Dissociation between pituitary GnRH binding sites and LH response to GnRH in vitro

Molecular and Cellular Endocrinology,

Ekvier

191

48 (1986) 191-197

Scientific Publishers Ireland, Ltd.

MCE 01560

Dissociation between pituitary GnRH binding sites and LH response to GnRH in vitro S.D. Abbot, S.I. Naik and R.N. Clayton * Department of Medieine, University

of Birmingham Queen Elizabeth Hospital, Birmingham (U.K.J

(Received 12 May 1986; accepted 29 July 1986)

Key worak

GuRH; pituitary; GnRH binding sites; LH responses.

Summary Experiments were performed to study gonadotroph responsiveness to gonadotrophin releasing hormone (GnRH) in vitro in dispersed pituitary cells from ovariectomised rats and mice when GnRH binding sites were increased or reduced, respectively. Maximal/basal LH release after GnRH treatment of intact female rat pituitary cells was 4.7 to 9.7-fold (range n = 3 expts.) compared to 3.4 to 5.0-fold for cells from ovariectomised rat donors. Both basal and maximal GnRH-stimulated LH release from ovariectomised (OVX) rat pituitary cells were 1.5 to 3-fold greater than from intact rat cells, which corresponded to increased LH content of the cells. There was no significant change in the GnRH EDso concentration (intact = 2.3 f 0.03 X lo- to M; OVX = 3.3 + 0.08 x lO_” M (mean f SEM, n = 3 expts.)), despite a 57-88% increase in GnRH binding sites in ovariectomised rat pituitary cells. Basal and maximal LH release from ovariectomised mouse pituitary cells was 1.5 to 3-fold greater than that from intact mouse pituitary cells. There was no change in the GnRH ED,, concentration (intact = 4.3 k 2.3 x 10-s M; OVX = 3.4 f 0.9 x 10e9 M (mean jc SEM, n = 3 expts.)), even though GnRH binding sites were reduced by 40-739;: in the cells from ovariectomised mice. These data indicate that changes in GnRH binding sites of the magnitude observed after ovariectomy play no part in the regulation of gonadotroph responsiveness to GnRH, which is determined by changes in post-receptor events, one of which is an increase in cellular LH.

Introduction Ovariectomy results in the removal of the negative feedback action of gonadal steroids on the hypothalamo-pituitary axis leading to an increase in GnRH in the pituitary stalk blood of rats (Sarkar and Fink, 1979; Sherwood and Fink, 1980).

* To whom correspondence should be addressed at present address: Clinical Research Centre, Watford Road, Harrow, Middlesex, U.K. 0303-7207/86/$03.50

In this species this leads to an increase in pituitary GnRH binding sites together with increased serum and pituitary LH content (Clayton and Catt, 1981a; Frager et al., 1981). In rats many studies have demonstrated a close correlation between GnRH binding site concentration and serum gonadotrophins in different physiological states. Significant changes in GnRH binding sites occur throughout the oestrous cycle (Park et al., 1976; Clayton et al., 1980; SavoyMoore et al., 1980), during development and pregnancy and lactation (Clayton and Catt, 1981b)

0 1986 Elsevier Scientific Publishers Ireland, Ltd.

