Received

2009; pu

G.T. has no

to disclo

C.C.-O.

This study

Univers

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Histolog

07070 A

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2716

Presence of the brain proteins cerebral cavernousmalformation-2 and cerebral cavernous malformation-3 in rat testes and their potential role in experimentalvaricocele

Gamze Tanriover, Ph.D.,a Leyla Sati, M.Sc.,a Merih Tekcan, B.Sc.,a Necdet Demir, Ph.D.,a Murat Gunel, M.D.,b

and Ciler Celik-Ozenci, D.D.S., Ph.D.a

a Department of Histology and Embryology, Akdeniz University, School of Medicine, Antalya, Turkey; and b Department of

Neurosurgery, Yale University School of Medicine, New Haven, Connecticut

Objective: To assess the distribution of cerebral cavernous malformation-2 (CCM2) and CCM3 proteins in a normaland experimentally induced varicocele model in rat testes.Design: Comparative and controlled study.Setting: University animal care and operation unit.Animal(s): Wistar male rats for experimental and control groups.Intervention(s): The control group underwent a sham operation. Rats in the experimental groups underwent partialligation of the renal vein to induce experimental varicocele, and left testicular tissues were analyzed.Main Outcome Measure(s): Tissues were fixed and processed for paraffin-embedded testicular tissues. Levels ofCCMs were assessed by immunohistochemistry and Western blot analysis.Result(s): In control and sham-operated testes, CCM2 expression was detected in acrosomes of round spermatidsat stages 6–8 and in the heads of elongating spermatids at stages 1–5 and 9–14. CCM3 expression was weakly lo-calized in pachytene spermatocytes at stages 8–12 and strongly immunolocalized in the cytoplasm of round sper-matids at stages 1–8 and in elongating spermatid cytoplasm at stages 9–12. Varicocele induction clearlydemonstrated histopathological changes due to germ cell degeneration. CCM2 and CCM3 expression increased sig-nificantly in varicocele-induced rat testes.Conclusion(s): The expression patterns of two brain proteins were clearly identified in rat testes, suggesting a roleduring normal and pathological spermatogenesis. This article represents the first demonstration that these proteinshave a role outside the central nervous system. (Fertil Steril� 2010;93:2716–22. �2010 by American Society forReproductive Medicine.)

Key Words: CCM2, CCM3, rat testes, experimental varicocele, spermatogenesis, spermiogenesis

Vascular malformations of the brain and spinal cord have typicallybeen classified into four major types: telangiectasias, venous anom-alies, cavernous malformations, and arteriovenous malformations.Cavernous malformations can occur anywhere in the body but usu-ally produce serious signs and symptoms only when they occur inthe central nervous system (CNS). Cavernous malformations maybe found in the spinal cord (1), while cutaneous (2), retinal (3), he-patic (4), and vertebral (5) lesions have been occasionally observedin patients concurrent with inherited cerebral cavernous malforma-tion (CCM). This disease is also called cavernous angiomas, caver-nomas, or sometimes simply angiomas. Cavernomas are collectionsof small blood vessels (capillaries) in the brain that are enlarged andirregular in structure and rarely seen vascular malformations of the

February 10, 2009; revised May 7, 2009; accepted June 15,

blished online July 31, 2009.

thing to disclose. L.S. has nothing to disclose. M.T. has nothing

se. N.D. has nothing to disclose. M.G. has nothing to disclose.

has nothing to disclose.

was partially supported by the Research Fund of Akdeniz

ity, Antalya, Turkey.

uests: Ciler Celik-Ozenci, Associate Professor, Department of

y and Embryology, Faculty of Medicine, Akdeniz University,

ntalya, Turkey (FAX: 90-242-227-44-86; E-mail: cilerozenci@

.edu.tr).

ertility and Sterility� Vol. 93, No. 8, May 15, 2010opyright ª2010 American Society for Reproductive Medicine, P

CNS. These capillaries have abnormally thin walls that are prone toleak (6).

CCM is inherited in an autosomal dominant manner, and thegenes mutated in three forms have recently been identified (7, 8).Krit1 (CCM1), the product of the Ccm1 gene, is known to interactwith ICAP-1 (9); malcavernin (CCM2), the product of the Ccm2gene, controls p38 MAP kinase (10); and the role of PDCD10(CCM3), the product of the Ccm3 gene, is unknown, although ithas been suggested that it may be involved in apoptotic processes(8). Even though it is currently unclear whether this effect onapoptosis is direct or indirect through modulation of the cell cycle,Chen et al. suggested that CCM lesions may form as a consequenceof aberrant apoptosis and also potentially alter the balance betweenthe endothelium and neural cells within the neurovascularunit (11). On the other hand, CCM genes play a role in CNSdevelopment and angiogenesis and specifically in assigning anarterial identity (12).

