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Embryonic poly(A) binding protein (ePAB)-deficient male mice have elevated MSY2 expression in testis
to assess if hydrodissection (HD) of the spermatic cord after MDSC would decrease the number of residual nerve fibers on the preserved arteries and veins without compromising blood flow. DESIGN: Prospective blinded randomized control trial: bilateral MDSC was performed on 22 adult rats (44 cords). Hydrodissection of the spermatic cord was performed on one side of each rat (side randomized) using the ERBEJET2 (ERBE Inc, Altanta, GA). The contralateral cord (no hydrodissection) was the control for each animal. MATERIALS AND METHODS: Blood flow through the vessels was monitored using a micro Doppler probe (Vascular Technology Inc, Nashua, NH). After completion of MDSC with or without HD, the animal was euthanized and a cross section of the residual cord sent to pathology (blinded to technique) for H&E staining and evaluation for small nerve (%1mm) density and signs of structural damage. RESULTS: The cord where HD had been performed had a significantly lower total median residual nerve count of 5 (0-10), compared to 8 (2-12) on the non-HD side (P¼0.007). No structural damage was seen in the vessels in the cord that had undergone HD (gross exam and histology). Blood flow had been maintained in the vessels when the ERBEJET2 was set to 87psi (based on separate dose titration study on 2 similar rats 4 MDSC). CONCLUSION: Hydrodissection of the spermatic cord at 87psi after MDSC significantly decreases residual nerve density without compromising vascular integrity in a rat model. Supported by: Research grant from ERBE Inc.
O-298 Wednesday, October 19, 2011 04:30 PM EMBRYONIC POLY(A) BINDING PROTEIN (EPAB)-DEFICIENT MALE MICE HAVE ELEVATED MSY2 EXPRESSION IN TESTIS. S. Ozturk, O. Guzeloglu-Kayisli, M. D. Lalioti, D. Sakkas, E. Seli. Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: Translational activation of mRNAs stored in the cytoplasm is a key process in the regulation of gene expression during spermatogenesis. Embryonic poly(A) binding protein (ePAB) is the predominant poly(A) binding protein during oocyte and early embryo development until ZGA; it stabilizes mRNAs and promotes their translation. We hypothesized that ePAB plays a key role in spermatogenesis and tested our hypothesis by generating and characterizing mice homozygous-deficient for ePAB. DESIGN: Experimental study. MATERIALS AND METHODS: ePAB knockout (ePAB-/-) mice were generated by targeted deletion of ePAB exon 2. Fertility of wild type (WT), heterozygous knockout (ePAB+/-), and homozygous knockout (ePAB-/-) male mice (n ¼ 12 for each genotype) was tested by mating with 5 adult WT female mice with proven fertility (male:female; 1:2), for a total of 20 weeks (minimum 4 oestrus cycles with each male). Testis weight and histology, and sperm count and morphology were evaluated. Apoptotic index in spermatozoa was determined by annexin V staining. Expression of RNA-binding proteins MSY2, PAIP2, PUM2, and PABPC1 in testis of WT and ePAB-/- mice (n ¼ 5 in each group) was determined by real-time polymerase chain reaction (qRT-PCR) and normalized to actin expression. RESULTS: Male ePAB-/- mice were viable, appeared phenotypically normal, and demonstrated normal fertility. Testis weight, seminiferous tubule diameter, sperm count, sperm morphology, or annexin V staining were not different between WT and ePAB-/- mice. MSY2 expression was increased by 50% in ePAB-/- mice (P<0.05). Levels of other RNA-binding proteins tested were also elevated (10-40%) in testes of ePAB-/- mice but the differences were not statistically significant. CONCLUSION: We found that male ePAB-/- mice are fertile, and have normal testis histology and sperm parameters. Elevated MSY2 and possibly other RNA-binding proteins seem to compensate for the required RNA-binding activity in testes of ePAB-/- mice. Supported by: NIH K08 HD046581 and R01 HD59909 (ES).
