Role for Primary Cilia in the Regulation ofMouse Ovarian FunctionEllen T. Johnson, Teodora Nicola,† Kevin Roarty, Bradley K. Yoder, Courtney J. Haycraft,‡ andRosa Serra*
Cilia are common organelles presenton nearly all cells. There are threebasic types: motile, primary, andnodal cilia. Primary cilia are nonmo-tile, solitary organelles projectingfrom the surface of cells (reviewed inDavenport and Yoder, 2005; Satir andChristensen, 2007). Their function onmost cells remains elusive. The pro-cess of intraflagellar transport (IFT) isresponsible for building and maintain-ing the structure and function of pri-mary cilia. Ift88/Tg737/Polaris is acore component of the complex B IFTparticle and disruption of Ift88 hasbeen shown to result in the loss of cilia(Pazour et al., 2000; Murcia et al.,2000; Yoder et al., 2002; Haycraft etal., 2007).
The presence of primary cilia oncells in the ovary has been docu-mented. Primary cilia have been de-tected on estrogen producing granu-losa cells of antral follicles as well ascells of the ovarian surface epithelium(Herman and Albertini, 1983; Teil-mann et al., 2005, 2006; Teilmann andChristensen, 2005). Furthermore, itwas shown that prolactin induced dif-ferentiation of granulosa cells re-sulted in a higher incidence of ciliawithin the population of cells (Her-man and Albertini, 1983). Motile ciliaare present on the epithelial cells ofthe oviduct. The functional unit of theovary is the ovarian follicle. Withinone ovary, follicles of varying stagecan be seen. The mature antral folliclecontains the oocyte, several layers of
granulosa cells, and a fluid filled spacecalled the antrum. Stromal cells sur-rounding the follicle form the thecalayers. Ovulation, the release of theoocyte, results in the collapses of thefollicle forming the corpus luteum.Maturation of the follicle and ovula-tion are controlled by a complex or-chestration of hormones from the hy-pothalamus/pituitary gland and theovary. This hormonal cycling is calledthe estrous cycle in rodents (Goldmanet al., 2007). Estrogen is required tomaintain a normal estrous cycle andmice with targeted deletion of estro-gen receptor alpha (ERKO) are acyclic(Couse and Korach, 1999). Neverthe-less, estrogen is not required for earlydevelopment of the follicle but it isrequired for ovulation and thus fertil-
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama†Dr. Nicola’s present address is Department of Pediatrics/ Division of Neonatology, University of Alabama at Birmingham.‡Dr. Haycraft’s present address is Department of Medicine/ Division of Nephrology, Medical University of South Carolina.*Correspondence to: Rosa Serra, Department of Cell Biology, University of Alabama at Birmingham, 1918 University Blvd.,MCLM 660, Birmingham, AL 35294-0005. E-mail: [email protected]
DOI 10.1002/dvdy.21612Published online 10 July 2008 in Wiley InterScience (www.interscience.wiley.com).
ity (Fisher et al. 1998; Toda et al.,2001; Britt and Findlay, 2002). Micewith targeted disruption of Cyp19, thegene for the primary estrogen-synthe-sizing enzyme, have normal folliclesat varying stages of maturation; how-ever, they do not demonstrate corpusluteum.
Mammary gland development in-volves the coordination of signals fromboth systemic and local factors as wellas coordination between the epithelialand stromal compartments (Reviewedin Imagawa et al., 1994; Silberstein,2001). At the onset of puberty, ovarianhormones, particularly estrogen,stimulate the appearance of terminalend buds (TEBs), specialized struc-tures where active cellular prolifera-tion and apoptosis occur (reviewed inHowlin et al., 2006). The TEBs ramifythrough the fat pad until approxi-mately 8 weeks of age when theyreach the limit of the fat pad and theTEBs disappear. Previously, ultra-structural examination of bovinemammary tissue revealed the pres-ence of primary cilia on epithelial andmyoepithelial cells (Nickerson, 1989)and on intralobular fibroblasts in hu-man tissue (Yamazaki and Eyden,1995).
