Cornell 1300 York
University
Medical
College,
Avenue, New York, New York 10021, USA
THE LH-hCG RECEPTOR OF HUMAN OVARY AT VARIOUS STAGES OF THE MENSTRUAL CYCLE
By S. Wardlaw, N. H. Lauersen and B. B. Saxena ABSTRACT
Receptors specific for hCG were found in human corpora lutea and follicles. hCG and LH were found to bind at a similar receptor site. The dissociation constant for hCG ranged from 10\m=-\10 to 10\m=-\11 mol/l in human corpora lutea. The number of binding sites for 125I-hCG ranged from 10\m=-\14 to 10\m=-\15 moles/mg protein in human corpora lutea. The binding of 125I-hCG to ovary was found to vary at different stages of the menstrual cycle. The binding of 125I-hCG to human ovaries increased on days 13\p=n-\15 of the cycle, then declined slightly, and increased again on days 22\p=n-\23. Following day 23, there was a slow decline until day 27 when binding activity could no longer be measured. No binding could be measured by the corpus luteum after the onset of menstruation or in corpora albicans.
Specific gonadotrophin receptors have previously been described in mouse, rat, pig, cow and human ovary (Haour 8c Saxena 1974; Rajaniemi 8c VanhaPerttula 1972; Rao 8c Saxena 1973; Lee et al. 1973; Lee 8c Ryan 1971; Kammerman et al. 1972; Cole et al. 1973; Channing 8c Kammerman 1973; Gospodarowicz 1913a,b). In most cases, maximum hormone-receptor binding was attained at pH 7.0 within 30 min at 37°C (Channing 8c Kammerman 1974). Supported by grants CA-13908 and HD-06543 Bethesda, Maryland, and the grant 670-0455A *
from the National Institutes of Health, from the Ford Foundation.
Career Scientist Awardee, Health Research Council of the Contract # 1-621.
City
of New York,
The high affinity of hormone for the receptor has been demonstrated by Kd values ranging from 10-9 to 10"11 mol/1. The number of binding sites/mg of fresh ovarian tissue has ranged from 10~14 to 10"13 moles in various species. The number of receptor sites in human oVary has been shown to change with various stages of the menstrual cycle (Lee el al. 1973; Cole et al 1973; Cole 8c Rice 1973). At 4°C, the hormone-receptor complex has been shown to be stable and the bound hormone is not dissociated up to 48 h, whereas at 37°C the bound hormone is dissociated from the receptor rapidly (Catt et al. 1972). The dissociated hormone has been shown to be immunologically and biolo¬ gically identical to native hormone (Dufau et al. 1972; Lee 8c Ryan 1973). It has been suggested that endogenous hormone bound to the ovarian receptor may affect the measurement of receptor sites by the use of exogenous labelled hormone. A study has, therefore, been designed to dissociate endogenous hor¬ mone without destroying receptor activity in human ovaries and to relate changes in the apparent and maximum number of binding sites with plasma LH levels and the stage of the menstrual cycle.
MATERIALS AND METHODS Collection of human ovarian tissue. Human luteal and follicular tissue was ob¬ tained in chilled 0.01 m Tris-HCl buffer of pH 7.8 containing 1 mM MgCL> and 1 mM dithiothreitol from patients, at various stages of the menstrual cycle, operated on at The New York Hospital primarily for uterine leiomyoma or benign ovarian cyst. The ovarian tissues were stored in liquid nitrogen until used. In each case, the endo¬ metrium was biopsied and a blood sample was drawn for the determination of plasma LH (Saxena et al. 1973) and progesterone (Thorneycroft Sc Stone 1972) by radio¬ -
immunoassay.
