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hCG receptor
Molecular and Cellular Endocrinology, 20 (1980) 145-156 0 Elsevier/North-Holland Scientific Publishers, Ltd.
145
HUMAN CHORIOGONADOTROPIN-INDUCED DESENSITIZATION OF GRANULOSA-CELL ADENYLATE CYCLASE TO GONADOTROPINS AND LOSS OF LH/hCG RECEPTOR .. .. Kalle JAASKELAINEN,
Tapani HYVijNEN
and Hannu RAJANIEMI
Department of Biochemistry and Anatomy, University of Kuopio, SF-70101 Kuopio 10, and Department of Anatomy, University of Oulu, SF-90220 Oulu 22 (Finland)
Received 8 May 1980; accepted 10 July 1980
Immature female rats that had been primed with pregnant-mare serum gonadotropin (PMSG) were injected intravenously with various doses of human choriogonadotropin (hCG) for the investigation of the relationship between adenylate cyclase activities and the concentrations of LH/hCG receptor in luteinizing granulosa cells. Injection of 1 pg of hCG induced a loss of LH and FSH sensitivities of adenylate cyclase within 6 h and a disappearance of free LH/ hCG receptors within 24 h. Basal adenylate cyclase activity has a transient maximum at 6 h after hCG injection. After injection of 100 pg of hCG the loss of LH sensitivity of adenylate cyclase and free LH/hCG receptors occurred immediately, but the changes in FSH-stimulated and basal activities followed the same time scale as after injection of 1 fig of hCG. When hCG was omitted from the injections the response of the animals to the endogenous gonadotropin surge varied. A complete desensitization of adenylate cyclase to LH and FSH stimulation and a 65% loss of free LH/hCG receptors were found at 24 h if the follicles were ovulated. These results suggest that occupation of a limited number of LH/hCG receptors in granulosa cells induces adenylate cyclase refractory to further stimulation by gonadotropins. The transient elevation of basal adenyiate cyclase activity and its desensitization to further stimulation by gonadotropins may have a role in physiological processes leading to ovulation and luteinization. Keywords:
adenylate cells.
cyclase desensitization;
LH/hCG-receptor
regulation; granulosa
Several hormones regulate the number of their own receptors in the target tissue. Exposure of target cells to elevated hormone concentrations leads to a decrease of receptors for insulin (Gavin et al., 1974), thyrotropin-releasing hormone (Hinkle and Tashjian, 1975), catecholamines (Mukherjee et al., 1975), growth hormone (Lesniak and Roth, 1976), human epidermal growth factor (Carpenter and Cohen, 1976) and LH/hCG (Sharpe, 1976; Conti et al,, 1976, 1977a, b; Haour and Saez, 1977; Rao et al., 1977). In some tissues, hormone-induced loss of receptors is associated with the loss of hormone sensitivity of adenylate cyclase (Mukherjee et al., 1975; Kebabian et al., 1975; Conti et al., 1976; Hsueh et al., 1977). However, at present the exact molecular mechanisms of the receptor loss and its relationship to
146
KaNe J&iskeltiinen, Tapani Hyvtinen, Hannu Rajaniemi
the desensitization of adenylate cyclase are not known. The maturation of the Graafian follicle is associated with the appearance of LH/ hCG receptors (Zeleznik et al., 1974; Richards et al., 1976; Nimrod et al., 1977; Rajaniemi et al., 1977). The existence of high LH/hCG- and FSH-receptor levels and the CQUpkIg of receptors with adenylate cyclase form a prerequisite for ovulation to occur. In preovulatory follicles, theta interna cells and peripheral granulosa cells contain the highest level of LH/hCG receptors, whereas FSH receptors are distributed evenly among the granulosa cells (Zeleznik et al., 1974; Louvet and Vaitukaitis, 1976; Rajaniemi et al., 1977). The loss of LH/hCG and FSH receptors has been reported to accompany ovulation induced by exogenous gonadotropins (Richards et al., 1976; Rao et al., 1977; Rajaniemi and Jaaskehiinen, 1979). Both exogenous hCG and endogenous ovulatory gonadotropin surges have been reported to render adenylate cyclase of intact pre-ovulatory follicles refractory to further stimulation by gonadotropins (Hunzicker-Dunn and Birnbaumer, 1976a, b); after luteinization of the granulosa cells the high LH/hCG receptor level and LH sensitivity of the adenylate cyclase are regained (Holt et al., 1976; Hunzicker-Dunn and Birnbaumer, 1976a, b). The present study was undertaken to investigate whether a desensitization of adenylate cyclase could be demonstrated in granulosa cells isolated from rat pre-ovulatory follicles after various times from hCG injections and to investigate the relationship between gonadotropin sensitivity of adenylate cyclase and the levels of unoccupied and occupied LH/hCG receptors. A few reports indicate that neither endogenous gonadotropin surges nor exogenous hCG injections render adenylate cyclase of granulosa cells refractory to LH stimulation (Nimrod et al., 1977; Hamberger et al., 1979). These results are in contrast with results in the present paper and reports on the behaviour of intact follicles.
