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O-028 Insulin and insulin-like growth factors stimulate proliferation of human ovarian theca-interstitial cells
Conclusion: We report absence of mutations in the SRYHMG box in our series of 21 subjects with 45,X/46,XY regardless of their variable gonadal phenotype. The complete failure of gonadal development in individuals with Y aneuploidy may be due to mutations at a locus other than SRY in the sex determining pathway.
O-026 Preferential Endometrial Stromal Cell Adhesion To Collagen IV and Collagen I Is Inhibited By Echistatin. 1C. A. Witz, 1R. V. Doucet, 1G. M. HonorS, 1I. A. Montoya, 2L. F. Bonewald and 1R. S. Schenken. 1Depts of OB/GYN and 2Medicine, The University of Texas Health Science Center at San Antonio, TX. Objective: Interactions between endometrial stromal cells (ESC), mesothelial cells, and extracellular matrix proteins (ECM) are involved in the pathogenesis of endometriosis and endometrial cancer. We have previously shown that ESC adhere to the ECM proteins collagen IV (C IV), collagen I (C I), fibronectin (FN), and laminin (LM). This study was conducted to determine whether a known inhibitor of integrin-mediated adhesion would impair attachment of ESC to ECM proteins. Methods: Endometrium was obtained from 6 women without endometriosis undergoing hysterectomy for benign conditions. Endometrium was enzymatically digested with collagenase and DNase. ESC were separated using a Percoll gradient and cultured in flasks. Purity of ESC cultures was determined morphologically and by immunohistochemical staining showing expression of vimentin and absence of cytokeratin and CD 45 staining. Following the second passage, cells were dispersed using trypsin/ EDTA and placed in six-well plates coated with C IV, C I, FN, and LM (n = 16). To evaluate the role of integrins in ESC adhesion, cell suspensions were incubated with echistatin (Echi), a disintegrin that blocks binding to the RGD oligopeptide sequence, at various concentrations (0.001-10 #g/ml) for 15 minutes prior to plating (n = 6). After one hour, non-adherent cells were removed by washing with Hank's buffered salt solution and the wells were fixed with 4% formaldehyde. The number of adherent cells was determined by counting five fields/quadrant of each well at 200×. Results: ESC preferentially adhered to C IV, therefore the binding of ESC to control wells with C IV was arbitrarily set at 1. Results are presented as a proportion of this maximum binding. Echi decreased ESC adhesion in a dose dependent fashion.
* Indicates significant difference from control in each group. Conclusions: These results demonstrate that ESC have preferential attachment to C IV and C I over FN and LM. Inhibition of this process by a disintegrin suggests the S14
Abstracts
adhesion process is mediated by integrins. Modulation of ESC binding to basement membrane C IV and then to the collagen rich peritoneal stroma may be involved in the invasive process of endometriosis.
0-027 Inhibition of Norepinephrine Synthesis With AlphaMethyl-Para-Tyrosine Does Not Alter Leptin Secretion. 1'2R. C. Zimmermann, 1A. Rahmanie, ~L. Krahn, 1M. Ferin, 1M. V. Sauer. 1Division of Reproductive Endocrinology and Department of Psychiatry, College of Physicians & Surgeons, Columbia University, New York, New York; 3Department of Psychiatry, Mayo Clinic, Rochester, Minnesota. Objective: The obesity gene has been recently cloned, and its product is a 16 kD protein called leptin. Its secretion is altered in underweight and overweight patients. The nature of the afferent signal to the adipocyte, which secretes leptin, is not known. A common denominator in genetic and hypothalamic forms of obesity is a diminished sympathetic tone. It is speculated that norepinephrine (NE) is involved in the regulation of leptin secretion. The purpose of this study was to create a state of diminished sympathetic tone using the catecholamine synthesis inhibitor alpha-methyl-para-tyrosine (AMPT), and study its effect on leptin secretion. Design: Randomized, double-blind, placebo controlled crossover design Material and Methods: Four lean subjects (2 women; 2 men) as determined by a normal BMI received a total of 5 × 1 g doses of AMPT or 5 × 50 mg promethazine (active placebo), which does not alter leptin secretion, over a 26hour period, separated by 4-6 weeks. Blood and urine samples for hormone measurements were obtained over 24hours (18 time points) on day 2 of each experiment. Hormone measurements were done using commercial assays for melatonin sulfate (MS), (Elias, USA; leptin, Linco Research Incorp., USA). 3-methoxy-4-hydroxyphenylglycol (MHPG) was measured by liquid chromatography. Results: The nocturnal rise in MS excretion, which is regulated by the sympathetic nervous system, was abolished with AMPT, but was maintained in the control group. Similarly MHPG excretion, which is major metabolite of NE was decreased (1.25 _+ 0.19 mg/24h to 0.50 _+ 0.08 mg/24h, p < 0.01). In contrast leptin secretion was not altered by AMPT. No circadian rhythm of leptin secretion was detected. As expected the leptin levels were higher in women compared to men. Conclusion: High dose AMPT created a state of low sympathetic tone abolishing the circadian rhythm of melatonin secretion and decreasing MHPG excretion. Although decreased binding of NE to /~3-adrenergic receptors and an increase in leptin secretion was anticipated, the lack of responsiveness indicates the role of a diminished sympathetic tone in the regulation ofleptin secretion by adipocytes remains undefined.
