0013-7227/90/1262-1264$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 2 Printed in U.S.A.
Estrous Cycle-Related Changes of High Affinity Luteinizing Hormone/Human Chorionic Gonadotropin Binding Sites in the Rat Uterus P. J. BONNAMY*, A. BENHAIM, AND P. LEYMARIE Laboratoire de Biochimie, CHU Cote de Nacre, CNRS URA-609, 14032 Caen, France
ABSTRACT. LH/human CG (LH/hCG) high affinity binding sites were detected in crude membrane preparations of rat uteri. There was little competition for receptor occupancy between hCG and ovine FSH (oFSH) (0.05%) and no competition between hCG and ovine PRL (less than 0.01%). No similar binding sites were detected in crude membrane preparation of heart, kidney, skeletal muscle, liver, and lung tissues. Concentrations of uterine unoccupied binding sites (RLH) were determined for each stage of the 4-day estrous cycle. The RLH were found in all preparations of metestrus uteri (n = 10) but only in some preparations from the other stages of the estrous cycle (1 of 7 on proestrus, 3 of 4 on estrus, 5 of 7 on diestrus). The concentration of uterine RLH varied throughout the estrous cycle with highest values during the metestrus (1.50 ± 0.15 fmol/mg protein) and lowest values during the proestrus (less than 0.2 fmol/
mg protein). The affinity constant for hCG of uterine RLH remained constant during the estrous cycle (about 0.8 x IO11 ivr l ) and was nearly identical to that of rat ovarian receptors. On metestrus, RLH concentration appeared to be approximately 35-fold lower in the uterus than in the ovaries when expressed per mg protein (1.50 ± 0.15 vs. 52.83 ± 3.61 fmol/mg protein) but only 20 times lower when expressed per organ (2.2 vs. 48.3 fmol/organ). The estrous cycle-related changes of uterine RLH concentration, together with our data establishing an in vitro influence of hCG on progesterone metabolism in rat uterus, suggest that some uterine functions could be directly regulated either by LH from the pituitary or during early pregnancy by an LH-like substance originating from the embryo. (Endocrinology 126: 1264-1269, 1990)
T
HERE IS increasing evidence for the existence of extraovarian LH/human CG (hCG) receptor sites in mammalian females. In the pig (1) and, more recently, in the rabbit (2) uterine high affinity LH/hCG binding sites (RLH) have been detected. The changes of their concentrations according to the hormonal status of the animal suggest their possible involvement in reproductive processes. But although a regulatory role in myometrium relaxation and uterine blood flow has been suggested, there is no decisive demonstration of any direct effect of LH/hCG upon uterine tissues. We have recently shown that hCG is able to modulate in vitro the progesterone content of the metestrus rat uterus via a cAMP-dependent mechanism (3). This effect was specific for the metestrus stage of the estrous cycle since no effect was observed in proestrus and estrus rat uteri. We concluded that 1) hCG should act upon metestrus uterus after binding to specific receptors and 2) the absence of RLH in proestrus and estrus uteri could have explained the inability of hCG to modulate progesterone content at these phases of the estrous cycle.
Received August 4,1989. *Supported by a fellowship from the C.N.R.S. and the Conseil Regional de Basse Normandie; to whom all correspondence and reprint requests should be addressed.
In this study, we have established the existence of high affinity LH/hCG binding sites in the rat uterus and measured their concentration throughout the estrous cycle.
