLRP5 Protein in theMacaque Ovary

To determine the subcellular localization of macaqueNLRP5, we performed immunofluorescence with antibodiesagainst mouse and human NLRP5 in the macaque ovary(Fig. 5). FITC-labeled NLRP5 is detected specifically inthe cytoplasm of the oocyte beginning at the primary folliclestage (Fig. 5A) and continuing to the preovulatory folliclestage (Fig. 5B). This observation is consistent with previousfindings in mice (Tong et al., 2004). No green fluorescencewas observed in normal rabbit serum treated ovariancryosections (Fig. 5C).

DISCUSSION

To date, approximately 20 NLRP family members havebeen identified in mice. Compared to the NLRP family inprimates, members of the mouse NLRP family are morediverged. For example, seven mouse NLRP proteins(named NLRP4A-G) are homologous to human NLRP4,and three mouse NLRP proteins (NLRP9A-C) show homol-ogy to human NLRP9. On the other hand, no close mouse

homologues to human NLRP7, 8, 11, and 13 have beenfound (Fig. 1). While macaque NLRP proteins share be-tween 84% and 98% amino acid identity with their humancounterparts, the identity between mouse and primateNLRP proteins varies from as low as 17% (mouse NLRP4Dvs. macaque NLRP4 variant 1) to as high as 83% (mouse vs.human or macaque NLRP3). Similar to previous findings inhumans, the majority of macaque NLRP genes (NLRP2, 4,5, 7, 8, 9, 11, 12, 13) are localized in a region on chromosome19 which is syntenic to a region on mouse chromosome 7where most mouse Nlrp genes reside (with an exception ofNlrp4f which is localized on mouse chromosome 13)(Tschopp et al., 2003; Hamatani et al., 2004).

To our surprise, all 14 NLRP transcripts are detectable inthe gonad in rhesus macaques. Although our RT-PCR datawere not quantitative, it is obvious that most NLRP mRNAs(except for NLRP6 and 12) are relatively enriched in eitherthe ovary or testis (Fig. 2). This gonad-preferential expres-sion pattern of the NLRP genes suggests important roles ofthe NLRP family in the reproductive system in nonhumanprimates. Since our research interest focuses on femalereproduction, we only analyzed eight NLRPs that are spe-cifically or preferentially transcribed in the ovary. Among the

Figure 3. In situ hybridization of selected NLRPs in the monkey ovary. Antisense probes of NLRP2, 4, 5,8, 9, 11, 13, and 14 were labeled with 35S-UTP, and hybridized with paraffin embedded monkeyovarian sections. Arrows denote oocytes at different follicular stages: PrF, primordial follicles; PF,primary follicles; SF, secondary follicles; AF: antral follicles. BF: bright field; DF: dark field.

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eight NLRP genes (i.e., NLRP2, 4, 5, 8, 9, 11, 13, and 14),homologues of NLRP8, 11, and 13 have not been discov-ered in rodents; however, limited reports on other NLRPgene homologues in lower species indicated their conservedoocyte-selective expression in the ovary during evolution(Dade et al., 2004; Hamatani et al., 2004; Horikawa et al.,2005; Ponsuksili et al., 2006). Furthermore, the temporalexpression and degradation of seven NLRP transcripts(NLRP4, 5, 8, 9, 11, 13, and 14) during early embryogenesiscoincide with the timing of oocyte-to-embryo transition inprimates, indicating their exclusive maternal origin. Thisexpression pattern is consistent with that of identifiedmaternal effect genes. Due to the current availability ofeffective antibodies against NLRP proteins, we only testedthe translation of NLRP5 in the macaque ovary. ConsideringNLRP5 predominantly functions in preimplantation embryodevelopment (in the mouse at least), translation of NLRP5gene begins fairly early during oogenesis in nonhumanprimates. Since NLRP family members share similar proteinmotifs, we speculate that the subcellular localization ofNLRP5 is representative.

Like human NLRP proteins (Tschopp et al., 2003), ma-caque NLRP family members share several common motifs:an amino (N)-terminal pyrin domain (PYD) followed by aNACHT domain and a carboxyl (C)-terminal leucine richrepeat domain (LRR), with an exception in NLRP10 whichlacks the C-terminal LRR. NLRP1 is the only NLRPprotein that contains a CARD at its C-terminal. PYD is aprotein–protein interaction module and has been identified inapproximately 20 human proteins that participate in stresssignaling pathways, leading either to stress kinase/nuclearfactor kB (NF-kB) activation or to apoptosis (Kohl andGrutter, 2004). Notably, unlike human and macaqueNLRP5, mouse NLRP5 does not possess a PYD at itsN-terminal. NACHT domain contains seven distinct motifs,including the ATP/GTPase-specific P-loop and Mg2þ-binding site. The NACHT in NLRP proteins is predicted tobind ATP (Koonin and Aravind, 2000). LRRs are 20–29-residue sequence motifs that may provide a versatile struc-tural framework for the formation of protein–proteininteractions (Kobe and Kajava, 2001), and have been iden-tified in many proteins with different functions.

