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OVER EXPRESSION OF AROMATASE CYP19 IN HUMAN TESTIS IS MOST LIKELY REASON FOR HYPOGONADISM IN MEN WITH KLINEFELTER SYNDROME
THE JOURNAL OF UROLOGY®
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
1885 MYOGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS IN VITRO AND LONG-TERM SURVIVAL IN A NUDE RAT MODEL FOR URINARY INCONTINENCE Gerhard Feil*, Adriana Drost, Simon Baumann, Jochen Schäfer, Sibylle Weng, Sabine Maurer, Jutta Krug, Robert Möhle, Arnulf Stenzl, Karl-Dietrich Sievert, Tuebingen, Germany INTRODUCTION AND OBJECTIVES: Mesenchymal stem cells (MSCs) have the capacity to differentiate into smooth and skeletal muscle cells. Thus, autologous MSCs might be an option for a functional treatment of urinary incontinence (UI). The aim was to study myogenic differentiation of human MSCs in vitro and to examine their survival in bladder neck tissue of athymic rats in a long-term study. METHODS: To induce myogenic differentiation in vitro bone marrow-derived human MSCs in culture passage (P) 1 were exposed to 5-azacytidine (AZA). In vitro differentiation was examined up to P6 by FACS analysis with stem cell markers and intracellular F-actin, RT-PCR with primers against skeletal muscle-specific regulatory factor myoD1 (MyoD1), and myosin heavy chain (MHC) and by immuncytochemistry with monoclonal antibodies against smooth muscle A-actin, A/G-actin, MyoD transcription factor, and skeletal slow muscle MHC. Native and AZA-exposed MSCs of P2, respectively, were directly injected into the bladder neck of two athymic rats in each case. For in vivo tracking MSCs were labeled with PKH26 red fluorescent cell linker. Integration of MSCs into host tissue was monitored histologically after 16 weeks of cell injection. RESULTS: FACS analysis of multiple MSC lines revealed that both native and AZA-exposed MSCs from P1-P6 were negative for the progenitor/endothelial antigen CD34, leukocytic CD45 and endothelial/ monocytic CD31. The MSC markers CD73, CD90, and CD105 increased over time in native, but not in AZA-exposed MSCs. Intracellular F-actin increased in AZA-exposed, but not in native MSCs until P3. Skeletal muscle mRNA expression decreased in native, but not in AZA-exposed MSCs from P1-P4. Native MSCs expressed A/G-actin and MyoD antigen only in P0 and P1, whereas A-actin was expressed in P0-P6. AZAexposed MSCs expressed these muscle antigens homogeneously in P2-P6. There was no reactivity for skeletal slow muscle MHC in both groups of MSCs. In bladder neck tissues from all rats, well-defined MSC clusters with further continuous dissemination of transplanted MSCs were detected independent of AZA-exposure. CONCLUSIONS: Exposure of human MSCs to AZA in vitro is associated with increased myogenic differentiation and persistent expression of muscle antigens. Vital integration of MSCs in bladder neck tissue might lead to final differentiation into functional muscle cells in vivo. This model emphasizes the use of autologous adult MSCs for a functional treatment of UI. Source of Funding: BioProfile program of the German Federal Ministry of Education and Research (PTJ-BIO 0313619)
Infertility: Physiology, Pathophysiology, Basic Research Moderated Poster 64 Wednesday, April 29, 2009
8:00 am - 10:00 am
1886 OVER EXPRESSION OF AROMATASE CYP19 IN HUMAN TESTIS IS MOST LIKELY REASON FOR HYPOGONADISM IN MEN WITH KLINEFELTER SYNDROME. Laurent Vaucher*, Edward Carreras, Anna Mielnik, Peter N Schlegel, Darius A Paduch, New York, NY INTRODUCTION AND OBJECTIVES: Klinefelter syndrome is the most common reason for hypergonadotropic hypogonadism. Most but not all men with KS develop progressive steroidogenic failure despite
681