Preparation of ovarian tissue. Follicular or luteal tissue was dissected, weighed, homogenized in the Tris-HCl buffer of pH 7.8 using twenty strokes in a teflon glass homogenizer to yield a final concentration of 1 g tissue/10 ml. An equivalent of 10 mg of homogenate was used in most of the experiments. An aliquot of the homo¬ genate was taken for protein determination (Lowry et al. 1951). In one case, plasma membranes were prepared from two corpora lutea. Ovarian homogenate was centrifuged for 60 min at 20 000 g in a Sorvall refrigerated centri¬ fuge. The pellet was suspended in Tris-HCl buffer and centrifuged again for 60 min at 20 000 g. The pellet was re-suspended in 1.0 ml of Tris-HCl buffer and layered on a continuous sucrose density gradient from 1.5-0.5 M and centrifuged at 100 000 g for 2 h using the Beckman Preparative Ultracentrifuge (Model L265, swinging bucket rotor, type SW 25-1). Fractions showing highest specific binding of 123I-hCG were examined by electron microscopy and were shown to contain plasma membranes (Haour Sc Saxena 1974; Rao 8c Saxena 1973). and
-
Preparation of radioactive hCG and LH. was performed by the procedure of
with 125I
Radioactive labelling of hCG and LH Hunter Se Greenwood (1962). Twenty pg
-
containing 12 000 IU/mgi) (obtained from Dr. R. E. Canfield, College of Physicians and Surgeons, Columbia University, New York, New York) or hLH con¬ taining 5292 IU 2nd IRP HMG/mg2), were dissolved in 20 ,«1 of 10 mM Tris-HCl buffer, pH 7.2, in the reaction vial. To the vial, 50 µ oí 0.5 M phosphate buffer, pH 7.4, 1.5 mCi of 123I (Cambridge Nuclear, Cambridge, Massachusetts), and 70 /ig of chloramine-T in 20 µ\ of 0.05 M phosphate buffer were added and mixed. After 15 seconds, the reaction was stopped by the addition of 240 /ig sodium metabisulphite in 100 µ\ of phosphate buffer. The crude reaction mixture was purified by gel filtra¬ tion through a 1x30 cm column of Sephadex G-100 eluted with 0.9% NaCl con¬ taining 0.1 % bovine serum albumin (BSA), pH 7.2. The purity and specific activity of the labelled hormones were determined by chromato-electrophoresis (Saxena et al. 1968). The specific activity of the labelled hCG1) and LH2) was 30 and 40 µCi/µg, respectively. One ml aliquots Dissociation of the endogenous hormone bound to the receptor. of ovarian homogenate containing 10 mg protein/ml were treated simultaneously by incubation at 20°C for 30 min in 10 ml of 1.0 m Tris-HCl buffer of either pH 7.2 or pH 10.0 containing 1 mM MgCW, 1 mM CaCL, and 0.1% BSA. Following incubation, the homogenates were centrifuged for 30 min at 20 000 g. The supernates were decanted, the pellets were washed with Tris-HCl buffer, pH 7.2, and centrifuged again for 30 min at 20 000 g. The pellets were re-suspended in 1 ml of Tris-HCl buffer, pH 7.2, and assayed for binding. A protein determination was made for every sample to correct for the losses during treatment and centrifugation. og hCG
-
Receptor binding assay. The binding of labelled hormone with 100 µ aliquots of the 20 000 g pellet, and in one case with 100 µ of the plasma membrane fraction, was measured by overnight incubation at 4°C in 200 µ of 10 mM Tris-HCl of pH 7.2 containing 1 mM MgCL, 1 mM CaCL, and 0.1% BSA. Bound and free hormones were separated by filtration under suction through a millipore filter of pore size 0.45 / ii Ü "3 | caS o
cj
Sca> —'
ca
ca 3
cû
so
OCCO-iJ-iclCOCNMOl
Ä Q
ho °
-a
X .SX
Oh Jœ
o
Binding
Table 2. of 123I-hCG with bovine corpus luteum homogenates treated incubation at varying pH1)
pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.2
%
by
specific binding 3.5 46 110 120 131 107 98 100
(control)2 8.0 9.0 10.0
115 118
136
0 One ml aliquots of homogenate were incubated at 20°C for 30 min in 10 ml of 0.1 M Tris-HCl buffer of pH 2.0-10.0. Following incubation, the homogenates were centri¬ fuged and binding of 125I-hCG measured as described in Materials and Methods. 2) Binding was considered as 100% for the untreated control preparation at pH 7.2.