Though NLRP family members are structurally related,there seems no functional redundancy in mice as the ab-sence of NLRP5 alone causes infertility (Tong et al., 2000a).To date, the most extensively studied NLRP proteins areNLRP1, 3, 5, and 7. Human NLRP1 mRNA was found inmultiple tissues. Though it was initially reported to induceapoptosis (Chu et al., 2001; Hlaing et al., 2001), recentlyaccumulating evidence shows that endogenous NLRP1holds a crucial role in the activation of proinflammatorycaspases (Chu et al., 2001). The expression of NLRP3 wasthought to be restricted to immune cells. Mutations withinNLRP3 gene have been linked to three human diseases, allinvolving multisystemic inflammation: familial cold auto-inflammatory syndrome, chronic infantile neurologic cuta-neous and articular syndrome and Muckle–Wells syndrome(Hoffman et al., 2001). NLRP5 (MATER) is required forembryogenesis beyond the 2-cell stage in the mouse, im-plying that it might be involved in embryo genome activation

Figure 4. The expression of selected NLRP genes during the oocyte-to-embryo transition. A: Schematic illustration of the oocyte-to-embryotransition in primates. Maternal (oocyte)-derived gene products sup-port the first several embryo cleavages, and the embryonic genomeactivation occurs between the 4-cell and the 8-cell stages upon whichtime the maternal transcripts begin to decay. B: RT-PCR data showingtemporal expression of oocyte-selective NLRPs during oocyte matura-tion, fertilization, and early embryo development. Total RNA wasextracted from germinal vesicle-intact (GV), metaphase I (MI) and II(MII) stage oocytes, zygotes (Zy), the 2-cell (2-C), 4-cell (4-C), 8-cell(8-C), morula (Mr) and blastocyst (Bl) stage embryos and reverselytranscribed into cDNA. The cDNAs were used as templates to amplifyeach individual NLRP gene. Cyclophilin A (PPIA) was used as internalloading control. C: Relative expression of selected NLRP genes inoocytes and early embryos based on our quantitative PCR (Q-PCR)data. Relative concentrations of the targeted genes were normalized by18S rRNA levels, and the ratios were log-transformed. [See colorversion online at www.interscience.wiley.com.]

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(Tong et al., 2000a). NLRP7 was the first identified maternaleffect gene in humans, and mutations of which have beenfound in recurrent hydatidiform moles and various formsof reproductive wastage such as spontaneous abortion,stillbirths and intrauterine growth retardation (Djuric et al.,2006; Murdoch et al., 2006; Qian et al., 2007). No mousehomologue of NLRP7 has been identified.

Interestingly, besides NLRP5 and 7, several uncharac-terized NLRP family members also appear to function infemale reproduction. Mouse Nlrp14 was identified as one ofthe differentially upregulated genes during the transitionfrom primordial to primary follicles and is only expressed inovaries and testes (Horikawa et al., 2005). Nlrp14, along withNlrp4b, 4c, and 4f, are downregulated in the absence ofeither FIGLA or NOBOX, both of which are crucial femalegermline survival factors (Choi et al., 2007; Joshi et al.,2007). Moreover, several mouse oocyte-specific Nlrpgenes (Nlrp4a, 9b, 14) that are homologous to humanNLRP4, 9, and 14 are highly expressed in ‘‘young’’oocytes (obtained from 5- to 6-week-old animals), but sig-nificantly decreased in ‘‘aged’’ oocytes (42–45-week-oldanimals) (Hamatani et al., 2004), suggesting a role of theseoocyte-specific Nlrp genes in female fertility as the oocytequality declines rapidly with aging. Indeed, knocking downNlrp14 in mouse fertilized eggs resulted in an developmentalarrest between the 2-cell and the 8-cell embryonic stages innearly 50% of the embryos (Hamatani et al., 2004). In aneffort to identify pivotal molecules for oogenesis and theoocyte-to-embryo transition in mice, Nlrp2, 4f, 5, and 14were found highly enriched in fully grown oocytes, butdiminished in the 2-cell embryos upon embryo genomeactivation (Evsikov et al., 2006). NLRP8 and NLRP9 were

also specifically expressed in the ovary and testis in cattle,and identified as oocyte markers in cows (Dalbies-Tranet al., 2005; Ponsuksili et al., 2006). Though functionallyunknown, bovine NLRP5, 8, and 9 are mapped to a quanti-tative traits locus (QTL) region for reproductive traits onchromosome 18.