Because mice with targeted germ-line disruptions of Ift88 die in midges-tation with multiple organ defects, atargeted approach to understandingcilia function is required (Murcia etal., 2000). We previously reported thegeneration of mice in which exons 4through 6 of the Ift88 locus wereflanked with loxP sites (Haycraft etal., 2007). When crossed to mouselines expressing Cre recombinase the
deletion was shown to result in a nullallele and depletion of cilia in the spe-cific targeted cells (Haycraft et al.,2007). In this report, we used thePrx1-Cre–expressing line to deletecilia in a specific array of cells. Prx1-Cre is most commonly used to deletespecific genes from very early limbmesenchyme and cranial mesoderm;however, Prx1-Cre is also expressed inthe ventral mesoderm of embryosstarting at E10.5 days (Logan et al.,2002). Furthermore, Prx1-Cre is ex-pressed in the follicle cells of the ovaryallowing us to use this line to examinethe role of cilia in ovarian function.
RESULTS
Ift88 and Prx-1 CreExpression in the Ovary
We previously showed that mice withtargeted deletion of Ift88 in Prx1-Cre–expressing cells have defects in skele-tal development including short limbs(Haycraft et al., 2007). Although themice are smaller than littermate con-trols they appear healthy in that theyinteract and groom normally. Thepresence of primary cilia on granulosacells has previously been reported(Herman and Albertini, 1983; Teil-mann et al., 2005, 2006; Teilmann andChristensen, 2005). Here, we deter-mined the expression pattern of Ift88in the adult ovary using a mouse linein which LacZ was inserted into theIft88 locus (Murcia et al., 2000). Cellsthat express Ift88 were detected byblue X-gal staining (Fig. 1A,B). Ift88was highly expressed in the granulosacells of antral follicles with less stain-
ing in immature follicles (Fig. 1B vs.1A). High levels of expression werealso observed in oocytes, the surfaceepithelium, and the epithelium of theoviduct, which contains motile cilia(Fig. 1A and data not shown). A low tomoderate level of expression was seenon theca cells and stromal cells (Fig.1B).
Next, we determined which celltypes in the ovary contain Prx1-Creactivity (Fig. 1E,F). The ROSA26 al-lele has been used to generate a Cre-reporter line that conditionally ex-presses LacZ in the presence of Credue to the excision of a stop codonplaced in front of the LacZ gene. Whencrossed to the Prx1-Cre line, cells thatexpress Cre as well as all of the prog-eny of the cells will be stained bluewith X-gal (Soriano, 1999). As previ-ously reported, Cre activity was ob-served in the oocyte (Fig. 1F; Logan etal., 2002). Cre activity was detected innewborn mice in the cuboidal epithe-lial cells of the immature follicle, cellsthat will differentiate into granulosacells as the ovary matures (Fig. 1E).This was confirmed in ovaries from6-week-old mice (Fig. 1F). Granulosacells in follicles of various stages werestained with X-gal indicating Cre ac-tivity at some point in the develop-ment of these cells. The intensity ofstaining varied among follicles. Nostaining was seen in the surface epi-thelium or in the ovary stroma (Fig.1F). Strong Cre activity was also de-tected in the epithelium of the oviduct,which contains motile cilia (data notshown). It is expected that Ift88 willbe deleted in all cells that contain Creactivity as well as all of the progeny of