O-299 Wednesday, October 19, 2011 04:45 PM STEROIDOGENIC POTENTIAL OF ADULT MOUSE AND HUMAN PROSTATE AND PENIS. K. Hwang, J. Choi, G. Ayala, M. Khera, L. I. Lipshultz, D. J. Lamb. Department of Urology, Baylor College of Medicine, Houston, TX; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX.
S88
Abstracts
OBJECTIVE: The major steroidogenic tissues in the male are well known. Expression of p450scc, 3b-HSD and StAR are well established in the adrenal gland and in the testis. Steroidogenic acute regulatory protein (StAR) represents the first and rate-limiting step for steroid biosynthesis leading to androgen production followed by the metabolism of cholesterol to pregnenolone by the cytochrome p450 side chain enzyme (p450scc). We tested the hypothesis that organs not usually considered to be steroidogenic, exhibit steroidogenic potential. We defined the expression of p450scc, 3b-HSD and StAR in other genital tract tissues. DESIGN: Controlled, in vitro studies on murine and human tissues. MATERIALS AND METHODS: The testes, prostate, penis, and adrenal gland were harvested from two 12 wk old c57Bl/6 mice. The tissue expression of these proteins was evaluated using immunohistochemistry. Expression of 3b-HSD, p450scc and StAR proteins was analyzed using microscopy. Human benign prostate tissue slides were purchased commercially and human penile tissue samples were taken from patients undergoing penile implant surgery with appropriate informed consent. RESULTS: Microscopic analysis of each tissue revealed the expected findings of significant levels of expression of p450scc, 3b-HSD and StAR in both the adrenal gland and in the testes, which served as positive controls. In mouse and human tissues, selected corpora cavernosal cells showed expression of p450scc, as well as cells of the tunica and prostate gland. Human prostate and penile tissue also displayed expression of p450scc, 3b-HSD and StAR. CONCLUSION: Steroidogenesis was confirmed by the expression of all three proteins in the adrenal gland and testis. Surprisingly, expression of 3b-HSD, p450scc and StAR were also confirmed in the normal prostate gland (without evidence of cancer) and penile tissue suggesting that both of these organs may produce androgens that could act locally during normal male genitourinary tract function in male mice and human tissue. Supported by: K12 DK0083014
O-300 Wednesday, October 19, 2011 05:00 PM GENOME-WIDE SPERM DNA METHYLATION ANALYSIS REVEALS A SUBSET OF PATIENTS WITH HIGHLY ABNORMAL METHYLATION PATTERNS AT IMPRINTED LOCI. K. I. Aston, V. Punj, L. Liu, D. T. Carrell. Andrology and IVF Laboratories, University of Utah School of Medicine, Salt Lake City, UT; Epigenome Center and Division of Hematology, University of Southern California, Los Angeles, CA. OBJECTIVE: To compare the genome-wide methylation status of sperm DNA derived from normozoospermic fertile controls, men with abnormal protamine 1/protamine 2 (P1/P2) ratios, and IVF patients that gave rise to poor embryo quality in the absence of known female factors. DESIGN: A case-control study. MATERIALS AND METHODS: Sperm DNA methylation at 27,578 CpGs was assessed in 15 normozoospermic, fertile control samples (F), 13 abnormal IVF embryo development samples (E), and 15 samples with abnormal P1/P2 ratios (P). Following bisulfite conversion of sperm DNA, samples were hybridized to Illumina HumanMethylation27 Beadchips. Data were analyzed by Spearman correlation, unsupervised clustering and, principal component analysis. Single locus associations were tested using t-tests. Subset analyses targeting differentially methylated regions (DMRs) were also performed. RESULTS: No significant single locus associations were observed for either of the groups, and discreet separation of phenotypes was not obtained by unsupervised clustering. Importantly, a very high correlation rate between the majority of cases and controls was observed (r2 > 0.94). In contrast to the the other 40 samples, one abnormal embryo and two abnormal protamine samples displayed remarkably different methylation patterns across a large number of CpGs, and these samples clustered independent of all others. Given the unique methylation profile of these three samples, the majority of additional analysis focused on them. Of 140 CpGs in DMRs representing 30 imprinted genes, 88 CpGs for 22 genes showed significantly abnormal hyper-methylation in the three samples compared with controls (bonferroni P < 0.05). Disrupted imprinting was observed in genes including H19, GNAS, IGF2, MEG3, MEST, PEG3, and PEG10 among others. CONCLUSION: These results indicate imprinted loci are particularly susceptible to disruptions in methylation, and nearly universal aberrations in sperm DNA methylation at imprinted loci may be a feature of some classes of male factor infertility.
