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ncreased Apoptosisf Spermatogenic Cells inryptorchidism Rat Model and

ts Correlation With Transformingrowth Factor Beta Type II Receptor

ian-Lin Yuan, Yun-Tao Zhang, and Yong Wang

BJECTIVES To investigate the role played by transforming growth factor beta receptor II (TGF�RII) incryptorchidism-induced spermatocyte apoptosis.

ETHODS A unilateral cryptorchidism rat model was surgically established in 20-day-old male SD rats. Testissamples were collected 0, 4, 7, 14, and 21 days after surgery. Histologic changes, apoptosis, TGF�RII/smad, and TGF�RII/mitogen-activated protein kinase activation were explored by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and western blotanalysis, respectively. TGF�RII was knocked down in GC-2 spg cells and the cells were then treatedwith hyperthermia. Western blot analysis was performed to detect TGF�RII, the phosphorylationstatus of smad2, smad3, and p38 and the cleavage status of caspase-3.

ESULTS Surgically induced cryptorchidism significantly impaired testis growth and spermatogenesis in uni-lateral undescended testes (UUTs) compared with contralateral descended testes 7, 14, and 21 daysafter surgery. The mean apoptotic index was significantly higher in UUTs than in contralateraldescended testes. Western blot analysis showed that TGF�RII and smad2 expression increased.Phosphorylation of smad2, smad3, and p38 and cleavage of caspase-3 increased in UUTs.TGF�RII knockdown in GC-2 spg cells reduced hyperthermia-induced apoptosis by inhib-iting smad2, smad3, and p38 phosphorylation as well as downstream caspase-3 cleavage.

ONCLUSIONS Cryptorchidism lowered the growth rate of testes by inducing apoptosis, via a mechanisminvolving the activation of the TGF�R/smad and TGF�R/mitogen-activated protein kinase

pathways. UROLOGY 75: 992–998, 2010. © 2010 Published by Elsevier Inc.

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ryptorchidism is one of the most common birthdefects of male genitalia. The incidence is 5.0%at birth and 1.8% at 3 months of age.1 This

efect results in reduced blood vessel volume of thendescended testes, disrupted production of germ cells,nd enhanced germ cell apoptosis.2 These features cannduce impairment of spermatogenesis and cause sper-atogenic cell maturation arrest. Although previous

tudies have shown that germ cell apoptosis in the testess an essential feature of cryptorchidism,3 the underlying

olecular mechanism remains to be determined.

his Project was granted by the Natural Science Fund of Shaan’xi Province, China.Jian-Lin Yuan and Yun-Tao Zhang contributed equally to this research.From the Department of Urology Surgery, Xijing Hospital, the Fourth Militaryedical University, Xi’an, Shaan’xi, China and Department of Urology Surgery,angdu Hospital, the Fourth Military Medical University, Xi’an, Shaan’xi, ChinaReprint requests: Yong Wang, Ph.D., Department of Urology Surgery, Tangduospital, the Fourth Military Medical University, Xi’an, Shaan’xi, China 710032.

p-mail: dryongwangfmmu@yahoo.com.cnSubmitted: December 4, 2008, accepted (with revisions): May 7, 2009

92 © 2010 Published by Elsevier Inc.

Many regulatory factors, such as transforming growthactor � (TGF�),4 are involved in the apoptosis of sper-atogenic cells. TGF� has diverse regulatory functions

n cell growth and differentiation in a variety of tissues.5

he different isoforms of TGF� bind to the extracellularortion of the transmembrane TGF� receptor type IITGF�RII), which then recruits TGF�RI, also a trans-embrane protein, to form heterodimers. The signal is

hen transduced into the cell through phosphorylation ofmads (small mothers against Dpp) and mitogen-acti-ated protein kinase (MAPK)/p38.6

TGF�RII is expressed in the testicular interstitium and isainly seen in elongated spermatids and spermatocytes,

ndicating a role of TGF� in the modulation of spermato-enesis and spermatocyte apoptosis.7 However, whetherGF�RII activation is important in cryptorchidism-in-uced apoptosis has not been determined. Therefore, in thistudy, we established a unilateral rat cryptorchidism modelnd investigated changes in TGF�RII expression and in the

hosphorylation of smads and MAPK.