Ovarian tissue from a 68-year-old woman with ovarian hyperplasia also was found to specifically bind 125I-hCG. However, a parathyroid-like hormone secreting medullary ovarian carcinoma was tested for binding and failed to specifically bind any gonadotrophin. The in vivo uptake of 125I-hCG by ovarian theca cell carcinoma in mice has previously been reported (Mizejewski et al 1971). An ovarian wedge from a 20-year-old girl with polycystic ovaries failed to bind LH or hCG; however, tissue from another patient with poly¬ cystic ovaries and a secretory endometrium indicating ovulation, did bind hCG (patient 11, Table 1). These observations suggest that gonadotrophinreceptor interaction play a role in various endocrine disorders of the ovary as well as in the growth of certain ovarian cancers.
ACKNOWLEDGMENTS We wish to thank Dr. Elmer Kramer (Professor of Obstetrics and Gynaecology and Clinical Professor of Pathology at the Cornell University Medical College-The New York Hospital) for analysis of ovarian biopsies and the dating of the endometrium, Dr. K. Sundaram (The Rockefeller University, New York, New York), and Dr. Ch. V. Rao (University of Louisville School of Medicine, Louisville, Kentucky) for the radio¬ immunoassay of progesterone in plasma samples.
REFERENCES Catt K. J., Tsuruhara T. Se Dufau M. L.: Biochim. biophys. Acta (Amst.) 279 (1972) 194. Channing C. P. &· Kammerman S.: Endocrinology 92 (1973) 531. Channing C. P. 8e Kammerman S.: Biol. of Reprod. 10 (1974) 179. Cole F. E. 8e Rice B. F.: Endocrine Society (1973) Abst. #315. Cole F. E., Weed J. C, Schneider G. T., Holland J. B., Geary W. L. Se Rice B. F.: Amer. J. Obstet. Gynec. 117 (1973) 87. Dufau M. L., Catt K. J. Se Tsuruhara T.: Proc. nat. Acad. Sci. (USA) 69 (1972) 2414. Gospodarowicz D.: J. biol. Chem. 248 (1973a) 5042. Gospodarowicz D.: J. biol. Chem. 248 (19736) 5050. Haour F. Se Saxena B. B.: J. biol. Chem. 249 (1974) 2195. Hunter W. M. Se Greenwood F. C: Nature (Lond.) 194 (1962) 495. Kammerman S., Canfield R. E., Kolena J. Se Channing C. P.: Endocrinology 91
(1972)
65.
Lee C. H., Coulam C. B., Jiang N. S. Se Ryan R. J.: J. clin. Endocr. 36 (1973) 148. Lee C. H. Sc Ryan R. J.: Endocrinology 89 (1971) 1515. Lee C. H. Se Ryan R. J.: Receptors for Reproductive Hormones. B. W. O'Malley and A. R. Means, Ltd., New York (1973) 419. Lowry O. H., Rosebrough N. ]., Farr A. L. Sc Randall R. J.: J. biol. Chem. 193 (1951) 265. Mizejewski G. J., Beierwaltes W. H. Se Quiñones J.: J. nucl. Med. 13 (1971) 101. Rajaniemi H. Se Vanha-Perttula T.: Endocrinology 90 (1972) 1. Rao C. V. Se Saxena B. B.: Biochim. biophys. Acta (Amst.) 313 (1973) 372. Reichert L. E., Jr., Leidenberger F. Se Trowbridge C. G.: Recent Progr. Hormone Res. 29 (1973) 497. Roth J.: Metabolism 22 (1973) 1059. Saxena B. B., Demura H.. Gandy H. M. Se Peterson R. E.: J. clin. Endocr. 28 (1968) 519.
Saxena B. B., Rathnam P. Se Rommler .: Endocr. Exp. 7 (1973) 19. Scatchard G.: Ann. N. Y. Acad. Sci. 51 (1949) 660. Thorneycroft J. H. Se Stone S. C: Contraception 5 (1972) 129. Received
on
September 9th,
1974.