192

which are generally reflected by parallel changes in serum gonadotrophin levels. Additionally, treatment of cultured pituitary cells with GnRH or agonist analogues (Loumaye and Catt, 1982,1983; Conn et al., 1984; Young et al., 1984, 1985) increases GnRH binding sites. These changes suggest that gonadotroph function may in part be regulated by variations in the concentration of GnRH binding sites perhaps by altering responsiveness of the pituitary to GnRH. Despite much evidence that GnRH positively regulates its own pituitary binding sites in the rat (Clayton and Catt, 1981b), and indeed in other species (Leung et al., 1984) studies in mice show a decline in GnRH binding sites post-gonadectomy when serum gonadotrophin levels are high (Naik et al., 1984a, b). The apparent paradoxical GnRH binding site response after ovariectomy of mice, in the presence of an appropriate serum LH response, implies that GnRH binding site changes of the magnitude observed (reduction by 50%) do not have deleterious consequences for gonadotroph function. In the present study we sought to determine firstly, whether the GnRH binding site changes found in pituitary gland homogenates after ovariectomy of rats and mice were maintained in dispersed cultured cells from their pituitaries and secondly, if these binding site changes altered gonadotroph sensitivity to GnRH in vitro. Materials and methods Animals and sample collection Adult Sprague-Dawley female rats (University of Birmingham) (200-250 g) and C,H/F, hybrid mice, obtained from the Department of Human Anatomy, University of Oxford (25-30 g), were housed under conditions of 14 h light and 10 h darkness and allowed food and water ad libitum, being the same for intact and ovariectomised animals. Ovariectomy was performed under light ether anaesthesia at undefined stages of the oestrous cycle. At lo-14 days post-ovariectomy animals were killed by decapitation; trunk blood was collected, and serum separated and stored at - 20°C until assayed. Intact animals were killed without reference to the oestrous cycle. Pituitary glands were removed and at least five pituitaries from each group were frozen rapidly in liquid

nitrogen and stored individually at - 70” C until assayed for pituitary GnRH binding site content. The remaining pituitaries (10-20) were collected in Dulbecco’s modified Eagle’s medium (DMEM) on ice and enzymatically dispersed immediately. Pituitary cell culture Pituitaries were dispersed by treatment with 0.2% collagenase (Worthington CLS II), 0.1% hyaluronidase (Sigma III) and 2.0 pg/ml DNase (Sigma) as described by Young et al. (1984). The cells were subsequently washed and resuspended in culture medium: DMEM supplemented with 10% horse serum, 2.5% foetal calf serum and 200 mM glutamine, containing 100 units/ml penicillin, 100 pg/ml streptomycin and 2.5 pg/ml amphotericin B (Fungizone, Squibb). Pituitary cells were plated into culture dishes (60 mm, Corning) at a density of approximately 0.2 X lo6 cells/well for LH responses to GnRH, and were maintained in culture at 37°C in 5% CO,/95% air for 70 h prior to treatment. The seeding density for cells from intact and ovariectomised animals, within an experiment, were the same. 1.5 X lo6 cells were used immediately after dispersion for the measurement of GnRH binding sites before plating for culture. Treatment of cultured pituitary cells Before treatment culture medium was removed, cells were washed twice with DMEM, and then incubated for 4 h with increasing concentrations of GnRH (10-l’ to 10e7 M) (kindly donated by Dr. J. Sandow, Hoechst, F.R.G.) in DMEM containing 0.3% bovine serum albumin and 1 mM bacitracin. The medium was removed from the wells and stored at - 20 o C until assayed for LH. The cells were washed with phosphate-buffered saline (PBS) pH 7.4 and subsequently frozen and thawed in Tris-HCI (pH 7.4) containing 0.5% Triton X-100 (Sigma) and 1 mM bacitracin which was then removed from the wells and stored at - 20°C for the measurement of intracellular LH content. Triplicate wells for determination of LH release at each GnRH concentration were obtained and the ED,, for each dose-response curve was calculated by a modified Allfit computer program (DeLean et al., 1978). Each experiment was per-

193

formed at least 3 times donor pituitary cells.