high circulating levels of LH. The molecular mechanism of hypogonadims in men with KS is unknown. AIM: to determine molecular basis for hypogonadism in men with KS. METHODS: 96 testicular samples from men with obstructive azoospermia (Norm), non-obstructive azoospermia (NOA), and Klinefelter syndrome (KS). The testicular tissues were incubated for 24 hours with medium only, 22(R)-hydroxycholesterol, and LH. Testosterone and estradiol production was measured using RIA. Total RNA was extracted from each sample and the expression of CYP19, 3-BHSD, vimentin (marker of Sertoli cells), DDX4 and DAZ (marker of germ cells), was measured using multicolor quantitative RT-PCR with hydrolysis probes and normalized to B-actin and HPRT. T-student test and ANOVA was used to compare normalized expression of CYP19, and cell specific markers. Prism (GraphPad) was used to analyze steroidogenic production which was adjusted for weigh of testicular tissue. Expression of CYP19 protein was measured using western blot and IHC. RESULTS: Men with KS had 25 % drop in testosterone production as compared to men with NOA after 24 hour incubation with 22(R)hydroxycholesterol or LH (p=0.035). This was statistically significant with and without adjustment for testicular weight. Men with KS had also lower rate of testosterone production at baseline. To answer if lower T in men with KS is secondary to problems with steroidogenesis or excessive metabolism of testosterone and its conversion to estradiol, the CYP 19 expression was compared between three groups. Men with KS had 4.15 times higher expression of CYP19 per mg of testicular tissue, after adjusting for number of Leydig cells, Sertoli cells and presence or lack of germ cells. The estradiol to testosterone ratio was statistically significant higher in men with KS than in NOA population (p=0.03) CONCLUSIONS: to our knowledge it is a first report which showed at molecular level that the lower testosterone production in men with KS is linked to significantly higher expression of CYP19 in testis, and increased conversion rate of testosterone to estradiol. Since estradiol lowers testosterone production through negative feedback, this study links clinical observation of hyperestrogenism in men with KS with expression data. Our data provides scientific rationale for use of aromatase inhibitors in men with KS. Source of Funding: Klinefelter Syndrome and Associates
1887 A NOVEL NON-INVASIVE, MOTILITY-INDEPENDENT SPERM SORTING METHOD AND TECHNOLOGY TO ISOLATE AND RETRIEVE VIABLE SPERM FROM NON-VIABLE SPERM, FOR USE WITH ISCI Maurice M Garcia*, San Francisco, CA; Aaron Ohta, Berkeley,, CA; Thomas J Walsh, Alan W Shindel, James F Smith, Tom F. Lue, San Francisco, CA INTRODUCTION AND OBJECTIVES: Selection of viable sperm for ICSI is challenging and relies primarily upon motility assessment. Current sperm viability assays are limited by subjectivity, limited sensitivity, and irreversible toxicity. Trypan Blue dye exclusion is a gold-standard viability assay. We evaluate the ability of a modified dielectrophoresis (DEP)-based cell-sorting platform to identify and isolate viable sperm from human ejaculate samples containing a natural mixture of viable and non-viable non-motile sperm, pre-incubated with Trypan Blue dye. METHODS: Fresh ejaculate specimens from 6 men were assessed. Specimen adequacy was confirmed by the presence of motile sperm. Specimens were incubated with 0.4% Trypan Blue and suspended in a sucrose/dextrose isotonic solution. Within 15 minutes, on a custom sorting chip, under 200X magnification, 55 sperm per specimen were assayed: Trypan Blue negative (N=25) and positive (N=25) non-motile sperm, and 5 motile sperm. Trypan stain (+ or -), and whether each sperm was “attracted”, “repulsed”, or “neutral” to the DEP force, was recorded. RESULTS: A total of 330 individual sperm were assayed. All (100%) motile sperm visualized in each specimen were Trypan (-), and all those assayed (N=25) were attracted to DEP. All (100%) sperm demonstrating an attractive response to DEP were Trypan (-); none (0%)
