expression of patched-1 and smoothened in testicular meiotic and post-meiotic cells

Expression of Patched-1 and Smoothened in TesticularMeiotic and Post-Meiotic CellsCARLOS R. MORALES,1* ANDREW FOX,1 MOHAMED EL-ALFY,1 XIAOYAN NI,1 AND W. SCOTTARGRAVES2*1Department of Anatomy and Cell Biology, McGill University, Montreal, Canada2Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425

KEY WORDS patched-1; smoothened; desert hedgehog; development; testis

ABSTRACT Desert hedgehog (Dhh) signaling plays an essential role in the normal develop-ment of the testis and in the process of spermatogenesis. Little is known about the involvement inspermatogenesis of the prototypic member of the family, Ptc1, which acts to suppress hedgehog sig-naling through Smoothened (Smo). Here, we have examined the expression of Ptc1, Smo, and Dhhin mouse and rat seminiferous epithelium. Our findings demonstrate that Ptc1 and Smo areexpressed by primary spermatocytes and by round and condensing spermatids whereas Dhh isexpressed by Sertoli cells. The findings suggest that Sertoli cells coordinate Dhh-dependent sper-matogenesis events via Ptc1 and Smo prior to the first meiotic division and in postmeiotic (haploid)cells, particularly during the first half of spermiogenesis. Microsc. Res. Tech. 72:809–815,2009. VVC 2009 Wiley-Liss, Inc.

INTRODUCTION

The mouse hedgehog gene family consists of threemembers, Sonic (Shh), Desert (Dhh), and Indianhedgehog (Ihh). These proteins bind to the receptorspatched-1 (Ptc1) and Ptc2 and release suppression ofthe transmembrane proto-oncogene protein smooth-ened (Smo) to mediate signal transduction (Marigoet al., 1996; Taipale et al., 2002). Hedgehog signalinggoverns a wide variety of embryonic and adult proc-esses through signaling networks described in severalreviews (Jiang and Hui, 2008; Zhao et al., 2007).

The three hedgehogs show little overlap in their pat-tern of tissue expression. Dhh is the only one expressedin the gonad (Bitgood and McMahon, 1995) and severalstudies have revealed the importance of Dhh signalingin reproduction, including roles in gonad developmentand spermatogenesis (Walterhouse et al., 2003). Forexample, Dhh signaling is a positive regulator of thedifferentiation of steroid-producing Leydig cells in thefetal testis (Yao et al., 2002). Furthermore, male micedeficient in Dhh are sterile with spermatogenesis beingblocked at the pachytene primary spermatocyte stage(Bitgood et al., 1996).

In the context of vertebrate reproduction, Ptc2 isexpressed in the testis by spermatocytes (Carpenteret al., 1998; Nieuwenhuis et al., 2006); however, Ptc2-deficient mice are fertile suggesting possible compensa-tion by other Ptc family members. Ptc3 is a candidategiven the fact that it is specifically expressed in roundspermatids and sperm, findings that suggest roles insperm development and sperm function (Fan et al.,2007). The possible contribution of Ptc1 to these proc-esses in vertebrates is not clear. What is known is thatduring embryonic development, Ptc1 is important inthe regulation of Dhh-dependent signaling required formurine Leydig cell differentiation (Yao et al., 2002).Furthermore, in Caenorhabditis elegans, Ptc1 isexpressed in germ line cells and Ptc1 null mutants are

sterile, displaying multinucleate germ cells thought toarise from a defect in cytokinesis (Kuwabara et al.,2000). The early embryonic lethality of Ptc1 null mice(Goodrich et al., 1997) prevents the use of this animalto assess the impact of Ptc1 deficiency on the process ofPtc1 in spermatogenesis. Here, we have assessed theexpression of Ptc1 protein and mRNA together withSmo and Dhh in the mouse testis during the process ofspermatogenesis. Our findings indicate that Ptc1 andSmo are coexpressed in meiotic and postmeiotic cells,apparently influenced by Dhh expressed by the Sertolicells.