Fig. 1. Ift88 and Prx1-Cre are expressed in ovary and mammary gland. A–D: Mice in which LacZ was inserted into the Ift88 locus were used todetermine the expression pattern of Ift88 in the ovary (A,B) and mammary gland (C,D). Blue X-gal staining in tissue from a 3-week-old mouse is shown.B: A mature antral (a) follicle. A: Less mature follicles. Staining was observed in oocytes (o) and granulosa cells (g) of the follicles (F; double arrow).A high level of staining was also observed in the surface epithelium (se). Less staining was seen in the theca cells (t) and stroma (s). C,D: The imagein C shows a whole mount of the mammary gland (3 weeks) with D showing sectioned tissue. Staining was seen in the ducts (d; arrow) as well as theperiductal stroma (s) and adipose (ad). TEB denotes a terminal end bud. Blue X-gal staining was not observed in wild-type control mice (not shown).E–H: Prx1-Cre mice were crossed to mice containing a Cre activatable LacZ in the ROSA26 locus. Cre activity was detected as blue X-gal stainingin the ovary (E,F) and mammary gland (G,H). E,F: Cre activity was detected in cuboidal follicle cells (fc) of the newborn ovary (E) as well as in the oocyte(o) and granulosa cells (g) of follicles in a 6-week-old mouse (F). E was counterstained with eosin. G,H: In the mammary gland, staining was seenthroughout the adipose (ad) and stroma in 2-week-old mice. H: Staining was not detected in the ducts (d). G shows a whole-mount stain and H showssections. Blue X-gal staining was not observed in the Cre-negative controls (not shown). I: DNA isolated from total mammary gland and ovary of 12-and 3-week-old control (Prx1-Cre;Ift88lox/wt (�;l/w) or Ift88 lox/lox (�;�/�)) as well as mutant (Prx1Cre;Ift88 lox/lox (�;l/l)) mice was subjected to PCRanalysis of the Ift88 locus. The top band represents the lox allele. The middle band represents the wild-type (wt) allele and the bottom band representsthe deleted allele (�). The deleted allele was present in both the ovary and mammary gland suggesting Cre was active in these organs. Tail DNA wasused as a control. Prx1-Cre is not expressed in the tail so the deleted allele was not detected in Cre� mice (�;l/w). J,K: Cilia were immunostainedin control (J) and mutant (K) granulosa cells using an anti acetylated tubulin antibody. Acetylated tubulin is shown in red. Cilia are denoted by smallarrows. The antrum is marked with an a. The nuclei of the cells are stained green with YoPro. Abundant cilia are detected in the control (J) granulosacells while only a few are seen in the mutant (K).
those cells including granulosa cells.Because we detect Cre activity in new-born mice, it is likely that Ift88 is de-leted in these very young mice beforethe onset of puberty.
To confirm Ift88 deletion in theovary, we used a polymerase chain re-action (PCR) -based assay (Fig. 1I).DNA was isolated from whole ovariesof 12- and 3-week-old mutant mice aswell as a Prx1-Cre;Ift88lox/wt andIft88lox/lox controls. DNA from tail,which does not express Prx1-Cre, wasused as an additional control. DNAwas used in a PCR assay that simul-taneously detected the lox, wild-type,and deleted alleles of Ift88 . When theCre was present in the ovary a bandrepresenting the deleted allele wasclearly visible, indicating Cre activityand deletion of Ift88 . The deleted al-lele was not detected in Cre-negativesamples or in the samples of tail DNAsuggesting that Ift88 was indeed de-leted from cells within the ovary inPrx1-Cre mutant mice. Because Creactivity is observed in the granulosacells, it is expected that Ift88 is de-leted from these cells (Fig. 1E,F). Weconfirmed depletion of cilia in thegranulosa cells of the mutant ovarycompared with control ovaries by im-munostaining with acetylated tubulin(Fig. 1J,K). Cilia were detected onmany granulosa cells within the folli-cles of wild-type mice (Fig. 1J). Veryfew cells contained structures thatlooked like cilia in follicles fromPrx1Cre:Ift88lox/lox mice (Fig. 1K).