Varicoceles are pathological dilations of the venous pampiniformplexus of the spermatic cord and occur more frequently on the leftside (13). Clinical varicocele is observed in 10%–20% of the generalmale population, whereas it is demonstrated in 19%–41% of menpresenting for infertility investigations (13, 14). Adult varicocelepresents a challenge for male reproductive specialists, andresearchers have yet to fully elucidate the pathophysiology of

0015-0282/$36.00ublished by Elsevier Inc. doi:10.1016/j.fertnstert.2009.06.028

varicocele. The effect of the varicocele varies but often results ina generalized impairment of sperm production, which is character-ized by abnormal sperm quality ranging from oligozoospermia tocomplete nonobstructive azoospermia.

The pathophysiology of the varicocele has received considerablestudy, both in humans and in animal models. In humans, testes biop-sies of varicocele patients are characterized by depressed spermato-genesis with a predominant picture of maturation arrest, sloughingof spermatogenic epithelium, an increase in Leydig cells, thickeningof tubular basement membranes and interstitial blood vessel wallswith narrowing of their lumina, and increased deposition of intersti-tial fibrous tissue (15). Mechanistic information is difficult to obtainfrom human subjects because study designs must not be invasive andthe subject population is variable. Because of these limitations, an-imal models of varicocele have been developed in several species,the most common being the rat. Experimental left varicocele canbe developed in the rat, and it induces increased testicular bloodflow and temperature in parallel with decreased testicular sperm out-put (16, 17). Thus, in the present study, we induced varicoceles inadult rats and were able to mimic the pathological perturbationspresent in humans. In our model, varicocele induction resulted inthe disruption of seminiferous tubules, causing maturation arrestand hypospermatogenesis.

Since CCM proteins have novel functions in CNS and their pos-sible involvement in reproductive organ functions is unknown, weinvestigated these proteins in the testicular tissue of adult rats. Thepresent study was organized to answer two questions: First, whatare the distributions of the CMM2 and CCM3 proteins in normalrat testicular tissue? Second, are their distribution and expressionlevels altered with experimentally induced varicocele? Thus, this ar-ticle describes for the first time the distribution of CCM2 and CCM3proteins in rat testes, suggesting their novel role during normal andpathological (varicocele) spermiogenetic maturation processes.

MATERIALS AND METHODSA total of 24 adult Wistar male rats were housed at 24�C under controlled

conditions with free access to standard rat chow and water. The experimental

protocol was approved by the Animal Care and Usage Committee of Akdeniz

University and was in accordance with the Declaration of Helsinki and Inter-

national Association for the Study of Pain guidelines.

Experimental VaricoceleTwenty-week-old adult animals were used for the control group. Animals un-

derwent sham operation at 7 weeks and were studied 13 weeks later (n ¼ 6).

Animals in the experimental group underwent partial ligation of the renal

vein at 7 weeks and were studied 13 weeks later (n¼ 12). The establishment

of the control and experimental groups are described elsewhere (17, 18).

Briefly, by careful blunt dissection, a tunnel was made in the fat and connec-

tive tissues surrounding the left renal vein, and the vein was cleared of adher-

ing tissue in a position medial to the insertion of the spermatic and adrenal

veins. A 4-0 silk suture was tied to partially occlude the left renal vein at

the point that the vessel was cleared of other tissue. The ligature was made

around a metal wire of 0.85 mm diameter. The wire was removed from the

ligature, and the vessel expanded to an external diameter of approximately

1 mm. After instillation of 10 mg/mL of gentamicin sulfate into the abdom-

inal cavity, midline incisions of abdominal wall and anterior abdominal mus-

cles were repaired separately. After identification of left varicocele by

observation of the dilatation of the internal spermatic veins, both testicles

then were harvested from the same incision. Sham-operated rats underwent

a similar procedure: the left renal vein was dissected free but was not ligated.