MATERIALS ANDMETHODSReagents and Antibodies

A rabbit polyclonal antiserum was produced againsta carboxy terminal polypeptide from mouse Ptc1(amino acid residues 1158–1434 in sequence accessionnumber Q61115). The Ptc1 polypeptide was expressedin bacteria as a GST fusion protein and the GST por-tion was removed by thrombin cleavage prior to immu-nization. IgG was purified from the resulting antise-rum (rb2160) by affinity chromatography on a columnof the GST-Ptc-1 fusion protein coupled to sepharosefollowed by protein G-sepharose. The antibody wasshown to react with a polypeptide corresponding to thesize of Ptc in extracts of CHO cells transfected toexpress full-length mouse Ptc. The antibody showed noreactivity in immunoblotting of extracts from non-

*Correspondence to: W. Scott Argraves, Department of Cell Biology and Anat-omy, Medical University of South Carolina, 173 Ashley Avenue, Charleston,South Carolina 29425. E-mail: [email protected] or Carlos R. Morales, Depart-ment of Anatomy and Cell Biology, McGill University, 3640 University Street,Montreal, Quebec, Canada, H3A 2B2. E-mail: [email protected]

Received 12 December 2008; accepted in revised form 2 April 2009

DOI 10.1002/jemt.20733

Published online 29 May 2009 in Wiley InterScience (www.interscience.wiley.com).

VVC 2009 WILEY-LISS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 72:809–815 (2009)

transfected CHO cells as described previously (Moraleset al., 2006). Mouse antibodies to Smo and Shh werepurchased from Santa Cruz Biotechnology (SantaCruz, CA). The use of these antibodies in immunocyto-chemistry has been previously described (Riobo et al.,2006; Sacedon et al., 2003). Dhh antibody was pur-chased from R&D Systems (Minneapolis, MN) and itsuse in immunocytochemistry has been described else-where (Sacedon et al., 2003). Goat antirabbit IgG andgoat antimouse IgG conjugated to horseradish peroxi-dase were from Cedarlane Labs (Hornby, ON) and nor-mal rabbit serum from Invitrogen (Burlington, ON).Other reagents used in immunological analyses werepurchased from Sigma Chemical Company (St. Louis,MO) unless otherwise specified.

Animals

Three male C57BL/6 mice (3 months old; 25 gweight) and three male retired breeder rats (3 monthsold; 350 g weight) were purchased from Charles RiverLaboratories (Montreal, QC). All animals were anes-thetized with an intraperitoneal injection of sodiumpentobarbital (MTC Pharmaceuticals, Hamilton, ON)prior to any surgical or experimental manipulation.

Tissue Preparation and ImmunohistochemicalStaining

Mouse testes were fixed over a 10-min interval byintracardiac perfusion with Bouin’s fixative via the leftventricle. Rat testes were fixed over a 10-min intervalvia the abdominal aorta with the same fixative. Follow-ing perfusion, testes were removed and immersed inBouin’s fixative for an additional 24 h. The tissueswere then dehydrated in graded ethanol and embeddedin paraffin. Sections (5 lm thick) were cut andmounted on glass slides. Immunostaining was accom-plished using a microwave antigen retrieval protocoldescribed by Morales and others (2006) in conjunctionwith the Zymed SP immunohistochemical staining kit(Zymed, San Francisco, CA). Briefly, paraffin sections(5 lm thick) were deparaffinized in toluene and rehy-drated with ethanol (90–50%). Endogenous peroxidaseactivity was blocked by incubation in 3% H2O2 in meth-anol for 30 min. After the antigen retrieval techniquewas performed and the slides were cooled, nonspecificbinding sites were blocked using 10% goat serum for 30min. The sections were then incubated for 90 min atroom temperature with antibody against Ptc1 and Smo(diluted to 0.16 lg IgG/lL). Sections were then washedin phosphate buffered saline (PBS) and incubated withbiotinylated secondary antibody for 60 min. Followingthe incubation, streptavidin-peroxidase was added tothe tissue for 10 min, allowing it to complex with thebiotinylated secondary antibody. Biotin/streptavidin-peroxidase complexes were then stained using thechromogen diaminobenzidine. Counterstaining wasperformed using hematoxylin for 30 sec. As controls,sections were incubated with normal rabbit immuneserum (diluted 1:100).

