FERTILITY AND SrEruLrry Copyright
@
Vol. 31, No.3, March 1979 Printed in U.s A.
1979 The American Fertility Society
PROLACTIN RECEPTORS IN THE OVARY*
ALFRED N. POINDEXTER, M.D., F.A.C.O.G.t:I: VEASY C. BUTTRAM, JR., M.D., F.A.C.O.G. PAIGE K. BESCH, PH.D. ROY G. SMITH, PH.D.
Department of Obstetrics and Gynecology and Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
The binding of prolactin (PRL) to the plasma membranes of bovine and human ovaries was investigated using both homologous and heterologous 125I-prolactin. Saturation and Scatchard analysis demonstrated that human prolactin binds to human ovarian membranes with a Kd of 2 X 10-10 M; to bovine ovarian membranes with a Kd of 1.9 X 10-10 M; and to bovine corpora lutea membranes with a Kd of 1.9 X 10-10 M. The concentrations of binding sites in bovine and human ovaries were 2.9 x 10-15 moleslmg of protein and 2.0 x 10-15 moleslmg of protein, respectively. The number of bindings sites in the bovine corpora lutea was 1.5 x 15-15 moleslmg of protein. Specificity studies with bovine PRL, ovine PRL, human luteinizing hormone, human follicle-stimulating hormone, and bovine growth hormone showed this binding to be specific. Comparison of binding of PRL to membranes of other target and nontarget tissues suggests that the ovary is one of the primary target tissues. These data suggest that prolactin plays a role in the ovarian cycle. Fertil Steril31:273, 1979
In 1938 Schooley and'Riddle l demonstrated that basophilic activity of the adenohypophysis increased with copulatory stimulation in the common pigeon and ring dove. From these data and from subsequent experiments prolactin was named "luteotrophic hormone" (LTH). However, since further investigations showed that maintenance of the corpus luteum by prolactin could not be demonstrated, the term LTH was discarded. More recently, primarily beca use of the availability of relatively pure prolactin and the
advent of a highly sensitive radioimmunoassay, the role of prolactin in reproduction has again been studied. Serum prolactin levels have now been measured in humans during the ovarian cycle. 2•s Apparently no significant change occurs in the serum concentration of immunoreactive prolactin during the follicular phase as opposed to the luteal phase of the cycle. This implies that prolactin plays no role in the human ovarian cycle. However, McNatty et al. 6 have measured prolactin and progesterone concentrations in follicular fluid and have shown that the prolactin concentration varies inversely to the progesterone concentration. The conclusion drawn from this more recent study is that prolactin may indeed play some role in the ovarian cycle. To investigate further the role of prolactin in the ovary, the molecular physiology must be investigated. Utilization of hormone receptor methodology may lead to a better understanding of the role of prolactin at the molecular level. It was the pur-
Received September 11,1978; revised October 23,1978; accepted October 27, 1978. *Supported in part by research funds from St. Luke's Episcopal Hospital and the Reproductive Research Laboratory Fund. tResearch completed during the tenure of a Postdoctoral Fellowship in Reproductive Endocrinology, Department of Obstetrics and Gynecology, Baylor College of Medicine. tReprint requests: A. N. Poindexter, M.D., Department of Obstetrics and Gynecology, Baylor College of Medicine.
273
274
POINDEXTER ET AL.