Testes were obtained from control, sham-operated, and varicocele-induced

adult rats. Animals were killed 13 weeks after renal vein ligation, and left tes-

ticular tissues were removed for immunohistochemistry and Western blot

Fertility and Sterility�

analysis. From each animal, left testicular tissues were fixed in Bouin’s fix-

ative for 4 hours, dehydrated in ethanol, and embedded in paraffin wax for

immunohistochemistry, and the left testes from another animal was frozen

in liquid nitrogen for Western blot analysis.

CCM2 and CCM3 Antibody ProductionA CCM3/PDCD10 (CCM3)-specific rabbit polyclonal antibody was

synthesized and affinity purified against the antigenic peptide

KIPDEINDRVRFLQTIKD (Open Biosystems, Huntsville, AL) as described

elsewhere (12). The CCM2 antibody synthesis, characterization, and valida-

tion have also been described elsewhere (19).

ImmunohistochemistryFor CCM2 and CCM3 immunohistochemistry, sections were deparaffinized

and blocked for endogenous peroxidase activity with methanol containing

3% H2O2 for 15 minutes and for nonspecific binding with universal blocking

reagent (BioGenex, San Ramon, CA) for 10 minutes at room temperature.

Anti-rabbit CCM2 and CCM3 diluted in dilution buffer 1/250 (LabVision,

Fremont, CA) were applied for 1 hour at room temperature. For negative con-

trols, the primary antibodies were replaced by normal rabbit IgG serum (Vec-

tor Laboratories, Burlingame, CA) at the same concentration. After several

washes in phosphate-buffered saline (PBS), sections were incubated with bi-

otinylated goat anti-rabbit IgG secondary antibody (1/400 dilution, Vector

Laboratories) for 30 minutes followed by LSAB streptavidin-peroxidase

complex (Dako, Carpenteria, CA) incubation for 30 minutes and rinsing

with PBS. Antibody complexes were visualized by incubation with diamino-

benzidine chromogen (BioGenex). Sections were counterstained with

Mayer’s hematoxylin (Dako), dehydrated, mounted, and examined on

a Zeiss-Axioplan, microscope (Oberkochen, Germany).

Western Blot AnalysisTotal protein from the tissues was extracted in a lysis buffer (10 mM Tris-

HCL, 1 mM EDTA, 2.5% SDS, 1 mM phenyl methyl sulfonylfluoride, 1

mg/mL leupeptin) supplemented with CompleteR protease inhibitor cocktail

(Boehringer, Mannheim, Germany). The protein concentration was deter-

mined using a standard bicinchoninic acid (BCA) assay (20, 21), and 50

mg protein was applied per lane. Before electrophoresis, samples were heated

for 5 minutes at 95�C. Samples were then subjected to SDS polyacrylamide

gel electrophoresis under standard conditions and then transferred onto pol-

yvinylidene fluoride (PVDF) membranes (BioRad, Hercules, CA) in a buffer

containing 0.2 mol/L glycine, 25 mM Tris, and 20% methanol overnight. The

membranes were blocked for 1 hour with 5% nonfat dry milk (BioRad) in

TBS-T for 1 hour to decrease nonspecific binding. Afterward, the membranes

were incubated with rabbit polyclonal antibody against human CCM2 and

CCM3 (both diluted 1/1000) in 5% nonfat dry milk in TBS-T for 1 hour.

The membranes were then incubated with horse peroxidase-labeled anti-rab-

bit IgG (dilution 1/10,000; Vector Laboratories) for 1 hour. Immunolabeling

was visualized using the chemiluminescence-based SuperSignal CL HRP

Substrate System (Pierce, Rockford, IL), and the membranes were exposed

to Hyperfilm (Amersham, Piscataway, NJ). After the membrane was stripped

using stripping solution (Pierce), equal loading of proteins in each lane was

confirmed by reprobing the membrane with mouse monoclonal anti-human

b-actin antibody (Abcam, Cambridge, UK). Human normal brain tissue sam-

ple from parietal cortex was used as a positive control.

The integrated optical density of the Western blot results were subse-

quently quantified using an Alpha Digi Doc 1000 gel documentation unit

(Alpha Innotech Corporation, San Leandro, CA).