with separate

groups

of

Assay of GnRH binding sites in whole pituitary glana5 and in dispersed cells The GnRH agonist analogue (D-ser-(tBu)6)des-Gly’O-GnRH N-ethylamide (GnRH-A, kindly supplied by Dr. J. Sandow, Hoechst), used in the receptor assay as radioligand, was iodinated with chloramine-T as previously described (Young et al., 1984). GnRH binding sites in rat and mouse glands were measured as described previously (Clayton and Catt, 1981a; Naik et al., 1984a). Briefly, GnRH binding sites were measured in an aliquot of an individual pituitary homogenate by equilibration at 4°C for 90 min with a nearsaturating concentration of GnRH-A as the ligand (0.1-0.2 nM tracer plus 0.9 nM unlabelled analogue). Non-specific binding, measured in duplicate tubes containing a 200-fold excess of unlabelled GnRH-A, was less than 4% of total counts added (50000-60000 counts). The difference between total and non-specific binding, the specific binding, for each individual pituitary allows quantitation of total binding site concentration which is expressed as femtomoles GnRH-A bound per mg protein. Previous studies (Young et al., 1983; Naik et al., 1984a, b) have shown that under different physiological conditions binding site affinity of mouse pituitaries does not alter. GnRH binding sites in acutely dispersed cells or in cells after 3 days of culture were measured using a similar method to the above (Young et al., 1984). After gentle scraping from the culture dish with a rubber policeman, washing in PBS and resuspending in buffer, approximately 3 x lo5 intact pituitary cells were incubated with 50000 counts ‘251-GnRH-A (0.2 nM) and non-specific binding was determined in the presence of a 200fold excess of unlabelled GnRH-A. The cells were incubated, in a total volume of 0.125 ml PBS/BSA, at room temperature for 80 min as previously described (Young et al., 1984). The assay provided triplicate estimates of specific binding for intact and ovariectomised cells per experiment. Differences in specific binding between cells from intact and ovariectomised animals were determined by Student’s t-test. The average SD of triplicate tubes from six experiments was less than

8%. Specific binding was corrected for of viable pituitary cells, assessed by exclusion at the end of the incubation expressed as femtomoles of GnRH-A lo6 viable cells.

the number trypan blue period, and bound per

Radioimmunoassays LH in serum, pituitary homogenates and culture medium was measured by double antibody RIA using reagents supplied by the National Pituitary Agency and the results expressed in terms of the RP-2 standard of rat LH. Intra-assay coefficient of variation was 3.7% and inter-assay coefficient of variation at B/B, = 0.5 was 15.8%.

Statistical analysis Statistical differences were dent’s t-test when appropriate.

analysed

by

Stu-

Results Rat and mouse pituitary cells cultured for 3 days showed good responses to increasing doses of GnRH over 4 h (Fig. 1) with cells from intact rats and mice showing a 4.7- to 9.7- (range n = 3 expts.) and 2.6- to 13.2-fold increase in maximal over basal release, respectively. Comparable ranges for cells from ovariectomised rats and mice were 3.4- to 5-fold and 2.4- to 7.4-fold (n = 3 expts.) (Table 1). Although in each experiment the maximal/ basal LH release was always higher for cells from intact rats and mice there was considerable variability between experiments such that this did not reach statistical significance when considering the mean values from three experiments. Both basal and maximal release of LH in cultures from ovariectomised donor pituitaries were elevated in rats and mice by between 1.5- and 3-fold. Ovariectomy had no effect on GnRH ED,, concentration in cells from either species (rat: intact = 2.3 f 0.3 x lo-” M; OVX = 3.3 f 0.08 X lo-” M; mouse: intact = 4.3 + 2.3 x 10e9 M; OVX = 3.4 * 0.9 x lob9 M) (mean f SEM, n = 3 expts.). However, the GnRH ED,, concentration was lo-fold greater for stimulation of LH release from both intact and ovariectomised mouse pituitary cells compared to that from rat cells in culture (Table 1). The increased basal and maximal LH release after ovariectomy is reflected by significant increases

pituitary glands prior to enzymatic dispersion by 2.2-fold in the rat and l.Cfold in the mouse. This was accompanied by a 3-fold and 5-fold increase in rat and mouse serum LH, respectively, compared with intact animal values (not shown). These LH changes were accompanied by divergence in GnRH binding site concentration in whole glands (Table 2). In the rat there was a 64-102% (range n = 3 expts.) increase in GnRH binding sites and in the mouse a 40-73% (range n = 4 expts.) reduction (Table 2). These relative GnRH binding site changes between pituitaries from ovariectomised and intact animals were reflected by similar changes when measured in acutely dispersed cells (Table 2) and in cells after 3 days in culture, where GnRH binding sites were increased by an average of 73 f 17% (mean* SEM, n =6) and decreased by 53 f 6% (mean f SEM, n = 8) in ovariectomised rat and mouse cells compared with cells from intact animals, respectively. However, there was some variability in the magnitude of the relative binding site change especially with rat pituitary cells after 3 days in culture. Nevertheless, the direction of change was invariably the same in dispersed rat and mouse cells. Insufficient cells were available for determination of GnRH binding site affinity but in whole glands after gonadectomy (Clayton and Catt, 1981a; Frager et al., 1981; Naik et al., 1984a) and in cultured pituitary cells from intact female rats following treatments which increase ‘251-GnRH-A binding (Loumaye and Catt, 1983; Young et al., 1984) no difference in Kd has been observed in many studies.

