Oxytocin and vasopressin binding sites in human and bovine ovaries Anna-Riitta Fuchs, DSc, Oliver Behrens, MD, Hanns Helmer, MD, Annette Vangsted, MD, Marina Ivanisevic, MD, Jamie Grifo, MD, PhD, Ciro Barros, PhD, and Michael Fields, PhD New York, New York, and Gainesville, Florida Human ovarian tissue and bovine ovarian stroma and follicles bound tritiated oxytocin and tritiated arginine vasopressin with similar affinity, whereas bovine corpora lutea bound tritiated oxytocin only. Competition for the binding of tritiated oxytocin by various agonists and antagonists was suggestive of receptor function. The number of oxytocin binding sites varied cyclically in all tissues. In bovine ovarian stroma and corpora lutea the concentrations were lowest on day 14 and highest on days 17, 19, and 21 after ovulation, with a striking peak in the luteal concentration on day 19. In human ovarian tissues the concentrations also were highest in samples obtained in late luteal phase. In large follicles the concentration of oxytocin binding sites was highest on the day of estrus and lowest on day 7. In bovine ovary the number of arginine vasopressin binding sites was -50% lower than oxytocin binding sites and the cyclic variations were not significant. Human ovarian tissue had similar numbers of oxytocin and arginine vasopressin binding sites. Because bovine ovaries produce oxytocin and argine vasopressin the results suggest a paracrine or autocrine role for these neuropeptides in luteolysis and ovulation. Although their synthesis in human ovaries is still controversial the presence of binding sites suggests a physiologic role in the regulation of human ovarian function as well. (AM J OBSTET GVNECOL 1990;163:1961-7.)
Key words: Oxytocin, vasopressin, receptors, ovaries, corpora lutea, follicles
Oxytocin has been found in the corpora lutea of many species including COW,I,2 sheep,' pig! rat,5 deer,3 human,6 and cynomolgus monkey.7 It is produced by the granulosa-derived large luteal cells" 8 and periovulatory granulosa cells'" 9. 10 The ability of oxytocin to affect luteal function has been demonstrated in several species including the ruminants l 1.12 and primates. 13 In species in which the uterus is essential for the regulation of corpus luteum life span, oxytocin is thought to act on the endometrium to cause the release of prostaglandin F2a\4 (PGF 2a ), which in turn acts on the corpus luteum to cause luteolysis. 15 , 16 In species in which the influence of the uterus on luteal life-span is not significant (e.g., the primates), oxytocin may have a paracrine role in the regulation of ovarian function, acting on ovarian tissues to release PGF2a , which then diffuses From the Department of Obstetrics and Gynecology, Cornell University Medical College, and the Department of Animal Science, University of Florida. Supported in part by a grant from Warner Lambert Foundation, Deutsche Forschungsgemeinschaft, The Lundbeck Foundation, The Danish Medical Research Council, the Fulbright Foundation, and grant US-1160-86C from BARD, the United States-Israel Binational Agricultural Research and Development Fund, Florida Agricultural Experimental Station journal no. R-OI093, Presented in part at the Thirty-seventh Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. Reprint requests: Anna-Riitta Fuchs, DSc, Room 412, Department of Obstetrics and Gynecology, Cornell University Medical College, 1300 York Ave., New York, NY 10021. 616124755
into the adjacent luteal cells to cause luteolysis. 17 To determine the validity of such a hypothesis, we examined the binding of oxytocin to ovarian tissues because the presence of receptors is a prerequisite for oxytocin action. Arginine vasopressin is produced in the ovaries of some species,' and specific binding sites for arginine vasopressin have been found in other tissues of the female reproductive tract. 18, 19 We therefore examined the tissues also for the presence of arginine vasopressin binding sites.