RT-PCR

Total RNAwas extracted from the testes of 5, 10, 15,20, 25, and 45 days postpartum mice (n 5 3 for eachage), using a RNeasy Mini Kit (Qiagen, Mississauga,ON). Total RNA was converted to cDNA using a

Omniscript RT Kit (Qiagen). The cDNAs were used astemplates in RT-PCR using primers based on mousePtc1 (accession number NM008957), forward primer50-CTTGATGTGGCCCTTGTT-30 and reverse primer50-AAGGAGCAGAGGCCCAAT-30. In addition, RT-PCRwas performed using primers to mouse Smo (accessionnumber NM176996) forward primer 50-TGCCACCAGAAGAACAAGCCA-30 and reverse primer was 50-GCCTCCATTAGGTTAGTGCGG-30. As a control RT-PCR was performed using primers based on mouseactin (accession number V01217) forward primer 50-CTCTCTTCCAGCCTTCCTTC-30 and reverse primer50-AGAGCCACCAATCCACACAG-30. PCR cyclingconditions for all primer pairs were 948C for 3 minfollowed by 30 cycles of 948C for 30 sec, 608C for 30 sec,and 728C for 30 sec, with a final extension for 10 minat 728C.

RESULTSImmunolocalization of Ptc1 in the Testis

In adult mouse testes, relatively high levels of Ptc1immunolabeling were observed in the cytoplasm oflate (diplotene) spermatocytes and secondary sperma-tocytes (stages X–XII of the cycle). High levels ofPtc1 immunolabeling were also evident in the cyto-plasm of round spermatids at steps 1–7 (stages I–VIIof the cycle). The relative level of Ptc1 immunolabel-ing was progressively lower in the cytoplasm of step8 spermatids (stage VIII) and elongating spermatidsat steps 10–12 (stages X-XII of the cycle). Ptc1 immu-nolabeling was relatively low in the cytoplasm of con-densing spermatids at step 13 (stage I of the cycle;Figs. 1 and 2). No Ptc1 immunolabeling was detectedin Sertoli cells, spermatogonia, primary spermato-cytes (except late pachytene and diplotene spermato-cytes), and condensing spermatids at steps 14–16(Fig. 1). High levels of Ptc1 immunolabeling werealso observed in the interstitial Leydig cells (Fig. 1inset). Sections incubated with nonimmune serumdid not produce any peroxidase reaction (data notshown). In parallel, immunohistological examinationof rat testis was performed and the findings wereclosely similar to those obtained with mouse testis,i.e., relatively high level of Ptc1 immunostaining wasobserved in late spermatocytes and round spermatidsand moderate levels in early condensing spermatids(Figs. 6A and 6B).

Immunolocalization of Smo in the Testis

Smo expression was observed in the cytoplasm oflate pachytene (diplotene) spermatocytes and in sec-ondary spermatocytes (stages X–XII of the cycle of theseminiferous epithelium). Similar levels of Smo immu-nolabeling were detected in the cytoplasm of roundspermatids and early elongating spermatids at steps1–9 (stages I–IX of the cycle). Relatively higher levelsof Smo immunolabeling were observed in the cyto-plasm of steps 10–16 spermatids (stages X–XVI of thecycle) particularly at the tips of their heads (Figs. 3A–3C and 4). No Smo immunolabeling was evident in Ser-toli cells, spermatogonia, primary spermatocytes(except diplotene spermatocytes; Figs. 3A–3C). Smoexpression was also observed in the cytoplasm of Ley-dig cells (Figs. 3A and 3B). The patterns of Smo immu-

Microscopy Research and Technique

810 C.R. MORALES ET AL.

nostaining observed in sections of rat testes were simi-lar to what was observed in the mouse testis (Fig. 6C).Sections incubated with nonimmune serum did notproduce a detectable level of peroxidase reaction (datanot shown).