University
Medical
College,
Avenue, New York, New York 10021, USA
THE LH-hCG RECEPTOR OF HUMAN OVARY AT VARIOUS STAGES OF THE MENSTRUAL CYCLE
By S. Wardlaw, N. H. Lauersen and B. B. Saxena ABSTRACT
Receptors specific for hCG were found in human corpora lutea and follicles. hCG and LH were found to bind at a similar receptor site. The dissociation constant for hCG ranged from 10\m=-\10 to 10\m=-\11 mol/l in human corpora lutea. The number of binding sites for 125I-hCG ranged from 10\m=-\14 to 10\m=-\15 moles/mg protein in human corpora lutea. The binding of 125I-hCG to ovary was found to vary at different stages of the menstrual cycle. The binding of 125I-hCG to human ovaries increased on days 13\p=n-\15 of the cycle, then declined slightly, and increased again on days 22\p=n-\23. Following day 23, there was a slow decline until day 27 when binding activity could no longer be measured. No binding could be measured by the corpus luteum after the onset of menstruation or in corpora albicans.
Specific gonadotrophin receptors have previously been described in mouse, rat, pig, cow and human ovary (Haour 8c Saxena 1974; Rajaniemi 8c VanhaPerttula 1972; Rao 8c Saxena 1973; Lee et al. 1973; Lee 8c Ryan 1971; Kammerman et al. 1972; Cole et al. 1973; Channing 8c Kammerman 1973; Gospodarowicz 1913a,b). In most cases, maximum hormone-receptor binding was attained at pH 7.0 within 30 min at 37°C (Channing 8c Kammerman 1974). Supported by grants CA-13908 and HD-06543 Bethesda, Maryland, and the grant 670-0455A *
from the National Institutes of Health, from the Ford Foundation.
Career Scientist Awardee, Health Research Council of the Contract # 1-621.
City
of New York,
The high affinity of hormone for the receptor has been demonstrated by Kd values ranging from 10-9 to 10"11 mol/1. The number of binding sites/mg of fresh ovarian tissue has ranged from 10~14 to 10"13 moles in various species. The number of receptor sites in human oVary has been shown to change with various stages of the menstrual cycle (Lee el al. 1973; Cole et al 1973; Cole 8c Rice 1973). At 4°C, the hormone-receptor complex has been shown to be stable and the bound hormone is not dissociated up to 48 h, whereas at 37°C the bound hormone is dissociated from the receptor rapidly (Catt et al. 1972). The dissociated hormone has been shown to be immunologically and biolo¬ gically identical to native hormone (Dufau et al. 1972; Lee 8c Ryan 1973). It has been suggested that endogenous hormone bound to the ovarian receptor may affect the measurement of receptor sites by the use of exogenous labelled hormone. A study has, therefore, been designed to dissociate endogenous hor¬ mone without destroying receptor activity in human ovaries and to relate changes in the apparent and maximum number of binding sites with plasma LH levels and the stage of the menstrual cycle.
MATERIALS AND METHODS Collection of human ovarian tissue. Human luteal and follicular tissue was ob¬ tained in chilled 0.01 m Tris-HCl buffer of pH 7.8 containing 1 mM MgCL> and 1 mM dithiothreitol from patients, at various stages of the menstrual cycle, operated on at The New York Hospital primarily for uterine leiomyoma or benign ovarian cyst. The ovarian tissues were stored in liquid nitrogen until used. In each case, the endo¬ metrium was biopsied and a blood sample was drawn for the determination of plasma LH (Saxena et al. 1973) and progesterone (Thorneycroft Sc Stone 1972) by radio¬ -
immunoassay.