pose of this investigation to determine whether there is significant, specific binding of prolactin to receptors in the ovary. MATERIALS AND METHODS
Preparation of Labeled Prolactin. Human prolactin (VLS-3) and bovine prolactin (NIH-P-B4) were obtained from the National Pituitary Agency, University of Maryland School of Medicine, Baltimore, Md., and The National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Md. All other peptide hormones used in this study were obtained from the same agencies. Prolactin was iodinated by the lactoperoxidase method. 7 This was accomplished by incubating 10 JLg of prolactin with 5 mCi of1 25INa in the presence of 150 JLg of hydrogen peroxide and 5 JLg of lactoperoxidase for 15 to 30 seconds at 37° C. The iodinated prolactin was separated from the free iodine by column chromatography using Sephadex G-100 in a 60 x 1 cm column. Fractions of 0.75 ml were collected. Three peaks were obtained. The first peak, consisting of aggregated prolactin and lactoperoxidase, 8 was discarded. 125I-prolactin in the second peak was considered to be biologically active in that it could be displaced from the receptor by unlabeled prolactin. The third peak was unbound radioactive iodine. The specific activities routinely obtained varied from 50 to 100 JLCi/JLg, as determined by trichloroacetic acid precipitation. 8 Preparation of Plasma Membrane. Bovine tissues were obtained from cows in reproductive years. Hum~n ovaries were obtained from hysterectomy specimens from premenopausal women. Bovine and human ovaries were dissected; corpus luteum was separated from the ovary. This ovarian tissue, which included stroma and follicles, is referred to as "ovarian tissue" to distinguish it from the corpus luteum. Membranes were obtamed by using a procedure similar to that described by Shiu et aJ.9 All tissue obtained for receptor assays was immediately placed in iced saline until further preparation could be carried out. At 4°C tissues were prepared by mincing with scissors and then by adding 5 parts of buffer A (10 mM Tris-10 mM calcium-0.3 M sucrose, pH 7.5) per gram of tissue. The minced tissue was homogenized briefly, first with a Brinkman Polytron and then with a Teflon-glass homogenizer. The tissue preparation was centrifuged at 13,000 x g for 30 minutes. The pellet, which contained debris, nuclei, and mitochondria,
March 1979
was discarded. The supernatant was then centrifuged at 105,000 x g for 90 minutes. The pellet, containing the plasma membranes, was resuspended in 5 volumes of buffer B (10 mM Tris-10 mM calcium, pH 7.5) and frozen at -80° C until assayed. Protein analysis, using the method of Lowry et al.,10 was carried out on membranes to determine the protein concentration. Incubation of 125I-Prolactin with Plasma Membranes. Plasma membrane protein (500 JLg) was incubated in Siliclad (Clay Adams, Parsippany, N. J.)-treated glass tubes with 125I-Iabeled prolactin in a 37° C metabolic shaker water bath for 6 hours. Each tube contained 400 JLg of bovine serum albumin and buffer B to give a total volume of 0.8 ml. Tubes with unlabeled prolactin or other unlabeled hormones were used for specific binding studies. Unlabeled hormones were added to the incubation in a 400- or 1OOO-fold excess over the labeled prolactin. All assays were carried out in triplicate. The incubation was stopped by adding 3 ml of cold buffer B followed by centrifugation at 1000 x g at 4° C for 30 minutes to separate membrane-bound prolactin from unbound prolactin. The supernatant was discarded. The pellet was counted in a Packard Autogamma counter. Affinity constants and the concentration of high-affinity binding sites were determined by the method of Scatchard. 11 Specific binding was measured by subtracting nonspecific binding from total binding. RESULTS AND DISCUSSION
Plasma membrane fractions isolated from bovine mammary gland, corpus luteum, ovary, pancreas, skeletal muscle, uterus, liver, and kidney were incubated with 125I-prolactin, and the numbers of binding sites specific for prolactin were determined. Human prolactin was used in preference to bovine prolactin for the binding studies, since preliminary displacement studies indicated that human prolactin was biologically more pure than the native hormone. Figure 1 shows that the mammary gland, which is a known target tissue for prolactin, contains the highest concentration of binding sites. Most important, both the corpus luteum and the ovary also contained high concentrations of binding sites as compared with pancreas, skeletal muscle, uterus, liver, and kidney. These data suggest that the ovarian tissues are target organs for prolactin. To determine whether the observed binding to ovarian tissue membranes is saturable, a constant amount of plasma membrane fractions was incu-
z
30
6.0
-
w
I
@
Vol. 31, No.3, March 1979 Printed in U.s A.