Materials and Methods Hormones Purified hCG (CR-121: 13,450 IU/mg) was provided by the National Hormone and Pituitary Program (NIDDK). Ovine LH (oLH-26), ovine FSH (oFSH-16), and ovine PRL (oPRL18) were furnished by the NIDDK. Commercial preparation of hCG (Pregnyl, 3,500-4,000 IU/mg) was supplied by Organon (Saint Denis, France). Animals Three- to four-month-old Sprague-Dawley female rats were kept on a schedule of 14 h of light, 10 h of darkness (lights on from 0500 h to 1900 h) with food and water ad libitum. Vaginal smears were recorded 6 days a week, and rats were used only after exhibiting at least two consecutive 4-day estrous cycles. Rats were killed by cervical dislocation between 1000 h and 1500 h after staging. Preparation of crude membrane fractions Uteri of rats were removed free of the oviducts and immediately placed in NaCl 0.9% (wt/vol), trimmed of connective
1264
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LH/hCG RECEPTORS IN RAT UTERUS tissue, weighed and slit longitudinally before rinsing in NaCl solution. Cell membrane fractions were obtained using the procedure described by Ziecick et al. (1) with some modifications. Briefly, after mincing with scissors, tissues were homogenized at 4 C with a Teflon-glass potter in THS buffer (25 mM Tris HC1, pH 7.4, containing 0.25 M sucrose, 1 ml/uterus) by four 10-sec bursts separated by cooling periods of 30 sec. The homogenate was centrifuged for 30 min at 500 x g at 4 C. The resulting pellet was resuspended in THS buffer (1 ml/uterus), submitted again to homogenization, and centrifuged as described above. The pooled supernatants were centrifuged further at 4 C for 1 h at 25,000 X g. The crude membrane pellet was resuspended in cold THM buffer. A fraction of the membrane preparation was assayed for protein determination by the method of Bradford (4). Then 9 volumes of the membrane preparation were mixed with 1 volume of THM buffer containing 1% BSA. The resulting suspension was used immediately for the measurement of LH/hCG receptor concentration or stored at —80 C for further experimentation. Iodination of hCG
hCG (CR-121) was labeled using the chloramide l,3,4,6-tetrachloro-3a,6a-diphenylglycouryl (Iodogen, Pierce, Rockford, IL) as previously described (5). Na-125I was furnished by Amersham (France). Separation of bound and free 125I was performed by chromatography on Sephadex G-50 (Pharmacia, Uppsala, Sweden) column (1 x 10 cm). Specific activities of labeled hCG were determined by self-displacement analysis on ovarian membrane preparations in the radioligand receptor assay (6), and varied from 29,000 to 45,000 cpm/ng. Determination of incubation conditions
1265
aspiration and 2 ml ice-cold incubation buffer were added. After centrifugation, and removal of the supernatants, the membrane pellets were counted for 125I in a Cobra Auto Gamma (Packard, France) with a counting efficiency of 75%. Nonspecific binding determined in the presence of 200 IU hCG Pregnyl was usually less than 2% of the total radioactivity added. Determination of binding characteristics of uterine LH/hCG receptor The concentration and affinity constant (Ka) of unoccupied binding sites were determined by Scatchard plot analysis (7). Six or seven subsaturing doses of [125I]hCG (in the range of 1,000-50,000 cpm) were used in duplicate or triplicate with each membrane preparation. The hormonal specificity of [125I]hCG binding on uterine membranes was determined by incubating samples from metestrus rats with a subsaturating dose of [125I] hCG (25,000 cpm) in the presence of increasing amounts of various nonradioactive hormones. Cross-reactivity was calculated from the relative amount of the tested hormone that inhibited the[125I]hCG binding by 50%. Organ specificity of the LH/hCG binding LH/hCG binding measurements were attempted with tissues from other organs of metestrus rats. Membrane fractions from ovary, lung, kidney, skeletal muscle, heart, and liver were prepared as described above for uterus. [125I]hCG binding to these membranes was determined by incubating 50 ng (ovary) to 1 mg (other organs) of membrane proteins in the presence of increasing amounts of [125I]hCG (1,000-50,0000 cpm) with or without 200 IU hCG. Statistics
125
The membrane suspension was incubated with [ I] hCG in glass tubes. The incubation volume was 0.5 ml and consisted of 0.1 ml of various amounts of [125I]hCG (1,000-50,000 cpm) in THM 0.1% BSA (incubation buffer), 0.3 ml of membrane preparation, (100-900 ng protein) in incubation buffer and 0.1 ml of THM (25 M Tris-HCl, pH 7.2, containing 5 mM MgCl2) and 0.1 % BSA (for measurement of total binding) or 0.1 ml THM and 0.1% BSA containing 200 IU Pregnyl (for nonspecific binding measurement). To determine the time and temperature dependence of specific [125I]hCG binding to uterine membranes, samples obtained from metestrus rats were incubated from 2 to 30 h at 4, 20, and 37 C in the presence of subsaturating dose of [125I]hCG. After this study was completed, 24-h incubation at room temperature (20 C) was chosen for subsequent assays. To separate receptor-bound from free [125I]hCG, 3 ml ice-cold incubation buffer were added at the end of the incubation period and the tubes were centrifuged at 3,000 X g for 10 min at 0 C. The supernatant was removed by
Values for the Ka and the total number of binding sites were determined by a computerized least squares fit of the Scatchard plot. Results are presented as mean + SEM. One-way analysis of variance (ANOVA) was used for statistical significance of differences.