MEAN f SE n-3 WELLS

.

INTACT ED,,-SX

1, ovx

ow 0 0.1

1O+

ED50-5X10-g

I 1

10

100

-I0 GnRH CONCENTRATION (X 10

1000

Discussion

MI

Fig. 1. Dose-response curves to GnRH in cells from intact and ovariectomised (OVX) rats (upper panel) and mice (lower panel). Cells were plated out at a density of 0.2 x lo6 cells/well. After 3 days they were washed and treated for 4 h in DMEM/BSA/bacitracin containing increasing doses of GnRH (lo-” to lo-’ M). Values are mean+ SE (n = 3 wells/dose). Where no error bar is shown this was within the symbol. A representative of three experiments is shown.

(P < 0.01) in total LH content in the cells from both rats and mice post-ovariectomy (Table 1). Ovariectomy also elevated the content of LH in

Changes in responsiveness of the pituitary gland to GnRH may result from changes in numbers of GnRH binding sites, the activity of the signal transduction mechanism(s), or both. That increased number of binding sites may mediate enhanced responsiveness to GnRH was suggested by studies of gonadectomy in both male and female rats where serum gonadotrophin levels increase together with GnRH binding sites (Clayton and Catt, 1981a; Frager et al., 1981). Conversely, binding site concentrations are reduced in lactating and hyperprolactinaemic rats, circumstances with suppressed basal LH levels (Clayton et al., 1980)

195 TABLE 1 A COMPARISON OF THREE DOSE-RESPONSE CURVES OBTAINED FROM CELLS FROM INTACT AND OVARIECTOMISED (OVX) RATS AND MICE Values are mean + SE, n is indicated at head of each column. Maximal basal ratio

GnRH Ed so (lo-s M)

LH release Basal (Wml) (n = 3 wells)

Maximal (Wml) (n = 3 wells)

intact OVX intact OVX intact OVX

0.2 0.2 0.2 0.5 0.3 0.3

6.6 rbO.6 9.8k2.2 a 8.2kO.8 26.2 &-2.7 a 7.7 f 0.2 ’ 21.9+1.1 ’

32.0+2.5 39.3 + 1.7 b 80.0+ 8.5 140.1+ 4.4 = 36.0+ 0.5 75.4+ 1.1 a

4.8 4.0 9.7 5.7 4.1 3.4

intact ovx intact ovx intact OVX

2.5 2.0 1.4 3.2 8.9 5.1

5.3 *0.4 8.5+0.8 b 8.2kO.8 21.5+1.6 b 14.7 + 4.8 24.111.4 b

13.2 1.7 2.6 2.4 5.4 2.9

LH content from cells after 3 days in culture (Wwell) (n=6wells)

Rat

Expt. 1 Expt. 2 Expt. 3

387.5 + 64.5 527.9+ 83.2 a 22.1+ 12.6 75.5+ 4.6 a 266 f 9.8 805 f21a

Moure

Expt. 1 Expt. 2 Expt. 3

0.4*0.01 1.1* 0.05 b 3.2 f 0.2 8.7 f 1.4 b 2.7kO.l 8.411.0 b

4.1+ 10.3f 8.2+ 19.7& 8.1+ 13.5+

1.5 3.4 a 2.0 5.8 a 1.1 1.6 a

’ P < 0.01; b P < 0.05 compared to intact by Student’s t-test.