CONCLUSION

In summary, we have identified homologues of 14 NLRPgene family members in rhesus macaque monkeys. Thespatial and temporal distribution of NLRP genes indicatesthat many members in the family are oocyte-selective innonhuman primates, suggesting important roles of NLRPs inoogenesis, oocyte maturation and early embryo develop-ment. Although the NLRP family is not well studied in thereproductive system, limited existing data imply their poten-tial functions in the survival of oocytes and early embryos.Further characterization of the oocyte-selective NLRPs inmultiple species along with the signaling pathway/interactiveprotein analysis may eventually unravel an oocyte-specificgenetic network that is crucial for female fertility.

MATERIALS AND METHODS

Animals and Husbandry

Tissues were collected from adult rhesus macaques at agesbetween 5 and 15 years old through a tissue distribution programprovided by Division of Animal Resources (DAR) at OregonNational Primate Research Center (ONPRC). All animal protocolsand procedures were approved by the Institutional Animal Care and

Figure 5. Subcellular localization of NLRP5 in the macaque oocyte. Frozen monkey ovarian sectionswere incubated with anti-NLRP5 peptide antisera (A,B, 1:200) or normal rabbit sera (C, 1:200)followed by fluorescein-conjugated goat-anti-rabbit IgG (1:200) (green fluorescence). The ovarysections were counterstained with DAPI (blue fluorescence). PF: primary follicles; SF: secondaryfollicles; AF: antral follicles. Arrows denote the oocyte inside the follicle.

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Use Committee (IACUC) of Oregon Health & Science University(OHSU) and were conducted in accordance with the NationalInstitute of Health’s Guide for the Care and Use of LaboratoryAnimals. All surgical procedures were conducted under generalanesthesia by trained personnel in the Surgery Department atONPRC, under the supervision of veterinarians in dedicated surgi-cal facilities using aseptic techniques and comprehensive physio-logical monitoring.

Database Mining and Sequence Deduction

Full-length human NLRP cDNAs were used to search theNational Center for Biotechnology Information (NCBI) GenBankdatabase (nonhuman, nonmouse genomic and transcripts) andRhesus Macaque Genome Resources database (http://www.ncbi.nlm.nih.gov/ projects/genome/guide/rhesus_macaque). Macaqueexpressed sequence tags (ESTs), predicted macaque genetranscripts and genomic fragments that contain individual NLRPhomologous sequences were downloaded and analyzed by com-paring the alignment with corresponding human NLRP cDNAsand searching for open reading frames (ORFs). The ORFs ofdeduced cDNAs were determined by Vector NTI software(Invitrogen, Carlsbad, CA). Translated protein from deduced indi-vidual macaque NLRP cDNAs were also aligned with homologoushuman NLRP proteins with Vector NTI software for comparison andverification. RT-PCR was performed to fill the gaps in thesequences.

Oocytes and Preimplantation Embryos Collection

For oocyte collection, the controlled ovarian stimulation (COS)and follicular aspiration procedure were performed as follows(Stouffer and Zelinski-Wooten, 2004): rhesus monkeys with regularmenstrual cycles were given 30 IU recombinant human follicularstimulating hormone (FSH) (product donation from Organon)intramuscularly (IM) twice daily for six consecutive days startingon the first 3 days of menses; on day 7 and day 8 of treatment, 30 IUFSH in combination with 30 IU of recombinant human luteinizinghormone (LH) (product donation from Ares-Serono) were IM in-jected once a day; on day 7 of treatment, a single injection of Acyline(NICHD, Contraceptive Branch), a potent suppressor of circulatinggonadotropin-releasing hormone (GnRH), was given sub-cutaneously (SC) at a dose of 0.75 mg/kg body weight to preventendogenous LH surge; approximately 36 hr prior to scheduledaspiration, a single dose of 1,000 IU human chorionic gonadotropin(hCG) was IM injected in the monkeys. Laparoscopic follicularaspiration was conducted on day 10 to obtain metaphase I (MI)and metaphase II (MII) oocytes. Germinal Vesicle (GV)-intactoocytes were collected through follicular aspiration on day 8 withouthCG treatment (Zheng et al., 2005). Mature oocytes (MII) werefertilized by intracytoplasmic sperm injection (ICSI), and zygotes,the 2-cell, 4-cell, 8-cell, morula and blastocyst stage embryos werecollected at appropriate times post fertilization.

