Journal of Pediatric Surgery 49 (2014) 312–316

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Journal of Pediatric Surgery

j ourna l homepage: www.e lsev ie r .com/ locate / jpedsurg

Molecular signals governing cremaster muscle development:Clues for cryptorchidism

Maciej Szarek a,b, Ruili Li a, Jaya Vikraman a,c, Bridget Southwell a,c, John M. Hutson a,b,c,d,⁎a Douglas Stephens Surgical Research Group, Murdoch Children’s Research Institute, Melbourne, Australiab Department of Anatomy and Neuroscience, University of Melbourne, Australiac Department of Paediatrics, University of Melbourne, Australiad Urology Department, Royal Children’s Hospital, Melbourne, Australia

a b s t r a c ta r t i c l e i n f o

Abbreviations: IR, immunoreactivity; CM, cremasterostnatal day; AR, androgen receptor; TCF, T-cell facctor; IGF-1, insulin like growth factor-1.⁎ Corresponding author at: Department of Paediatrepartment of Urology, Royal Children’s Hospital, Fl052, Australia. Tel.: +61 3 9345 5805; fax: +61 3 9

E-mail address: john.hutson@rch.org.au (J.M. Hutso

0022-3468/$ – see front matter © 2014 Elsevier Inc. Alhttp://dx.doi.org/10.1016/j.jpedsurg.2013.11.049

Article history:

Received 6 November 2013Accepted 10 November 2013

Key words:CryptorchidismGubernaculumsBeta-cateninTestesWnt signalingCremaster muscleDifferentiation

Background/Aim: Cryptorchidism affects 2-4% of newborn boys. Testicular descent requires the gubernaculumto differentiate into cremaster muscle (CM) during androgen-mediated inguino-scrotal descent, but thecellular mechanisms regulating this remodeling remain elusive. β-Catenin, a marker of canonical Wntsignaling, promotes myogenic genes and cellular adhesion. We aimed to determine if androgen receptor (AR)blockade altered β-catenin and its downstream myogenic proteins within the CM.Method: Gubernacula from male rats (n = 12) and rats treated with anti-androgen, flutamide (n = 12) atE19, D0, D2 were processed for immunohistochemistry. Antibodies against β-catenin, embryonic myosin, andmyogenin were visualized by confocal microscopy.Results: At E19, β-catenin immuno-reactivity (IR) localized to the CM membrane. By D2, cytoplasmicβ-catenin-IR was noted with overall β-catenin-IR decreasing. Myogenic proteins resided primarily in cellscontaining β-catenin on their plasma membrane. Embryonic myosin-IR was high at E19 and then decreased

by D2, while myogenin-IR increased. AR blockade increased cytoplasmic β-catenin at D2 and reduced levelsof both myogenic proteins.Conclusion:Myogenic proteins are present in CM cells containing β-catenin. AR blockade did not alter cellularadhesion via β-catenin. In contrast, blocking AR prevented β-catenin entering the nucleus and impaired CMmyogenesis. Mutations in this pathway may result in idiopathic cryptorchidism.

© 2014 Elsevier Inc. All rights reserved.

Cryptorchidism is one of the most common genital anomalies thataffect 2–4% of newborn males [1]. Testicular descent into the scrotalposition relies on a ligament (the gubernaculum) that is composed ofprimitive mesenchymal cells. These cells respond to hormonal cuesthat cause proliferation and differentiation into striated muscle thatestablishes the cremaster muscle (CM). Androgen is one of the pri-mary hormones governing CM development and regulates theremodeling of the gubernaculum during CM formation; however,the exact molecular mechanisms controlling mesenchymal cell differ-entiation into the CM remain unknown.

During the second, or inguino-scrotal phase of testicular descent,the rodent gubernaculum remodels in response to androgen causingthe repositioning of the loose mesenchyme [2]. During this phase, themesenchymal cells undergo differentiation and proliferation that

muscle; E, embryonic day; D,tor; LEF, Lymphoid enhancing

ics, University of Melbourne,emington Road, Parkville, VIC345 7997.n).

pfa

D3

l rights reserved.

results in the formation of striatedmuscle that constitutes the CM. Thegubernaculum then everts into the inguinal subcutaneous tissue andmigrates towards the scrotum, pulling the testes along with it. Recentresearch using microarray analysis has suggested that Wnt signal-ing working in conjunction with androgen receptor (AR) may con-tribute to CM formation and testicular descent, as a defect in the Wntsignaling pathway and AR both produce intra-abdominal testis, whichis a phenotype commonly associated with patients presenting withcomplete androgen insensitivity syndrome [3,4].