Vol. 96., No. 3, Supplement, Sepetmber 2011
O-298 Wednesday, October 19, 2011 04:30 PM EMBRYONIC POLY(A) BINDING PROTEIN (EPAB)-DEFICIENT MALE MICE HAVE ELEVATED MSY2 EXPRESSION IN TESTIS. S. Ozturk, O. Guzeloglu-Kayisli, M. D. Lalioti, D. Sakkas, E. Seli. Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: Translational activation of mRNAs stored in the cytoplasm is a key process in the regulation of gene expression during spermatogenesis. Embryonic poly(A) binding protein (ePAB) is the predominant poly(A) binding protein during oocyte and early embryo development until ZGA; it stabilizes mRNAs and promotes their translation. We hypothesized that ePAB plays a key role in spermatogenesis and tested our hypothesis by generating and characterizing mice homozygous-deficient for ePAB. DESIGN: Experimental study. MATERIALS AND METHODS: ePAB knockout (ePAB-/-) mice were generated by targeted deletion of ePAB exon 2. Fertility of wild type (WT), heterozygous knockout (ePAB+/-), and homozygous knockout (ePAB-/-) male mice (n ¼ 12 for each genotype) was tested by mating with 5 adult WT female mice with proven fertility (male:female; 1:2), for a total of 20 weeks (minimum 4 oestrus cycles with each male). Testis weight and histology, and sperm count and morphology were evaluated. Apoptotic index in spermatozoa was determined by annexin V staining. Expression of RNA-binding proteins MSY2, PAIP2, PUM2, and PABPC1 in testis of WT and ePAB-/- mice (n ¼ 5 in each group) was determined by real-time polymerase chain reaction (qRT-PCR) and normalized to actin expression. RESULTS: Male ePAB-/- mice were viable, appeared phenotypically normal, and demonstrated normal fertility. Testis weight, seminiferous tubule diameter, sperm count, sperm morphology, or annexin V staining were not different between WT and ePAB-/- mice. MSY2 expression was increased by 50% in ePAB-/- mice (P<0.05). Levels of other RNA-binding proteins tested were also elevated (10-40%) in testes of ePAB-/- mice but the differences were not statistically significant. CONCLUSION: We found that male ePAB-/- mice are fertile, and have normal testis histology and sperm parameters. Elevated MSY2 and possibly other RNA-binding proteins seem to compensate for the required RNA-binding activity in testes of ePAB-/- mice. Supported by: NIH K08 HD046581 and R01 HD59909 (ES).
O-299 Wednesday, October 19, 2011 04:45 PM STEROIDOGENIC POTENTIAL OF ADULT MOUSE AND HUMAN PROSTATE AND PENIS. K. Hwang, J. Choi, G. Ayala, M. Khera, L. I. Lipshultz, D. J. Lamb. Department of Urology, Baylor College of Medicine, Houston, TX; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX.