Ovarian Function Is Alteredin Prx1-Cre Mice
To begin to determine whether therewere defects in ovarian function in thePrx1-Cre mice, we examined the es-trous cycle in control and mutant mice
(Fig. 2A). The estrous cycle is regu-lated by ovarian hormones includingestrogen (Couse and Korach, 1999).Mice normally cycle over 5 daysthrough four stages: estrus, metestrus,diestrus, and proestrus. The mouseestrous cycle can be staged by vaginalcytology. Each stage is distinguishedby the cell types present in the swab(Goldman et al., 2007). Vaginal swabswere taken from three 12-week-oldcontrol and mutant mice everyday for16 days. As expected, in control mice 2days of diestrus preceded proestrusand estrus. In contrast, the estrouscycle was altered in mutant mice.Each stage was hard to define exceptdiestrus, which lasted a minimum of 5to 7 days. The results suggest alter-ations in ovarian function in the mu-tant mice.
Because we were never able to ob-tain litters from the mutant mice, wewanted to determine whether themice were able to ovulate. To this endwe sectioned through four control andthree mutant ovaries and hematoxy-lin and eosin (H&E) stained everythird section. Comparison of ovarianhistology in Prx1Cre;Ift88lox/lox miceindicated that all the stages of folliclematuration, including primary to an-tral follicles, were present in controland mutant mice (Fig. 2B). We did notdetect corpus lutea in mutant ovaries;however, there were many observed inthe controls (Fig. 2B). The results sug-gest that ovulation in the mutant miceis impaired. Next, we compared thenumber of ovulated oocytes in the ovi-ducts of control (n � 2) and mutant(n � 2) mice after superovulation withpregnant mare serum gonadotropin(PMSG) followed by human chorionicgonadotropin (hCG). Oocytes sur-rounded by cumulus cells were easily
recovered from control mice; however,we were not able to recover any oo-cytes from the oviducts of mutantmice. We then compared the histologyof ovaries from wild-type and mutantmice after superovulation to deter-mine whether corpus lutea werepresent (Fig. 2B). Again many corpuslutea were observed in the controlmice (Fig. 2B). Corpus lutea were alsodetected in the superovulated mutantmice although to a lesser extent. Theresults indicate the mice are capableof forming corpus luteum whentreated with exogenous hormones.The formation of corpus luteum undersuperovulatory conditions was also re-ported for ERKO mice (Rosenfeld etal., 2000). Together the results sug-gest that ovulation in Prx1-Cre;Ift88lox/lox mice is impaired.
Because the formation of TEBs inthe developing mammary gland is reg-ulated by ovarian hormones, primar-ily estrogen, we compared mammarygland development in control andIft88 mutant mice (Fig. 2C). Mam-mary glands were removed from con-trol (Ift88lox/lox, Ift88lox/wt, or Prx1-Cre;Ift88lox/wt) and mutant (Prx1-Cre;Ift88lox/lox) mice at 3 weeks, 5 weeks, 8weeks, and 12 weeks of age andstained with Carmine red to observethe overall ductal structure of thegland (Fig. 2C). At 3 weeks of age, theducts have not begun to extendthrough the fat pad and the TEBs arejust starting to develop in the controlmice. In mutant mice, the ducts hadnot extended through the fat pad andno TEBs were detected. At 5 weeks ofage, the control mice demonstrated atypical ductal pattern. The ductal treehad extended approximately halfwaythrough the fat pad and large TEBswere observed. In contrast, the mu-
Fig. 2. Ovarian function is altered in mutant mice. A: Vaginal swabs were taken everyday for 16 days from three control and three mutant mice todetermine the estrous cycling pattern of the mice. Swabs from a representative control and mutant mouse are shown. The control mice cycled asexpected with two days of diestrus (D) in between 1 day each of estrus (E), metestrus (M) and proestrus. Diestrus is characterized by a large numberof small dark staining leucocytes (l). Proestrus contains primarily nucleated epithelial cells (n) with some leukocytes and some cornified anuclear cells(c). Estrus consists mainly of large cornified (anucleated) cells with no leukocytes. Leucocytes appear with the cornified epithelial cells duringmetestrus. The stage of estrous was difficult to determine in the mutant mice but it appeared as if the mice stayed in diestrus (D; characterized by alarge number of small dark staining leukocytes) for a minimum of 5 to 7 days. B: Sections of ovaries from control and Prx1Cre;Ift88lox/lox mice werestained with hematoxylin and eosin (H&E). Sections from the ovaries of 12 week old control (a,c) and mutant mice either untreated (a,b) orsuperovulated (c,d) are shown. Follicle cells (f) are small and stain dark purple. Cells in the corpus luteum (cl) stain lighter than follicle cells and are largerwith more cytoplasm. C:Carmine red stained mammary glands from control and Prx1-Cre;Ift88lox/lox mice at 3, 5, 8, and 12 weeks of age. Mutant micedemonstrate a severe delay in mammary gland development. Insets show the end buds in control mice and the lack of end bud structures in mutantmice. By 8 weeks of age mammary ducts fill the fat pad in control mice. In contrast, the fat pad is not filled in mutant mice as late as 12 weeks of age(length of most terminal duct shown with arrows) and end buds are still not visible. L denotes the lymph node.