RESULTSHistological Evaluation of the Varicocele ModelLight microscopic histologic evaluation of our experimental modelrevealed that in the varicocele group disintegration of the germinalepithelium was clearly observed (Fig. 1J; Table 1). In some tubules,there were noncohesive and less orderly germ cells that sloughedinto the lumen of seminiferous tubules, and spermatogenesis was

2717

FIGURE 1

Localization of the CCM2 protein in control, sham, and varicocele-induced adult rat testes (A–N). (A, E, J) Low magnification images of control,

sham, and varicocele-induced rat testicular tissue sections are presented. (B, C) Note that CCM2 first appears in the acrosomal caps of roundspermatid acrosome and is specific to tubules in spermatogenic stages 6–8 (arrows). Also, elongating spermatid heads are CCM2

immunopositive in stages 1–5 (F, arrows) and 9–14 (D, G, arrows). (K, E) In the varicocele group, expression of CCM2 is localized in symplasts

(arrows) and vessels in the interstititum (arrowheads). (H) Negative control section; note the absence of CCM2 immunostaining. (N) Ductus

epididiymis section localized at the periphery of the varicocele-induced testes. Note the presence of abnormal gametes passing through theduct. Arrows indicate round spermatids with CCM2 expression. Scale bar represents 50 mm.

Tanriover. CCM2, CCM3 expressions in rat testes. Fertil Steril 2010.

2718 Tanriover et al. CCM2, CCM3 expressions in rat testes Vol. 93, No. 8, May 15, 2010

TABLE 1Summary of the localization patterns of CCM2 and CCM3 in normal and varicocele-induced rat testes.

CCM2 expression CCM3 expression

Normal rat testes Heads of all postmeiotic germ cells; roundspermatid acrosome (stages 6–8); elongating

spermatid acrosome (stages 1–5 and 9–14)

Cytoplasm of round spermatids (stages 1–8);elongating spermatid cytoplasm (stages 9–14);

cytoplasm of pachytene spermatocytes (stages

8–12); cytoplasm of diplotene spermatocytes

(stage 13); cytoplasm of meiotic figures (weakexpression, stage 14)

Varicocele-induced rat testes Increased immunoreaction in round and elongating

spermatid acrosomes; Leydig cells and vessels

Increased immunoreaction in the cytoplasm of

round spermatids (stages 1–8); microvessels of

the interstitium (strong expression)

Tanriover. CCM2, CCM3 expressions in rat testes. Fertil Steril 2010.

arrested. Moreover, in some tubules there were only one to threelayers of germ cells, and a considerable number of tubules exhibitedonly Sertoli cells. Symplasts, the nuclei of all spermatids of a cloneat certain stages of the spermatogenic cycle, joined together to formmultinucleate masses (22) in some tubules. The nuclei of degenerateround spermatids showed a halo appearance. The formation of thesemultinucleated giant cells was a consistent feature of varicocele-induced tubular degeneration.

ImmunohistochemistryTo clarify the cell types that express CCM2 and CCM3 proteins,immunohistochemical studies were performed with rat testiculartissue.

Cellular Localization of CCM2 in Normal and Varicocele-Induced Rat TestesIn the seminiferous epithelium, male germ cells at different develop-mental phases are arranged in defined associations or stages. Alongthe seminiferous tubules, these stages follow each other in a regular,consecutive, linear fashion, giving rise to the wave of the seminifer-ous epithelium in most mammals. The CCM2 protein was detectedin the heads of all postmeiotic germ cells, particularly in the roundspermatid and elongating spermatid acrosomes. The localization ofCCM2 in the round spermatid acrosome was specific to tubules inspermatogenic stages 6–8 (Fig. 1B, 1C arrows), whereas, elongatingspermatid heads were CCM2 immunopositive in stages 1–5V(Fig. 1F) and 9–14 (Fig. 1D, 1G, arrows).

In the varicocele group, expression of CCM2 was increased in theround spermatid and elongating spermatid acrosomes. These post-meiotic germ cells at different stages of the spermatogenic cyclerevealed that the level of immunostaining was increased in stages1–14. Additionally, interstitial Leydig cells and vessels (Fig. 1M,arrowheads) were immunopositive for CCM2.

Cellular Localization of CCM3 in Normal and Varicocele-Induced Rat TestesImmunohistochemical localization of CCM3 expression was prom-inent in cytoplasm of round spermatids at spermatogenic stages 1–8(Fig. 2B, 2F), with increased expression at stage 8. Also, at stages9–14, elongating spermatid cytoplasm exhibited CCM3 expression(Fig. 2C, arrows). This expression was strongest at stages 9–12,with a sharp decrease in the immunoreactivity at stages 8 and 14(Fig. 2G). Moreover, cytoplasm of pachytene spermatocytes in

Fertility and Sterility�

stages 8–12, cytoplasm of diplotene spermatocytes in stage 13,and cytoplasm of meiotic figures in stage 14 showed weak expres-sion of CCM3 protein. In testicular sections preincubated with anexcess of the immunogen, no staining was observed (Fig. 2H).