1979 The American Fertility Society
PROLACTIN RECEPTORS IN THE OVARY*
ALFRED N. POINDEXTER, M.D., F.A.C.O.G.t:I: VEASY C. BUTTRAM, JR., M.D., F.A.C.O.G. PAIGE K. BESCH, PH.D. ROY G. SMITH, PH.D.
Department of Obstetrics and Gynecology and Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
The binding of prolactin (PRL) to the plasma membranes of bovine and human ovaries was investigated using both homologous and heterologous 125I-prolactin. Saturation and Scatchard analysis demonstrated that human prolactin binds to human ovarian membranes with a Kd of 2 X 10-10 M; to bovine ovarian membranes with a Kd of 1.9 X 10-10 M; and to bovine corpora lutea membranes with a Kd of 1.9 X 10-10 M. The concentrations of binding sites in bovine and human ovaries were 2.9 x 10-15 moleslmg of protein and 2.0 x 10-15 moleslmg of protein, respectively. The number of bindings sites in the bovine corpora lutea was 1.5 x 15-15 moleslmg of protein. Specificity studies with bovine PRL, ovine PRL, human luteinizing hormone, human follicle-stimulating hormone, and bovine growth hormone showed this binding to be specific. Comparison of binding of PRL to membranes of other target and nontarget tissues suggests that the ovary is one of the primary target tissues. These data suggest that prolactin plays a role in the ovarian cycle. Fertil Steril31:273, 1979
In 1938 Schooley and'Riddle l demonstrated that basophilic activity of the adenohypophysis increased with copulatory stimulation in the common pigeon and ring dove. From these data and from subsequent experiments prolactin was named "luteotrophic hormone" (LTH). However, since further investigations showed that maintenance of the corpus luteum by prolactin could not be demonstrated, the term LTH was discarded. More recently, primarily beca use of the availability of relatively pure prolactin and the
advent of a highly sensitive radioimmunoassay, the role of prolactin in reproduction has again been studied. Serum prolactin levels have now been measured in humans during the ovarian cycle. 2•s Apparently no significant change occurs in the serum concentration of immunoreactive prolactin during the follicular phase as opposed to the luteal phase of the cycle. This implies that prolactin plays no role in the human ovarian cycle. However, McNatty et al. 6 have measured prolactin and progesterone concentrations in follicular fluid and have shown that the prolactin concentration varies inversely to the progesterone concentration. The conclusion drawn from this more recent study is that prolactin may indeed play some role in the ovarian cycle. To investigate further the role of prolactin in the ovary, the molecular physiology must be investigated. Utilization of hormone receptor methodology may lead to a better understanding of the role of prolactin at the molecular level. It was the pur-
Received September 11,1978; revised October 23,1978; accepted October 27, 1978. *Supported in part by research funds from St. Luke's Episcopal Hospital and the Reproductive Research Laboratory Fund. tResearch completed during the tenure of a Postdoctoral Fellowship in Reproductive Endocrinology, Department of Obstetrics and Gynecology, Baylor College of Medicine. tReprint requests: A. N. Poindexter, M.D., Department of Obstetrics and Gynecology, Baylor College of Medicine.
273
274
POINDEXTER ET AL.