Androgens are thought to act in a Wnt-dependent manner bystabilizing the key effector β-catenin [5,6]. During striated muscledevelopment, β-catenin is known to participate in 2 molecular roles:gene transcription and cell-cell adhesion [7]. Gene transcriptionoccurs in the nucleus. When aWnt ligand binds to a canonical Frizzledreceptor β-catenin stabilizes within the cytoplasm where it thentranslocates through the nuclear pore, bound to be liganded AR. Oncein the nucleus it then interacts with T-cell factor and lymphoid en-hancing factor (TCF/LEF) to facilitate the transcription of Wnt-responsive genes. The Wnt-responsive genes transcribed throughthis pathway have been implicated in all facets of cellular homeostasisthat include cellular proliferation, differentiation as well as initiatingevents that contribute to myogenesis [8]. Secondly, β-catenin is also

Table 1Primary and secondary antibodies.

Company/cataloguenumber

Raised in/clonality

Working conc.(vol/vol)

Primary antibodyAnti-β-catenin Abcam, ab2365 Rabbit/polyclonal 1/200Myogenin Abcam, ab1835 Mouse/monoclonal 1/200Embryonicmyosin

DSHB, BF-45 Mouse/monoclonal 1/200

Secondary antibodyAlex 594 Invitrogen A-11001 Donkey/mouse 1/400Alex 488 Invitrogen A-11008 Donkey/rabbit 1/400DAPI Sigma-Aldrich, D9564 N/A 1/1000

313M. Szarek et al. / Journal of Pediatric Surgery 49 (2014) 312–316

known to regulate cellular adhesion during myogenesis by regulatingboundary formation between cells. This achieved by the membrane-bound protein, cadherin [9]. The cytoplasmic portion of the cadherinprotein has β-catenin binding sites allowing β-catenin and cadherinproteins to form active cadherin-catenin complexes. This complexbinds in the plasma membrane of cells in conjunction with α-catenin,in turn allowing neighboring cells to fuse together in a coordinatedfashion. This process enables multilayered muscle spindles to form,facilitating myogenesis [10].

During the formation of the CM, conditional knock down ofβ-catenin caused a failure in CM myogenesis resulting in intra-abdominal cryptorchidism [11]; however, how β-catenin regulatesmesenchymal cell differentiation into the CM is not completelyknown. As previous authors have implicated β-catenin as the majorprotein regulating CM formation, we aimed to determine the preciselocalization of β-catenin during inguino-scrotal descent and decipherits role in relation to its spatiotemporal location and assess the effectof AR blockade on its downstream myogenic targets.

1. Methods

Sprague-Dawley rats were purchased from a commercial supplierand housed in the institute's Animal Research Laboratory in standardshoebox cages. Animals were maintained in a temperature-controlledatmosphere with a 12-hour light-dark cycle and fed commercial ratchow and water ad libitum. Ethical approval was gained from theInstitutional Ethics Committee (license number A644) and withcorrect care taken in accordancewith the National Health andMedicalResearch Council animal ethics guidelines.

Male fetuses were collected at embryonic day 19 (E19; n = 4)(E0 = day vaginal plug found). One group of dams were time-matedand allowed to deliver where male offspring were collected at birth(D0; n = 4) and D2 (n = 4). A separate group of dams (n = 3)received the anti-androgen, flutamide (75 mg/kg bodyweight) ongestational days E16 to E19 inclusive (12 male pups from flutamidegroup collected in total at the same time points mentioned above).Animals were sacrificed by Lethabarb injection (2 mL/kg; Vertak pk,Australia) after being anesthetised with 5% isofluorane. Pups wereremoved by hysterectomy, placed on ice for 10–15 minutes and

Table 2Comparative statistical analysis of fluorescence intensity in each gubernacula group.

n SD

Fig. 2 E19 Control & D2 Control 8 0.0919Fig. 4A E19 Control & E19 Flutamide 8 0.0977