Preparation of ovarian tissue. Follicular or luteal tissue was dissected, weighed, homogenized in the Tris-HCl buffer of pH 7.8 using twenty strokes in a teflon glass homogenizer to yield a final concentration of 1 g tissue/10 ml. An equivalent of 10 mg of homogenate was used in most of the experiments. An aliquot of the homo¬ genate was taken for protein determination (Lowry et al. 1951). In one case, plasma membranes were prepared from two corpora lutea. Ovarian homogenate was centrifuged for 60 min at 20 000 g in a Sorvall refrigerated centri¬ fuge. The pellet was suspended in Tris-HCl buffer and centrifuged again for 60 min at 20 000 g. The pellet was re-suspended in 1.0 ml of Tris-HCl buffer and layered on a continuous sucrose density gradient from 1.5-0.5 M and centrifuged at 100 000 g for 2 h using the Beckman Preparative Ultracentrifuge (Model L265, swinging bucket rotor, type SW 25-1). Fractions showing highest specific binding of 123I-hCG were examined by electron microscopy and were shown to contain plasma membranes (Haour Sc Saxena 1974; Rao 8c Saxena 1973). and
-
Preparation of radioactive hCG and LH. was performed by the procedure of
with 125I
Radioactive labelling of hCG and LH Hunter Se Greenwood (1962). Twenty pg
-
containing 12 000 IU/mgi) (obtained from Dr. R. E. Canfield, College of Physicians and Surgeons, Columbia University, New York, New York) or hLH con¬ taining 5292 IU 2nd IRP HMG/mg2), were dissolved in 20 ,«1 of 10 mM Tris-HCl buffer, pH 7.2, in the reaction vial. To the vial, 50 µ oí 0.5 M phosphate buffer, pH 7.4, 1.5 mCi of 123I (Cambridge Nuclear, Cambridge, Massachusetts), and 70 /ig of chloramine-T in 20 µ\ of 0.05 M phosphate buffer were added and mixed. After 15 seconds, the reaction was stopped by the addition of 240 /ig sodium metabisulphite in 100 µ\ of phosphate buffer. The crude reaction mixture was purified by gel filtra¬ tion through a 1x30 cm column of Sephadex G-100 eluted with 0.9% NaCl con¬ taining 0.1 % bovine serum albumin (BSA), pH 7.2. The purity and specific activity of the labelled hormones were determined by chromato-electrophoresis (Saxena et al. 1968). The specific activity of the labelled hCG1) and LH2) was 30 and 40 µCi/µg, respectively. One ml aliquots Dissociation of the endogenous hormone bound to the receptor. of ovarian homogenate containing 10 mg protein/ml were treated simultaneously by incubation at 20°C for 30 min in 10 ml of 1.0 m Tris-HCl buffer of either pH 7.2 or pH 10.0 containing 1 mM MgCW, 1 mM CaCL, and 0.1% BSA. Following incubation, the homogenates were centrifuged for 30 min at 20 000 g. The supernates were decanted, the pellets were washed with Tris-HCl buffer, pH 7.2, and centrifuged again for 30 min at 20 000 g. The pellets were re-suspended in 1 ml of Tris-HCl buffer, pH 7.2, and assayed for binding. A protein determination was made for every sample to correct for the losses during treatment and centrifugation. og hCG
-
Receptor binding assay. The binding of labelled hormone with 100 µ aliquots of the 20 000 g pellet, and in one case with 100 µ of the plasma membrane fraction, was measured by overnight incubation at 4°C in 200 µ of 10 mM Tris-HCl of pH 7.2 containing 1 mM MgCL, 1 mM CaCL, and 0.1% BSA. Bound and free hormones were separated by filtration under suction through a millipore filter of pore size 0.45 / ii Ü "3 | caS o
cj
Sca> —'
ca
ca 3
cû
so
OCCO-iJ-iclCOCNMOl
Ä Q
ho °
-a
X .SX
Oh Jœ
o
Binding
Table 2. of 123I-hCG with bovine corpus luteum homogenates treated incubation at varying pH1)
pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.2
%
by
specific binding 3.5 46 110 120 131 107 98 100
(control)2 8.0 9.0 10.0
115 118
136
0 One ml aliquots of homogenate were incubated at 20°C for 30 min in 10 ml of 0.1 M Tris-HCl buffer of pH 2.0-10.0. Following incubation, the homogenates were centri¬ fuged and binding of 125I-hCG measured as described in Materials and Methods. 2) Binding was considered as 100% for the untreated control preparation at pH 7.2.