MATERIALS
AND METHODS
Animal treatment and radioiodination of hCG Immature 25-day-old female Wistar rats were injected S.C. at 09.00 h with 15 IU of pregnant-mare serum gonadotropin (PMSG) (Gestyl, Organon) to initiate and synchronize the follicular development. 2 days later at 13.00-14.00 h the animals received, under light ether anaesthesia, a single tail-vein injection of 1 or 100 1.18of hCG (7700 IU/mg, Diosynth) in 0.2 ml of 0.01 M phosphate-buffered 0.14 M NaCl (PBS) (PH 7.0). The activities of adenylate cyclase and the levels of free LH/hCGreceptor sites were measured before and at 15 min, 1, 6 and 24 h after the injections. In control experiments, hCG was omitted from the injection. In experiments on uptake of hormone in vivo each dose of hCG was mixed with 0.7 or 3.3 nCi of 1251labelled hCG before the injection. Highly purified hCG (11500 IU/mg), donated by Dr. A. Rees Midgley Jr., University of Michigan, was radio-iodinated as described previously (Greenwood et al., 1963; Markkanen et al., 1980). The specific activity of the preparations averaged 25 /.K!i/pg determined by the method of Ireland and Richards (1978).
Regulation of adenylate cyclase and gonadotropin receptors
141
Adenylate cyclase activity For measurement of adenylate cyclase activity 3 rats were decapitated at each time, ovaries were removed and granulosa cells were isolated by rupturing follicles and expressing cells at 4” into a buffer consisting of 27% sucrose (BDH Chemicals), 1 mM EDTA (Merck) and 10 mM Tris-HCI (Sigma) (pH 7.5). Granulosa cells of the same time point were pooled, a portion of the cell suspension was taken for measurement of available LH/hCG-binding sites and the rest was homogenized by 4 strokes in a ground-glass tissue grinder. Homogenization was carried out during 1 h from killing of the animals, and the homogenates were assayed for adenylate cyclase within 15 min from preparation. Adenylate cyclase activities were measured in triplicate assays by a modification of the method of Nakai and Brooker (1975). The fmal assay mixture contained 50 mM Tris-HCI (pH 7.5), 4 mM MgC12 (Merck), 0.45 mM EDTA, 0.2 mM ATP (Sigma), [cY-~*P]ATP (Radiochemical Centre, Amersham) to give a final specific activity of 100-300 cpm/pmole, 0.5 mM cyclic AMP (Sigma), 20 mM creatine phosphate (Sigma) and creatine phosphokinase, 50 U/ml (Sigma). When present, the final concentration of KF (Baker) was 10 mM. Responses to gonadotropins were determined by addition of ovine LH (NIH-LHS16) or ovine FSH (NIH-FSH-S9) to a final concentration of 20 @g/ml. Reaction was started by adding 20 ~1 of granulosa cell homogenate containing 0.1-0.2 mg of protein to prewarmed incubation tubes; the final assay volume was 50 fl. The linearity of the reaction with regard to homogenate protein concentration was tested by measuring the fluoride-stimulated enzymic activity, and the linearity with regard to time was tested by measuring basal, LH- and FSH-stimulated activities. The incubation temperature was 30’. Unless otherwise stated, the reaction time was 2Cl min. The reaction was stopped by addition of 1 ml of 1 M HCI; most of the remaining ATP was destroyed by heating the samples at 90’ for 10 min, and the samples were then neutralized by adding 0.5 ml of 2 M KOH. Thereafter the samples were precipitated by adding 0.5 ml of 0.15 M Ba(OH)2 (Merck) and 0.5 ml of 5% ZnSO4 . 7 Hz0 (Merck), and were centrifuged. In this procedure, ATP, ADP, AMP and phosphate adsorb to the precipitate and cyclic AMP remains in the supernatant. The precipitation was repeated twice by taking 1 ml of the supernatarit. 1 ml of the final supernatant was taken for the liquid-scintillation counting. Measurement of unoccupied LH,hCG-binding sites For measurement of available LH/hCG-binding sites, portions of pooled granuloss cell suspensions were taken before homogenization. The binding assays were conducted in triplicate in polypropylene tubes precoated with bovine serum albumin. The assay tubes contained an aliquot of granulosa cell suspension equivalent to 0.1-0.2 mg of protein (20 d), [1251]hCG, 200 000 cpm (10 PI), an excess of unlabelled crude hCG (10 A) (30 IU) (Pregnyl, Organon) or an equivalent volume of buffer and 0.1% bovine serum albumin in PBS (pH 7.0) to give a final volume of 120 ~1. After incubation of the mixture for 4 h at 22” with continuous shaking, 3 ml of ice-cold PBS was added, and the granulosa cells were immediately sedimented by
148
Kalle J,i~skeEnen,
Tapani HyvBnen, Hannu Rajaniemi
centrifugation at 1200 X g for 15 min. The rinse of the cells with ice-cold PBS was repeated, and pellets were counted in an automatic gamma counter with 50% efiiciency. [ r2’I]hCG at about 100000-150000 cpm was sufficient to saturate all available binding sites in receptor assays; higher concentrations of [12’I]hCG did not increase the amount of bound hormone. Measurement of uptake of hCG in vivo After injection of the rats with the mixture of [‘251]hCG and unlabelled hCG 2 rats were decapitated at each time, blood was collected and an aliquot of serum counted in the gamma counter. Ovaries were removed and granulosa cells were isolated by rupturing follicles and expressing cells into PBS (pH 7.0); the cells were washed once with PBS and counted for 1251 radioactivity in the gamma counter. The amounts of hormone in serum and in granulosa cells were calculated from the known specific activities of the injection mixtures. Protein determination Protein was determined by the method serum albumin as a standard.