D2 Control & D2 Flutamide 8 0.144Fig. 4B E19 Control & E19 Flutamide 8 0.2646

D2 Control & D2 Flutamide 8 0.1552Fig. 4C E19 Control & E19 Flutamide 8 0.0432

D2 Control & D2 Flutamide 8 0.0509

*SD = standard deviation, IQR = interquartile range, M = median.

bisected at the level of the umbilicus with the lower body fixed in 4%paraformaldehyde for 48 hours at 4 °C. Specimens were paraffinprocessed, cut, and prepared for immunohistochemistry according toa previously described protocol [12]. Single- and double labelling ofgubernacular sections occurred with the antibodies described inTable 1. Slides were mounted using Mowiol and stored in the darkat 4 °C. A Leica LSM-2 confocal microscope (Leica Microsystems,Wetzlar, Germany) was used to obtain fluorescent images.

Images were assessed using ImageJ (ImageJ version 1.43u, WayneRasband, National Institute of Health, USA). The fluorescence valuesfor each protein in the CM were calculated for images taken at ×63magnification. Values were tabulated as the ratio against 4′,6-diamidino-2-phenylindole (DAPI) that labels cell nuclei and allowsthe fluorescence intensity to be related to the number of cells withineach image. Ratios were initially calculated in Microsoft Excel andthen transferred into SPSS (V.21) for statistical analysis. Data werecompared using the Mann-Whitney U test with significance wasassigned at P b 0.05. Data were then expressed as median and inter-quartile range (IQR) (Table 2 for all data sets).

2. Results

2.1. β-Catenin localises to the developing CM on the cell surface

Labelling experiments showed that β-catenin immuno-reactivity(IR) was most prevalent in the CM with β-catenin-IR residing on thewhole plasma membrane of CM cells in E19 (n = 4) and D0 spe-cimens (n = 4). β-catenin-IR appeared cytoplasmic in D2 specimensthat labelled ubiquitously throughout the entire CM (n = 4).Additionally, β-catenin-IR appeared to fragment by D2, a timewhen the gubernaculum is everting before migration into thescrotum (Fig. 1). The overall IR pattern in the time-points inves-tigated suggests that β-catenin is highest at E19, but declines by D2(n = 12, Table 2, Fig. 2).

2.2. β-Catenin-positive CM cells express the embryonic myosin protein

In E19 specimens, β-catenin-IR and embryonic myosin-IR co-localise at the cell-cell contact sites with this diminishing by D2.Embryonic myosin labeled only within the cytoplasm of differentiat-ing CM cells containing β-catenin on their cell surface. Cells thatlacked β-catenin on the plasma membrane showed no embryonicmyosin-IR (Fig. 3).

2.3. Androgen blockade causes increased intra-cytoplasmic β-catenin IRat D2

The localization of β-catenin-IR at the plasma membrane at E19did not change when AR was blocked. However, in D2 specimens, asignificant increase in β-catenin-IR was observed in the cytoplasm ofCM cells when AR was inhibited (n = 4 per group, Table 2, Fig. 4A).

IQR M Mann-Whitney U P

0.1687 0.1725 1 b .050.1426 0.3049 7 .7730.2307 0.2005 0 b .050.4996 0.438 0 b .050.2282 0.0361 1 b .050.0476 0.0114 0 b .050.0863 0.0274 0 b .05

Fig. 1. β-Catenin in control CM. (A) Control CM at E19 showing β-catenin localising to the plasma membrane (arrow). (B) D2 CM cell displaying β-catenin-IR beginning to fragmentat the same time the gubernaculum begins to evert into the scrotum (arrow). Scale bar 10 μm.

314 M. Szarek et al. / Journal of Pediatric Surgery 49 (2014) 312–316

2.4. Androgen blockade inhibits embryonic myosin IR

Embryonicmyosin-IR was reduced in all time-points in AR blockedspecimens when compared to controls. By D2, embryonic myosin-IRwas severely decreased and markedly less than in control animals(n = 4 per group, Table 2 Fig. 4B).

2.5. Myogenin IR declines in flutamide-treated CM

Myogenin-IR increased in the nuclei by D2 in control animals.However, when AR was blocked there was little myogenin-IR acrossall ages. By D2, a stage where myogenin-IR was seen to increase incontrols, there was significantly less myogenin-IR in flutamide-treated CM (n = 4 per group, Table 2, Fig. 4C).