Immunolocalization of Dhh in the Testis

Dhh antibodies detected Dhh in the cytoplasm ofSertoli cells and in the cytoplasm of late condensingspermatids (Figs. 5A–5C). Other spermatogenic cellsshowed no immunoreactivity. Stage-specific differencesin the relative intensity of anti-Dhh immunolabelingwere not evident in Sertoli cells at different stages ofthe cycle (Figs. 5A–5C). Dhh expression was alsodetected in the cytoplasm Leydig cells (Figs. 5A–5C).Anti-Dhh immunostaining of the rat testes mirroredthe results obtained in the mouse testes (Fig. 6D). Bycontrast, no Shh was detected in testicular tissuesusing a Shh antibody that detected Shh in the efferentducts (Morales et al., 2006) under the same fixationconditions (data not shown).

Ptc1 and Smo mRNA Expression in the Testis

Ptc1 and Smo transcripts were detected in total tes-ticular RNA isolated from 5, 10, 15, 20, 25, and 45 dayspostpartum mice (Fig. 7A). Expression of both Ptc1 andSmo in the testis was observed to increase between 5and 20 days postpartum corresponding to the develop-mental appearance of late spermatocytes and roundspermatids (Fig. 7B). Levels of Ptc1 and Smo tran-scripts were significantly reduced in testis from 25 and45 days postpartum mice consistent with the appear-ance of condensing spermatids, which do not transcribemRNAs.

DISCUSSION

Dhh signaling regulates the normal development ofthe testis as well as spermatogenesis (Szczepny et al.,2006). Dhh-null mice are infertile due to a primaryspermatocyte arrest (Bitgood et al., 1996). Since a pre-vious study reported expression of Ptc1 in Leydig cellsand peritubular cells but not in cells of the seminifer-ous epithelium (Bitgood et al., 1996), Dhh signalingwas believed to occur via Ptc2 receptors which havebeen localized in primary spermatocytes (Carpenteret al., 1998). We decided to re-examine the expressionof Ptc1 by immunohistochemistry using a polyclonalantibody raised against the carboxy terminal segmentof mouse Ptc1 (Morales et al., 2006). As a result, wefound Ptc1 expression in primary spermatocytes at theend of the prophase of the first meiotic division (diplo-tene stage). The relative expression of Ptc1 greatlyincreased in postmeiotic round spermatids (steps 1–7)and gradually decreased in condensing spermatids(steps 8–13), i.e., during nuclear condensation andcytoplasmic elongation. No Ptc1 expression was appa-rent in more mature condensing spermatids (steps 14–16). Similar results were observed in rat seminiferousepithelium indicating that in rodents, the expression ofPtc1 is prevalent in late primary spermatocytes and inround spermatids. Thus, it is likely that Ptc1 plays animportant role in Dhh-dependent signaling prior to thefirst meiotic division and in postmeiotic (haploid) cells,particularly during the first half of spermiogenesis.

Other than Leydig cells, Ptc1 expression has notbeen reported in cells of the seminiferous epithelium.Here, we used immunohistochemistry to evaluate theexpression of the Ptc1 protein in the seminiferous epi-thelium of mice during the first round of spermatogen-esis. In addition to Leydig cells, we found that Ptc1

Fig. 2. Diagrammatic representation of Ptc1 expression indeveloping spermatogenic cells. The figure summarizes the relativeintensity of Ptc1 immunostaining observed in spermatogenic cells.The intensity of immunostaining was evaluated using a relative scor-ing scale: 1, apparently positive, low level of staining relative to noprimary antibody control sections;11, unequivocally positive, moder-ate level of staining; 111, high level of staining; 1111, highestlevel of expression. This relative scoring system was applied to multi-ple sections from separate animals to obtain a consensus. A, type Aspermatogonium; B, type B spermatogonium; PL, preleptotene sper-matocyte; L-Z, leptotene or zygotene spermatocyte; EP-MP, early pri-mary or late primary spermatocyte; Di, diplotene spermatocyte; 1–8,round spermatids; 9–16 condensing spermatids.