0-028 Insulin and Insulin-like Growth Factors Stimulate Proliferation of Human Ovarian Theca-Interstitial
Cells. A. J. Duleba, R. Z. Spaczynski, and D. L. Olive. Department of OB/GYN, Yale University School of Medicine, New Haven, CT. Objective: Polycystic ovary syndrome (PCOS) is associated with excessive androgen synthesis and histologically demonstrable thecal and stromal hyperplasia. Furthermore, patients with PCOS are usually insulin resistant and hyperinsulinemic. This study was designed to evaluate the effects of insulin and insulin-like growth factors I and II (IGF-I and II) on proliferation of h u m a n thecainterstitial (T-I) cells. Design: In vitro assay determining DNA synthesis of h u m a n T-I cells. Materials and Methods: Ovarian fragments were obtained from three premenopausal patients (aged 33-40) undergoing oophorectomy for benign conditions. T-I cells were purified by Percoll density gradient centrifugation and anti-CD45 immunomagnetic beads separation. T-I cells were cultured in chemically defined media for 48-96 hours with or without insulin (1-100 nM), IGF-I (0.3-30 nM) and IGF-II (0.3-30 nM). Proliferation of T-I cells was evaluated during the last 24 hours by determination of [3H] thymidine incorporation into the cells. The effect of treatments on T-I cells proliferation was confirmed by counting the steroidogenically active (stained positive for 3fl-hydroxysteroid dehydrogenase-3fl-HSD) and inactive (3fl-HSD negative) cells. Statistical analysis consisted of analysis of variance followed by post-hoc comparison of means using Bonferroni correction. Results: In all patients, insulin, IGF-I, and IGF-II stimulated T-I proliferation. Insulin produced maximal effect at 10 and 100 nM (up to 2.1 fold; p < 0.01). IGF-I and IGF-II exerted dose-dependent stimulatory effects on thymidine incorporation with significant effects at 10-30 nM (for IGF-I up to 4.4-fold above control, p < 0.05; and for IGF-II up to 7-fold above control, p < 0.05). Total cell count was increased in the presence of 10 nM IGF-I (by 22%, N.S.) and 10 nM insulin (by 16%, N.S.). The proportions of steroidogenically active cells were not altered by any of the treatments. Conclusion: The present results support the hypothesis that insulin, IGF-I, and IGF-II stimulate proliferation of T-I cells. Such actions may represent a mechanism by which hyperplasia of the thecal-stromal compartment occurs in PCOS. O-029 I n t r a c e l l u l a r S i g n a l i n g Via E x t r a c e l l u l a r S i g n a l Rel a t e d K i n a s e (ERK) in L u t e i n i z e d H u m a n G r a n u l o s a Cells (GC). H. H. Kim and J. Yeh; Brigham and Women's Hospital-Harvard Medical School; Boston, MA Objectives: Evidence suggests that Epidermal Growth Factor (EGF) works in synergy with Follicle Stimulating Hormone (FSH) during follicular development to promote GC proliferation and steroidogenesis. The mechanism by which EGF enhances FSH-action is poorly understood. ERKs are key proteins in intracellular signal transduction. After activation, ERKs are translocated from the cytoplasm into the nucleus where they modulate gene tran-
scription. We hypothesize that EGF signaling in the GC occurs via activation of ERK. To determine whether ERKmediated signaling occurs in the luteinized human GC, we sought to identify ERK proteins and assess ERK activation in response to EGF stimulation. Design: The presence and activity of ERK was examined in cell lysates prepared from luteinized human GC cultured in the presence or absence of EGF. Immunoblotting was performed with an anti-ERK antibody to test for the presence of ERK, and ERK activity was assayed using myelin basic protein (MBP) as an in vitro substrate for ERK. Materials and Methods: H u m a n GC were collected at the time of oocyte retrieval for in vitro fertilization and purified to remove blood cells. GC proteins were resolved by SDS-PAGE and transferred to Immobilon-P membrane. Immunoblotting was performed with antibody specific for ERK2, a 42kd isoform. ERK activity was assayed in 5 GC samples. GC were cultured overnight in serum-free media and incubated for 30 minutes with or without EGF (100 