Results Validation of the LH/hCG binding assay The specific binding of [125I]hCG to metestrus rat uterine receptors was time and temperature dependent (Fig. 1). A maximal level of specific binding was reached for 37 C and 20 C, respectively, after 12 and 24 h. The results demonstrated that LH/hCG binding to uterine membranes was a saturable process. For subsequent determinations, we considered that apparent equilibrium was reached after 24-h incubation at room temperature (20 C). The amount of [125I]hCG bound was linear with the amount of membrane suspension in the range of 0.1-1 mg of protein (r = 0.96, data not shown). Scatchard analysis of the data obtained at equilibrium
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1266
LH/hCG RECEPTORS IN RAT UTERUS
O.1Ch
Endo • 1990 Voll26«No 2
Ka, ( 0.1 mg protein ) = 1.25x10* I M " 1 Ka 2 ( 0.2 mg protein ) = 0.88X101 'NT 1 Ka 3 ( 0.5 mg protein ) = l.
£ 0.05F
INCUBATION TIME ( HOURS )
FIG. 1. Time and temperature dependence of specific ( ) and nonspecific ( ) [125I]hCG binding to uterine membranes of metestrus rats in a representative experiment. Crude membrane preparation (800 Mg protein) was incubated with 40,000 cpm [125I]hCG at 4 C (A), 20 C (•), and 37 C (•). Means of duplicates are shown.
gave for metestrus uterine samples (six to eight pooled uteri for each Ka determination) high values of affinity constant (Ka in the range of 0.41-1.4 lO11]^"1) and low binding capacities (0.88-2.48 fmol/mg protein) (Fig. 2). The specificity of [125I]hCG binding to metestrus rat uterine membrane preparation was then investigated (Fig. 3). oLH as well as hCG could completely inhibit the binding of [125I]hCG. There was little competition between hCG and ovine FSH (oFSH) (0.05%) and no competition between hCG and ovine PRL (oPRL). The specificity of [125I]hCG binding to metestrus uterine membranes was similar to that obtained with a membrane preparation from metestrus ovary (data not shown). The concentration of specific [125I]hCG binding sites in various metestrus rat tissues as compared to the uterus is shown in Fig. 4. There was no measurable specific binding of [125I]hCG on five different tissues other than the ovary.
0.3
O.9
O.6
hCG BOUND(pM) FIG. 2. Estimation of the number and affinity constant (Ka) of LH/ hCG binding sites in three different amounts of crude membrane from three different preparations of metestrus rat uteri. Means of duplicates or triplicates are shown. B/F, bound to free ratio. 100-r oPRL
oFSH
0.1
1
10
100
1000
10000
HORMONE(NG )
Affinity constant and capacity of uterine LH/hCG binding sites throughout the estrous cycle Uterine concentration of LH/hCG binding sites varied significantly during the estrous cycle (Fig. 5). Concentration of LH/hCG receptors in metestrus uteri (1.50 ± 0.15 fmol/mg protein) was significantly higher (P < 0.01) than those in estrus (0.53 ± 0.19) and in diestrus uteri (0.70 ± 0.05). Moreover LH/hCG receptors were found in all preparations (n = 10) of metestrus uteri but not in all uterine preparations from other stages of the estrous
FlG. 3. Hormonal specificity of [125I]hCG binding to rat uterine crude membrane preparation. Crude membrane preparation (450 ^g protein) was incubated with 25,000 cpm [125I]hCG in the presence of increasing amounts of hormones. Means of duplicates are shown.
cycle. Positive determinations (e.g. LH/hCG receptor concentrations equal to or higher than 0.2 fmol/mg protein, which was the limit of sensitivity of our assay) were found in 3 out of 4 preparations from estrus, 5 out of 7 from diestrus and only 1 out of 7 from proestrus. However, only this later proportion differed from the others
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LH/hCG RECEPTORS IN RAT UTERUS
1267
100
HI
1-
Vol. 126, No. 2 Printed in U.S.A.