S88
Abstracts
OBJECTIVE: The major steroidogenic tissues in the male are well known. Expression of p450scc, 3b-HSD and StAR are well established in the adrenal gland and in the testis. Steroidogenic acute regulatory protein (StAR) represents the first and rate-limiting step for steroid biosynthesis leading to androgen production followed by the metabolism of cholesterol to pregnenolone by the cytochrome p450 side chain enzyme (p450scc). We tested the hypothesis that organs not usually considered to be steroidogenic, exhibit steroidogenic potential. We defined the expression of p450scc, 3b-HSD and StAR in other genital tract tissues. DESIGN: Controlled, in vitro studies on murine and human tissues. MATERIALS AND METHODS: The testes, prostate, penis, and adrenal gland were harvested from two 12 wk old c57Bl/6 mice. The tissue expression of these proteins was evaluated using immunohistochemistry. Expression of 3b-HSD, p450scc and StAR proteins was analyzed using microscopy. Human benign prostate tissue slides were purchased commercially and human penile tissue samples were taken from patients undergoing penile implant surgery with appropriate informed consent. RESULTS: Microscopic analysis of each tissue revealed the expected findings of significant levels of expression of p450scc, 3b-HSD and StAR in both the adrenal gland and in the testes, which served as positive controls. In mouse and human tissues, selected corpora cavernosal cells showed expression of p450scc, as well as cells of the tunica and prostate gland. Human prostate and penile tissue also displayed expression of p450scc, 3b-HSD and StAR. CONCLUSION: Steroidogenesis was confirmed by the expression of all three proteins in the adrenal gland and testis. Surprisingly, expression of 3b-HSD, p450scc and StAR were also confirmed in the normal prostate gland (without evidence of cancer) and penile tissue suggesting that both of these organs may produce androgens that could act locally during normal male genitourinary tract function in male mice and human tissue. Supported by: K12 DK0083014
O-300 Wednesday, October 19, 2011 05:00 PM GENOME-WIDE SPERM DNA METHYLATION ANALYSIS REVEALS A SUBSET OF PATIENTS WITH HIGHLY ABNORMAL METHYLATION PATTERNS AT IMPRINTED LOCI. K. I. Aston, V. Punj, L. Liu, D. T. Carrell. Andrology and IVF Laboratories, University of Utah School of Medicine, Salt Lake City, UT; Epigenome Center and Division of Hematology, University of Southern California, Los Angeles, CA. OBJECTIVE: To compare the genome-wide methylation status of sperm DNA derived from normozoospermic fertile controls, men with abnormal protamine 1/protamine 2 (P1/P2) ratios, and IVF patients that gave rise to poor embryo quality in the absence of known female factors. DESIGN: A case-control study. MATERIALS AND METHODS: Sperm DNA methylation at 27,578 CpGs was assessed in 15 normozoospermic, fertile control samples (F), 13 abnormal IVF embryo development samples (E), and 15 samples with abnormal P1/P2 ratios (P). Following bisulfite conversion of sperm DNA, samples were hybridized to Illumina HumanMethylation27 Beadchips. Data were analyzed by Spearman correlation, unsupervised clustering and, principal component analysis. Single locus associations were tested using t-tests. Subset analyses targeting differentially methylated regions (DMRs) were also performed. RESULTS: No significant single locus associations were observed for either of the groups, and discreet separation of phenotypes was not obtained by unsupervised clustering. Importantly, a very high correlation rate between the majority of cases and controls was observed (r2 > 0.94). In contrast to the the other 40 samples, one abnormal embryo and two abnormal protamine samples displayed remarkably different methylation patterns across a large number of CpGs, and these samples clustered independent of all others. Given the unique methylation profile of these three samples, the majority of additional analysis focused on them. Of 140 CpGs in DMRs representing 30 imprinted genes, 88 CpGs for 22 genes showed significantly abnormal hyper-methylation in the three samples compared with controls (bonferroni P < 0.05). Disrupted imprinting was observed in genes including H19, GNAS, IGF2, MEG3, MEST, PEG3, and PEG10 among others. CONCLUSION: These results indicate imprinted loci are particularly susceptible to disruptions in methylation, and nearly universal aberrations in sperm DNA methylation at imprinted loci may be a feature of some classes of male factor infertility.
Vol. 96., No. 3, Supplement, Sepetmber 2011