Oocytes used for RT-PCR and ICSI were collected in TalpHepes medium (Macklon et al., 2006) and maintained at 37�C.Fertilized eggs (indicated by formation of two pronuclei) werecultured in hamster embryo culture medium 9 (HECM-9) in anatmosphere of 5% CO2, 5% O2, 90% N2 until the 4-cell stage. The4-cell embryos were transferred to HECM-9 medium containing 5%fetal bovine serum (FBS) and cultured until the blastocyst stage.Embryo culture media were changed every 48 hr (Wolf et al., 2004).

RNA Isolation, RT-PCR, and Taqman QuantitativePCR (Q-PCR)

Total RNA was extracted from monkey tissues using Trizolreagent (Invitrogen) following the manufacturer’s manual. For RNAextraction from monkey oocytes and embryos, Absolutely RNA

Nanoprep kit (Stratagene, La Jolla, CA) was applied according tothe manufacturer’s instructions. Oocytes and early embryos werewashed briefly in PBS at room temperature and transferred to thelysis buffer supplied in the kit. Total RNAs isolated from macaquetissues, oocytes and preimplantation embryos were treatedwith RNase-free DNase (Promega, Madison, WI) and reverse-transcribed into cDNA with random hexamer and Superscript IIIReverse Transcriptase (Invitrogen). PCR was carried out withspecific primers listed in Table S1 (Supplementary Material).Gene-specific primers were designed based on the sequence atthe common region if more than one transcript may exist. All PCRproducts were verified by DNA sequencing.

Q-PCR primers and probes (Table S2, Supplementary Material)were designed using PrimerExpress software (Applied Bio-systems, Foster City, CA), and purchased from Invitrogen andApplied Biosystems, respectively. Amplifications were conduct-ed in a 10 ml final volume containing 250 nmol/L TaqMan NLRPprobe, 300 nmol/L NLRP forward and reverse primers, 250 nmol/LTaqMan 18S probe, 100 nmol/L forward and reverse 18S primers,1� TaqMan Universal PCR master mix (Applied Biosystems), andoocyte (or early embryos) cDNA. The Q-PCR reactions wereconducted in an ABI PRISM 7500 fast Real time PCR System(Applied Biosystems). The internal standard curve, used for relativemRNA quantification, was generated from five two-fold dilutions ofpooled monkey oocyte cDNA. The threshold cycle (CT) values forunknown samples were used to extrapolate the amount of RNAequivalents from the internal standard curve. The RNA equivalentvalues were then normalized by 18S RNA equivalent values derivedfrom the same internal standard curve.

In Situ Hybridization

Each macaque NLRP gene-specific amplicon obtained by RT-PCR as described above was cloned into the pGEMT-vector(Promega Corp., Madison, WI). The orientation of the insert wasdetermined by DNA sequencing, and the specificity of the sequencewas verified by NCBI nonredundant necleotide database searchusing BLAST (Basic Local Alignment Search Tool). Monkey ovarieswere fixed in 4% paraformaldehyde, embedded in paraffin, andsectioned at 5 mm intervals. a-[35S]UTP-labeled antisense andsense probes were generated by the Riboprobe T7/SP6 combina-tion systems (Promega Corp., Madison, WI). Hybridization wascarried out as previously described (Wu et al., 2003, 2004).

Immunofluorescence

Freshly collected monkey ovaries were directly embedded inOCT medium on dry ice blocks. The 5 mm-thick ovarian cryosec-tions were fixed in ice-cold acetone for 15 min, then blocked withPBS buffer containing 5% normal goat serum (Vector Laboratories,Burlingame, CA) for 30 min at room temperature (RT) and incubat-ed with rabbit anti-NLRP5 (a gift from Drs. Lawrence Nelson andZhi-bin Tong at National Institute of Health) at 1:200 dilution for 1 hrat RT (Tong et al., 2002, 2004). After 30 min wash with PBS at RT,the ovarian sections were incubated with fluorescein-conjugatedgoat-anti-rabbit IgG (Vector Laboratories) at 1:200 dilution for 1 hrat RT. The sections were counter stained with 10 mg/ml DAPI andmounted with ProLong Gold antifade reagent (Invitrogen).

ACKNOWLEDGMENTS

The authors thank the following core facilities at Oregon NationalPrimate Research Center: the Assistant Reproductive Tech-nologies (ART) core for assisting in monkey oocyte and earlyembryo collection; the Molecular and Cellular Biology (MCB) corefor sequencing; and the Imaging and Morphology (IM) core fortissue processing and photographing. We are grateful to

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Dr. Lawrence Nelson and Dr. Zhi-bin Tong for providing rabbit anti-NLRP5 peptide antisera. The study is supported by a start-up fundprovided by Oregon National Primate Research Center, OregonHealth & Science University to Dr. Xuemei Wu.

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