Estrous Cycle-Related Changes of High Affinity Luteinizing Hormone/Human Chorionic Gonadotropin Binding Sites in the Rat Uterus P. J. BONNAMY*, A. BENHAIM, AND P. LEYMARIE Laboratoire de Biochimie, CHU Cote de Nacre, CNRS URA-609, 14032 Caen, France
ABSTRACT. LH/human CG (LH/hCG) high affinity binding sites were detected in crude membrane preparations of rat uteri. There was little competition for receptor occupancy between hCG and ovine FSH (oFSH) (0.05%) and no competition between hCG and ovine PRL (less than 0.01%). No similar binding sites were detected in crude membrane preparation of heart, kidney, skeletal muscle, liver, and lung tissues. Concentrations of uterine unoccupied binding sites (RLH) were determined for each stage of the 4-day estrous cycle. The RLH were found in all preparations of metestrus uteri (n = 10) but only in some preparations from the other stages of the estrous cycle (1 of 7 on proestrus, 3 of 4 on estrus, 5 of 7 on diestrus). The concentration of uterine RLH varied throughout the estrous cycle with highest values during the metestrus (1.50 ± 0.15 fmol/mg protein) and lowest values during the proestrus (less than 0.2 fmol/
mg protein). The affinity constant for hCG of uterine RLH remained constant during the estrous cycle (about 0.8 x IO11 ivr l ) and was nearly identical to that of rat ovarian receptors. On metestrus, RLH concentration appeared to be approximately 35-fold lower in the uterus than in the ovaries when expressed per mg protein (1.50 ± 0.15 vs. 52.83 ± 3.61 fmol/mg protein) but only 20 times lower when expressed per organ (2.2 vs. 48.3 fmol/organ). The estrous cycle-related changes of uterine RLH concentration, together with our data establishing an in vitro influence of hCG on progesterone metabolism in rat uterus, suggest that some uterine functions could be directly regulated either by LH from the pituitary or during early pregnancy by an LH-like substance originating from the embryo. (Endocrinology 126: 1264-1269, 1990)
T
HERE IS increasing evidence for the existence of extraovarian LH/human CG (hCG) receptor sites in mammalian females. In the pig (1) and, more recently, in the rabbit (2) uterine high affinity LH/hCG binding sites (RLH) have been detected. The changes of their concentrations according to the hormonal status of the animal suggest their possible involvement in reproductive processes. But although a regulatory role in myometrium relaxation and uterine blood flow has been suggested, there is no decisive demonstration of any direct effect of LH/hCG upon uterine tissues. We have recently shown that hCG is able to modulate in vitro the progesterone content of the metestrus rat uterus via a cAMP-dependent mechanism (3). This effect was specific for the metestrus stage of the estrous cycle since no effect was observed in proestrus and estrus rat uteri. We concluded that 1) hCG should act upon metestrus uterus after binding to specific receptors and 2) the absence of RLH in proestrus and estrus uteri could have explained the inability of hCG to modulate progesterone content at these phases of the estrous cycle.
Received August 4,1989. *Supported by a fellowship from the C.N.R.S. and the Conseil Regional de Basse Normandie; to whom all correspondence and reprint requests should be addressed.
In this study, we have established the existence of high affinity LH/hCG binding sites in the rat uterus and measured their concentration throughout the estrous cycle.