Fig. 1. Immunolocalization of Ptc1. Shown is Ptc1-immunostain-ing of late pachytene spermatocytes in stages XII (late) and XIII(SXII, SXIII), and round spermatids in stages V and VII (SV, SVII) ofthe cycle of the seminiferous epithelium. Leydig cells in the intersti-tial space also show Ptc1 immunoreactivity (arrows and inset). Otherspermatogenic cells (spermatogonia and condensing spermatids) aswell as the somatic Sertoli cells showed no anti-Ptc1 immunoreactiv-ity. The bar equals 100 lm. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]


811EXPRESSION OF PTC1 AND SMO

was expressed by diplotene spermatocytes and second-ary spermatocytes, and at highest levels in round sper-matids. Using RT-PCR, we showed that Ptc1 mRNAwas expressed in the testes at 5, 10, 15, 20, 25, and 45days postpartum. Its expression increased from 5 to 20days and then declined. The period of maximal Ptc1expression corresponds to the developmental appear-ance of late spermatocytes and round spermatids.

The pattern of Smo mRNA expression in the testisover the 5- to 45-day postpartum period was similar tothat of Ptc1. Previously, Szczepny et al. (2006) used insitu hybridization to show that Smo transcripts wereexpressed in spermatogonia, spermatocytes, and to alesser extent in round spermatids. We used immuno-histochemistry to evaluate the expression of the Smoprotein in the seminiferous epithelium of adult miceduring the first round of spermatogenesis. We observeda moderate level of expression of Smo in late pachytene(diplotene) spermatocytes. The level of immunoreactiv-ity was relatively higher in round spermatids and stillgreater in condensing spermatids. Relatively high lev-els of Smo immunoreactivity were evident in the headof condensing spermatids. Similar results wereobserved in rat testes. Taken together, our findingssuggest that Ptc1 and Smo play roles in hedgehog sig-naling during meiosis and spermiogenesis.

Dhh transcripts have previously been shown to beexpressed in Sertoli cells in developing and adultmouse testes (Bitgood et al., 1996). Using immunohis-tochemistry, we detected Dhh in the cytoplasm of Ser-toli cells at all stages of the cycle in both mice and rats.Dhh expression by Sertoli cells appeared uniformthroughout the various stages. Leydig cells were alsofound to express Dhh together with Ptc1 and Smo sug-gesting that these cells are capable of autocrine Dhh-signaling.

Inactivation of murine Dhh results in hypogonadismand male infertility (Bitgood et al., 1996). The infertil-ity is due to incomplete spermatogenesis whereby pri-

Fig. 3. Immunolocalization of Smo in the mouse testis. A Smo im-munostaining of diplotene spermatocytes (arrows), dividing sperma-tocytes (arrowhead), and step 11 condensing spermatids at stages XIof the cycle of the seminiferous epithelium. B Smo immunostaining ofstep 1 round spermatids (arrows), and step 13 condensing spermatidsat stage I. C Smo imunostaining of round spermatids (step 6) and con-densing spermatids (step 15) at stage VI. Other spermatogenic cells(spermatogonia, early and mid primary spermatocytes) as well as thesomatic Sertoli cells show no Smo immunoreactivity. Strong anti-Smoimmunoreactivity was observed around the heads of condensing sper-matids. The bar equals 50 lm and applies to all micrographs. [Colorfigure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 4. Diagrammatic representation of Smo expression in devel-oping spermatogenic cells. The figure summarizes the relative inten-sity of Smo immunostaining observed in spermatogenic cells applyingthe relative scoring system described in the legend of Figure 2. A,type A spermatogonium; B, type B spermatogonium; PL, preleptotenespermatocyte; L-Z, leptotene or zygotene spermatocyte; EP-MP, earlyprimary or late primary spermatocyte; Di, diplotene spermatocyte;1–8, round spermatids; 9–16 condensing spermatids.



mary spermatocytes form, but not postmeiotic sperma-tids (Bitgood et al., 1996). Dhh deficiency results inblockage in spermatogenesis during and after the dip-lotene stage of the first meiotic division. This phase inspermatogenesis is coincident with the expression ofPtc1 and not Ptc2, whose expression appears to be at