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igure 1. Cyrptorchidism-induced histologic changes in UUTs (21 days). (A-D) Hematoxylin and eosin staining of CDTs and UUTs.A) In the CDT group (�100), seminiferous tubules are round and clear and the interstitium surrounding them is clearlyiscernable. The black arrow highlights the interstitium. The clearly observed cells are Leydig cells. (B) CDTs of the 21-day group�400). Many primary spermatocytes, spermatocytes, and round as well as elongated spermatids are observed in eacheminiferous tubule. Spermatocytes of different stages can be seen. The white arrow indicates a primary spermatocyte; grayrrow indicates a spermatocyte; red arrow indicates a round spermatid; and black arrow indicates an elongated spermatid. (C)

UTs of the 21-day group. Seminiferous tubules are significantly shrunken (�100) (Color version available online).

ROLOGY 75 (4), 2010 993

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ATERIAL AND METHODS

ell Culture and Animal Tissue Preparationhe hyperthermia-sensitive GC-2 spg cell line was purchased

rom the National Institute of Family Planning of China. TheC-2 spg cells were cultured in 5% CO2, at 37°C in Ham’s F-10

rowth medium (Invitrogen, Beijing, China) supplementedith 10% fetal bovine serum. Cells were seeded into 6-cmishes before small interfering RNA (siRNA) transfection.The average life span of the Sprague-Dawley (SD) rat is 2.5

ears. Male SD rats become sexually mature at around 65 days.herefore, a cryptorchidism model established between 20 and 41ays after birth best mimics the effect of cryptorchidism on ado-escent testis development.8 We, therefore, used 20-day-old maleD rats obtained from the Animal Center of the Fourth Militaryedical University (Animal experiments were approved by the

ourth Military Medical University Animal Bioscience Commit-ee). The rats were individually housed in an animal house at 22°Cnder a light/dark cycle of 12/12 hours, with standard food andater provided ad libitum. To induce unilateral cryptorchidism,

ats were anesthetized with soluble pentobarbitone (40 mg/kg). Aidline incision was made in the abdomen.9 The gubernaculums

n the right side was cut to displace the testis into the abdomen.he inguinal canal was sutured on the right side to prevent the

estis from descending. The contralateral testis was sham-operatedo act as control; the gubernaculums was cut and the inguinal canaleconstructed by suturing the tunica albuginea. The inguinal skinas then closed with 4-0 Vicryl sutures. The rats were randomlyssigned to 5 groups (0, 4, 7, 14, and 21 days), with 5 rats in eachroup. At 0, 4, 7, 14, and 21 days after surgery, the testes wereesected after anesthesia and weighed. The testes were fixed in 4%araformaldehyde for 24 hours, and processed for paraffin embed-ing. Serial 5-�m sections were cut for immunohistochemistry,ematoxylin-eosin staining, and for the terminal deoxynucleotidylransferase dUTP nick-end labeling (TUNEL) assay.

iRNA Transfection and Hyperthermia Treatmenthe GC-2 spg cells were incubated at 37°C under 5% CO2 for4 hours until 50%-80% confluence. This took approximately8 hours after seeding 6-cm dishes with 3 � 105 cells/dish.hen, 100 nM TGF�RII siRNA (5=-AAAGUCGGUUAA-AGCGAUCUdTdT-3=, and 3=-AGAUCGCUGUUAAC-GACUUUdTdT-5, Dharmacon, Chicago, IL) or nonsilenc-

ng control siRNA was transfected using oligofectamine,ccording to the manufacturer’s instructions (Invitrogen).ryptorchidism may induce spermatocyte cell apoptosis by hy-erthermia; therefore, we treated the nonsilencing controlNSC) and TGF�RII knockdown cells to a high temperature byncubation at 41°C for 1 hour before protein extraction.10

permatogenic cells and cell layers in each tubule are decreased.re Leydig cells that are not very clear, and are swollen. The wh1-day group. Both round and elongated spermatids are rarely semaller than that of the CDT group (�400). (E) Weight of UUTs aesected and weighed (n � 5). At 7, 14, and 21 days, the weighlightly between days 14 and 21, although this decrease was not sUTs. At all time points, testes are stained with hematoxylin-eos0 randomly selected tubules. Seminiferous tubule diameters in the Student t test shows that this difference is significant at day

