Neuroendocrinology 26: 118-128 (1978)
Luteinizing Hormone Secretion and Gonadotropin Releasing Hormone Binding in Heifer and Steer Anterior Pituitaries J.C. Z olm an and L.J. V alenta Department of Medicine, University of California at Irvine, Orange, Calif.
Key Words. LH • GnRH • Heifer • Steer • Anterior pituitary Abstract. Luteinizing hormone (LH) release and tissue content were studied in explanted bovine pituitaries from heifer and steer during superfusion in vitro. Compared to steer, heifer pituitaries contained significantly more LH and released more LH into the incubation medium. Quantitative response in LH release to gonadotropin releasing hor mone (GnRH) stimulation was about equal in heifer and steer, but relative increase was more pronounced in steer. Specific 125I-GnRH binding to dispersed anterior pituitary (AP) cells and isolated plasma membranes was also analyzed. Concentration dependent binding curve of 125I-GnRH to dispersed cells exhibited sigmoid characteristics suggesting positive cooperativity. There was a sex dependent difference, the binding curve for heifer being shifted to the left and reaching saturation at lower GnRH concentrations than that for steer. ‘Multisigmoid’ concentration dependent GnRH binding curves were obtained using isolated plasma membranes, revealing the presence of multiple binding sites. One of the binding sites, present in heifer, was absent in the steer derived materials, but could be induced by pretreatment of the steer plasma membranes with 17-/3 estradiol as low as 50 pg/ml. It is concluded that the differences in GnRH binding may be responsible, at least in part, for the observed differences in LH secretion pattern between heifer and steer.
Received: August 2nd, 1977; revised MS accepted: January 10th, 1978.
Downloaded by: Boston University 128.197.229.194 - 1/14/2019 11:03:35 AM
There are striking differences in the secretion of gonadotropin between sexes which are manifested especially by the cyclic changes in gonadotropins in females. In addition, in the female the responsiveness of luteinizing (LH) and follicle stimulating hormone (FSH) to gonadotropin releasing hormone (GnRH) varies during the reproductive cycle in both humans [Y en et al., 1972] and animals [Z olm an e t al., 1974], This response is most pronounced in the preovulatory phase when the plasma estrogen levels are also high.
ZOLMAN/V ALENTA
119
Similarly, the responsiveness of the pituitary to GnRH may be augmented by pretreatment with exogenous estrogen [A rim u ra and S c h a l l y , 1971; D e b e u u k et al., 1972], On the other hand, estrogen may also suppress pi tuitary responsiveness to GnRH under certain conditions [N eg ro -V ila r et al., 1973], However, no unanimity exists today as to whether the negative feedback mechanism of estrogens on gonadotropin secretion involves only the hypothalamus or anterior pituitary (AP) as well. Estrogen pretreatment may modify not only the secretion of gonadotro pins but other pituitary hormones as well, such as prolactin or thyroid stim ulating hormone [K a l r a et al., 1973]. The nature of action of the estrogen on feedback mechanisms is unknown. In the present work we describe data suggesting that, at least in part, this may be due to the modulation of binding of GRH to the GnRH receptor substance in the anterior pituitary. Estrogen dependent modulation of GnRH binding has also been described recently by S axena [1977],
Materials and Methods
Specific binding o f GnRH. Specific binding of l25I-GnRH was studied using dispersed AP cells or isolated plasma membranes. Dispersed cells were obtained by treatment of the bovine AP tissue (approximately 200 mg/ml) with 0.2% collagenase, 0.15% hyaluronidase, and 0.02% soybean trypsin inhibitor in TC 199, at 37°C for 1 h in a DubnofT me tabolic shaker. The mixture was then filtered through cheesecloth to remove the debris and
Downloaded by: Boston University 128.197.229.194 - 1/14/2019 11:03:35 AM
L H secretion in vitro. The method of an in vitro superfusion of bovine AP tissue slices was described and validated earlier [Zolman and C onvey, 1972], Briefly, bovine APs from steer and heifer were obtained in the abbattoir. They were immersed in the tissue culture medium (TC 199) at 37 °C, transported to the laboratory and used within 2 h. The AP tissue was sliced into ribbons 1 x 1 x 8 mm and randomly distributed in the incubation chambers. Approximately 60 mg tissue was incubated in vitro per chamber of a decastaltic pump, and perfused by oxygenated TC 199 containing the tested substances. The experi mental conditions were reproduced in 4 parallel channels of the pump. The perfusion rate was 0.5 ml/min. The effluent (fractions of about 1 ml) was collected at time intervals as indicated. LH was measured in the collected fractions and pituitary tissue by a specific bovine LH radioimmunoassay [Zolman and C onvey, 1972]. Stimulated LH release was studied after exposure of the tissue to synthetic GnRH. Following 2 h preincubation, the tissue was exposed for 20 min to 4 x 10- 9 m GnRH. LH concentration of the effluent was monitored during the whole incubation period. The method of analysis of variance of various degree of complexity [G ill et al., 1971] was used to analyze the LH secretion data. The effect of 17-/3 estradiol (E2) was studied by addition of E2 to the medium, in con centration of 10 pg, 50 pg, 10 ng and 500 ng per ml.