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
1885 MYOGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS IN VITRO AND LONG-TERM SURVIVAL IN A NUDE RAT MODEL FOR URINARY INCONTINENCE Gerhard Feil*, Adriana Drost, Simon Baumann, Jochen Schäfer, Sibylle Weng, Sabine Maurer, Jutta Krug, Robert Möhle, Arnulf Stenzl, Karl-Dietrich Sievert, Tuebingen, Germany INTRODUCTION AND OBJECTIVES: Mesenchymal stem cells (MSCs) have the capacity to differentiate into smooth and skeletal muscle cells. Thus, autologous MSCs might be an option for a functional treatment of urinary incontinence (UI). The aim was to study myogenic differentiation of human MSCs in vitro and to examine their survival in bladder neck tissue of athymic rats in a long-term study. METHODS: To induce myogenic differentiation in vitro bone marrow-derived human MSCs in culture passage (P) 1 were exposed to 5-azacytidine (AZA). In vitro differentiation was examined up to P6 by FACS analysis with stem cell markers and intracellular F-actin, RT-PCR with primers against skeletal muscle-specific regulatory factor myoD1 (MyoD1), and myosin heavy chain (MHC) and by immuncytochemistry with monoclonal antibodies against smooth muscle A-actin, A/G-actin, MyoD transcription factor, and skeletal slow muscle MHC. Native and AZA-exposed MSCs of P2, respectively, were directly injected into the bladder neck of two athymic rats in each case. For in vivo tracking MSCs were labeled with PKH26 red fluorescent cell linker. Integration of MSCs into host tissue was monitored histologically after 16 weeks of cell injection. RESULTS: FACS analysis of multiple MSC lines revealed that both native and AZA-exposed MSCs from P1-P6 were negative for the progenitor/endothelial antigen CD34, leukocytic CD45 and endothelial/ monocytic CD31. The MSC markers CD73, CD90, and CD105 increased over time in native, but not in AZA-exposed MSCs. Intracellular F-actin increased in AZA-exposed, but not in native MSCs until P3. Skeletal muscle mRNA expression decreased in native, but not in AZA-exposed MSCs from P1-P4. Native MSCs expressed A/G-actin and MyoD antigen only in P0 and P1, whereas A-actin was expressed in P0-P6. AZAexposed MSCs expressed these muscle antigens homogeneously in P2-P6. There was no reactivity for skeletal slow muscle MHC in both groups of MSCs. In bladder neck tissues from all rats, well-defined MSC clusters with further continuous dissemination of transplanted MSCs were detected independent of AZA-exposure. CONCLUSIONS: Exposure of human MSCs to AZA in vitro is associated with increased myogenic differentiation and persistent expression of muscle antigens. Vital integration of MSCs in bladder neck tissue might lead to final differentiation into functional muscle cells in vivo. This model emphasizes the use of autologous adult MSCs for a functional treatment of UI. Source of Funding: BioProfile program of the German Federal Ministry of Education and Research (PTJ-BIO 0313619)
Infertility: Physiology, Pathophysiology, Basic Research Moderated Poster 64 Wednesday, April 29, 2009
8:00 am - 10:00 am
1886 OVER EXPRESSION OF AROMATASE CYP19 IN HUMAN TESTIS IS MOST LIKELY REASON FOR HYPOGONADISM IN MEN WITH KLINEFELTER SYNDROME. Laurent Vaucher*, Edward Carreras, Anna Mielnik, Peter N Schlegel, Darius A Paduch, New York, NY INTRODUCTION AND OBJECTIVES: Klinefelter syndrome is the most common reason for hypergonadotropic hypogonadism. Most but not all men with KS develop progressive steroidogenic failure despite
681