of Lowry et al. (1951) with bovine
RESULTS Linearity of the adenylate cyclase assay with protein concentration and time The time course of the adenylate cyclase reaction is shown in Fig. 1. The concentration of granulosa cell homogenate protein in each assay tube was 208 pg/50 ~1. The linearity of the basal adenylate cyclase activity was maintained for 25 min and the linearities of LH- and FSH-stimulated activities for 20 min. Fluoride-stimulated adenylate cyclase activity as a function of granulosa cell homogenate protein is shown in Fig. 2. The incubation time was 20 min. The enzymic activity was linearly proportional to homogenate protein concentration over a range up to 250 pg of protein in 50 /_d of assay volume. The ATP-regenerating system is essential for the linearity of the reaction (Nakai and Brooker, 1975). We found that, after an incubation of 20 min, 80% of [a-32P]ATP remained intact in the assay mixture. Adenylate cyclase and unoccupied LH/hCG receptors Rats pretreated with PMSG were injected intravenously with 1 or 100 pg of hCG l-2 h before the onset of endogenous gonadotropin surge. The adenylate cyclase activities of the granulosa cell homogenates after injection of 100 pg of hCG are shown in Fig. 3. The ability of LH to stimulate adenylate cyclase disappeared within 15 min. The loss of enzymic response to FSH was slower. After 1 h, FSH stimulation was still 2-fold. FSH receptors were evidently responsible for the stimulation of adenylate cyclase activity by. FSH. After luteinization the adenylate cyclase activity of rat luteal homogenates could be stimulated by hCG and by LH
ofaden~~~e cyclase and gonadotropin receptors
Regulation
5
10 15 INCUBATION TIME
20 irncni
25
149
,ug
HOMOGENATE
PROTEIN
Fig. 1. Adenylate cyclase activities of granulosa cell homogenates as a function of incubation time. The assays were performed as described in Materials and Methods. The drugs were present at the fo~ow~~ concelltrations: LH, 20 &g/ml: FSH, 20 fig/ml. The concentration of the homogenate protein in each assay tube was 208 @g/SOPI. Each point represents the mean (+SElM) value of triplicate determinations. Fig. 2. Fluoride-stimulated adenylate cyclase activity as a function of the quantity ofgranulosa celI homogenate protein in the assay volume of 50 pi. The assays were performed as described in Materials and Methods. The concentration of KF was IO mM. Each point represents the mean (+SEM) vaIue of triplicate determinations.
but not by the FSH concentration (20 p&/ml) used in our adenylate cyclase assays (results not shown). Adenylate cyclase activity measured in the presence of fluoride ion fell by about 40% within 1 h. Basal activity reached a maximum at 6 h, but after 24 h it had returned to the initial level. Fig. 4 shows the adenylate cydase activities after injection of 1 ,ug of hCG. A decrease in both LH and FSH responses was seen after 1 h; after 6 h both responses had disappeared. Fluoride-stimulated activity fell by about 30%. Basal activity had a maximum at 6 h, but after 24 h it had declined to the initial level. To obtain an insight into the me~h~isms of the desensjt~~ation we measured the levels of free LH/hCG receptors of the same granulosa cells we used in measuring the adenylate cyclase activities. Receptor levels compared with initial values are shown in Fig. 5. Injection of 100 pp of hCG saturated all measurable LH/hCG receptor sites within a few minutes. After administration of 1 ,ug of hCG the level of free ~~/hCG receptors had declined at 1 h by 35% and at 6 h by 90%. With the Iatter dose, loss of LH sensitivity of adenylate cyclase seemed to be slightly faster than the loss of free LH/hCG receptors. To control the possible desensitizing effect of endogenous gonadotropins on adenylate cyclase, PMSG-treated rats were injected with PBS instead of hCG. The results from one injection group to another varied. Adenylate cyclase activities of one group of animals are shown in Fig. 6. Basal activity had a slight maximum after 6 h from PBS injections. After 24 h, gonadotropin responses had disappeared. The
150
K&e J&iskeli:inen, Tapani ffyvhen, Hannu Rajaniemi
t
lh
hCG~lK)uy
2h IV) TIME
3h
&h
AFTER
5h
6h
2Lh
INJECTION
Fig. 3. Adenylate cyclase activities of granulosa cell homogenates before and after an intravenous injection of 100 pg of hCG. Triplicate assays were performed as described in Materials and Metbods. The drugs were present at the following co~c~n~at~ons: LH, 20 &g/ml; FSH, 20 &g/ml; KF, 10 mM. Each point represents the mean (+SEM) value of a cell pool collected from the ovaries of 3 rats.