2056 JOHNSON ET AL.
Fig. 2.
tant gland consisted of a rudimentaryductal tree similar to that seen in pre-pubescent mice. TEBs were not ob-served. The phenotype resembled thatseen in mice deleted for estrogen re-ceptor alpha (ERKO; Korach et al.,1996; Mueller et al., 2002; Mallepell etal., 2006; Feng et al., 2007). By 8weeks of age, the fat pad of controlmice was completely filled with epi-thelium. In mutant mice at 8 and 12weeks of age, the ductal tree had onlyextended through approximately halfof the fat pad and TEBs were not ob-served. The results indicate a severedelay in extension of the ductsthrough the fat pad, likely a result ofdefects in the formation of TEBs,which are under the control of ovarianhormones.
Ift88 and Cre Expression inthe Mammary Gland
Because Prx1-Cre was expressed inthe precursors of the mammary mes-enchyme, we wanted to determinewhether Ift88 was expressed in themammary gland and if Cre activitywas also detected there. Previousstudies demonstrated cilia on epithe-lial, myoepithelial, and stromal fi-broblasts in bovine and human
mammary tissue by electron micros-copy (Nickerson, 1989; Yamazakiand Eyden, 1995). To determine theexpression pattern of Ift88 in themammary gland, we used the Ift88-LacZ mice described above (Fig.1C,D). Strong blue X-gal stainingwas detected in the ducts of 3-week-old mice (Fig. 1C,D). Weaker stain-ing was observed in the periductalstroma and fat adjacent to the ducts.Expression was reduced or absent inthe stroma further away from theducts. Using the ROSA26 reporterline, Cre activity was detected in themammary stroma (Fig. 1G,H). Creactivity was not detected in the epi-thelium, indicating that Ift88 andcilia are intact in the mammary ep-ithelium of Prx1-CreIft88 mutantmice. The results suggest that Ift88 ,and subsequently cilia, are expectedto be deleted only from the stroma ofthe mammary gland in the mutantmice. We also used a PCR-based as-say to show that Ift88 is deleted inthe mammary gland (Fig. 1I). Be-cause Cre activity is only detected inthe stroma, the level of deletion ob-served in the PCR assay is expectedto be due to loss of the Ift88 gene instromal cells. Recently, null alleles
of the estrogen receptor were gener-ated and it was shown that estrogenacts directly on the mammary epi-thelium to regulate normal develop-ment (Mallepell et al., 2006; Feng etal., 2007). Although glands in Ift88mutant mice had defects that resem-bled those seen in mice with alter-ations in estrogen signaling, the factthat Prx1-Cre did not target themammary epithelium suggests thatthe glands are able to respond to es-trogen but that development is de-layed due to a systemic hormonal in-sufficiency, likely as a result ofdefects in ovarian function.