94

mmunohistochemistryissue sections were de-waxed using routine methods and endog-nous peroxidases were blocked with 0.3% methanol-H2O2. Theections were incubated with a primary TGF�RII antibody (1:200,anta Cruz Biotechnology, CA) and then with the ChemMateAKO EnVision Detection reagent (DAKO, Tokyo, Japan), fol-

owed by DAB (3,3=-diaminobenzidinetetrahydrochloride, Sigma,oulder, CO) according to the manufacturer’s instructions. Neg-tive control sections were prepared using 0.1% phosphate-buff-red saline/bovine serum albumin (PBS/BSA) and 5% nonim-une rabbit serum. The sections were then examined under a 40�

bjective and photographed. TGF�RII-positive cells in a mini-um of 20 random fields were counted and the mean � standard

eviation (x � s) per field was used to represent the numbers ofGF�RII-positive cells per unit.

UNEL Assayhe TUNEL assay was performed with a TUNEL assay kit

Boster, Wuhan, China). Paraffin sections were de-waxed, re-ydrated, and blocked with 0.3% methanol-H2O2. The sectionsere then incubated in a moist chamber with proteinase K

1:200) at 37°C and then washed with TBS; they were thenncubated for 2 hours with TUNEL mix 0.3 U/�L (calf thymuserminal deoxynucleotidyl transferase, 7 pmol/�L biotin dUTP,nd 1 mM cobalt chloride in reaction buffer). After washing, theections were sequentially incubated in blocking buffer, extravidin peroxidase antibody, followed by diaminobenzidineDAB). Negative control slides were prepared using Tris-buff-red saline (TBS) instead of terminal deoxynucleotidyl trans-erase (TdT) buffer. Positive control slides (lung tissue) wererovided by the manufacturer. At least 50 randomly selectedeminiferous tubules from each group were observed, and theumbers of TUNEL-positive and TUNEL-negative cells wereecorded. The seminiferous tubules with apoptotic cells wereefined as positive seminiferous tubules. The positive seminif-rous tubule rate and the apoptotic index11 (AI, number ofpoptotic cells per 1000 cells) was calculated.

estern Blot Analysisntibodies used were rabbit polyclonal anti-TGF�RII (Santaruz Biotech, CA), polyclonal anti-smad2 (Abcam, Cam-ridge, UK), polyclonal anti smad3 (BioVision, Palo Alto),hosphosmad2, phosphosmad3 (Bio-Lab, London, UK), andhosphop38 (New England Biolabs, MA). Total lysates fromnilateral undescended testes (UUTs) and contralateral de-cended testes (CDTs) and from GC-2 spg cells transfectedith TGF�RII and NSC siRNA, respectively, were preparedith radioimmunoprecipitation assay buffer supplemented withrotease inhibitor Mix (Roche, NJ) and phosphatase inhibitor

black arrow indicates the interstitium; the cells in the interstitiumrow indicates a shrunken seminiferous tubule. (D) UUTs of thethe seminiferous tubules and the diameter of the tubule is muchTs. At 0, 4, 7, 14, and 21 days, testes of different groups were

UTs was less than that of CDTs. The weight of UUTs decreasedtically significant. (F) Seminiferous tubule diameters in CDTs ande seminiferous tubule diameters were determined by examiningUT groups were all smaller compared to those in the CDT groups.�.05), and very significant at days 14 and 21 (P �.01).

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ix (Sigma, CO) 48 hours after siRNA transfection. Lysates ofoth UUTs and CDTs were cleaned and enriched with therotein Clean Up and Enrichment Kit (Pierce, Rockford, IL).rotein lysates were separated by 7.5% SDS-PAGE (sodiumodecyl sulfate-polyacrylamide gel electrophoresis) and trans-erred to 0.45-�m nitrocellulose membranes (Sigma, CO).fter blocking, membranes were incubated with primary

ntibodies overnight. Further, membranes were incubatedith secondary antirabbit horseradish peroxidase-labeled anti-ody (stock solution: 400 �g mL�1; 1:50.000; Santa Cruz, CA)or 1 hour at room temperature. Bound antibodies were detectedith ECL plus (GE Healthcare, London, UK) and subsequentlyetected using Kodak BioMax sensitive film (Sigma, CO). Filmsere scanned and quantified by ImageJ software (available free

igure 2. Cryptorchidism inhibited testis growth, and inpoptosis-positive seminiferous tubule rate, and the apopDTs and UUTs were determined after TGF�RII immunohisrous tubule. TGF�RII expression in CDTs was higher thannd 21-day groups. (B) Apoptosis-positive seminiferouseminiferous tubule rates in UUTs were much higher comppermatogenic cells. The apoptotic indexes of spermatogen4 and 21 compared to those in the respective CDTs.