lh 2h hCG(l,ug iv) TIME
t
3h 4h 5h AFTER INJECTION
Gh
Uh
J
Fig. 4. Adenylate cyclase activities of granulosa cell homogenates before and after an intravenous injection of 1 pg of hCG. Triplicate assays were performed as described in Materials and Methods. The drugs were present at the following concentrations: LH, 20 pg/ml; FSH, 20 rrg/ ml; KF, 10 mM. Each point represents the mean (*SEM) value of a cell pool collected from the ovaries of 3 rats.
Regulation of adenylate cyclase and gonadotropin receptors
Lh
.3h hCGllv
I
TIME
AFTER
5h INJECTION
Gh
151
24h
Fig. 5. Unoccupied LM/hCG-binding sites of granulosa cells after intravenous injections of hCG or PBS. Triplicate binding assays were performed as described in Materials and Methods. The results are expressed as percentage of initial binding sites calculated on a protein basis. Each point represents the mean (iSEM) value of a cell pool collected from the ovaries of 3 rats.
level of free LH/hCG receptors after 24 h was still 35% of the initial level (Fig. 5). Stereomicroscopic examination of the ovaries from this injection group showed that a majority of the follicles had ovulated. Within another group of rats, 7 from the total of 9 had ovaries that showed no ovulation at 24 h. The basal, LH- and FSH-stimulated adenylate cyclase activities of the granulosa cell homogenates from
f4 PBS
6h iv
TIME
AFTER
24h
INJECTION
Fig. 6. Adenylate cyclase activities of granulosa cell homogenates before and after an intravenous injection of PBS. Triplicate assays were performed as described in Materials and Methods. The drugs were present at the following concentrations: LH, 20 pg/ml; FSH, 20 pg/ml; KF, 10 mM. Each point represents the mean (+SEM) value of a cell pool collected from the ovaries of 3 rats.
Kalle JciCskelCinen, Tapani HyvBnen, Hannu Rajaniemi
152
lh hCGllv1
2h 3h Lh 5h TIME AFTER INJECTION
Fh
24h
Fig. 7. Serum concentrations of hCG after intravenous injections of 0.7 &i of ‘2SI-labelled hCG together with 1 pg of unlabelled hCG or 3.3 nCi of 1 2 5 I-labelled hCG with 100 ng of unlabelled hCG. Each point represents the mean (+SEM) value for 2 rats.
these unovulated follicles at 24 h after PBS injections were 3.1, 10.3 and 10.5 pmoles CAMP/mm X mg homogenate protein, resp. The level of free LH/hCG receptors was 50% higher than the initial receptor level. The value of basal adenylate cyclase activity after 6 h from PBS injections was 2.7 pmoles cAMP/min X mg homogenate protein. Kostyk et al. (1978) reported that the responses of immature rats to exogenous gonadotropin treatment are dependent upon the type and quantity of injected gonadotropin. The percentage of animals ovulating at 72 h after a single S.C.injection of 15 IU of PMSG was about 35.
z B z 20 x
A k! 10
/‘%CG
PLUS
‘,,cJ OF
‘L&C
KC
3 3 2
2
:-_:t
hCGllv
lh
1
2h TIME
3h 4h 5h AFTER INJECTION
Gh
24h
Fig. 8. Occupancy of granulosa cells by hCG after intravenous injections of 0.7 nCi of 1251labelled hCG together with 1 gg of unlabelled hCG or 3.3 PCi of 1251-labelled hCG with 100 Mg of unlabelled hCG. The specific radioactivities of the gonadotropin fractions bound to granulosa cells were assumed to be the same as the specific radioactivities of the injection mixtures. Each point represents the mean (+SEM) value for 2 rats.
Regulation of adenylate cyclase and gonadotropin
receptors
153
Serum and granulosa cell hCG levels Serum and granulosa-cell hCG levels after intravenous injections of 1 or 100 pg hCG together with 1251-labelled hCG are shown in Figs. 7 and 8. By gel filtration on Sepharose 6B, 90-95% of the serum radioactivity was identified to be intact [‘251]hCG (results not shown). The radioactivity of the granulosa cells showed a maximum after 2 h from the injection of hCG (Fig. 8). By comparing the hCG concentrations bound to the granulosa cells (Fig. 8) and the concentrations of free receptors (Fig. 5) one can conclude that occupation of a small portion of LH/hCG receptors induces the loss of all high-affinity LH/hCG-receptor sites. After injection of 1 pug of hCG, the loss of free receptors seemed to be quantitatively more important than the loss of the occupied receptor sites. By gel filtration on Sepharose 6B, 70-90% of the radioactivity of the granulosa cells was identified as receptor-[1251]hCG complex.