Ovarian tissue from a 68-year-old woman with ovarian hyperplasia also was found to specifically bind 125I-hCG. However, a parathyroid-like hormone secreting medullary ovarian carcinoma was tested for binding and failed to specifically bind any gonadotrophin. The in vivo uptake of 125I-hCG by ovarian theca cell carcinoma in mice has previously been reported (Mizejewski et al 1971). An ovarian wedge from a 20-year-old girl with polycystic ovaries failed to bind LH or hCG; however, tissue from another patient with poly¬ cystic ovaries and a secretory endometrium indicating ovulation, did bind hCG (patient 11, Table 1). These observations suggest that gonadotrophinreceptor interaction play a role in various endocrine disorders of the ovary as well as in the growth of certain ovarian cancers.
ACKNOWLEDGMENTS We wish to thank Dr. Elmer Kramer (Professor of Obstetrics and Gynaecology and Clinical Professor of Pathology at the Cornell University Medical College-The New York Hospital) for analysis of ovarian biopsies and the dating of the endometrium, Dr. K. Sundaram (The Rockefeller University, New York, New York), and Dr. Ch. V. Rao (University of Louisville School of Medicine, Louisville, Kentucky) for the radio¬ immunoassay of progesterone in plasma samples.
REFERENCES Catt K. J., Tsuruhara T. Se Dufau M. L.: Biochim. biophys. Acta (Amst.) 279 (1972) 194. Channing C. P. &· Kammerman S.: Endocrinology 92 (1973) 531. Channing C. P. 8e Kammerman S.: Biol. of Reprod. 10 (1974) 179. Cole F. E. 8e Rice B. F.: Endocrine Society (1973) Abst. #315. Cole F. E., Weed J. C, Schneider G. T., Holland J. B., Geary W. L. Se Rice B. F.: Amer. J. Obstet. Gynec. 117 (1973) 87. Dufau M. L., Catt K. J. Se Tsuruhara T.: Proc. nat. Acad. Sci. (USA) 69 (1972) 2414. Gospodarowicz D.: J. biol. Chem. 248 (1973a) 5042. Gospodarowicz D.: J. biol. Chem. 248 (19736) 5050. Haour F. Se Saxena B. B.: J. biol. Chem. 249 (1974) 2195. Hunter W. M. Se Greenwood F. C: Nature (Lond.) 194 (1962) 495. Kammerman S., Canfield R. E., Kolena J. Se Channing C. P.: Endocrinology 91
(1972)
65.
Lee C. H., Coulam C. B., Jiang N. S. Se Ryan R. J.: J. clin. Endocr. 36 (1973) 148. Lee C. H. Sc Ryan R. J.: Endocrinology 89 (1971) 1515. Lee C. H. Se Ryan R. J.: Receptors for Reproductive Hormones. B. W. O'Malley and A. R. Means, Ltd., New York (1973) 419. Lowry O. H., Rosebrough N. ]., Farr A. L. Sc Randall R. J.: J. biol. Chem. 193 (1951) 265. Mizejewski G. J., Beierwaltes W. H. Se Quiñones J.: J. nucl. Med. 13 (1971) 101. Rajaniemi H. Se Vanha-Perttula T.: Endocrinology 90 (1972) 1. Rao C. V. Se Saxena B. B.: Biochim. biophys. Acta (Amst.) 313 (1973) 372. Reichert L. E., Jr., Leidenberger F. Se Trowbridge C. G.: Recent Progr. Hormone Res. 29 (1973) 497. Roth J.: Metabolism 22 (1973) 1059. Saxena B. B., Demura H.. Gandy H. M. Se Peterson R. E.: J. clin. Endocr. 28 (1968) 519.
Saxena B. B., Rathnam P. Se Rommler .: Endocr. Exp. 7 (1973) 19. Scatchard G.: Ann. N. Y. Acad. Sci. 51 (1949) 660. Thorneycroft J. H. Se Stone S. C: Contraception 5 (1972) 129. Received
on
September 9th,
1974.