ng/ml). GC lysates were incubated with P32-1abeled ATP and MBP for 10 minutes. Since only active ERK phosphorylates MBP, P32 incorporated into MBP reflects ERK activity. P32 was measured with a scintillation counter. Baseline incubations omitted GC lysate. Results: Immunostaining with anti-ERK2 revealed a band corresponding to 42kd, consistent with ERK2. Luteinized h u m a n GC appear to have basal ERK activity. Lysate from control GC, cultured with no EGF, produced a 3.5 (_+0.6) fold increase in ERK activity compared with baseline. EGF stimulated even further increases in ERK activity (p < 0.047). Lysate from the EGF-stimulated GC produced a 5.4 (_+1.1) fold increase in ERK activity compared with baseline conditions. Conclusions: Our data demonstrate that ERK protein is present and biologically active in luteinized h u m a n GC and that the addition of EGF further increases ERK activity. These findings support our hypothesis that EGF enhances FSH-mediated GC proliferation and steroidogenesis via stimulation of ERK activity. Full understanding of ERK-mediated signaling will provide new insights into the intracellular events responsible for follicular development. O-030 Induction of Vascular Endothelial Growth Factor a n d I n t e r l e u k i n - 6 E x p r e s s i o n in H u m a n E n d o m e t r i osis S t r o m a l Cell C u l t u r e s by I n t e r l e u k i n - 1 Beta. D. I. Lebovic, I.P. Ryan and R. N. Taylor. Reproductive Endocrinology Center, Dept. of Obstetrics, Gynecology & Reproductive Sciences, University of California School of Medicine, San Francisco, CA 94143 Objectives: Activated macrophages and ectopic endometrim implants coexist in the peritoneal cavity of women with endometriosis. We and others have postulated that macrophage-derived cytokines, such as interleukin (IL)lfl, may participate in an inflammatory cascade leading to clinical signs and symptoms in these patients. Vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells and promotes angiogenesis. We have reported the production of VEGF in endometriosis
Abstracts
$15
O-026 Preferential Endometrial Stromal Cell Adhesion To Collagen IV and Collagen I Is Inhibited By Echistatin. 1C. A. Witz, 1R. V. Doucet, 1G. M. HonorS, 1I. A. Montoya, 2L. F. Bonewald and 1R. S. Schenken. 1Depts of OB/GYN and 2Medicine, The University of Texas Health Science Center at San Antonio, TX. Objective: Interactions between endometrial stromal cells (ESC), mesothelial cells, and extracellular matrix proteins (ECM) are involved in the pathogenesis of endometriosis and endometrial cancer. We have previously shown that ESC adhere to the ECM proteins collagen IV (C IV), collagen I (C I), fibronectin (FN), and laminin (LM). This study was conducted to determine whether a known inhibitor of integrin-mediated adhesion would impair attachment of ESC to ECM proteins. Methods: Endometrium was obtained from 6 women without endometriosis undergoing hysterectomy for benign conditions. Endometrium was enzymatically digested with collagenase and DNase. ESC were separated using a Percoll gradient and cultured in flasks. Purity of ESC cultures was determined morphologically and by immunohistochemical staining showing expression of vimentin and absence of cytokeratin and CD 45 staining. Following the second passage, cells were dispersed using trypsin/ EDTA and placed in six-well plates coated with C IV, C I, FN, and LM (n = 16). To evaluate the role of integrins in ESC adhesion, cell suspensions were incubated with echistatin (Echi), a disintegrin that blocks binding to the RGD oligopeptide sequence, at various concentrations (0.001-10 #g/ml) for 15 minutes prior to plating (n = 6). After one hour, non-adherent cells were removed by washing with Hank's buffered salt solution and the wells were fixed with 4% formaldehyde. The number of adherent cells was determined by counting five fields/quadrant of each well at 200×. Results: ESC preferentially adhered to C IV, therefore the binding of ESC to control wells with C IV was arbitrarily set at 1. Results are presented as a proportion of this maximum binding. Echi decreased ESC adhesion in a dose dependent fashion.