Material and methods Human subjects. Ovarian tissue was obtained from nine premenopausal women who had operations for the following nonmalignant causes: ovarian cysts (three cases), endometriosis (three), benign ovarian tumor (one), fibroids of the uterus (one), and hydrosalpinx (one). The contralateral ovary was used in all instances but one, and the tissue was histologically normal in all instances. All patients gave their consent and the project was approved by the Institutional Human Rights Committee. The tissues were kept on ice until the pathologists had examined the ovarian tissue and cut a portion of macroscopically normal tissue without the capsule and corpora lutea. The tissue was then wrapped tightly in foil and frozen at - 70° to - 80° C until assayed. Because insufficient tissue was available from individual patients to permit saturation analysis, tissues were pooled according to the stage in menstrual cycle 1961
1962
Fuchs et al.
when the operation was performed. This was determined from the menstrual history and verified by histologic examination of the endometrium. The following pools were obtained: 1, proliferative phase, days 6 and 7 (two pools); 2, midcycle, days 14 to IS (two pools); 3, late luteal phase, days 19 to 2S (one pool). Animals. Eighteen Hereford cows from the herd of the Animal Science Department of the University of Florida were killed on days 7,14,17,19, and 21 after behavioral estrus (three cows each day). The tissues were removed as quickly as possible, the ovaries were freed of peritoneum, large follicles and corpora lutea were dissected, and the stroma, follicles, and corpora lutea were frozen separately and kept at - 70° to - SOO C until assayed. Tissue preparation. The tissues were homogenized in 10 mmol/L Tris buffer containing 3 mmollL ethylenediaminetetraacetic acid, 0.5 mmollL dithiothreitol, and 0.002% of a protease inhibitor, phenylmethylsulfonyl fluoride (Sigma, St. Louis). Four 5-second pulses with a Polytron tissue grinder (Brinkman, Westbury, N.Y.) with 30-second intervals were used followed by four up-and-down strokes with a hand-held glass tissue grinder (Kontes, Vineland, N.J.). All steps were performed in an ice bath. A crude membrane preparation precipitated between 1000 g (10 minutes) and 160,000 g (30 minutes) at 4° C was used to maximize binding. The membrane preparations were washed repeatedly in 3 mmollL ethylenediaminetetraacetic acid buffer to dissociate all endogenous oxytocin or arginine vasopressin from their binding sites. This is an important step because corpora lutea may contain significant amounts of oxytocin, which would interfere with the receptor assay, because of the relatively low specific activity of the available tritiated oxytocin (37 mCi/mmol). Membranes were suspended in calciumfree Hanks physiologic salt solution for storage at -70° to - SOO C in a concentration of I mg 1m!. Binding assay. The assay measures the total number of binding sites in the particulate membrane fractions. The final protein concentration was 1: 1.5 mg/ml in the assay buffer; the total volume of 220 IJ.lItube was increased to 400 IJ.lItube when >330 IJ.g protein per tube was used. The assay buffer consisted of 0.05 mollL Tris maleate, 5 mmollL manganese chloride, 0.002% phenylmethylsulfonyl fluoride, and 0.1 % bovine serum albumin pH 7.4. The labeled ligands were tritiated oxytocin (specific activity, 37 mCi/mmol) and tritiated arginine vasopressin (specific activity, 57.S mCilmmol) from New England Nuclear (Boston). The assay conditions were as previously described!8. !9: incubation with shaking for 60 minutes at 22° C terminated by adding 5 ml ice-cold buffer and rapid filtration through Whatman CF/F glass fiber filters using a cell harvester (Brandel, Gaithersburg, Md.). The unlabeled
December 1990 Am J Obstet Gynecol
ligands (purity: >99% by high-performance liquid chromatography verified by capillary electrophoresis) were obtained from Bachem (Torrance, Calif.). Other peptides were obtained from Peninsula, and other chemicals were from Sigma. Saturation analysis. Analysis was done by use of six to eight different concentrations oflabeled ligand ranging from 10- 10 to 10- 8 mollL. Nonspecific binding was determined in the presence of 1.1 X 10- 6 mollL concentration of unlabeled ligand. All assays were done in duplicate. Displacement and competition assays. These were performed with pooled bovine ovarian stromal membranes prepared from tissues obtained on days 17 and 21 of cycle. The following agonists were used: oxytocin; arginine vasopressin; isotocin, a highly selective oxytocin agonist; I-deamino-S-D-arginine vasopressin (ddAVP), a highly selective antidiuretic agonist; and two selective oxytocin antagonists, I deamino[2D-tyrosine (O-ethyl)-S-ornithine]oxytocin RWJ (provided by Dr. D. W. Hahn, Ortho Pharmaceutical Corp., Raritan, N.].) and