Fig. 5. Immunolocalization of Dhh in the mouse testis. Shown isDhh immunostaining in Sertoli cells (arrows) at selected stages of thecycle of the seminiferous epithelium. A Stage I, B stage II, and Cstage VII. In C, relatively strong Dhh immunolabeling is apparent inthe basal cytoplasm of the somatic cells and moderate labeling isapparent in the apical processes (arrowheads). At stage VII, relativelystrong Dhh immunolabeling is apparent in the cytoplasm of Sertolicells around the forming residual bodies (C). After judging the lowlevel of Dhh immunolabeling apparent in the cytoplasmic lobules ofcondensing spermatids to be nonspecific, spermatogenic cells weredetermined to be negative for Dhh expression. The bar equals 50 lmand applies to all micrographs.

Fig. 6. Immunolocalization of Ptc1, Smo and Dhh in the rat testis.A Ptc1 immunostaining at early stage XIV. Note the relatively strongimmunostaining in diplotene spermatocyte (Di) and dividing primaryspermatocytes (M1). B Ptc1 staining at late stage XIV of the cycle ofthe seminiferous epithelium. Note the immunostaining in dividingsecondary spermatocytes (M2) and step 1 round spermatids. C Smoimmunostaining at stage XIII. Note Smo immunostaining of diplotenespermatocytes and step 13 condensing spermatids. D Dhh immunos-taing selected stage VII. Note the strong immunostaining in thecytoplasm of Sertoli cells.



earlier phases, i.e., in early and mid primary spermato-cytes (Carpenter et al., 1998), which are cells that arepresent in the testes of Dhh-deficient mice. Further-more, the peak levels of expression of Ptc1 and Smoduring spermatogenesis also correlate with the expres-sion of other hedgehog target genes including the tran-

scription factor Gli1 and Fused (Fu), which areexpressed in spermatocytes, and to a lesser extent inround spermatid (Szczepny et al., 2006). Excessive Gli1activation is detrimental to spermatogenesis as evi-denced by the findings that Gli1-overexpressing trans-genic mice result in spermatogenic failure at thepachytene stage. The phenotypic similarity betweenthe Dhh null and Gli1 overexpressing mice suggeststhat Gli1, acting through a negative feedback loop, sup-presses Dhh expression. This is consistent with find-ings showing that mRNA encoding Suppressor of fused(Sufu), a negative regulator of hedgehog signaling, isprominent in late round spermatids and elongatingspermatids, suggesting that it is part of the mechanismby which Sufu hedgehog signaling in haploid (postmei-otic) germ cells is switched off (Szczepny et al., 2006).

Based on our observations and on findings of others(Kogerman et al., 1999; Kroft et al., 2001; Muroneet al., 2000; Szczepny et al., 2006) a model of hedgehogsignaling in the rat and mouse seminiferous epithe-lium is beginning to take shape. Dhh is synthesized bySertoli cells and secreted into the adluminal compart-ment of the seminifeous epithelium where it binds toPtc1 expressed by diplotene spermatocytes and roundspermatids, and relieves the repression of Smo func-tion. In turn, Smo initiates a signaling cascade, allow-ing cytoplasmic Gli1 to enter the nucleus of round sper-matids where it presumably activates target genes.Prior to, or during spermatid elongation, Dhh signalingis suppressed in late round and condensing spermatidsvia the action of Sufu, which functions to sequesterGli1 in the cytoplasm. Indeed, the localization of Gli1has been shown to shift from the nucleus to the cyto-plasm in elongating spermatids (Kroft et al., 2001).Inactivation of Dhh signaling may in turn affect thegrowth of diplotene spermatocyte, round spermatids,and condensing spermatids causing spermatogonialarrest at a primary spermatocyte stage.

In conclusion, we report that Ptc1 is expressed inrodent meiotic and postmeiotic spermatogenic cells.The developmental stage-specific expression of Ptc1suggests that it may function to regulate Dhh signalingin late spermatocytes and round spermatids.

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expression of patched-1 and smoothened in testicular meiotic and post-meiotic cells

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