120
Z o l m a n /V a l e n t a
undigested tissue. Isolated cells were collected by centrifugation at 70 for 5 min and washed several times to remove the enzymes. The final suspension in TC 199 was counted for the number of cells in a counting chamber. The number of viable cells was determined by intravital staining. More than 90% of the cells were viable. The suspension was diluted to obtain about 10s cells per ml. Plasma membranes were isolated from the tissue homogenate (Waring Blender, high speed, 30 sec), as described [Poirier et a!., 1974], Briefly, homogenization was in 20 volumes (v/w) of 1 mM bicarbonate - 5 him /2-mercaptoethanol buffer pH 7.4. The ho mogenate was centrifuged at 2,000 x g for 30 min. The pellet was resuspended in 10 volumes of the buffer and centrifuged at 100 for 30 min. The supernatant was pelleted at 30,000 for 30 min. The top brownish layer of the pellet was collected and fractionated on a discontinuous sucrose gradient (densities of 1.14, 1.16, 1.18, 1.20, and 1.22). Centrif ugation was for 2 h at 120,000 xg, at 4°C in the rotor 27 of a Spinco L 65 B centrifuge. Opalescent layers at the interphases were identified by electron microscopy and enzyme markers as, from the top to the bottom: (1) large membrane fragments (2) small membrane fragments (3) rough microsomes (4) smooth microsomes (5) secretory granules in the pellet. Plasma membranes, i.e. fractions 1 and 2, were pooled and used for the binding studies.
GnRH binding studies. The mixture for the binding assay contained 125I-GnRH of the order of 10-9 to I0 _1 om, and 105 dispersed cells or 7.5 fig protein (by L owry method) of the isolated plasma membranes, in 0.1 m Tris-HCl pH 7.4, final volume 1.0 ml. After incubation at 37 °C for 30 min, the bound and free hormone were separated by microfiltra tion on fiberglass filters (Whatman). Parallel samples contained 10- 7 m nonlabelled GnRH to assess nonsaturable (nonspecific) binding. This was a linear function of GnRH concen tration and was of the order of 10% total binding. The nonspecific binding was subtracted from the total binding and all data here illustrated indicate specific binding. Specificity of the binding was further demonstrated by the absence of specific binding in the unrelated plasma membrances (from human placenta), and by the inability of excess of unrelated hormones (10_ 7 m thyrotropin releasing hormone, somatostatin, Lys-vasopressin or oxy tocin) to inhibit I25I-GnRH binding.
Downloaded by: Boston University 128.197.229.194 - 1/14/2019 11:03:35 AM
GnRH labelling and biological activity. GnRH was labelled with 125I by a modified chloramine T technique, with 100 times less chloramine T than in the original method [H unter et al., 1962], and omitting metabisulfite. Equimolar contrations of 125I-iodide as N al were employed. Monoiodinated 125I-GnRH was mostly obtained and was purified by column chromatography on Biogel P-2, 0.7x30 cm. Its specific radioactivity was 1,500 Ci/mmole. Its biological activity was compared with that of the unlabelled hormone using stimulation of LH release in vitro as a biological response. This was done by exposing randomized bovine AP tissue slices in parallel channels of a 10 channel polystatic pump [Zolman and Convey, 1972] to equal concentrations of 125I-GnRH or nonlabelled GnRH. The effluent media were analyzed for the LH concentration by a specific radioimmuno assay. Duplicates were run for each hormone concentration, and the entire experiment was repeated 4 times. The results (fig. 1) represent mean ± SE of all values. It is obvious that there is no difference in LH release in response to equal concentrations of I25I-GnRH and unlabelled, native hormone and that the original and labelled hormone exhibit the same dose-response relationship.