In the varicocele-induced testes, CCM3 expression was onlyincreased in cytoplasm of round spermatids at stages 1–8. Addition-ally, there was a strong positive immunoreactivity for CCM3 inmicrovessels of the interstitium (Fig. 2M).

Confirmation of Increased Expressions of CCM2 and CCM3by Western BlotCCM2 and CCM3 expression was analyzed by Western blottingfrom normal rat testicular tissue. The blots revealed clear bandsfor CCM2 and CCM3 corresponding to 47 and 25 kDa, respectively.Equal loading and normalization of the results were confirmed bydividing CCM2 and CCM3 signals with b-actin (43 kDa) signal(Fig. 3A). As a positive control, human normal brain tissue wasused to confirm the bands observed in testicular lysates. Identical re-sults were obtained in all 12 samples. The protein levels of CCM2and CCM3 molecules were significantly higher in the varicocele-induced groups than in the control and sham-operated groups(Fig. 3B).

DISCUSSIONThe present study investigates the presence of CCM2 and CCM3 innormal and varicocele-induced adult rat testes by immunohisto-chemistry and Western blot analysis. CCM proteins, CCM2 andCCM3, are related to cavernomas in the CNS (6). These proteinscan occur sporadically or as an autosomal dominant conditionwith variable expression and incomplete penetrance (6, 23). Familialforms have been linked to three chromosomal loci identified asCcm1, Ccm2, and Ccm3.

We have identified the CCM2 protein associated with the roundspermatid acrosome at stages 6–8 where the acrosome attachesand expands laterally by surrounding the cell nucleus. Moreover,at stages 1–5 and 9–14, CCM2 was only present in elongating sper-matid heads, indicating that CCM2 might be involved in acrosomebiogenesis. During spermatogenesis, in addition to dramaticchanges in the germ cell, membrane domains of the spermatid un-dergo significant redistribution with the formation of acrosome(24). Although the morphological changes involved in acrosomeformation have been well described, the underlying molecularevents required for acrosome biogenesis remain unclear (25).Although there is currently limited knowledge, it was previously

2719

FIGURE 2

Representative pictures of CCM3 protein expression in control, sham, and varicocele-induced adult rat testes (A–N). (A, E, J) The lowmagnification image of the rat testicular tissue section is presented in control, sham, and varicocele-induced groups. An intense

immunostaining of CCM3 protein in round spermatid cytoplasm is shown in spermatogenic stages 1–8 (B, F), with increased expression in

stage 8 (F). (C, D, F) Elongating spermatid cytoplasm exhibited strong CCM3 expression in spermatogenic stages 9–14, whereas weak

immunoreactivity is observed in primary spermatocytes (C, double arrow). (H) In testicular sections preincubated with an excess of theimmunogen, no staining is observed. (K) Immunohistochemical localization of CCM3 expression is prominent in symplasts in varicocele-

induced groups (arrows). (M) In addition, there is a strong positive immunoreactivity for CCM3 in microvessels of the interstitium (arrowheads).

(N) Ductus epididymis section localized at the periphery of the varicocele-induced testes. Arrows indicate round spermatids with cytoplasmicCCM3 expression. Scale bar represents 50 mm.

Tanriover. CCM2, CCM3 expressions in rat testes. Fertil Steril 2010.

2720 Tanriover et al. CCM2, CCM3 expressions in rat testes Vol. 93, No. 8, May 15, 2010

FIGURE 3

Western blot results of CCM2 and CCM3 proteins in control,

sham, and varicocele-induced rat testes. (A) Immunoblots oftestes extracts using anti-CCM2 and anti-CCM3 bands (47 and 25

kDa, respectively) are detected in positive control (brain; lane B)

and testes samples (lane C, control; lane S, sham; lane V,

varicocele). The immunoexpression of b-actin (43 kDa) is used asan internal standard. (B) The protein levels of CCM2 and CCM3

molecules are significantly higher in varicocele-induced groups

than the levels in the control and sham-operated groups.