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ROLOGY 75 (4), 2010

tatistical Analysiseights of UUTs and CDTs were compared using the matched

aired t test using SPSS. Numbers of TGF�RII-positive cells pernit, apoptosis positive seminiferous tubule rate, and AI wereompared using the Mann-Whitney U test. Differences wereonsidered statistically significant when P �.05 and very sig-ificant when P �.01.

ESULTS

ryptorchidism Suppressed Testiculareight Increase and Induced Histologic Changesesticular weights in cryptorchidism and contralateral

d apoptosis. The number of TGF�RII-positive cells, theindex of TGF�RII-positive cells per seminiferous tubule inemistry. (A) Number of TGF�RII-positive cells per seminif-in UUTs in the day 4 group and much higher in the 7-, 14-,le rate in UUT and CDT groups. The apoptosis-positiveto those in CDTs at all time points. (C) Apoptotic index in

ells in UUTs were higher at day 7 and much higher at days

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Fig. 1A). The weight increase of CDTs was more rapidnd steady than that of UUTs (� 5- to 6-fold at 21 day).DT weights were approximately � 0.15 g on days 0 and, � 0.23 g on day 7, � 0.53 g on day 14, and � 0.86 gn day 21. In contrast, the weight of UUTs increasednly about 2-fold, from � 0.15 g on day 4 to � 0.32 g onay 21. A slight decrease in weight was noted from day 14� 0.36 g) to day 21 (� 0.32 g), although this decreaseas not statistically significant. At day 14, the weight of

he UUT testes was significantly less than that of CDTs.t day 21, the difference was very significant.Four days after surgery, no remarkable histologic differ-

nces were observed among CDTs, UUTs, and the controlestes. Fourteen and 21 days after surgery, the diameter ofeminiferous tubules of UUTs was smaller than that ofDTs (Fig. 1B). On day 21, the CDTs had clear primary

permatocytes, spermatocytes of different stages, spermatids,nd elongated spermatids in the seminiferous tubules (Figs.C and D), and the different stages of spermatogenic epi-helium were clearly present. In contrast, the seminiferousubules of UUTs presented only spermatogonia and a smalluantity of primary spermatocytes, but no spermatids (Figs.E and F), which indicates maturation arrest. No obviousifference in Sertoli and Leydig cells was observed betweenUTs and CDTs.

permatocyte Apoptosis Wasncreased in Cryptorchidism Testesrevious reports have indicated that spermatocytes and amall quantity of spermatogonia were the main cell typeshat undergo apoptosis during spermatogenesis.12 On day, more apoptotic spermatogonia cells were observed inUTs than in CDTs. The percentage of apoptotic cells

n UUTs increased up to day 14 and then decreased onay 21 (Fig. 2A). The percentage of apoptotic cells inDTs parallels that of UUTs, that is, the number of

poptotic cells was highest on day 14. However, at allime points, both the seminiferous tubule apoptosis pos-tive rate (Fig. 2B) and the AI (Fig. 2C) of the UUTroups were much higher (3- to 4-fold higher or more)ompared to that of the CDT groups.

xpression of TGF�RII and Phosphorylationf smads and p38 Was Increased in UUTso explore activation of the TGF�RII pathway, we per-

ormed western blot analysis. TGF�RII expression wasncreased in UUTs compared with CDTs (Fig. 3A),hich is consistent with a previous report.13 TGF�RII isnown to activate the smad2/smad3 and MAPK path-ays, which transduce the signal into the nucleus. We,

herefore, assayed the phosphorylation changes in smadsnd p38 in UUT and CDT testes. The expression andhosphorylation status of smad2, smad 3, and p38 arehown in Figures 3B, 3C, and 3D, respectively. Thexpression of smad2 and smad3 was upregulated and thehosphorylation of smad2 and smad3 in UUTs of differ-