Sex Differences in LH Release
121
Results
Fig. 1. Biological activity of GnRH, expressed as a dependence of the LH response front bovine AP slices in vitro on the dose of GnRH. The biological activity (LH release) of 125I-GnRH was tested in the superfusion in vitro system as described in Materials and Methods, and compared with equal concentrations of nonlabelled LnRH. Each column corresponds to the mean ( ± SE) of an in duplicate performed experiment, and represents total amount of LH liberated at 20 min period following GnRH stimulation. LH release occurs with as little as 4 x !0 - 1 om GnRH, and is compared here to the control (0, no GnRH). There is a dose-response relationship with increasing concentrations. It is obvious that I25I-GnRH had the same effect on LH release as nonlabelled GnRH.
Downloaded by: Boston University 128.197.229.194 - 1/14/2019 11:03:35 AM
LH Secretion in vitro In respect to pituitary content of LH and its release into the incubation medium, the following difference between heifer and steer derived tissues were observed: (1) The LH content of the pituitary tissue obtained from heifer was significantly (p < 0.01) higher than that from the steer (table I). (2) During the first 60 min of incubation of the heifer pituitaries, there was a phase of rapid release of LH into the incubation medium. During the same time period, the LH release rate from steer pituitary did not change signifi cantly (fig. 2). (3) At any time period, absolute amount of LH released from heifer pituitaries into the incubation medium was more than twice (p 0.05) and it may be assumed that during that phase LH release was steady. (5) LH release from both heifer and steer pituitary tissue was stimulated by GnRH. First, the tissue was preincubated in TC 199 alone, and then, when the LH release became stabilized, it was exposed to GnRH. Significant increase in
122
ZOLM AN/VALENTA
Table /. Bovine LH synthesis during superfusion in vitro Pituitary LH content (//g/mg)
Probability
AP source
120 min incubation
360 min incubation
Steer Heifer
0.868 1.147
0.871 1.442
NS 0.006
Randomized pituitary tissue slices from steer or heifer were superfused by TC 199 in vitro. Approximately Vi of each tissue sample was removed at time 120 min of incuba tion. The remaining tissue was incubated for another 240 min, i.e., total of 360 min. The tissue was homogenized and its LH content was measured by specific bovine LH radio immunoassay. The results are expressed in /
Luteinizing Hormone Secretion and Gonadotropin Releasing Hormone Binding in Heifer and Steer Anterior Pituitaries J.C. Z olm an and L.J. V alenta Department of Medicine, University of California at Irvine, Orange, Calif.
Key Words. LH • GnRH • Heifer • Steer • Anterior pituitary Abstract. Luteinizing hormone (LH) release and tissue content were studied in explanted bovine pituitaries from heifer and steer during superfusion in vitro. Compared to steer, heifer pituitaries contained significantly more LH and released more LH into the incubation medium. Quantitative response in LH release to gonadotropin releasing hor mone (GnRH) stimulation was about equal in heifer and steer, but relative increase was more pronounced in steer. Specific 125I-GnRH binding to dispersed anterior pituitary (AP) cells and isolated plasma membranes was also analyzed. Concentration dependent binding curve of 125I-GnRH to dispersed cells exhibited sigmoid characteristics suggesting positive cooperativity. There was a sex dependent difference, the binding curve for heifer being shifted to the left and reaching saturation at lower GnRH concentrations than that for steer. ‘Multisigmoid’ concentration dependent GnRH binding curves were obtained using isolated plasma membranes, revealing the presence of multiple binding sites. One of the binding sites, present in heifer, was absent in the steer derived materials, but could be induced by pretreatment of the steer plasma membranes with 17-/3 estradiol as low as 50 pg/ml. It is concluded that the differences in GnRH binding may be responsible, at least in part, for the observed differences in LH secretion pattern between heifer and steer.
Received: August 2nd, 1977; revised MS accepted: January 10th, 1978.
Downloaded by: Boston University 128.197.229.194 - 1/14/2019 11:03:35 AM
There are striking differences in the secretion of gonadotropin between sexes which are manifested especially by the cyclic changes in gonadotropins in females. In addition, in the female the responsiveness of luteinizing (LH) and follicle stimulating hormone (FSH) to gonadotropin releasing hormone (GnRH) varies during the reproductive cycle in both humans [Y en et al., 1972] and animals [Z olm an e t al., 1974], This response is most pronounced in the preovulatory phase when the plasma estrogen levels are also high.