pose of this investigation to determine whether there is significant, specific binding of prolactin to receptors in the ovary. MATERIALS AND METHODS
Preparation of Labeled Prolactin. Human prolactin (VLS-3) and bovine prolactin (NIH-P-B4) were obtained from the National Pituitary Agency, University of Maryland School of Medicine, Baltimore, Md., and The National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Md. All other peptide hormones used in this study were obtained from the same agencies. Prolactin was iodinated by the lactoperoxidase method. 7 This was accomplished by incubating 10 JLg of prolactin with 5 mCi of1 25INa in the presence of 150 JLg of hydrogen peroxide and 5 JLg of lactoperoxidase for 15 to 30 seconds at 37° C. The iodinated prolactin was separated from the free iodine by column chromatography using Sephadex G-100 in a 60 x 1 cm column. Fractions of 0.75 ml were collected. Three peaks were obtained. The first peak, consisting of aggregated prolactin and lactoperoxidase, 8 was discarded. 125I-prolactin in the second peak was considered to be biologically active in that it could be displaced from the receptor by unlabeled prolactin. The third peak was unbound radioactive iodine. The specific activities routinely obtained varied from 50 to 100 JLCi/JLg, as determined by trichloroacetic acid precipitation. 8 Preparation of Plasma Membrane. Bovine tissues were obtained from cows in reproductive years. Hum~n ovaries were obtained from hysterectomy specimens from premenopausal women. Bovine and human ovaries were dissected; corpus luteum was separated from the ovary. This ovarian tissue, which included stroma and follicles, is referred to as "ovarian tissue" to distinguish it from the corpus luteum. Membranes were obtamed by using a procedure similar to that described by Shiu et aJ.9 All tissue obtained for receptor assays was immediately placed in iced saline until further preparation could be carried out. At 4°C tissues were prepared by mincing with scissors and then by adding 5 parts of buffer A (10 mM Tris-10 mM calcium-0.3 M sucrose, pH 7.5) per gram of tissue. The minced tissue was homogenized briefly, first with a Brinkman Polytron and then with a Teflon-glass homogenizer. The tissue preparation was centrifuged at 13,000 x g for 30 minutes. The pellet, which contained debris, nuclei, and mitochondria,
March 1979
was discarded. The supernatant was then centrifuged at 105,000 x g for 90 minutes. The pellet, containing the plasma membranes, was resuspended in 5 volumes of buffer B (10 mM Tris-10 mM calcium, pH 7.5) and frozen at -80° C until assayed. Protein analysis, using the method of Lowry et al.,10 was carried out on membranes to determine the protein concentration. Incubation of 125I-Prolactin with Plasma Membranes. Plasma membrane protein (500 JLg) was incubated in Siliclad (Clay Adams, Parsippany, N. J.)-treated glass tubes with 125I-Iabeled prolactin in a 37° C metabolic shaker water bath for 6 hours. Each tube contained 400 JLg of bovine serum albumin and buffer B to give a total volume of 0.8 ml. Tubes with unlabeled prolactin or other unlabeled hormones were used for specific binding studies. Unlabeled hormones were added to the incubation in a 400- or 1OOO-fold excess over the labeled prolactin. All assays were carried out in triplicate. The incubation was stopped by adding 3 ml of cold buffer B followed by centrifugation at 1000 x g at 4° C for 30 minutes to separate membrane-bound prolactin from unbound prolactin. The supernatant was discarded. The pellet was counted in a Packard Autogamma counter. Affinity constants and the concentration of high-affinity binding sites were determined by the method of Scatchard. 11 Specific binding was measured by subtracting nonspecific binding from total binding. RESULTS AND DISCUSSION
Plasma membrane fractions isolated from bovine mammary gland, corpus luteum, ovary, pancreas, skeletal muscle, uterus, liver, and kidney were incubated with 125I-prolactin, and the numbers of binding sites specific for prolactin were determined. Human prolactin was used in preference to bovine prolactin for the binding studies, since preliminary displacement studies indicated that human prolactin was biologically more pure than the native hormone. Figure 1 shows that the mammary gland, which is a known target tissue for prolactin, contains the highest concentration of binding sites. Most important, both the corpus luteum and the ovary also contained high concentrations of binding sites as compared with pancreas, skeletal muscle, uterus, liver, and kidney. These data suggest that the ovarian tissues are target organs for prolactin. To determine whether the observed binding to ovarian tissue membranes is saturable, a constant amount of plasma membrane fractions was incu-
z
30
6.0
-
w
I