nt groups was increased compared with the respective

96

igure 3. Cryptorchidism increased expression of TGF�RII,mad2, smad3, and p38 and increased phosphorylation ofmad2, smad3, and p38 in UUTs compared with CDTs. (A)GF�RII expression assay. TGF�RII expression graduallyncreased in UUTs with time after surgery. In CDTs it re-ains almost at the same level. (B) smad2 expression andhosphorylation assay. In UUTs, at days 4, 7, 14, and 21,n increase in smad2 phosphorylation was found in the UUTroup. (C) smad3 expression and phosphorylation assay.fter operation, smad3 expression was increased in UUTsroups. After 21 days, there is a significant increase inmad3 phosphorylation in UUTs. The increase in smad3hosphorylation was not accompanied by upregulation ofmad3 expression. (D) p38 expression and phosphoryla-ion assay. Although the level of p38 expression did nothange significantly by cryptorchidism, the phosphorylationf p38 was significantly increased in the UUTs. In CDTs,38 expression decreased slightly but not significantly.E) Procaspase 3 and cleaved caspase 3 assay. At allimepoints after operation, obvious increased expression ofrocaspase 3 could not be found. But the increase ofleaved caspase 3 was obvious. Parallel with the decreasef AI and smad2 at 21st day, there was also a decrease ofleaved caspase 3 at 21st day, suggesting a close corre-

UROLOGY 75 (4), 2010

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DT groups. Phosphorylation of smad2 and smad3 wasarkedly enhanced compared with controls, whereas the

verall levels of smad2 and smad3 expression were onlylightly enhanced. The ratio of phosphorylated total pro-ein indicated that there was an increase in phosphory-ation from day 4 to day 14 for smad2, while a slightecrease was noted on day 21. Furthermore, although theevel of p38 expression did not change following surgery,38 phosphorylation increased in UUTs, while in CDTst remained constant, which indicated that the MAPKathway in UUTs was also activated.To further explore whether TGF�RII could activate

mad2, smad3, and p38, we used siRNA to knock downGF�RII expression in GC-2 spg cells, a hyperthermia

ensitive spermatocyte cell line.13 We found that success-ul TGF�RII knockdown significantly (P �.05) reduced

igure 4. Knockdown of TGF�RII inhibited smad2, smadatocyte GC-2 spg cells. NSC�: nonsilencing control s

encing control siRNA-transfected group with hyperthermiaiRNA with hyperthermia. (A) Apoptotic index of GC-2 syperthermia treatment in both NSC and TGF�RII siRNA-brogated the hyperthermia-induced apoptosis. (B) Wehosphorylated smad2 (p-smad2), smad3 (p-smad3),xpression was obviously inhibited. Hyperthermia did nnockdown and hyperthermia did not have obvious effeyperthermia treatment, there was an obvious increasenockdown, smad2, smad3, and p38 phosphorylation w

he hyperthermia-induced apoptosis (Fig. 4A). Further- c

ROLOGY 75 (4), 2010

ore, western blot analysis showed that smad2, smad3,nd p38 were phosphorylated by hyperthermia treatment,hile TGF�RII knockdown partially abrogated this hy-erthermia-induced increase in apoptosis (Fig. 4B).

OMMENTGF�(s) is a multifunctional regulator of cell function,hich regulates the inhibition of cell proliferation and dif-

erentiation during reproduction.14 TGF�1 can inhibit prolif-ration and induce apoptosis in different tissues.15,16 However,he role of TGF�RII in cryptorchidism is still unclear.

It was previously reported that TGF� receptor acti-ation significantly increased DNA fragmentation inonocytes.17 In our experiment, we also found thatpermatocyte and spermatogonia apoptosis was signifi-

nd p38 phosphorylation in temperature-sensitive sper--transfected group without hyperthermia. NSC�: nonsi-F�: TGF�RII siRNA without hyperthermia. TGF�: TGF�RIIells. There was an obvious increase in apoptosis aftersfected groups. However, TGF�RII significantly (P �.05)n blot analysis of TGF�RII, smad2, smad3, p38 and(p-p38). In TGF�RII siRNA-transfected group, TGF�RIIve any obvious effect on TGF�RII expression. TGF�RII

n smad2, smad3, and p38 expression. However, aftermad2, smad3, and p38 phosphorylation. After TGF�RIIbviously inhibited.