and reduced responsiveness to exogenous GnRH (Lu et al., 1976; Muralidhar et al., 1977). In addition, GnRH rapidly induces its own binding sites, serum, and pituitary gonadotrophin levels in hypogonadotropic hypogonadal (hpg) mice whose pituitaries have never previously been exposed to GnRH in vivo (Young et al., 1983). Intermittent injections or continuous infusions of low doses of GnRH or GnRH agonist analogues induce pituitary GnRH binding sites in rats and increase basal serum LH concentrations (Clayton et al., 1980; Clayton, 1982). Despite all this evidence of a positive correlation there are many instances where a disparity between changes of GnRH binding site and serum gonadotrophin levels has been observed. No change in number and affinity of GnRH binding sites is observed prior to the LH surge on the day of pro-oestrus in rats (Clayton et al,, 1980; Savoy-Moore et al., 1980), cows (Leung et al., 1984) or hamsters (Adams and Spies, 1981), although there is increasing responsiveness to GnRH throughout the day of pro-oestrus ap-

proaching the LH surge (Amirura et al., 1971; Aiyer et al., 1974; Cooper et al., 1974; Leung et al., 1984). Further, GnR_H binding sites remain unchanged during the oestradiol-induced surge of gonadotropbins in ewes (Wagner et al., 1979) though work in ovariectomised monkeys has shown that the oestradiol-induced LH surge is preceded by increased number of G&H binding sites (Adams et al., 1981). The most striking divergence is observed after gonad~tomy of mice where GnRH binding sites decrease despite elevation of serum LH and FSH (Naik et al., 1984a, b). Thus, data from the mouse in vivo, where GnRH binding sites do not correlate with serum LH levels or responsiveness to GnRH, question the relevance of binding site regulation as a major factor in modulation of gonadotroph responsiveness in this species. The present study shows that the species differences in GnRH binding sites after ovariectomy are maintained in both acutely dispersed and S-day cultured cells. Despite these binding site changes

196 TABLE

2

GnRH RECEPTOR LEVELS IN WHOLE PITUITARY GLANDS IN ACUTELY DISPERSED CELLS AND IN CELLS AFTER 3 DAYS IN CULTURE Values are the mean+ SE; n = 5-6 for whole triplicate determinations of specific binding culture/group for the cells. GnRH-R in whole glands (fmol/mg protein) (n = 5-6) Rut Expt. 1 Expt. 2 Expt. 3

GnRH-R

glands from

measured

and one

in cells

Acutely dispersed (fmol/106 cells)

After 3 days in culture (fmol/106 cells)

intact ovx intact ovx intact ovx

28.3 * 1.2 57.1* 4.4 = 36.6 * 6.8 68.7 + 2.9 b 26.7kO.8 43.8k 3.2 a

4.7 & 0.8 8.1 *O.l b 5.5 *0.3 10.4 + 0.4 a 17.3 i 0.7 23.5*0.5 b

4.9&0.1 12.3 +0.2 a 13.OkO.6 18.8 f 0.4 b 6.9kO.7 9.9kO.l b

intact ovx intact ovx intact ovx intact ovx

14.2 + 2.4 5.6+0.8’ 14.0*0.9 8.4kO.9 = 18.0* 1.5 4.9*0.4 b 28.Ok 1.2 12.9* 1.7 a

11.9+1.2 7.Ok 0.2 b 12.9 + 0.1 4.1 kO.3 a 11.4*0.8 8.2+0.4’ 13.OkO.6 5.21tO.8 b

13.8 + 1.1 5.6 + 0.7 32.0 + 2.9 9.o*o.li 8.2i0.8 3.4,O.g 13.2k 1.1 8.2kO.2

M0U.W Expt. 1 Expt. 2 Expt. 3 Expt. 4

a P < 0.001; b P < 0.01; ’ P < 0.05 vs. intact by Student’s

b b b b t-test.