L365209 (provided by Dr. D. Pettibone, Merck, West Point, N.Y.). The binding specificity was tested by competition with human chorionic gonadotropin (94% purity, Steris Laboratories, Phoenix, Ariz.), and thyrotropin releasing hormone (Thypinone, Abbott Laboratories, North Chicago, II!.). Protein assays were performed according to the method of Lowry et al. 20 with bovine serum albumin as standard, and deoxyribonucleic acid (DNA) assays were performed according to the method of Burton 2 ! with calf thymus DNA as standard. Data analysis. Analysis of the data was performed with a computerized nonlinear iterative curve-fitting program, LIGAND, which calculates the parameters Ka (association constant), Kd (dissociation constant), K. (dissociation constant of inhibitor), and Bma. (maximum binding) (Biosoft, Elsevier Science Publishers, New York). Statistical analysis. The significance of the cyclic variations was assessed by analysis of variance followed by Dunnett's test using log-transformed data, and Student's t test was used to compare differences between two groups; values of p < 0.05 were considered significant. Values are given as mean ± SE unless otherwise indicated; n indicates the number of cows or number of pools from human ovaries, respectively.
Results Preliminary results with bovine ovarian tissues indicated that the binding of tritiated oxytocin and tritiated arginine vasopressin was proportional to the protein concentration up to 1.5 mg/ml; at 2 mg/ml nonspecific interactions began to occur. Saturation analysis with 0.4 and O.S mg/ml of protein from the same bovine ovarian
Volume 16:, ",umber 6. Part I
stromal membrane preparation resulted in identical Kd and B""" values, with SEs < 10%. Specific binding reached equilibrium at 22° C in approximately 45 minutes and was stable until 90 minutes; at + 4° C the equilibrium was reached in 2 hours. At 37° C the results were erratic and specific binding had declined to low levels at 90 minutes. The presence of a divalent cation was essential for binding; calcium was ineffective and manganese was more effective than magnesium. Nonspecific binding constituted from 50% to 80% of total binding, depending on the concentration of labeled ligand and the concentration of protein in the saturation assay. Thyrotropin releasing hormone or human chorionic gonadotropin, in concentrations up to 10- 6 mol/L, did not compete for the binding of labeled oxytocin. The precision of the assay was determined by measuring the binding to aliquots of a similar membrane fraction prepared from bovine endometrium obtained on the day of estrus. Scatchard plots on the binding data obtained with these quality control samples in 15 assays yielded similar parameters as the original analysis on the same tissue. 19 High-affinity, saturable binding sites for tritiated oxytocin were found in all ovarian tissues, including the human pooled ovarian tissues, bovine ovarian stroma, corpora lutea, and follicles (Fig. I). Tritiated arginine vasopressing binding was found in all tissues examined except the bovine corpora lutea, which showed no binding. The affinities were similar for both peptides and for all tissues with no significant variations between tissues or days of cycle (Table I). Unlabeled oxytocin displaced the labeled ligand with a Kd of 0.5 x 10- 8 mol/L, and unlabeled arginine vasopressin competed for tritiated oxytocin with high affinity, K, 0.8 x 10-'1 mol/L Labeled arginine vasopressin was displaced by arginine vasopressin with a Kd of 1.4 x 10- 8 and oxytocin competed with tritiated arginine vasopressin with a K, of 0.8 x 10-", indicating a high degree of cross-reactivity at the binding sites. The selective oxytocin agonist isotocin competed for tritiated oxytocin with high affinity, whereas the selective antidiuretic agonist ddAVP competed with much lower affinity. The two selective oxytocin antagonists RWJ22165 and L365209 also competed for tritiated oxytocin binding sites with similar affinity as in myometrial and endometrial membranes. 18 Fig. 2 shows the logit-Iog transformed competition curves for the tested agonists and antagonists. The sizes of the follicles and corpora lutea in the ovaries of the experimental cows are shown in Table II together with plasma progesterone levels at the time of slaughter and the luteal oxytocin content, which was measured in corpora lutea of other cows from the same herd slaughtered on the same days of cycle. The measurements were performed by radioimmunoassay with
Oxytocin and arginine vasopressin receptors in ovaries
1963
B/F 0.009
o
o 0.005
o
0.000
O.OOE+OO
7.66E-12
1. 53E-11
Bound
2.30E-ll
(M)
Fig. 1. Scatchard plot of data obtained in a saturation experiment with tritiated oxytocin binding to 400 fLg of pooled bovine ovarian stromal membranes per tube. The ovaries were from days 17 and 21 of the estrous cycle.