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hat in CDTs. Although the apoptotic positive tubuleate at day 21 was slightly lower compared to that at day4, the number of apoptotic positive cells in the 7- and4-day CDT or UUT groups were much higher than thatf the 0- and 4-day groups, which indicates a peak ofpermatocyte apoptosis at day 14. The apoptosis rate alsoncreased in CDTs. This may be correlated with thedolescent developmental stage, which peaks 34 daysostnatally in rats. However, the peak of apoptosis at day4 in the UUT groups is likely to be due to the sheddingf cells and to a lack of spermatocytes in day 21 UUTs.n addition, both 14 and 21 days after surgery, the UUTroup presented maturation arrest, which may also con-ribute to hypospermatogenesis of UUTs.

A previous research has suggested that TGF� plays anmportant role in testis development and spermatogene-is.18 However, the effects of TGF� on spermatogenicell apoptosis in vivo have not been reported. In this ratryptorchidism model, we found that TGF�RII was ex-ensively expressed in Leydig and spermatogenic cells atifferent development stages in the seminiferous tubulesf normal testes (CDTs). TGF�RII expression was mark-dly increased in UUTs compared to that in CDTs. Theelative increase in TGF�RII protein levels in spermat-genic cells of UUTs was accompanied by a greaterercentage of apoptotic spermatogenic cells.The smad and MAPK pathways play important roles in

ransducing TGF�RII signals to the nucleus.19 TGF�inds to specific cell surface receptors, such as type II andype I serine/threonine kinase receptors. Receptor-regu-ated smads (R-smads), such as smad2 and smad3, arehen recruited to the receptor20 and are phosphorylatedfter TGF�RII activation. They then form heterocom-lexes with common-partner smads (Co-smads). Theseomplexes then translocate into the nucleus. By directinding with DNA, interaction with DNA-binding pro-eins, and recruitment of coactivators or corepressors,hey can regulate the transcription of target genes.

TGF� activation can activate the MAPK pathway inancer cells.21 However, to date, the role of the p38APK pathway in testicle development is not clear. In

ur rat cryptorchidism model, we found that increasedxpression of TGF�RII in UUTs was correlated withncreased phosphorylation of smad2, smad3, and p38 andith the upregulation of smad2 expression. This indi-ated that the activation of the TGF�RII/MAPK signal-ng pathway was involved in the excessive apoptosis ofpermatogenic cells in cryptorchidism. Furthermore, knock-own of TGF�RII in the hyperthermia sensitive spermato-yte GC-2 spg cell line could decrease hyperthermia-in-uced apoptosis by inhibition of smad2, smad3, and p38hosphorylation. These results suggest that TGF� may playritical roles in the pathologic processes of cryptorchidism.he activation of TGF� and its downstream signals, includ-

ng smads and MAPK pathways, could render spermato-enic cells more susceptible to nonphysiological apoptosis,

uch as cryptorchidism-induced apoptosis.

98

Taken together, our data indicate that in our rat model,ryptorchidism leads to decreased testicular weight increase,aturation arrest, increased spermatocyte apoptosis, in-

reased TGF�RII expression, and activation of TGF�RII/APK pathways. These data may help clarify the mo-

ecular mechanism of cryptorchidism-induced apoptosisn the testicle.

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torchidism: a prospective study of 7500 consecutive male births.Arch Dis Child. 1992;67:892-899.

2. Heninger NL, Staub C, Blanchard TL, et al. Germ cell apoptosis inthe testes of normal stallions. Theriogenology. 2004;62:283-297.

3. Vigueras-Villaseñor RM, Molina-Ortiz D, et al. Effect of allopuri-nol on damage caused by free radicals to cryptorchid testes. ActaHistochem. 2009;111:127-137.

4. Konrad L, Keilani MM, Laible L, et al. Effects of TGF-betas and aspecific antagonist on apoptosis of immature rat male germ cells invitro. Apoptosis. 2006;11:739-748.

5. Rahimi RA, Leof EB. TGF-beta signaling: a tale of two responses.J Cell Biochem. 2007;102:593-608.

6. Kurisaki A, Kose S, Yoneda Y, et al. Transforming growth factor-beta induces nuclear import of Smad3 in an importing-beta1 andRan-dependent manner. Mol Biol Cell. 2001;12:1079-1091.

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6. Barendrecht MM, Mulders AC, van der Poel H, et al. Role oftransforming growth factor beta in rat bladder smooth muscle cellproliferation. J Pharmacol Exp Ther. 2007;322:117-122.

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8. Olaso R, Pairault C, Boulogne B, et al. Transforming growth factorbeta1 and beta2 reduce the number of gonocytes by increasingapoptosis. Endocrinology. 1998;139:733-740.

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