DISCUSSION The results of the present study suggest that intravenous injections of microgram quantities of hCG, in 6 h, render adenylate cyclase of rat pre-ovulatory granulosa cells refractory to further stimulation by LH or FSH. Conti et al. (1976) found that, after 6 h from an S.C. injection of 20 IU of hCG (2 pg of hCG), the adenylate cyclase of luteinized rat ovaries can no longer be stimulated by LH. The loss of LH/ hCG receptors is complete after 24 h. Later, Conti et al. (1977a) reported that the lowest dose of hCG to cause a measurable loss in LH/hCG receptors of luteinized ovaries is about 20 ng/lOO g of body weight, which gives a serum concentration of hCG within the physiological serum concentration of LH. Conti et al. (1977b) also reported that, in desensitized rat luteal cells, the decrease in LH/hCG-receptor level is accompanied by a lesion in the progesterone biosynthetic pathway beyond the level of cyclic AMP production. The results of our control experiments (PBS instead of hCG injections) were not reproducible at 24 h from one injection group of rats to another. However, the desensitization of the adenylate cyclase to gonadotropins found at 24 h after injection correlated positively with the follicular ovulation. In addition, the control experiments showed that the desensitization of the adenylate cyclase to gonadotropins occurs without a total .loss of the receptors. Hunzicker-Dunn and Birnbaumer (1976b) report that, after 3 days from treating prepubertal rats with PMSG, a synchronous ovulation has occurred with a concomitant loss in the ability of LH and FSH to stimulate adenylate cyclase of ovarian homogenates. Adenylate cyclase activities measured in homogenates of ovarian follicles of cycling rats also become desensitized to LH and FSH by the morning of oestrus (Hunzicker-Dunn and Birnbaumer, 1976b). The present results are contradictory to the results of Nimrod et al. (1977) and to the results of Hamberger et al. (1979). Nimrod et al. (1977) used as a model sys-
154
Kalle Jriiiskehiinen, Tapani HyvSnen, Hannu Rajaniemi
tern normal cycling rats and they injected the rats i.p. at 14.00 h on the day of prooestrus with 50 IU of hCG. They did not find a loss of LH sensitivity of adenylate cyclase in granulosa cells collected at 24.00 h either from follicles of hCG-treated rats or from follicles of untreated rats. Hamberger et al. (1979) used PMSG-treated rats and collected the granulosa cells either before or after the endogenous gonadotropin peak. Adenylate cyclase of granulosa cells collected a few hours after the endogenous LH-FSH peak did not show-a desensitization to LH stimulation. Intraperitoneal injections of LH or FSH (10 pg/rat) did not desensitize adenylate cyclase to LH or FSH when granulosa cells were collected after 2 h from the injections. We think it is important to follow the time course of adenylate cyclase activities for longer times than 10 h after endogenous surges or exogenous gonadotropin injection. In the control experiment of Fig. 6 the desensitization to gonadotropin stimulation was seen at 24 h but not yet at 6 h after PBS injections. It is also possible that intraperitoneally injected hormones are effectively taken up by the liver and catabolized (Markkanen et al., 1979). We found that, after injection of 1 pg of hCG, occupancy of a limited portion of LH/hCG receptors initiated desensitization of adenylate cyclase and loss of occupied as well as of free LH/hCG receptors. The,rapidity of the loss of free LH/hCG receptors suggests that aggregation or clustering of the receptors may form one step in their inactivation. Especially after endogenous gonadotropin surge, the desensitization of adenylate cyclase to LH seemed to be faster than the loss of free LH/hCG receptors, which suggests that, besides the disappearance of receptors, other mechanisms may be involved in the desensitization process. Su et al. (1979) incubated human astrocytoma cells with low concentrations of isoproterenol and compared the decrease in /3-adrenergic receptor density with a reduction in isoproterenolstimulated adenylate cyclase activity. 