CONCLUSIONS: High-dose CPP resulted in lower mouse sur-vival. While no histologic differences were seen in the xenograftedtestis, there were dramatic changes in expression profiles with down-regulation of genes involved in germ line specific and the MAPK/ERKpathway, involved in gonadotropin signaling. Methylation status corre-sponded with expression changes, suggesting a mechanism for CPPinduced gonadotoxicity. Using CPP as an example, we have an excel-lent model for examining the multi-dimensional (survival, histology,epigenetic, gene expression) effects of other gonadotoxins on humangerm cells.

Source of Funding: None

2036DOWN-REGULATION OF HUSP26 IN HORMONALLY-RECEPTIVECANCERS SUGGESTS A ROLE IN CANCER DEVELOPMENT

Matthew Wosnitzer*, Anna Mielnik, Peter Schlegel, Darius Paduch,New York, NY

INTRODUCTION AND OBJECTIVES: Human ubiquitin specificprotease 26 (hUSP26), an X-linked gene, was initially reported to betestis specific. We have previously shown the changes in USP26sequence are associated with infertility and low testosterone produc-tion. The aims of this study were to develop and express a GSP-USP26construct for tissue expression analysis and pull-down experiments toidentify USP26 binding proteins using MALDI-TOF and to compareexpression of hUSP26 in normal and neoplastic human tissues.

METHODS: hUSP26 was cloned into pF1K and pFN2A GSTFlexi vectors, sequenced and expressed in BL21 (DEC3) E. coli.Anti-GST beads were used for purification and AcTev proteinase wasused to cleave the construct. Anti-GST and commercial anti-USP26antibodies were used to select the optimal antibody using Westernblots. Dig-dUTP -labeled DNA probe USP26 was used for in situhybridization. Three human tissue arrays were analyzed using ImageJafter background and linearity correction. Expression was confirmedusing protein lysates.

RESULTS: N-terminal, polyclonal antibody detecting 100 kDais the most sensitive and specific antibody for hUSP26. USP26 isexpressed in other tissues than testis, including prostate, ovary, cervix,and breast. Thyroid, adrenal, and stomach cancer highly overexpressUSP26, and breast cancer, prostate and ovarian cancers have lowerexpression of USP26 (Figure 1). USP26 was highly overexpressed inthyroid cancer and down-regulated in breast cancer. Within human andmurine testis, USP26 was expressed in Leydig cells and early sper-matogonia, as expected from our previously published mutational anal-ysis showing that mutations in USP26 lead to infertility and low testos-terone.

CONCLUSIONS: hUSP26 is not testis specific. Some andro-gen dependent cancers show expression. Since USP26 regulatesandrogen receptor (AR) transcriptional activity, this gene may have arole in carcinogenesis. Our results have potential applications in pros-tate cancer and other cancers in which androgen receptor plays asignificant role in progression. Further studies in these tumors areunderway.

Source of Funding: None

2037AGE DEPENDENT DEGENERATION OF SERTOLI AND LEYDIGCELLS IN ADOLESCENTS AND MEN WITH KLINEFELTERSYNDROME: A HISTOLOGICAL ANALYSIS

Michael Funaro*, Akanksha Mehta, Russell Simons,Alexander Bolyakov, Anna Mielnik, Maria Shevchuk, Peter Schlegel,Darius Paduch, New York, NY

INTRODUCTION AND OBJECTIVES: Klinefelter syndrome(KS) is the most common chromosomal disorder leading to maleinfertility and hypogonadism. Limited clinical observations suggest thatmeiotic divisions during puberty lead to progressive loss of germ cellsand testicular atrophy in KS. Sertoli (SC) and Leydig (LC) cells dividemitotically during puberty and are believed to be non-dividing cells inadulthood. Little is known about the effects of age on Sertoli and Leydigcell in KS patients after adolescence. This study aimed to correlate therelationship between patient age and testicular morphology on testisbiopsy in 47,XXY men.

METHODS: Open testicular biopsies from 17 KS patients werepreserved in Bouin’s solution, embedded in paraffin, sectioned (0.5um), and fixed on slides stained with H&E. The diameter of roundseminiferous tubules (SFTs), LC internuclear distance, and number ofSC per SFT were measured. At least 10 cross-sections of SFTs and100 LCs were measured for each slide using light microscopy andmorphometry software (Nikon NIS Elements®).

RESULTS: Testicular biopsies from six adolescents (mean age15.8, 95%CI: 14.9-16.6), and 11 adults (mean age 34.7, 95%CI:33.3-36) were available for analysis. There was an age-associated

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decrease in SFT diameter (R2�0.35, p�0.009), LC size (R2�0.28,p�0.03), and number of SCs per SFT (R2�0.53, p�0.0009) (Fig. 1).Mean number of SCs per SFT was lower in adults compared toadolescents: (8.5 vs. 35.6, p�0.0006). In men older than 20, decreasesin number of SCs per SFT correlated with decrease in SFT diameter(R2�0.82, p�0.0001). LCs in adolescents were larger than in adults(19 um vs. 15.6 um, p�0.04).

CONCLUSIONS: Progressive testicular atrophy appears to cor-relate with loss in number of SCs in adult men with KS. Our findingsstrongly suggest that Sertoli cells undergo further morphological dete-rioration after puberty as opposed to Leydig cells, which, althoughsmaller in adults, seem to be less affected by age. Age-related changesin testicular morphology may guide interventions for maximizing fertilitypotential in KS patients.

Source of Funding: 1. The Robert Dow Foundation2. Mr. and Mrs. Howard and Irena Laks.