ZOLMAN/V ALENTA
119
Similarly, the responsiveness of the pituitary to GnRH may be augmented by pretreatment with exogenous estrogen [A rim u ra and S c h a l l y , 1971; D e b e u u k et al., 1972], On the other hand, estrogen may also suppress pi tuitary responsiveness to GnRH under certain conditions [N eg ro -V ila r et al., 1973], However, no unanimity exists today as to whether the negative feedback mechanism of estrogens on gonadotropin secretion involves only the hypothalamus or anterior pituitary (AP) as well. Estrogen pretreatment may modify not only the secretion of gonadotro pins but other pituitary hormones as well, such as prolactin or thyroid stim ulating hormone [K a l r a et al., 1973]. The nature of action of the estrogen on feedback mechanisms is unknown. In the present work we describe data suggesting that, at least in part, this may be due to the modulation of binding of GRH to the GnRH receptor substance in the anterior pituitary. Estrogen dependent modulation of GnRH binding has also been described recently by S axena [1977],
Materials and Methods
Specific binding o f GnRH. Specific binding of l25I-GnRH was studied using dispersed AP cells or isolated plasma membranes. Dispersed cells were obtained by treatment of the bovine AP tissue (approximately 200 mg/ml) with 0.2% collagenase, 0.15% hyaluronidase, and 0.02% soybean trypsin inhibitor in TC 199, at 37°C for 1 h in a DubnofT me tabolic shaker. The mixture was then filtered through cheesecloth to remove the debris and
Downloaded by: Boston University 128.197.229.194 - 1/14/2019 11:03:35 AM
L H secretion in vitro. The method of an in vitro superfusion of bovine AP tissue slices was described and validated earlier [Zolman and C onvey, 1972], Briefly, bovine APs from steer and heifer were obtained in the abbattoir. They were immersed in the tissue culture medium (TC 199) at 37 °C, transported to the laboratory and used within 2 h. The AP tissue was sliced into ribbons 1 x 1 x 8 mm and randomly distributed in the incubation chambers. Approximately 60 mg tissue was incubated in vitro per chamber of a decastaltic pump, and perfused by oxygenated TC 199 containing the tested substances. The experi mental conditions were reproduced in 4 parallel channels of the pump. The perfusion rate was 0.5 ml/min. The effluent (fractions of about 1 ml) was collected at time intervals as indicated. LH was measured in the collected fractions and pituitary tissue by a specific bovine LH radioimmunoassay [Zolman and C onvey, 1972]. Stimulated LH release was studied after exposure of the tissue to synthetic GnRH. Following 2 h preincubation, the tissue was exposed for 20 min to 4 x 10- 9 m GnRH. LH concentration of the effluent was monitored during the whole incubation period. The method of analysis of variance of various degree of complexity [G ill et al., 1971] was used to analyze the LH secretion data. The effect of 17-/3 estradiol (E2) was studied by addition of E2 to the medium, in con centration of 10 pg, 50 pg, 10 ng and 500 ng per ml.
120
Z o l m a n /V a l e n t a
undigested tissue. Isolated cells were collected by centrifugation at 70 for 5 min and washed several times to remove the enzymes. The final suspension in TC 199 was counted for the number of cells in a counting chamber. The number of viable cells was determined by intravital staining. More than 90% of the cells were viable. The suspension was diluted to obtain about 10s cells per ml. Plasma membranes were isolated from the tissue homogenate (Waring Blender, high speed, 30 sec), as described [Poirier et a!., 1974], Briefly, homogenization was in 20 volumes (v/w) of 1 mM bicarbonate - 5 him /2-mercaptoethanol buffer pH 7.4. The ho mogenate was centrifuged at 2,000 x g for 30 min. The pellet was resuspended in 10 volumes of the buffer and centrifuged at 100 for 30 min. The supernatant was pelleted at 30,000 for 30 min. The top brownish layer of the pellet was collected and fractionated on a discontinuous sucrose gradient (densities of 1.14, 1.16, 1.18, 1.20, and 1.22). Centrif ugation was for 2 h at 120,000 xg, at 4°C in the rotor 27 of a Spinco L 65 B centrifuge. Opalescent layers at the interphases were identified by electron microscopy and enzyme markers as, from the top to the bottom: (1) large membrane fragments (2) small membrane fragments (3) rough microsomes (4) smooth microsomes (5) secretory granules in the pellet. Plasma membranes, i.e. fractions 1 and 2, were pooled and used for the binding studies.