2 different reactions were found to’ be involved in the desensitization process: initially a loss of adenylate cyclase activity occurs with little loss of fl-adrenergic receptors, and a slower reaction results in a marked decrease of fl-adrenergic receptors. Su et al. (1979) suggest that the loss of fl-adrenergic receptors occurs as a result of the initial uncoupling of the P-receptorlinked adenylate cyclase. In pre-ovulatory follicles, theta interna cells and peripheral granulosa cells contam the highest level of LH/hCG receptors, and FSH receptors are distributed evenly among the granulosa cells (Zeleznik et al., 1974; Louvet and Vaitukaitis, 1976; Rajaniemi et al., 1977). The ability of FSH to stimulate the adenylate cyclase of the cell homogenates indicates that the cell pools obtained consist mainly of granulosa cells. Desensitization of adenylate cyclase to FSH was relatively slow and followed a similar pattern after both doses of hCG (Figs. 3 and 4). Perhaps intercellular communication through gap junctions (Lindner et al., 1977; Gilula et al., 1978) is a prerequisite for desensitization of innermost granulosa cells to FSH stimulation. Different results have been obtained for the hormone specificity of the desensiti-
Regulation of adenylate cyclase and gonadotropin receptors
155
zation of ovarian adenylate cyclase. Zor et al. (1976) found that culture of preovulatory rat follicles with LH, FSH or prostaglandin Ez for 24 h desensitized adenylate cyclase to homologous hormone but not to heterologous hormones. Richards et al. (1979) found that iv. injection of hCG to hypophysectomized, oestradiol-, and FSH-primed rats renders adenylate cyclase refractory to FSH stimulation in 2 h. In the reverse experiment, FSH rendered adenylate cyclase refractory to hCG stimulation in 2 h. In our experiments, adenylate cyclase of granulosa cells became refractory to FSH stimulation in 6 h after hCG injections and in 24 h after PBS injections (control experiment of Fig. 6). After hCG injection, and also in the control experiment of Fig. 6, basal adenylate cyclase activity reached a transient maximum at 6 h. Bauminger et al. (1978) found that the cyclic AMP level of rat pre-ovulatory follicles increases by about 40% after 3-5 h from the onset of endogenous gonadotropin surge and that the cyclic AMP level returns to the initial level at the time of ovulation. The results of the present study suggest that the activation of granulosa cell adenylate cyclase by endogenous LH-FSH surge and its further desensitization to gonadotropins cause the fluctuations in the cyclic AMP levels, and that these changes have a physiological role in the positive ovulatory response of rat Graatian follicles.
ACKNOWLEDGMENTS We thank Prof. A.R. Midgley Jr., University of Michigan, for a donation of highly purified hCG. The skilful technical and secretarial assistance of Miss Arja Venakiinen and Sirkka Pennanen is greatly appreciated. Financial support was given by the Academy of Finland and by the Cultural Foundation of Finland.
REFERENCES Bauminger, S., Koch, Y., Kahn, I., Hillensjo, T., Nilsson, L., and Ah&, K. (1978) J. Reprod. Fertil. 52, 21-23. Carpenter, G.;and Cohen, S. (1976) J. Cell Biol. 71, 159-171. Conti, M., Harwood, J.P., Hsueh, A.J.W., Dufau, M.L., and Catt, K.J. (1976) J. Biol. Chem. 251,7729-7731. Conti, M., Harwood, J.P..Dufau, ML., and Catt, K.J. (1977a) Mol. Pharmacol. 13, 1024-1032. Conti, M., Harwood, J.P., Dufau, M.L., and Catt, K.J. (1977b) J. Biol. Chem. 252,8869-8874. Gavin, J.R., Roth, J., Neville Jr., D.M., De Meyts, P., and Baell, D.N. (1974) Proc. Natl. Acad. Sci. (U.S.A.) 71,84-88. Gilula, N.B., Epstein, M.L., and Beers, W.H. (1978) J. Cell Biol. 78,58-75. Greenwood, F.E., Hunter, W.M., and Clover, J.S. (1963) Biochem. J. 89, 114-123. Hamberger, L., Nilsson, L., Nordenstrom, K., and Sjogren, A. (1979) in: Ovarian Follicular and Corpus Luteum Function, Eds.: C.P. Channing, J. Marsh and W.A. Sadler (Plenum, New York) pp. 105-112. Haour, F., and Saez, J.M. (1977) Mol. Cell. Endocrinol. 7, 17-24. Hinkle, P.M., and Tashjian Jr., A.H. (1975) Biochemistry 14, 3845-3851.