high circulating levels of LH. The molecular mechanism of hypogonadims in men with KS is unknown. AIM: to determine molecular basis for hypogonadism in men with KS. METHODS: 96 testicular samples from men with obstructive azoospermia (Norm), non-obstructive azoospermia (NOA), and Klinefelter syndrome (KS). The testicular tissues were incubated for 24 hours with medium only, 22(R)-hydroxycholesterol, and LH. Testosterone and estradiol production was measured using RIA. Total RNA was extracted from each sample and the expression of CYP19, 3-BHSD, vimentin (marker of Sertoli cells), DDX4 and DAZ (marker of germ cells), was measured using multicolor quantitative RT-PCR with hydrolysis probes and normalized to B-actin and HPRT. T-student test and ANOVA was used to compare normalized expression of CYP19, and cell specific markers. Prism (GraphPad) was used to analyze steroidogenic production which was adjusted for weigh of testicular tissue. Expression of CYP19 protein was measured using western blot and IHC. RESULTS: Men with KS had 25 % drop in testosterone production as compared to men with NOA after 24 hour incubation with 22(R)hydroxycholesterol or LH (p=0.035). This was statistically significant with and without adjustment for testicular weight. Men with KS had also lower rate of testosterone production at baseline. To answer if lower T in men with KS is secondary to problems with steroidogenesis or excessive metabolism of testosterone and its conversion to estradiol, the CYP 19 expression was compared between three groups. Men with KS had 4.15 times higher expression of CYP19 per mg of testicular tissue, after adjusting for number of Leydig cells, Sertoli cells and presence or lack of germ cells. The estradiol to testosterone ratio was statistically significant higher in men with KS than in NOA population (p=0.03) CONCLUSIONS: to our knowledge it is a first report which showed at molecular level that the lower testosterone production in men with KS is linked to significantly higher expression of CYP19 in testis, and increased conversion rate of testosterone to estradiol. Since estradiol lowers testosterone production through negative feedback, this study links clinical observation of hyperestrogenism in men with KS with expression data. Our data provides scientific rationale for use of aromatase inhibitors in men with KS. Source of Funding: Klinefelter Syndrome and Associates
1887 A NOVEL NON-INVASIVE, MOTILITY-INDEPENDENT SPERM SORTING METHOD AND TECHNOLOGY TO ISOLATE AND RETRIEVE VIABLE SPERM FROM NON-VIABLE SPERM, FOR USE WITH ISCI Maurice M Garcia*, San Francisco, CA; Aaron Ohta, Berkeley,, CA; Thomas J Walsh, Alan W Shindel, James F Smith, Tom F. Lue, San Francisco, CA INTRODUCTION AND OBJECTIVES: Selection of viable sperm for ICSI is challenging and relies primarily upon motility assessment. Current sperm viability assays are limited by subjectivity, limited sensitivity, and irreversible toxicity. Trypan Blue dye exclusion is a gold-standard viability assay. We evaluate the ability of a modified dielectrophoresis (DEP)-based cell-sorting platform to identify and isolate viable sperm from human ejaculate samples containing a natural mixture of viable and non-viable non-motile sperm, pre-incubated with Trypan Blue dye. METHODS: Fresh ejaculate specimens from 6 men were assessed. Specimen adequacy was confirmed by the presence of motile sperm. Specimens were incubated with 0.4% Trypan Blue and suspended in a sucrose/dextrose isotonic solution. Within 15 minutes, on a custom sorting chip, under 200X magnification, 55 sperm per specimen were assayed: Trypan Blue negative (N=25) and positive (N=25) non-motile sperm, and 5 motile sperm. Trypan stain (+ or -), and whether each sperm was “attracted”, “repulsed”, or “neutral” to the DEP force, was recorded. RESULTS: A total of 330 individual sperm were assayed. All (100%) motile sperm visualized in each specimen were Trypan (-), and all those assayed (N=25) were attracted to DEP. All (100%) sperm demonstrating an attractive response to DEP were Trypan (-); none (0%)