Mammary GlandDevelopment Resumes Afterthe Addition of Estradiol
To determine whether exogenouslyadded estradiol could restore the de-velopment of the TEBs in the mutantmice, wild-type and mutant mice wereinjected with cyclodextrin encapsu-lated estradiol or cyclodextrin alonefor 7 days (Fig. 3A–D). In wild-typemice the efficacy of the estradiol treat-ment could be seen as increased ex-tension of the duct through the fat padin treated mice versus untreated con-trol littermates (Fig. 3A,B). Estradiol-treated mutant mice demonstratedlarge TEBs as well as increased exten-sion of the ducts through the fat padrelative to untreated mutant mice(Fig. 3C,D). The experiment was re-peated three times. The results indi-cate that the mutant mammary glandcan respond to estradiol. BecauseTEBs and ductal extension throughthe fat pad resume in mutant mice inthe presence of additional estradiol,the mammary TEB phenotype ob-served is most likely secondary to al-terations in ovarian function.
DISCUSSION
In this study, we used a targeted ap-proach to delete Ift88 from cells in theovary. We show that Ift88 is expressedand that Prx1-Cre activity is detectedin the follicle cells of the ovary. Dele-tion of Ift88 was confirmed using aPCR-based assay. Prx1Cre Ift88 mu-tant mice demonstrated abnormalitiesin the estrous cycle, alterations inovulation, and a delay in mammarygland development, characterized by a
Fig. 3. Estradiol rescues the terminal end bud (TEB) phenotype in mutant mammary glands. A–D:Five-week-old wild-type (A,B) and mutant (C,D) mice were injected ip with 20 �g of cyclodextrinencapsulated E2/ mouse everyday for 7 days (B,D). A,C: Control mice were injected with cyclo-dextrin alone. Mammary glands were removed and stained with Carmine red. In wild-type mice,treatment with estrogen resulted in increased expansion of the ducts through the fat pad asindicated by the large arrows. The lymph node was used as a structural landmark (L). The resultindicated that the estrogen injections were working. Likewise, mutant mice demonstrated in-creased extension of the ducts through the fat pad as well as formation of large terminal end buds(small arrows). The results suggest that the gland can respond to estradiol and that the delay inmammary development is likely due to defects in ovarian function. A representative of threeexperiments is shown.
2058 JOHNSON ET AL.
lack of TEBs, which are regulated byovarian hormones, most notably estro-gen. Alterations in ovarian functioncan be inferred from these pheno-types. We then showed that the for-mation of TEBs and extension of theduct through the fat pad resumed inmutant mice after treatment with es-tradiol. The results suggest that themutant glands could still respond toestrogen and that delayed mammarydevelopment was a consequence of de-fects in ovarian function.
How deletion of Ift88 results in al-terations in ovarian function in themouse is not clear. We did not detectactivation of Prx1-Cre in the hypo-thalamus or pituitary supporting theidea that the primary defect is in theovary. Because cilia can act to senseand coordinate external chemical sig-nals, it is possible that the responsive-ness of granulosa cells to factors thatregulate the synthesis of estrogen isaltered. However, because ovaries ofmutant mice develop secondary andmature follicles it is unlikely that re-sponsiveness to follicle-stimulatinghormone is affected. It is also possiblethat Ift88 has a direct role in regulat-ing the synthesis or activity of the en-zymes that make estrogen. It has beenshown that the Hedgehog signalingpathway regulates ovarian functionand that granulosa cells are potentialtargets of Hedgehog signaling in theovary (Russell et al., 2007). Cilia arerequired to mediate both the activa-tion of Hedgehog signaling and thesignal repressor functions of Gli3 (re-viewed in Satir and Christensen,2007; Serra, 2008). Treatment withShh was shown to increase growthand proliferation in cultured granu-losa cells but estrogen synthesis wasnot affected. Because estrogen res-cued the mammary phenotype, it issuggested that estrogen synthesis orsecretion is affected by the loss of Ift88in the granulosa cells, although wecannot exclude additional alterationsin the regulation of other ovarian hor-mones.