1.56
Kalle Jb%kel&en,
Tapani Hyvdnen, Hannu Rajaniemi
Halt, J.A., Richards, J.S., Midgley Jr., A.R., and Reichert Jr., L.E. (1976) Endocrinology 98, 1005-1013. Hsueh, A.J.W., Dufau, M.L., and Catt, K.J. (1977) Proc. Natl. Acad. Sci. (U.S.A.) 74,592-595. Hunzicker-Dunn, M., and Birnbaumer, L. (1976a) Endocrinology 99, 185-197. Hunzicker-Dunn, M., and Birnbaumer, L. (1976b) Endocrinology 99, 198-210. Ireland, J.J., and Richards, J.S. (1978) Endocrinology 102, 876-883. Kebabian, J.W., Zatz, M., Romero, J.A., and Axelrod, J. (1975) Proc. Natl. Acad. Sci. (U.S.A.) 72,3735-3739. Kostyk, S.K., Dropcho, E.J., Moltz, H., and Swartwout, J.R. (1978) Biol. Reprod. 19, 11021107. Lesniak, M.A., and Roth, J. (1976) J. Biol. Chem. 251, 3720-3729. Lindner, H.R., Amsterdam, A., Salomon, Y., Tsafriri, A., Nimrod, A., Lamprecht, S.A., Zor, U., and Koch, Y. (1977) J. Reprod. Fertil. 51, 215-235. Louvet, J.-P., and Vaitukaitis, J.L. (1976) Endocrinology 99, 758-764. Lowry, O.H., Rosebrough, N.J., Farr, A.L.. and Randall, R.J. (1951) J. Biol. Chem. 193,265275. Markkanen, S., Tdllikkd, K., Vanha-Perttula, T., and Rajaniemi, H. (1979) Endocrinology 104, 1540- 1547. Markkanen, S., Tollikko, K., Jaaskelainen, K., and Rajaniemi, H. (1980) Hormone Res. 12,3245. Mukherjee, C., Caron, M.G., and Lefkowitz, R.J. (1975) Proc. Natl. Acad. Sci. (U.S.A.) 72, 1945-1949. Nakai, C., and Brooker, G. (1975) Biochim. Biophys. Acta 391,222-239. Nimrod, A., Bedrak, E., and Lamprecht, S.A. (1977) Biochem. Biophys. Res. Commun. 78, 977-984. Rajaniemi, H., and Jaaskelainen, K. (1979) in: Ovarian Follicular and Corpus Luteum Function, Eds.: C.P. Channing, J. Marsh and W.A. Sadler (Plenum, New York), pp. 129-135. Rajaniemi, H.J., Midgley Jr., A.R., Duncan, J.A., and Reichert Jr., L.E. (1977) Endocrinology 101,898-910. Rao, M.C., Richards, J.S., Midgley Jr., A.R., and Reichert Jr., L.E. (1977) Endocrinology 101, 512-523. Richards, J.S.. Ireland, J.J., Rao, M.C., Bernath, G.A., Midgley Jr., A.R., and Reichert Jr., L.E. (1976) Endocrinology 99, 1562-1570. Richards, J.S., Uilenbroek, Th.J., and Jonassen, J.A. (1979) in: Ovarian Follicular and Corpus Luteum Function, Eds.: C.P. Channing, J. Marsh and W.A. Sadler (Plenum, New York), pp. 11-26. Sharpe, R.M. (1976) Nature (London) 264,644-646. Su, Y.-F., Harden, T.K., and Perkins, J.P. (1979) J. Biol. Chem. 2.54, 38-41. Zeleznik, A.J., Midgley Jr., A.R., and Reichert Jr., L.E. (1974) Endocrinology 95, 818-825. Zor, U., Lamprecht, S.A., Misulovin, Z., Koch, Y., and Lindner, H.R. (1976) Biochim. Biophys. Acta 428, 761-765.
145
HUMAN CHORIOGONADOTROPIN-INDUCED DESENSITIZATION OF GRANULOSA-CELL ADENYLATE CYCLASE TO GONADOTROPINS AND LOSS OF LH/hCG RECEPTOR .. .. Kalle JAASKELAINEN,
Tapani HYVijNEN
and Hannu RAJANIEMI
Department of Biochemistry and Anatomy, University of Kuopio, SF-70101 Kuopio 10, and Department of Anatomy, University of Oulu, SF-90220 Oulu 22 (Finland)
Received 8 May 1980; accepted 10 July 1980
Immature female rats that had been primed with pregnant-mare serum gonadotropin (PMSG) were injected intravenously with various doses of human choriogonadotropin (hCG) for the investigation of the relationship between adenylate cyclase activities and the concentrations of LH/hCG receptor in luteinizing granulosa cells. Injection of 1 pg of hCG induced a loss of LH and FSH sensitivities of adenylate cyclase within 6 h and a disappearance of free LH/ hCG receptors within 24 h. Basal adenylate cyclase activity has a transient maximum at 6 h after hCG injection. After injection of 100 pg of hCG the loss of LH sensitivity of adenylate cyclase and free LH/hCG receptors occurred immediately, but the changes in FSH-stimulated and basal activities followed the same time scale as after injection of 1 fig of hCG. When hCG was omitted from the injections the response of the animals to the endogenous gonadotropin surge varied. A complete desensitization of adenylate cyclase to LH and FSH stimulation and a 65% loss of free LH/hCG receptors were found at 24 h if the follicles were ovulated. These results suggest that occupation of a limited number of LH/hCG receptors in granulosa cells induces adenylate cyclase refractory to further stimulation by gonadotropins. The transient elevation of basal adenyiate cyclase activity and its desensitization to further stimulation by gonadotropins may have a role in physiological processes leading to ovulation and luteinization. Keywords:
adenylate cells.