Table I. Affinity constants of oxytocin and arginine vasopressin receptors in human ovarian tissue and bovine ovarian stroma, follicles, and corpora lutea obtained from saturation experiments Affinity constant (nmollL) Oxytocin
Human ovary, pooled
1.50 ± 0.4
1.4
(n = 5)
(n = I) 2.10 ± 0.55 (n = 10)
Bovine ovarian stroma
1.53 ± 0.23
Bovine follicles, pooled
0.92 ± 0.13
(n = 18) (n = 4)
Bovine corpora lutea
Arginine vasopressin
2.55 ± 0.30 (n = 10)
0.45 (11 = I)
No binding
Values are mean ± SE.
0.4 mol/L acetic acid extracts that were purified by absorption on Florisil columns with subsequent elution with 90% acetone water. Human pooled ovarian tissues. The concentration oftritiated oxytocin binding sites per milligram of DNA rose progressively during the menstrual cycle from low levels in the proliferative phase (days 6 and 7), 71.7 ± 5 fmol/mg DNA, to 157 ± 60 mol/mg DNA in midcycle (days 14 to 18), and a peak of (652 mol/mg DNA) in the luteal phase (days 19 to 28). This value was more than three times the standard deviation above the mean for the proliferative phase and midcycle samples (Fig. 3). Expressed per milligram of protein, the variations during the cycle were similar; the concentration in the luteal phase was significantly higher than the combined mean for the proliferative phase and the midcycle val-
1964
Fuchs et al.
December 1990 Am J Obstet Gyneco1
.OT 5 4
• AVP • IT
•
o L365209 A RW22165
3
2
!:::
Key words: Oxytocin, vasopressin, receptors, ovaries, corpora lutea, follicles
Oxytocin has been found in the corpora lutea of many species including COW,I,2 sheep,' pig! rat,5 deer,3 human,6 and cynomolgus monkey.7 It is produced by the granulosa-derived large luteal cells" 8 and periovulatory granulosa cells'" 9. 10 The ability of oxytocin to affect luteal function has been demonstrated in several species including the ruminants l 1.12 and primates. 13 In species in which the uterus is essential for the regulation of corpus luteum life span, oxytocin is thought to act on the endometrium to cause the release of prostaglandin F2a\4 (PGF 2a ), which in turn acts on the corpus luteum to cause luteolysis. 15 , 16 In species in which the influence of the uterus on luteal life-span is not significant (e.g., the primates), oxytocin may have a paracrine role in the regulation of ovarian function, acting on ovarian tissues to release PGF2a , which then diffuses From the Department of Obstetrics and Gynecology, Cornell University Medical College, and the Department of Animal Science, University of Florida. Supported in part by a grant from Warner Lambert Foundation, Deutsche Forschungsgemeinschaft, The Lundbeck Foundation, The Danish Medical Research Council, the Fulbright Foundation, and grant US-1160-86C from BARD, the United States-Israel Binational Agricultural Research and Development Fund, Florida Agricultural Experimental Station journal no. R-OI093, Presented in part at the Thirty-seventh Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. Reprint requests: Anna-Riitta Fuchs, DSc, Room 412, Department of Obstetrics and Gynecology, Cornell University Medical College, 1300 York Ave., New York, NY 10021. 616124755
into the adjacent luteal cells to cause luteolysis. 17 To determine the validity of such a hypothesis, we examined the binding of oxytocin to ovarian tissues because the presence of receptors is a prerequisite for oxytocin action. Arginine vasopressin is produced in the ovaries of some species,' and specific binding sites for arginine vasopressin have been found in other tissues of the female reproductive tract. 18, 19 We therefore examined the tissues also for the presence of arginine vasopressin binding sites.