Materials and Methods Hormones Purified hCG (CR-121: 13,450 IU/mg) was provided by the National Hormone and Pituitary Program (NIDDK). Ovine LH (oLH-26), ovine FSH (oFSH-16), and ovine PRL (oPRL18) were furnished by the NIDDK. Commercial preparation of hCG (Pregnyl, 3,500-4,000 IU/mg) was supplied by Organon (Saint Denis, France). Animals Three- to four-month-old Sprague-Dawley female rats were kept on a schedule of 14 h of light, 10 h of darkness (lights on from 0500 h to 1900 h) with food and water ad libitum. Vaginal smears were recorded 6 days a week, and rats were used only after exhibiting at least two consecutive 4-day estrous cycles. Rats were killed by cervical dislocation between 1000 h and 1500 h after staging. Preparation of crude membrane fractions Uteri of rats were removed free of the oviducts and immediately placed in NaCl 0.9% (wt/vol), trimmed of connective
1264
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LH/hCG RECEPTORS IN RAT UTERUS tissue, weighed and slit longitudinally before rinsing in NaCl solution. Cell membrane fractions were obtained using the procedure described by Ziecick et al. (1) with some modifications. Briefly, after mincing with scissors, tissues were homogenized at 4 C with a Teflon-glass potter in THS buffer (25 mM Tris HC1, pH 7.4, containing 0.25 M sucrose, 1 ml/uterus) by four 10-sec bursts separated by cooling periods of 30 sec. The homogenate was centrifuged for 30 min at 500 x g at 4 C. The resulting pellet was resuspended in THS buffer (1 ml/uterus), submitted again to homogenization, and centrifuged as described above. The pooled supernatants were centrifuged further at 4 C for 1 h at 25,000 X g. The crude membrane pellet was resuspended in cold THM buffer. A fraction of the membrane preparation was assayed for protein determination by the method of Bradford (4). Then 9 volumes of the membrane preparation were mixed with 1 volume of THM buffer containing 1% BSA. The resulting suspension was used immediately for the measurement of LH/hCG receptor concentration or stored at —80 C for further experimentation. Iodination of hCG
hCG (CR-121) was labeled using the chloramide l,3,4,6-tetrachloro-3a,6a-diphenylglycouryl (Iodogen, Pierce, Rockford, IL) as previously described (5). Na-125I was furnished by Amersham (France). Separation of bound and free 125I was performed by chromatography on Sephadex G-50 (Pharmacia, Uppsala, Sweden) column (1 x 10 cm). Specific activities of labeled hCG were determined by self-displacement analysis on ovarian membrane preparations in the radioligand receptor assay (6), and varied from 29,000 to 45,000 cpm/ng. Determination of incubation conditions
1265
aspiration and 2 ml ice-cold incubation buffer were added. After centrifugation, and removal of the supernatants, the membrane pellets were counted for 125I in a Cobra Auto Gamma (Packard, France) with a counting efficiency of 75%. Nonspecific binding determined in the presence of 200 IU hCG Pregnyl was usually less than 2% of the total radioactivity added. Determination of binding characteristics of uterine LH/hCG receptor The concentration and affinity constant (Ka) of unoccupied binding sites were determined by Scatchard plot analysis (7). Six or seven subsaturing doses of [125I]hCG (in the range of 1,000-50,000 cpm) were used in duplicate or triplicate with each membrane preparation. The hormonal specificity of [125I]hCG binding on uterine membranes was determined by incubating samples from metestrus rats with a subsaturating dose of [125I] hCG (25,000 cpm) in the presence of increasing amounts of various nonradioactive hormones. Cross-reactivity was calculated from the relative amount of the tested hormone that inhibited the[125I]hCG binding by 50%. Organ specificity of the LH/hCG binding LH/hCG binding measurements were attempted with tissues from other organs of metestrus rats. Membrane fractions from ovary, lung, kidney, skeletal muscle, heart, and liver were prepared as described above for uterus. [125I]hCG binding to these membranes was determined by incubating 50 ng (ovary) to 1 mg (other organs) of membrane proteins in the presence of increasing amounts of [125I]hCG (1,000-50,0000 cpm) with or without 200 IU hCG. Statistics
125
The membrane suspension was incubated with [ I] hCG in glass tubes. The incubation volume was 0.5 ml and consisted of 0.1 ml of various amounts of [125I]hCG (1,000-50,000 cpm) in THM 0.1% BSA (incubation buffer), 0.3 ml of membrane preparation, (100-900 ng protein) in incubation buffer and 0.1 ml of THM (25 M Tris-HCl, pH 7.2, containing 5 mM MgCl2) and 0.1 % BSA (for measurement of total binding) or 0.1 ml THM and 0.1% BSA containing 200 IU Pregnyl (for nonspecific binding measurement). To determine the time and temperature dependence of specific [125I]hCG binding to uterine membranes, samples obtained from metestrus rats were incubated from 2 to 30 h at 4, 20, and 37 C in the presence of subsaturating dose of [125I]hCG. After this study was completed, 24-h incubation at room temperature (20 C) was chosen for subsequent assays. To separate receptor-bound from free [125I]hCG, 3 ml ice-cold incubation buffer were added at the end of the incubation period and the tubes were centrifuged at 3,000 X g for 10 min at 0 C. The supernatant was removed by
Values for the Ka and the total number of binding sites were determined by a computerized least squares fit of the Scatchard plot. Results are presented as mean + SEM. One-way analysis of variance (ANOVA) was used for statistical significance of differences.