gonadotroph sensitivity to GnRH was unaltered since the ED,, concentration remained the same after ovariectomy. This is in agreement with in vitro studies by O’Connor et al. (1980) and Tang et al. (1979) using cells from castrated male and female rats. Thus, after ovariectomy GnRH binding site changes of the magnitude observed (> 50% increase or > 50% decrease compared with intact) were of little consequence for gonadotroph function. In the only other experiments in which changes in GnRH binding sites and GnRH responsiveness have been examined in the same cells, a 25-50s increase in binding was associated with a reduction by about 50% in the GnRH ED,, concentration (Loumaye and Forni, 1982; Tang et al., 1982) in cells pretreated with 17/3-oestradiol. Thus, in oestrogen-treated cells a true increase in sensitivity to GnRH suggests that the increased GnRH binding sites may be functional receptors,

although post-receptor events may also be altered by oestrogens. That the increased binding site concentration after ovariectomy of rats did not reduce the GnRH ED,, concentration may have several possible explanations. Firstly, the bioassay system may not be sufficiently sensitive to detect a small left-shift in dose-response curves. Secondly, the increased binding sites may not be functionally coupled to the signal transduction mechanisms required for LH release. There is now good evidence for rapid breakdown of membrane polyphosphoinositides after GnRH receptor activation and changes in ‘true’ receptors might only be implied by showing a direct correlation between polyphosphoinositide turnover and receptor occupation. This assumes that polyphosphoinositide turnover rates show a linear relationship with LH release, which has not yet been demonstrated. Thirdly, it may be that the initial rate of LH release was increased but measurement of LH accumulation 4 h after GnRH stimulation would not detect this. In cells derived from ovariectomised animals of both species maximal LH output was increased, in agreement with studies of O’Connor et al. (1980) using castrated male and female rats. It is likely that this reflects a rise in the amount of LH available for release since the content of LH was increased in cells from ovariectomised animals of both species. A consistent observation in these studies was the reduced sensitivity of mouse pituitary cells to GnRH, whether derived from intact or ovariectomised animals. This could be explained if the mouse pituitary GnRH receptor has a lower affinity for GnRH. Although not measured in the cells in this study, because of limited availability, there is evidence that the Kd for GnRH analogue binding to mouse pituitary preparations (= 6 X 10-i’ M) (Naik et al., 1984a, b; Naik, 1985) is about 3-fold higher than to similar preparations from rat pituitaries (K, = 2 X lo-” M) (Clayton and Catt, 1981a, b; Clayton, 1982). Assuming that a similar difference in binding affinity applies also to GnRH, this could partly account for the reduced sensitivity of mouse cells. Pertinent to this point is the relative resistance of mouse gonadotrophs to desensitisation by GnRH agonists (Bex and Corbin, 1982). Furthermore, the positive rela-

197

tionship between the K, of GnRH analogue binding and the zbiological responsiveness of the gonadotrophs provides more evidence that the GnRH analogue binding sites are indeed physiologically relevant ‘receptors’. Although changes in hormone milieu alter the number of pituitary GnRH binding sites, it is clear from the present studies that modulation of this initial step in GnRH action exerts only a minor effect, if any, on cellular sensitivity to the ligand. Thus, changes in as yet unidentified postreceptor events such as receptor coupling to signal transduction mech~isms and/or other post-receptor events seem to be of greater significance for regulation of gonadotroph function. What, therefore, is the relevance of changes in GnRH binding sites that have been reported in a wide variety of circumstances? They are probably a consequence of gonadotroph activation and/or suppression by hormonal and biochemical stimuli. Acknowledgements

This work was supported by grants from the MRC (to R.N.C.) and the Govemment of Pakistan (to S.I.N.). S.D.A. is in receipt of an MRC studentship. References Adams, T.E. and Spies, H.G. (1981) Endocrinology 108, 2245-2253. Adams, T.E., Norman, R.L. and Spies, H.G. (1981) Science 231,1388-1390. Aiyer, M.S., Fink, G. and Grieg, F. (1974) J. Endocrind. 60, 47-52. Amirura, A. and Schally, A.V. (1971) Proc. Sot. Exp. Biol. Med. 136,290-293. Bex, F.J., Corbin, A and France, E. (1982) Life Sci. 30, 1263-1269. Clayton, R.N. (1982) Endocrindogy 111,152-161. Clayton, R.N. and Catt, K.J. (1981a) Endocrinology 108, 887-895. Clayton, R.N. and Catt, K.J. (1981b) Endocr. Rev. 2, 186-209.

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