GnRH binding studies. The mixture for the binding assay contained 125I-GnRH of the order of 10-9 to I0 _1 om, and 105 dispersed cells or 7.5 fig protein (by L owry method) of the isolated plasma membranes, in 0.1 m Tris-HCl pH 7.4, final volume 1.0 ml. After incubation at 37 °C for 30 min, the bound and free hormone were separated by microfiltra tion on fiberglass filters (Whatman). Parallel samples contained 10- 7 m nonlabelled GnRH to assess nonsaturable (nonspecific) binding. This was a linear function of GnRH concen tration and was of the order of 10% total binding. The nonspecific binding was subtracted from the total binding and all data here illustrated indicate specific binding. Specificity of the binding was further demonstrated by the absence of specific binding in the unrelated plasma membrances (from human placenta), and by the inability of excess of unrelated hormones (10_ 7 m thyrotropin releasing hormone, somatostatin, Lys-vasopressin or oxy tocin) to inhibit I25I-GnRH binding.
Downloaded by: Boston University 128.197.229.194 - 1/14/2019 11:03:35 AM
GnRH labelling and biological activity. GnRH was labelled with 125I by a modified chloramine T technique, with 100 times less chloramine T than in the original method [H unter et al., 1962], and omitting metabisulfite. Equimolar contrations of 125I-iodide as N al were employed. Monoiodinated 125I-GnRH was mostly obtained and was purified by column chromatography on Biogel P-2, 0.7x30 cm. Its specific radioactivity was 1,500 Ci/mmole. Its biological activity was compared with that of the unlabelled hormone using stimulation of LH release in vitro as a biological response. This was done by exposing randomized bovine AP tissue slices in parallel channels of a 10 channel polystatic pump [Zolman and Convey, 1972] to equal concentrations of 125I-GnRH or nonlabelled GnRH. The effluent media were analyzed for the LH concentration by a specific radioimmuno assay. Duplicates were run for each hormone concentration, and the entire experiment was repeated 4 times. The results (fig. 1) represent mean ± SE of all values. It is obvious that there is no difference in LH release in response to equal concentrations of I25I-GnRH and unlabelled, native hormone and that the original and labelled hormone exhibit the same dose-response relationship.
Sex Differences in LH Release
121
Results
Fig. 1. Biological activity of GnRH, expressed as a dependence of the LH response front bovine AP slices in vitro on the dose of GnRH. The biological activity (LH release) of 125I-GnRH was tested in the superfusion in vitro system as described in Materials and Methods, and compared with equal concentrations of nonlabelled LnRH. Each column corresponds to the mean ( ± SE) of an in duplicate performed experiment, and represents total amount of LH liberated at 20 min period following GnRH stimulation. LH release occurs with as little as 4 x !0 - 1 om GnRH, and is compared here to the control (0, no GnRH). There is a dose-response relationship with increasing concentrations. It is obvious that I25I-GnRH had the same effect on LH release as nonlabelled GnRH.
Downloaded by: Boston University 128.197.229.194 - 1/14/2019 11:03:35 AM
LH Secretion in vitro In respect to pituitary content of LH and its release into the incubation medium, the following difference between heifer and steer derived tissues were observed: (1) The LH content of the pituitary tissue obtained from heifer was significantly (p < 0.01) higher than that from the steer (table I). (2) During the first 60 min of incubation of the heifer pituitaries, there was a phase of rapid release of LH into the incubation medium. During the same time period, the LH release rate from steer pituitary did not change signifi cantly (fig. 2). (3) At any time period, absolute amount of LH released from heifer pituitaries into the incubation medium was more than twice (p 0.05) and it may be assumed that during that phase LH release was steady. (5) LH release from both heifer and steer pituitary tissue was stimulated by GnRH. First, the tissue was preincubated in TC 199 alone, and then, when the LH release became stabilized, it was exposed to GnRH. Significant increase in
122
ZOLM AN/VALENTA
Table /. Bovine LH synthesis during superfusion in vitro Pituitary LH content (//g/mg)
Probability
AP source
120 min incubation
360 min incubation
Steer Heifer
0.868 1.147
0.871 1.442
NS 0.006
Randomized pituitary tissue slices from steer or heifer were superfused by TC 199 in vitro. Approximately Vi of each tissue sample was removed at time 120 min of incuba tion. The remaining tissue was incubated for another 240 min, i.e., total of 360 min. The tissue was homogenized and its LH content was measured by specific bovine LH radio immunoassay. The results are expressed in /