* Indicates significant difference from control in each group. Conclusions: These results demonstrate that ESC have preferential attachment to C IV and C I over FN and LM. Inhibition of this process by a disintegrin suggests the S14
Abstracts
adhesion process is mediated by integrins. Modulation of ESC binding to basement membrane C IV and then to the collagen rich peritoneal stroma may be involved in the invasive process of endometriosis.
0-027 Inhibition of Norepinephrine Synthesis With AlphaMethyl-Para-Tyrosine Does Not Alter Leptin Secretion. 1'2R. C. Zimmermann, 1A. Rahmanie, ~L. Krahn, 1M. Ferin, 1M. V. Sauer. 1Division of Reproductive Endocrinology and Department of Psychiatry, College of Physicians & Surgeons, Columbia University, New York, New York; 3Department of Psychiatry, Mayo Clinic, Rochester, Minnesota. Objective: The obesity gene has been recently cloned, and its product is a 16 kD protein called leptin. Its secretion is altered in underweight and overweight patients. The nature of the afferent signal to the adipocyte, which secretes leptin, is not known. A common denominator in genetic and hypothalamic forms of obesity is a diminished sympathetic tone. It is speculated that norepinephrine (NE) is involved in the regulation of leptin secretion. The purpose of this study was to create a state of diminished sympathetic tone using the catecholamine synthesis inhibitor alpha-methyl-para-tyrosine (AMPT), and study its effect on leptin secretion. Design: Randomized, double-blind, placebo controlled crossover design Material and Methods: Four lean subjects (2 women; 2 men) as determined by a normal BMI received a total of 5 × 1 g doses of AMPT or 5 × 50 mg promethazine (active placebo), which does not alter leptin secretion, over a 26hour period, separated by 4-6 weeks. Blood and urine samples for hormone measurements were obtained over 24hours (18 time points) on day 2 of each experiment. Hormone measurements were done using commercial assays for melatonin sulfate (MS), (Elias, USA; leptin, Linco Research Incorp., USA). 3-methoxy-4-hydroxyphenylglycol (MHPG) was measured by liquid chromatography. Results: The nocturnal rise in MS excretion, which is regulated by the sympathetic nervous system, was abolished with AMPT, but was maintained in the control group. Similarly MHPG excretion, which is major metabolite of NE was decreased (1.25 _+ 0.19 mg/24h to 0.50 _+ 0.08 mg/24h, p < 0.01). In contrast leptin secretion was not altered by AMPT. No circadian rhythm of leptin secretion was detected. As expected the leptin levels were higher in women compared to men. Conclusion: High dose AMPT created a state of low sympathetic tone abolishing the circadian rhythm of melatonin secretion and decreasing MHPG excretion. Although decreased binding of NE to /~3-adrenergic receptors and an increase in leptin secretion was anticipated, the lack of responsiveness indicates the role of a diminished sympathetic tone in the regulation ofleptin secretion by adipocytes remains undefined.
0-028 Insulin and Insulin-like Growth Factors Stimulate Proliferation of Human Ovarian Theca-Interstitial
Cells. A. J. Duleba, R. Z. Spaczynski, and D. L. Olive. Department of OB/GYN, Yale University School of Medicine, New Haven, CT. Objective: Polycystic ovary syndrome (PCOS) is associated with excessive androgen synthesis and histologically demonstrable thecal and stromal hyperplasia. Furthermore, patients with PCOS are usually insulin resistant and hyperinsulinemic. This study was designed to evaluate the effects of insulin and insulin-like growth factors I and II (IGF-I and II) on proliferation of h u m a n thecainterstitial (T-I) cells. Design: In vitro assay determining DNA synthesis of h u m a n T-I cells. Materials and Methods: Ovarian fragments were obtained from three premenopausal patients (aged 33-40) undergoing oophorectomy for benign conditions. T-I cells were purified by Percoll density gradient centrifugation and anti-CD45 immunomagnetic beads separation. T-I cells were cultured in chemically defined media for 48-96 hours with or without insulin (1-100 nM), IGF-I (0.3-30 nM) and IGF-II (0.3-30 nM). Proliferation of T-I cells was evaluated during the last 24 hours by determination of [3H] thymidine incorporation into the cells. The effect of treatments on T-I cells proliferation was confirmed by counting the steroidogenically active (stained positive for 3fl-hydroxysteroid dehydrogenase-3fl-HSD) and inactive (3fl-HSD negative) cells. Statistical analysis consisted of analysis of variance followed by post-hoc comparison of means using Bonferroni correction. Results: In all patients, insulin, IGF-I, and IGF-II stimulated T-I proliferation. Insulin produced maximal effect at 10 and 100 nM (up to 2.1 fold; p < 0.01). IGF-I and IGF-II exerted dose-dependent stimulatory effects on thymidine incorporation with significant effects at 10-30 nM (for IGF-I up to 4.4-fold above control, p < 0.05; and for IGF-II up to 7-fold above control, p < 0.05). Total cell count was increased in the presence of 10 nM IGF-I (by 22%, N.S.) and 10 nM insulin (by 16%, N.S.). The proportions of steroidogenically active cells were not altered by any of the treatments. Conclusion: The present results support the hypothesis that insulin, IGF-I, and IGF-II stimulate proliferation of T-I cells. Such actions may represent a mechanism by which hyperplasia of the thecal-stromal compartment occurs in PCOS. O-029 I n t r a c e l l u l a r S i g n a l i n g Via E x t r a c e l l u l a r S i g n a l Rel a t e d K i n a s e (ERK) in L u t e i n i z e d H u m a n G r a n u l o s a Cells (GC). H. H. Kim and J. Yeh; Brigham and Women's Hospital-Harvard Medical School; Boston, MA Objectives: Evidence suggests that Epidermal Growth Factor (EGF) works in synergy with Follicle Stimulating Hormone (FSH) during follicular development to promote GC proliferation and steroidogenesis. The mechanism by which EGF enhances FSH-action is poorly understood. ERKs are key proteins in intracellular signal transduction. After activation, ERKs are translocated from the cytoplasm into the nucleus where they modulate gene tran-
scription. We hypothesize that EGF signaling in the GC occurs via activation of ERK. To determine whether ERKmediated signaling occurs in the luteinized human GC, we sought to identify ERK proteins and assess ERK activation in response to EGF stimulation. Design: The presence and activity of ERK was examined in cell lysates prepared from luteinized human GC cultured in the presence or absence of EGF. Immunoblotting was performed with an anti-ERK antibody to test for the presence of ERK, and ERK activity was assayed using myelin basic protein (MBP) as an in vitro substrate for ERK. Materials and Methods: H u m a n GC were collected at the time of oocyte retrieval for in vitro fertilization and purified to remove blood cells. GC proteins were resolved by SDS-PAGE and transferred to Immobilon-P membrane. Immunoblotting was performed with antibody specific for ERK2, a 42kd isoform. ERK activity was assayed in 5 GC samples. GC were cultured overnight in serum-free media and incubated for 30 minutes with or without EGF (100 ng/ml). GC lysates were incubated with P32-1abeled ATP and MBP for 10 minutes. Since only active ERK phosphorylates MBP, P32 incorporated into MBP reflects ERK activity. P32 was measured with a scintillation counter. Baseline incubations omitted GC lysate. Results: Immunostaining with anti-ERK2 revealed a band corresponding to 42kd, consistent with ERK2. Luteinized h u m a n GC appear to have basal ERK activity. Lysate from control GC, cultured with no EGF, produced a 3.5 (_+0.6) fold increase in ERK activity compared with baseline. EGF stimulated even further increases in ERK activity (p < 0.047). Lysate from the EGF-stimulated GC produced a 5.4 (_+1.1) fold increase in ERK activity compared with baseline conditions. Conclusions: Our data demonstrate that ERK protein is present and biologically active in luteinized h u m a n GC and that the addition of EGF further increases ERK activity. These findings support our hypothesis that EGF enhances FSH-mediated GC proliferation and steroidogenesis via stimulation of ERK activity. Full understanding of ERK-mediated signaling will provide new insights into the intracellular events responsible for follicular development. O-030 Induction of Vascular Endothelial Growth Factor a n d I n t e r l e u k i n - 6 E x p r e s s i o n in H u m a n E n d o m e t r i osis S t r o m a l Cell C u l t u r e s by I n t e r l e u k i n - 1 Beta. D. I. Lebovic, I.P. Ryan and R. N. Taylor. Reproductive Endocrinology Center, Dept. of Obstetrics, Gynecology & Reproductive Sciences, University of California School of Medicine, San Francisco, CA 94143 Objectives: Activated macrophages and ectopic endometrim implants coexist in the peritoneal cavity of women with endometriosis. We and others have postulated that macrophage-derived cytokines, such as interleukin (IL)lfl, may participate in an inflammatory cascade leading to clinical signs and symptoms in these patients. Vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells and promotes angiogenesis. We have reported the production of VEGF in endometriosis
Abstracts
$15