3. Discussion

Emerging evidence suggests that the key effector of the canonicalWnt pathway, β-catenin, is essential for testicular descent [13] andthat it interacts with AR to enable expression of downstreammyogenic proteins. β-Catenin is highly conserved throughoutevolution, and the intracellular cascade that regulates its transloca-tion to either the nucleus or the plasma membrane hinges on themolecular machinery that stabilizes it in the cytoplasm. Ligandssuch as Wnt1, Wnt3a and Wnt5a, initiate the stabilization ofβ-catenin [14]. This stabilized β-catenin then functions in bothmyogenic gene transcription as well as cellular adhesion; enabling

Fig. 2.Mean β-catenin-IR in CM control specimens. β-Catenin-IR is highest at E19 witha significant decline by D2 (p b 0.05).

cells to adhere to one another in a coordinated fashion ensuringmuscle fiber maturation.

This novel study clearly shows that β-catenin localizes to theplasma membrane of CM cells at E19 then membranous β-catenindecreases and fragments when the gubernaculum begins to evert andmigrate towards the scrotal position. This localization was indepen-dent of androgen. However, blocking AR resulted in changes inβ-catenin levels in the cytoplasm of CM cells by D2. This occurred atthe same time the myogenic proteins embryonic myosin andmyogenin were reduced. Thereby showing that CM maturation isinhibited concurrently with β-catenin accumulation, providing aninsight into the molecular mechanisms contributing to CM mesen-chymal cell differentiation.

In this study, β-catenin labelling was carried out at E19, D0 andD2, being the start of the inguino-scrotal stage of descent, todetermine β-catenin’s spatial-temporal arrangement within thegubernaculum. β-catenin-IR was present in the developing CM butnot the undifferentiated mesenchymal cells of the gubernacularcore. We observed that β-catenin localized to the plasma membrane

Fig. 3. Embryonic myosin in control CM. At E19 in control specimens, β-catenin-IR(green) and embryonic myosin-IR (red) in CM show a possible correlation. When β-catenin is present between cell-cell junctions both neighboring cells contain colocalizedmyosin-IR (yellow arrow). Whereas, when β-catenin is lacking, this coordinatedimmuno-reactivity pattern is absent between neighbouring cells (white arrow).

Fig. 4. Comparison between control and flutamide-treated CM. (A) β-catenin-IR significantly increased in D2 flutamide-treated specimens (P b .05) appearing cytoplasmic (datanot shown). (B) Embryonic myosin decreased in all flutamide-treated CM. (C) Myogenin-IR decreased significantly in AR blocked specimens when compared to controls at both E19and D2 (P b .05).

315M. Szarek et al. / Journal of Pediatric Surgery 49 (2014) 312–316

of CM cells residing between the cell-cell contact sites, most likelyto be the cellular junction abundant in adhesive proteins, the ad-herens junctions.

Previous reports have suggested that β-catenin localization to theplasma membrane indicates active adherens junctions. Finnermannet al. observed that once gene transcription had ceased in proli-ferating premyogenic cells, β-catenin localized to the adherensjunctions [15]. They hypothesized that cadherin proteins and α-catenin form a dimer and sequester cytoplasmic β-catenin and link itto the actin cytoskeleton where it then enables cellular coordination.At E19, localization of β-catenin on the plasma membrane isconsistent with its role in cellular adhesion and suggests activeadherens junctions have formed during CM myogenesis. Moreover,decreased β-catenin-IR by D2 on the cell membranes, accompaniedby a fragmenting appearance of β-catenin fluorescence suggests thatadhesion between CM cells is breaking down. We hypothesize thatactive adherens junctions are separating to allow gubernaculareversion and migration to the scrotum, as this loss in adhesionoccurs at the correct time to enable the gubernaculum to evert.

Additionally, double-labelling experiments showed that β-cate-nin co-localized with the primary muscle cell marker, myosin. AtE19, β-catenin localization to the plasma membrane appears todictate the expression of embryonic myosin, with CM cells thatlacked β-catenin on the cell surface lacking the embryonic myosinprotein within their cytoplasm. It is tempting to speculate thatcommitted cells expressing the myosin protein influence thepathway of differentiation of their uncommitted neighbors, aidedby β-catenin at the adherens junctions. It is hypothesized that oncecells express the correct maturation markers, muscle cells wouldthen communicate with their microenvironment and adhere to one

another to form the multinucleated, maturing CM fibers. This ap-pears to be independent of AR as when AR was blocked, β-catenin onthe cell surface was unaffected. Taken together, this suggests that bysending intercellular signals through adhesive receptors, such asβ-catenin, CM cells are able to elicit the spatially coordinating eventsthat are required for coordinated myogenesis.