Material and methods Human subjects. Ovarian tissue was obtained from nine premenopausal women who had operations for the following nonmalignant causes: ovarian cysts (three cases), endometriosis (three), benign ovarian tumor (one), fibroids of the uterus (one), and hydrosalpinx (one). The contralateral ovary was used in all instances but one, and the tissue was histologically normal in all instances. All patients gave their consent and the project was approved by the Institutional Human Rights Committee. The tissues were kept on ice until the pathologists had examined the ovarian tissue and cut a portion of macroscopically normal tissue without the capsule and corpora lutea. The tissue was then wrapped tightly in foil and frozen at - 70° to - 80° C until assayed. Because insufficient tissue was available from individual patients to permit saturation analysis, tissues were pooled according to the stage in menstrual cycle 1961
1962
Fuchs et al.
when the operation was performed. This was determined from the menstrual history and verified by histologic examination of the endometrium. The following pools were obtained: 1, proliferative phase, days 6 and 7 (two pools); 2, midcycle, days 14 to IS (two pools); 3, late luteal phase, days 19 to 2S (one pool). Animals. Eighteen Hereford cows from the herd of the Animal Science Department of the University of Florida were killed on days 7,14,17,19, and 21 after behavioral estrus (three cows each day). The tissues were removed as quickly as possible, the ovaries were freed of peritoneum, large follicles and corpora lutea were dissected, and the stroma, follicles, and corpora lutea were frozen separately and kept at - 70° to - SOO C until assayed. Tissue preparation. The tissues were homogenized in 10 mmol/L Tris buffer containing 3 mmollL ethylenediaminetetraacetic acid, 0.5 mmollL dithiothreitol, and 0.002% of a protease inhibitor, phenylmethylsulfonyl fluoride (Sigma, St. Louis). Four 5-second pulses with a Polytron tissue grinder (Brinkman, Westbury, N.Y.) with 30-second intervals were used followed by four up-and-down strokes with a hand-held glass tissue grinder (Kontes, Vineland, N.J.). All steps were performed in an ice bath. A crude membrane preparation precipitated between 1000 g (10 minutes) and 160,000 g (30 minutes) at 4° C was used to maximize binding. The membrane preparations were washed repeatedly in 3 mmollL ethylenediaminetetraacetic acid buffer to dissociate all endogenous oxytocin or arginine vasopressin from their binding sites. This is an important step because corpora lutea may contain significant amounts of oxytocin, which would interfere with the receptor assay, because of the relatively low specific activity of the available tritiated oxytocin (37 mCi/mmol). Membranes were suspended in calciumfree Hanks physiologic salt solution for storage at -70° to - SOO C in a concentration of I mg 1m!. Binding assay. The assay measures the total number of binding sites in the particulate membrane fractions. The final protein concentration was 1: 1.5 mg/ml in the assay