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Home , Vasectomy reversal

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ISOLATION OF A DNA FRAGMENT ENCODING THE 5'­ LOCALIZATION AND REGULATION OF INDOLEAMINE UPSTREAM REGION OF THE MEP 17 GENE 2,3-DIOXYGENASE EXPRESSIONIN THE . K. Suzuki .... , J.-J. Lareyre•', D.E. Ong• ..", R.J. Matusik•�•. S. Kasper•�•. Y. Y. Gu*, J. Chen*, L. Keskintepe*, S. J. Conway*, IMMAG, and M.-C. Orgebin-Crist", Departments of Obstetrics and Gynecology', Araki'", Medical College of Georgia, Augusta, GA, 309 12 Biochemistry" and Urologic Surgery', Center for Reproductive Biology Research', Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-2633, lndoleamine 2,3-dioxygenase (IDO) is the initial and rate-limiting lnstitut National de Ia Recherche Agronomique', Rennes, France 35042 enzyme that catabolizes tryptophan and other indole derivatives to It is well known that the epididymis provides a unique intraluminal kynurenine. It's activity is found in various mouse tissues with the environment that allows maturation to occur. Our laboratory highest enzyme activity in the epididymis. However, the pattern of has previously found that a murine epididymal retinoic acid-binding its expression and its regulation in epididymis is currently unknown. protein (mE-RABP), belonging to the lipocalin superfamily, is secreted by the epithelium of the mid/distal caput epididymidis. Functional In the present study, we investigated IDO expression in the studies, using transgenic mouse, revealed that a 5 kb DNA fragment epididymis and at different ages, and the hormonal of the 5'-flanking region of the mE-RABP gene contains all the regulation of its expression in epididymis.. We found that IDO information required for the hormonal regulation and the spatial and expression in the epididymis starts at 2 weeks post partum and temporal expression of the mE-RABP gene in the epididymis. We have recently identified within the 5 kb DNA fragment a novel gene, reaches its peak at week 8, and is kept at moderate levels the rest of named MEP 17, highly homologous to the mE-RABP gene. life. Localization of IDO by in situ hybridization showed that it is Preliminary studies using in situ hybridization analysis revealed that absent in testis; highest expression in distal part of the caput; weak MEP 17 mRNA was localized only in the principal cells of the initial expression in the cauda and vas deferens; and very little expression segment, whereas mE-RABP mRNA was expressed in the distal caput in the corpus. IDO expression is confined to the epididymal epididymidis. In order to characterize the 5'-flanking region of the gene, a 2.5 kb EcoRI and a 7 kb EcoRV restriction fragments epithelial cells. Our data indicates that regulates IDO overlapping with the 5'-flanking region of the mE-RABP gene were expression in the epididymis, as when testosterone is removed from isolated from the genomic BAC clone used to isolate the mE-RABP. the hormone supply by , there is a large decline of IDO A computer based analysis of the DNA sequence of the 2.5 kb DNA expression. Significantly replacement of the hormone can rescue fragment revealed the presence of a putative androgen receptor binding site (ARBS), several putative AP-I and c-Ets cis-DNA regulatory IDO expression. Sperm and other testicular factors have no effect on elements as well as several potential Sry-related transcription factors IDO expression within the epididymis. In conclusion, IDO is binding sites. These data provide a framework forfurther analysis of differentially distributed within the epididyrnis, its expression in the the regulation of MEP 17 expression and structure-function of the epididymis is age-dependent and androgen-dependent. protein. (Supported by NIH grant HD36900 and a grant from the Rockfeller Foundation/Ernst Schering Foundation.) (This work is supported by NIHgrant HL60714 to S.J.C.)

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OXIDATIVE STRESS AND IL-6 LEVELS IN PATIENTS UNDERGOING EXPRESSION OF P34H, A SPERM PROTEIN, ALONG THE REVERSAL EPIDIDYMIS OF V ASECTOMIZED MEN INVAS DEFERENS R.K. Sharma, F.F. Pasqualotto, H. Kobayashi, A.J. Thomas, Jr. and A. Agarwal. AND Christine Legare and Robert Sullivan. Centre de Recherche en Biologie Center for Advanced Research in Human Reproduction and , * Department of , The Cleveland Clinic Foundation, Cleveland, OH. de la Reproduction, Departement d'Obstetrique-Gynecologie, Faculte de medecine, Universite Laval, Ste-Foy, PQ, Canada. Oxidative stress as a result of elevated levels of reactive oxygen species (ROS) and depressed levels of antioxidants is associated with . Cytokines We have previously described P34H, a human epididymal sperm protein, are also involved in the production of reactive oxygen species. It is known that proposed to be involved in sperm-zona pellucida interactions. P34H IL-6 levels are increased in the seminal plasma of infertile men. The role of mRNA (9 12bp) has been shown to be expressed by the principal cells of oxidative stress in men following vasectomy reversal is unclear. The purpose of the human corpus epididymis. Recovery of following our study was to determine whether men undergoing vasectomy reversal have anastomosis of the vas deferens with testicular tubules, vas efferens or high seminal oxidative stress using three measures of oxidative stress: reactive along the epididymis has challenged the physiological significance of the oxygen species (ROS), total antioxidant capacity (TAC), and a composite ROS­ TAC score. In addition we also measured the IL-6 concentration in the seminal epididymal transit in humans. In order to understand sperm maturation in plasma. Semen and seminal plasma samples were obtained from 23 men these pathological situations, we have studied the expression of P34H in following 6 to 8 months after vasectomy reversal and 11 normal donors. the vas deferens and along the epididymis of vasectomized men. Northern

Vasectomy reversal patients were further divided into infertile (n = 13) and fertile blot analysis revealed a single 912bp P34H mRNA expressed at similar (n = 11). ROS and TAC production in the semen specimens was measured by the levels in the human vas deferens as well as in the corpus epididymidis. In chemiluminescence assay. A composite ROS-TAC score was generated. IL-6 situ hybridization experiments showed high label ling of epithelial cells concentration in the seminal plasma was measured by the enzyme-linked bordering the vas deferens lumen. P34H expression was also studied on immunoassay. Vasectomy reversal infertile patients had higher ROS levels (mean epididymal segments from vasectomized man. Hybridization signal was log [ROS I] 2.46 ± 0.28) compared to vasectomy reversal fertile patients ( 1.84 + detectable in proximal caput of vasectomized tissues at a much higher ± 0.29; P = 0.2), and controls (1.25 ± 0.3 1; P = 0.006). TAC levels were intensity than the one characterizing tissues from normal men. Some comparable in the three groups. The ROS-TAC scores in infertile men were reduced (46.8 ± 4.28) compared to those in the controls (50.0 ± 3.02). IL-6 levels histological modifications of the vasectomized men epididymis have also were significantly elevated in both vasectomy reversal infertile group (1.89 ± been observed. The epididymal lumen diameter, as an example, increases 0.37; p <0.01) and fertile vasectomy reversal group (1.76 ± 0.24; p <0.02) from the distal caput to the distal cauda while the bordering principal compared to controls (0.83 ± 0.3 1). Infertility following vasectomy reversal is cells were smaller in height. Thus, obstruction of vas deferens may alter associated with increased level of ROS and elevated IL-6. Oxidative stress in the expression along the human epididymis of mRNA encoding for these patients may decrease over time following increased interval after their proteins necessary for sperm maturation. Furthermore, our results suggest reversal. [Supported by a research grant from The Cleveland Clinic Foundation.] that the vas deferens may play an important function in these processes. This work was supported by MRC-Canada.

25th annual meeting of the amerlcan society of andrology I 39