Furthermore, the increase in cytoplasmic β-catenin in the CMobserved in D2 control specimens is consistent with its role as amyogenic transcript promoter and suggests that in D2 CM, β-cateninis beginning to accumulate to initiate the transcription of myogenicproteins to enable CM maturation. However, the generation ofmyogenic transcripts relies on β-catenin reaching the nucleus toform a complex with TCF/LEF. This nuclear translocation of β-catenin is hypothesized to be dependent on interactions with AR, asbinding to AR allows β-catenin to travel through the nuclear pore[16]. When flutamide specifically blocked AR before the start ofinguino-scrotal descent, β-catenin-IR increased in the cytoplasm ofthe CM cells at D2 when compared to controls. This suggests that β-catenin is not able to bind to AR, reducing nuclear translocation,causing it to remain in the cytoplasm. This increased cytoplasmicaccumulation could lead to impaired transcription of myogenicgenes within the CM.

In support of this, after flutamide treatment, embryonic myosin-and myogenin-IR were severely reduced; suggesting primarymyofiber formation and differentiation of mesenchymal cells wasdelayed. This delay coincides with altered β-catenin levels at D2. Wetherefore hypothesize that embryonic myosin and myogenin arereliant on signals provided by β-catenin, AR and TCF/LEF in the nucleiof CM cells to initiate myogenic transcription and when this processis interrupted, CM maturation is inhibited.

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The hypothesized interactions between β-catenin and AR remainsa topic of debatewith some reports suggesting that insulin-like growthfactor-1 (IGF-1) plays a part in β-catenin nuclear translocation [17].Verras et al. aimed to determine which molecules were implicated inenhancingβ-catenin translocation in prostate cancer cells in culture bymanipulating culture conditions to observe the effects of IGF-1. Theyobserved that β-catenin still translocates to the nucleus bound toIGF-1. However, the difference observed between the experimentscompleted by Verras et al., and the current study may be due to themanipulated culture conditions, as well as the specific cell line usedand hence, extrapolation to the CM may not be warranted and mayrequire further experimentation to apply to CM myogenesis.

This study shows the canonical Wnt pathway is active in thegubernaculum during inguino-scrotal descent, as indicated by thepresence of β-catenin in the CM. Blocking AR leads to β-cateninaccumulating in the cytoplasm and reduced myogenic proteins sug-gesting androgen controls translocation of β-catenin to the nucleusand myogenic gene transcription before eversion and migrationtowards the scrotum. β-catenin is also involved in CM cellularadhesion, enabling cremaster cells to adhere to one another duringgubernacular remodeling. These results suggest canonical Wnt pro-teins interact with androgens during inguino-scrotal descent, provid-ing clues to the complex etiology of congenital cryptorchidism.

References

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Discussion

Discussant: Paolo De Coppi (Great Ormond Street Hospital, London):

Have you looked at controlling the muscle with an anti-androgenas a factor (or an effect?) in other skeletal muscles and secondly,using muscle when there is no differentiation there is prolifera-tion – did you look at proliferation of these cells?

Response: Mr. Szarek: We did not do a control muscle because thecremaster muscle is different from other skeletal muscles in thebody and matures quite late, and differs in the firing frequency inits contractions. I wasn’t going to use the anterior abdominal wallas a control because I don’t think that would be a good solidcontrol. A previous study from our lab has used Ki67 to markproliferation. The gubernaculum does have a proliferation zone,which is the progress zone, but because I looked at the cremaster,this is where the cells should be differentiated and there shouldn’tbe proliferation.

Discussant: Clare Rees (Royal London Hospital): How do any of thegenetic pathways explain unilateral cryptorchidism which is themost common form that we see?

Response: Mr. Szarek: There are two gubernacula, one on the left andone on the right, so there is a balance and when there is a problemon the key signalling pathway, this has gone wrong.