Results Validation of the LH/hCG binding assay The specific binding of [125I]hCG to metestrus rat uterine receptors was time and temperature dependent (Fig. 1). A maximal level of specific binding was reached for 37 C and 20 C, respectively, after 12 and 24 h. The results demonstrated that LH/hCG binding to uterine membranes was a saturable process. For subsequent determinations, we considered that apparent equilibrium was reached after 24-h incubation at room temperature (20 C). The amount of [125I]hCG bound was linear with the amount of membrane suspension in the range of 0.1-1 mg of protein (r = 0.96, data not shown). Scatchard analysis of the data obtained at equilibrium
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 04:30 For personal use only. No other uses without permission. . All rights reserved.
1266
LH/hCG RECEPTORS IN RAT UTERUS
O.1Ch
Endo • 1990 Voll26«No 2
Ka, ( 0.1 mg protein ) = 1.25x10* I M " 1 Ka 2 ( 0.2 mg protein ) = 0.88X101 'NT 1 Ka 3 ( 0.5 mg protein ) = l.
£ 0.05F
INCUBATION TIME ( HOURS )
FIG. 1. Time and temperature dependence of specific ( ) and nonspecific ( ) [125I]hCG binding to uterine membranes of metestrus rats in a representative experiment. Crude membrane preparation (800 Mg protein) was incubated with 40,000 cpm [125I]hCG at 4 C (A), 20 C (•), and 37 C (•). Means of duplicates are shown.
gave for metestrus uterine samples (six to eight pooled uteri for each Ka determination) high values of affinity constant (Ka in the range of 0.41-1.4 lO11]^"1) and low binding capacities (0.88-2.48 fmol/mg protein) (Fig. 2). The specificity of [125I]hCG binding to metestrus rat uterine membrane preparation was then investigated (Fig. 3). oLH as well as hCG could completely inhibit the binding of [125I]hCG. There was little competition between hCG and ovine FSH (oFSH) (0.05%) and no competition between hCG and ovine PRL (oPRL). The specificity of [125I]hCG binding to metestrus uterine membranes was similar to that obtained with a membrane preparation from metestrus ovary (data not shown). The concentration of specific [125I]hCG binding sites in various metestrus rat tissues as compared to the uterus is shown in Fig. 4. There was no measurable specific binding of [125I]hCG on five different tissues other than the ovary.
0.3
O.9
O.6
hCG BOUND(pM) FIG. 2. Estimation of the number and affinity constant (Ka) of LH/ hCG binding sites in three different amounts of crude membrane from three different preparations of metestrus rat uteri. Means of duplicates or triplicates are shown. B/F, bound to free ratio. 100-r oPRL
oFSH
0.1
1
10
100
1000
10000
HORMONE(NG )
Affinity constant and capacity of uterine LH/hCG binding sites throughout the estrous cycle Uterine concentration of LH/hCG binding sites varied significantly during the estrous cycle (Fig. 5). Concentration of LH/hCG receptors in metestrus uteri (1.50 ± 0.15 fmol/mg protein) was significantly higher (P < 0.01) than those in estrus (0.53 ± 0.19) and in diestrus uteri (0.70 ± 0.05). Moreover LH/hCG receptors were found in all preparations (n = 10) of metestrus uteri but not in all uterine preparations from other stages of the estrous
FlG. 3. Hormonal specificity of [125I]hCG binding to rat uterine crude membrane preparation. Crude membrane preparation (450 ^g protein) was incubated with 25,000 cpm [125I]hCG in the presence of increasing amounts of hormones. Means of duplicates are shown.
cycle. Positive determinations (e.g. LH/hCG receptor concentrations equal to or higher than 0.2 fmol/mg protein, which was the limit of sensitivity of our assay) were found in 3 out of 4 preparations from estrus, 5 out of 7 from diestrus and only 1 out of 7 from proestrus. However, only this later proportion differed from the others
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LH/hCG RECEPTORS IN RAT UTERUS
1267
100
HI
1-