buffer; the total volume of 220 IJ.lItube was increased to 400 IJ.lItube when >330 IJ.g protein per tube was used. The assay buffer consisted of 0.05 mollL Tris maleate, 5 mmollL manganese chloride, 0.002% phenylmethylsulfonyl fluoride, and 0.1 % bovine serum albumin pH 7.4. The labeled ligands were tritiated oxytocin (specific activity, 37 mCi/mmol) and tritiated arginine vasopressin (specific activity, 57.S mCilmmol) from New England Nuclear (Boston). The assay conditions were as previously described!8. !9: incubation with shaking for 60 minutes at 22° C terminated by adding 5 ml ice-cold buffer and rapid filtration through Whatman CF/F glass fiber filters using a cell harvester (Brandel, Gaithersburg, Md.). The unlabeled
December 1990 Am J Obstet Gynecol
ligands (purity: >99% by high-performance liquid chromatography verified by capillary electrophoresis) were obtained from Bachem (Torrance, Calif.). Other peptides were obtained from Peninsula, and other chemicals were from Sigma. Saturation analysis. Analysis was done by use of six to eight different concentrations oflabeled ligand ranging from 10- 10 to 10- 8 mollL. Nonspecific binding was determined in the presence of 1.1 X 10- 6 mollL concentration of unlabeled ligand. All assays were done in duplicate. Displacement and competition assays. These were performed with pooled bovine ovarian stromal membranes prepared from tissues obtained on days 17 and 21 of cycle. The following agonists were used: oxytocin; arginine vasopressin; isotocin, a highly selective oxytocin agonist; I-deamino-S-D-arginine vasopressin (ddAVP), a highly selective antidiuretic agonist; and two selective oxytocin antagonists, I deamino[2D-tyrosine (O-ethyl)-S-ornithine]oxytocin RWJ (provided by Dr. D. W. Hahn, Ortho Pharmaceutical Corp., Raritan, N.].) and L365209 (provided by Dr. D. Pettibone, Merck, West Point, N.Y.). The binding specificity was tested by competition with human chorionic gonadotropin (94% purity, Steris Laboratories, Phoenix, Ariz.), and thyrotropin releasing hormone (Thypinone, Abbott Laboratories, North Chicago, II!.). Protein assays were performed according to the method of Lowry et al. 20 with bovine serum albumin as standard, and deoxyribonucleic acid (DNA) assays were performed according to the method of Burton 2 ! with calf thymus DNA as standard. Data analysis. Analysis of the data was performed with a computerized nonlinear iterative curve-fitting program, LIGAND, which calculates the parameters Ka (association constant), Kd (dissociation constant), K. (dissociation constant of inhibitor), and Bma. (maximum binding) (Biosoft, Elsevier Science Publishers, New York). Statistical analysis. The significance of the cyclic variations was assessed by analysis of variance followed by Dunnett's test using log-transformed data, and Student's t test was used to compare differences between two groups; values of p < 0.05 were considered significant. Values are given as mean ± SE unless otherwise indicated; n indicates the number of cows or number of pools from human ovaries, respectively.