Tanriover. CCM2, CCM3 expressions in rat testes. Fertil Steril 2010.

suggested that CCM2 regulates p38 MAP kinase activity (10).The activation of the p38 MAP kinase cascade usually results inan inflammatory response or apoptosis, but it also regulates othercellular functions including cell proliferation and differentiation(26, 27). Recently, p38 MAP kinase was found to be involved inthe acrosome reaction of human sperm (28).

Moreover, the preferential elongating spermatid head expressionof CCM2 in testes suggests that this protein may play a role in sper-miogenesis processes. It has been found that rat elongating sperma-tids express a native form of p38 MAP kinase (29). The activation ofthe p38 MAPK is known to regulate cell adhesion and migration bymodulating actin dynamics (30). Significantly, p38 MAP kinase isinvolved in apical ectoplasmic specialization dynamics, which isrestricted to the Sertoli-spermatid interface in the seminiferousepithelium (31).

On the other hand, we observed the CCM2 immunoreactivity tobe present in Leydig cells, and its expression was increased by var-icocele induction. It is known that Leydig cells have important rolesin the regulation of T production and release (32). Thus, we specu-late that CCM2 may also have important roles in Leydig cell func-tion, in addition to its possible role in acrosomal biogenesis.

Another novel aspect of the study reported here is that we havelocalized the CCM3 protein in the cytoplasm of primary spermato-

Fertility and Sterility�

cytes and round spermatids. It is possible that the weak expression ofCCM3 in primary spermatocytes might be related to the meiotic di-vision processes. Moreover, the dense expression pattern in roundspermatid cytoplasm and in the cytoplasm of elongating spermatidssuggests that CCM3 might be related to spermiogenetic events, suchas cytoplasmic remodeling and shaping of the rat sperm.

While the function of the CCM3 protein is unknown, it has re-cently been found that a large portion of the CCM3 protein resideswithin a novel large multiprotein assembly, referred to as the STRI-PAK (striatin-interacting phosphatase and kinase) complex (33).The STRIPAK assembly establishes mutually exclusive interactionswith the CTTNBP2 (cortactin binding protein-2) proteins, which in-teract with the cytoskeletal protein cortactin (33). Cortactin hasemerged as an essential protein in regulation of the cortical actin cy-toskeleton through its ability to influence cytoskeletal reorganiza-tion in response to extra- and intracellular stimuli. By interactingwith a variety of binding partners, cortactin bridges the actin cyto-skeleton to many effector proteins that control actin-based cellularprocesses (34).

The shaping of the mammalian sperm involves the elongationand condensation of the spermatid nucleus, the development ofthe acrosome, and the transient appearance of the microtubularmanchette (35). Phosphorylated cortactin is present in the acrop-laxome, an F-actin/keratin-5-containing cytoskeletal plate thatanchors the developing acrosome to the spermatid nucleus, thussupporting a role of this F-actin remodeling pathway during sper-matid head shaping (36). In light of the recent findings discussedabove, we may speculate that CCM3, as a large portion of theSTRIPAK complex present in round and elongating spermatid cy-toplasm, could interact with cortactin and have an important rolein shaping the spermatid head. Moreover, the peripheral presenceof the CCM3 protein in the marginal cytoplasm of symplasts,which we observed because of the varicocele pathology, may in-dicate its role during abnormal cytoplasmic remodeling, leadingto multinuclear giant cell formation and absent spermatid headshaping.

According to our results, round spermatids at stages 6–8 and elon-gating spermatids at stages 9–14 showed immunoreactivity withboth CCM2 and CCM3 proteins. It is possible that both proteinsare involved in spermiogenesis events, such as acrosome biogenesisand/or cytoplasmic remodeling of round and elongating spermatids.Weak CCM3 expression in pachytene and diplotene spermatocytesis followed by both CCM2 and CCM3 expression in round and elon-gating spermatids. Therefore, round spermatids are the critical cellsof the testes in understanding molecular interactions between theseCCM proteins.

In conclusion, this is the first study that attempts to investigate thepossible roles of the CNS proteins CCM2 and CCM3 in normal andpathological rat testes. Our results indicate that both proteins arenecessary during normal spermatogenesis and that their expressionsare increased by varicocele induction. It is clear that further studiesof CCM2 and CCM3 in mammalian testes, their possible contribu-tion to other testicular pathologies, and their role in the formation ofabnormal gametes are needed.

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