2038DIFFERENTIAL DIAGNOSIS OF AZOOSPERMIA WITHPROTEOMIC BIOMARKERS QUANTIFIED IN SEMINAL PLASMA

Andrei Drabovich*, Brendan Mullen, Eleftherios Diamandis,Keith Jarvi, Toronto, Canada

Withdrawn

2039DIET RICH IN POLYUNSATURATED FATTY ACID INDUCESOVERWEIGHT BUT IMPROVES THE TESTICULARMORPHOLOGY IN ADULT RATS

Pamella Silva, Angelica Furriel, Nathalia Machado, Diogo Souza,Carla Gallo, Bianca Gregório*, Francisco Sampaio, Waldemar Costa,Rio de Janeiro, Brazil

INTRODUCTION AND OBJECTIVES: The obesity and its met-abolic complications affecting the endocrine system and multiple or-gans such as testis. Therefore, the objective of this study was toevaluate the effects of different high-fat diets on body weight andtesticular morphology in rats at 7 months-old.

METHODS: 39 male Wistar rats (3 months-old) were dividedinto 4 groups: SC (standard chow), HF-S (diet rich in saturated fattyacids), HF-P (diet rich in polyunsaturated fatty acids), HF-SP (diet richin saturated and polyunsaturated fatty acids). During the experiment,food intake and body weight were measured daily and weekly, respec-tively. At 7 months-old, the animals were sacrificed. Next, blood sam-ples were collected by cardiac puncture for analysis of serum glucose,insulin, total cholesterol and triglycerides. Testis were collected andprocessed for histomorphometrical and immunohistochemical ana-

lyzes. In the same way, epididymal, retroperitoneal and subcutaneousfat deposits were removed. Data were analyzed by one-way ANOVAand Bonferroni post-test, considering p � 0.05.

RESULTS: Different high-fat diets promoted an increase inbody mass when compared to SC group (p � 0.0001). Accordingly,epididymal, retroperitoneal and subcutaneous fat deposits were higherin high-fat groups (HF-S, HF-P, HF-SP) than SC group (p � 0.0004;p � 0.0001; p � 0.0001, respectively). As for serum parameters, thegroups that received different high fat diets showed hyperglycemia,hyperinsulinemia, and hypercholesterolemia at the end of the experi-ment (p � 0.0055, p �0.05, p � 0.0021, respectively). The levels ofserum triglycerides did not differ among the groups. Regarding thetestis, seminiferous epithelium height was less in HF-S group than SCgroup. However, HF-P group and HF-SP group presented an increasein this parameter in comparison to HF-S group resembling SC group(p�0.0003). Already cells proliferation was lower in HF-S group thanSC group. However, only the HF-P group presented an increase in thisparameter in comparison to HF-S group resembling SC group(p�0.0043). There was a decrease in seminiferous tubule diameter inthe HF-SP group when compared SC group (p�0.0022). Among thegroups, no difference was observed in the testicular volume.

CONCLUSIONS: The high-fat diet administration, independentof the lipid quality, promoted overweight in adult rats. Nevertheless, dietrich in saturated fatty acids (lard) promoted an adverse remodelingupon the testicular morphology that was not observed in the group thatreceived a diet rich in polyunsaturated fatty acids (canola oil).

Source of Funding: FAPERJ, CAPES and CNPq.

2040A SYSTEMATIC REVIEW AND META-ANALYSIS OFANEJACULATION IN SPINAL CORD INJURED PATIENTS INFAVOUR OF A SPINAL GENERATOR OF EJACULATION

Clement Chehensse, Montigny-le-Bretonneux, France;Stephane Bahrami, Pierre Denys, Garches, France; Pierre Clement,Montigny le Bretonneux, France; Francois Giuliano*, Garches,France

INTRODUCTION AND OBJECTIVES: After spinal cord injury(SCI) most men cannot ejaculate, although ejaculation can be obtainedwith medical assistance. There are gaps in the understanding of theneurophysiology of ejaculation and the pathophysiology of anejacula-tion in SCI males. Notably, the existence of a spinal generator ofejaculation, which has been identified in the rat, is not substantiated inthe human. Studies reporting ejaculation after SCI have been reviewedin the light of recent advances on the physiology of ejaculation, espe-cially regarding the spinal neurophysiology.

METHODS: Using 4 databases since 1955, 513 studies wereidentified and 45 fulfilled the following inclusion criteria 1) characteri-sation of the SCI (completeness and either upper limit or somatic reflexfunction below this upper limit that allowed to estimate lower limit ofSCI) and 2) antegrade ejaculation rate. A total of 3795 patients werepooled together. Subgroups analyses were performed depending on i)the method used to elicit ejaculation: ecological stimulation i.e. mas-turbation or coïtus, penile vibratory stimulation (PVS) with or withoutoral midodrine before stimulation and masturbation following acetylcho-line esterase (AchE) inhibitors it prostigmine or sc physostigmine ii) SCIcompleteness according to ASIA Impairment Scale and iii) SCI upperand lower limit.

RESULTS: Ejaculation rate in patients with complete SCI was11.8%, 45.7% and 54.7% respectively during masturbation or coïtus, inresponse to penile vibratory stimulation (PVS) and during masturbationafter AchE inhibitors. Complete injury of the sympathetic centres (spinalsegments T12 to L2) or of the parasympathetic and somatic centres(spinal segments S2 to S4) totally disrupted ejaculation in response toPVS. AchE inhibitors followed by masturbation elicited ejaculation in 5and 31% of men with complete injury of the T12 to L2 and S2 to S4segments respectively. Controlling for the number of T12 to L2 seg-ments completely injured, ejaculation rate was significantly lower when

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