Results Preliminary results with bovine ovarian tissues indicated that the binding of tritiated oxytocin and tritiated arginine vasopressin was proportional to the protein concentration up to 1.5 mg/ml; at 2 mg/ml nonspecific interactions began to occur. Saturation analysis with 0.4 and O.S mg/ml of protein from the same bovine ovarian
Volume 16:, ",umber 6. Part I
stromal membrane preparation resulted in identical Kd and B""" values, with SEs < 10%. Specific binding reached equilibrium at 22° C in approximately 45 minutes and was stable until 90 minutes; at + 4° C the equilibrium was reached in 2 hours. At 37° C the results were erratic and specific binding had declined to low levels at 90 minutes. The presence of a divalent cation was essential for binding; calcium was ineffective and manganese was more effective than magnesium. Nonspecific binding constituted from 50% to 80% of total binding, depending on the concentration of labeled ligand and the concentration of protein in the saturation assay. Thyrotropin releasing hormone or human chorionic gonadotropin, in concentrations up to 10- 6 mol/L, did not compete for the binding of labeled oxytocin. The precision of the assay was determined by measuring the binding to aliquots of a similar membrane fraction prepared from bovine endometrium obtained on the day of estrus. Scatchard plots on the binding data obtained with these quality control samples in 15 assays yielded similar parameters as the original analysis on the same tissue. 19 High-affinity, saturable binding sites for tritiated oxytocin were found in all ovarian tissues, including the human pooled ovarian tissues, bovine ovarian stroma, corpora lutea, and follicles (Fig. I). Tritiated arginine vasopressing binding was found in all tissues examined except the bovine corpora lutea, which showed no binding. The affinities were similar for both peptides and for all tissues with no significant variations between tissues or days of cycle (Table I). Unlabeled oxytocin displaced the labeled ligand with a Kd of 0.5 x 10- 8 mol/L, and unlabeled arginine vasopressin competed for tritiated oxytocin with high affinity, K, 0.8 x 10-'1 mol/L Labeled arginine vasopressin was displaced by arginine vasopressin with a Kd of 1.4 x 10- 8 and oxytocin competed with tritiated arginine vasopressin with a K, of 0.8 x 10-", indicating a high degree of cross-reactivity at the binding sites. The selective oxytocin agonist isotocin competed for tritiated oxytocin with high affinity, whereas the selective antidiuretic agonist ddAVP competed with much lower affinity. The two selective oxytocin antagonists RWJ22165 and L365209 also competed for tritiated oxytocin binding sites with similar affinity as in myometrial and endometrial membranes. 18 Fig. 2 shows the logit-Iog transformed competition curves for the tested agonists and antagonists. The sizes of the follicles and corpora lutea in the ovaries of the experimental cows are shown in Table II together with plasma progesterone levels at the time of slaughter and the luteal oxytocin content, which was measured in corpora lutea of other cows from the same herd slaughtered on the same days of cycle. The measurements were performed by radioimmunoassay with
Oxytocin and arginine vasopressin receptors in ovaries
1963
B/F 0.009
o
o 0.005
o
0.000
O.OOE+OO
7.66E-12
1. 53E-11
Bound
2.30E-ll
(M)
Fig. 1. Scatchard plot of data obtained in a saturation experiment with tritiated oxytocin binding to 400 fLg of pooled bovine ovarian stromal membranes per tube. The ovaries were from days 17 and 21 of the estrous cycle.
Table I. Affinity constants of oxytocin and arginine vasopressin receptors in human ovarian tissue and bovine ovarian stroma, follicles, and corpora lutea obtained from saturation experiments Affinity constant (nmollL) Oxytocin
Human ovary, pooled
1.50 ± 0.4
1.4
(n = 5)
(n = I) 2.10 ± 0.55 (n = 10)
Bovine ovarian stroma
1.53 ± 0.23
Bovine follicles, pooled
0.92 ± 0.13
(n = 18) (n = 4)
Bovine corpora lutea
Arginine vasopressin
2.55 ± 0.30 (n = 10)
0.45 (11 = I)
No binding
Values are mean ± SE.
0.4 mol/L acetic acid extracts that were purified by absorption on Florisil columns with subsequent elution with 90% acetone water. Human pooled ovarian tissues. The concentration oftritiated oxytocin binding sites per milligram of DNA rose progressively during the menstrual cycle from low levels in the proliferative phase (days 6 and 7), 71.7 ± 5 fmol/mg DNA, to 157 ± 60 mol/mg DNA in midcycle (days 14 to 18), and a peak of (652 mol/mg DNA) in the luteal phase (days 19 to 28). This value was more than three times the standard deviation above the mean for the proliferative phase and midcycle samples (Fig. 3). Expressed per milligram of protein, the variations during the cycle were similar; the concentration in the luteal phase was significantly higher than the combined mean for the proliferative phase and the midcycle val-
1964
Fuchs et al.
December 1990 Am J Obstet Gyneco1
.OT 5 4
• AVP • IT
•
o L365209 A RW22165
3
2
!:::