Even though estrogen has beenshown to act directly on mammary ep-ithelium (Mallepell et al., 2006; Fenget al., 2007), it is known that themammary stroma is critical to normalmammary development (reviewed inParmar and Cunha, 2004). This studydoes not exclude a direct role for Ift88
and cilia in the mammary stroma. It ispossible that there are both systemicand inherent effects on mammary de-velopment that are dependent on Ift88and cilia. For example, during postna-tal mammary development Gli2 andGli3, downstream effectors of Hedge-hog signaling, are expressed in themammary stroma (reviewed in Hat-sell and Cowin, 2006; Hatsell andFrost, 2007). It has been shown thatGli2 is required for normal postnatalmammary development and trans-plant experiments suggested that Gli2acted primarily in the mammarystroma (Lewis et al., 2001). Character-ization of mice with conditional dele-tion of Ift88 in specific cell typeswithin the mammary gland will beused in the future to distinguish be-tween systemic and inherent roles ofIft88 in mammary development.
In summary, we have shown thatdeletion of Ift88 in the ovary results indefects in ovarian function as mea-sured by alterations in the estrouscycle, ovulation, and mammary de-velopment. Future experiments willdetermine the mechanism by whichIFT and cilia affect ovarian function.
EXPERIMENTALPROCEDURES
Maintenance of the MouseColonies
All the mice used in this study werehandled following the guidelines ofthe Institutional Animal Care andUse Committee of the University ofAlabama at Birmingham. Genera-tion and identification of Prx1-Cre;Ift88lox/lox mice were recently de-scribed (Haycraft et al., 2007). Primersfor detecting each of the three possiblealleles are as follows: common 5� GCCTCC TGT TTC TTG ACA ACA GTG;3� flox and wild-type GGT CCT AACAAG TAA GCC CAG TGT T; 3� de-leted allele CTG CAC CAG CCA TTTCCT CTA AGT CAT GTA. The lymphnode was removed from mammaryglands before DNA was extracted forPCR.
X-gal Staining
Whole tissue was stained by X-gal aspreviously described (Alvarez et al.,2002). After post-fixing in paraformal-
dehyde the tissue was transferred to5% sucrose in PBS at room tempera-ture followed by 30% sucrose in PBSat 4°C overnight. An equal amount ofOCT was added and mixed until ho-mogenous. Tissue was embedded inOCT and cryosectioned.
Immunofluorescence
Cilia were stained using an antibodydirected to acetylated tubulin as pre-viously described (Song et al., 2007).
Vaginal Cytology
The estrous cycle was staged as de-scribed in Goldman et al. (2007).
Superovulation Protocol
Two control and two mutant micewere intraperitoneally injected withPMSG followed by hCG 48 hr later.Eighteen hours after the last injectionthe cumulus masses were removedfrom the oviducts and the ovarieswere fixed in 4% paraformaldehydefor standard histological analysis(Hogan et al., 1994).
Carmine Red Staining
Whole mammary glands weremounted onto glass slides and fixed inCarnoy’s fix (25% acetic acid/ 75% eth-anol) for 1 hr, stained with carminealum for 12 hr, destained in increas-ing gradations of alcohol (70%–100%),defatted in acetone, and cleared in xy-lenes.
Estradiol Injections
Mice were injected intraperitoneallywith water-soluble, cyclodextrin en-capsulated �-estradiol (Sigma CO9265G). Controls were injected with cyclo-dextrin alone. Mice were given 20 �g/day for 7 days.
ACKNOWLEDGMENTSWe thank Dr. Michael Miller (Univer-sity of Alabama at Birmingham) forhelpful advice and discussion aboutovarian function and Dr. Andra Frost(University of Alabama at Birming-ham) for help with ovary histology.We also thank Julie Kiessling andMeghan Knight for their assistancewith the project.
ROLE OF CILIA IN THE OVARY 2059
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