cyclase desensitization;
LH/hCG-receptor
regulation; granulosa
Several hormones regulate the number of their own receptors in the target tissue. Exposure of target cells to elevated hormone concentrations leads to a decrease of receptors for insulin (Gavin et al., 1974), thyrotropin-releasing hormone (Hinkle and Tashjian, 1975), catecholamines (Mukherjee et al., 1975), growth hormone (Lesniak and Roth, 1976), human epidermal growth factor (Carpenter and Cohen, 1976) and LH/hCG (Sharpe, 1976; Conti et al,, 1976, 1977a, b; Haour and Saez, 1977; Rao et al., 1977). In some tissues, hormone-induced loss of receptors is associated with the loss of hormone sensitivity of adenylate cyclase (Mukherjee et al., 1975; Kebabian et al., 1975; Conti et al., 1976; Hsueh et al., 1977). However, at present the exact molecular mechanisms of the receptor loss and its relationship to
146
KaNe J&iskeltiinen, Tapani Hyvtinen, Hannu Rajaniemi
the desensitization of adenylate cyclase are not known. The maturation of the Graafian follicle is associated with the appearance of LH/ hCG receptors (Zeleznik et al., 1974; Richards et al., 1976; Nimrod et al., 1977; Rajaniemi et al., 1977). The existence of high LH/hCG- and FSH-receptor levels and the CQUpkIg of receptors with adenylate cyclase form a prerequisite for ovulation to occur. In preovulatory follicles, theta interna cells and peripheral granulosa cells contain the highest level of LH/hCG receptors, whereas FSH receptors are distributed evenly among the granulosa cells (Zeleznik et al., 1974; Louvet and Vaitukaitis, 1976; Rajaniemi et al., 1977). The loss of LH/hCG and FSH receptors has been reported to accompany ovulation induced by exogenous gonadotropins (Richards et al., 1976; Rao et al., 1977; Rajaniemi and Jaaskehiinen, 1979). Both exogenous hCG and endogenous ovulatory gonadotropin surges have been reported to render adenylate cyclase of intact pre-ovulatory follicles refractory to further stimulation by gonadotropins (Hunzicker-Dunn and Birnbaumer, 1976a, b); after luteinization of the granulosa cells the high LH/hCG receptor level and LH sensitivity of the adenylate cyclase are regained (Holt et al., 1976; Hunzicker-Dunn and Birnbaumer, 1976a, b). The present study was undertaken to investigate whether a desensitization of adenylate cyclase could be demonstrated in granulosa cells isolated from rat pre-ovulatory follicles after various times from hCG injections and to investigate the relationship between gonadotropin sensitivity of adenylate cyclase and the levels of unoccupied and occupied LH/hCG receptors. A few reports indicate that neither endogenous gonadotropin surges nor exogenous hCG injections render adenylate cyclase of granulosa cells refractory to LH stimulation (Nimrod et al., 1977; Hamberger et al., 1979). These results are in contrast with results in the present paper and reports on the behaviour of intact follicles.
MATERIALS
AND METHODS
Animal treatment and radioiodination of hCG Immature 25-day-old female Wistar rats were injected S.C. at 09.00 h with 15 IU of pregnant-mare serum gonadotropin (PMSG) (Gestyl, Organon) to initiate and synchronize the follicular development. 2 days later at 13.00-14.00 h the animals received, under light ether anaesthesia, a single tail-vein injection of 1 or 100 1.18of hCG (7700 IU/mg, Diosynth) in 0.2 ml of 0.01 M phosphate-buffered 0.14 M NaCl (PBS) (PH 7.0). The activities of adenylate cyclase and the levels of free LH/hCGreceptor sites were measured before and at 15 min, 1, 6 and 24 h after the injections. In control experiments, hCG was omitted from the injection. In experiments on uptake of hormone in vivo each dose of hCG was mixed with 0.7 or 3.3 nCi of 1251labelled hCG before the injection. Highly purified hCG (11500 IU/mg), donated by Dr. A. Rees Midgley Jr., University of Michigan, was radio-iodinated as described previously (Greenwood et al., 1963; Markkanen et al., 1980). The specific activity of the preparations averaged 25 /.K!i/pg determined by the method of Ireland and Richards (1978).
Regulation of adenylate cyclase and gonadotropin receptors
141
Adenylate cyclase activity For measurement of adenylate cyclase activity 3 rats were decapitated at each time, ovaries were removed and granulosa cells were isolated by rupturing follicles and expressing cells at 4” into a buffer consisting of 27% sucrose (BDH Chemicals), 1 mM EDTA (Merck) and 10 mM Tris-HCI (Sigma) (pH 7.5). Granulosa cells of the same time point were pooled, a portion of the cell suspension was taken for measurement of available LH/hCG-binding sites and the rest was homogenized by 4 strokes in a ground-glass tissue grinder. Homogenization was carried out during 1 h from killing of the animals, and the homogenates were assayed for adenylate cyclase within 15 min from preparation. Adenylate cyclase activities were measured in triplicate assays by a modification of the method of Nakai and Brooker (1975). The fmal assay mixture contained 50 mM Tris-HCI (pH 7.5), 4 mM MgC12 (Merck), 0.45 mM EDTA, 0.2 mM ATP (Sigma), [cY-~*P]ATP (Radiochemical Centre, Amersham) to give a final specific activity of 100-300 cpm/pmole, 0.5 mM cyclic AMP (Sigma), 20 mM creatine phosphate (Sigma) and creatine phosphokinase, 50 U/ml (Sigma). When present, the final concentration of KF (Baker) was 10 mM. Responses to gonadotropins were determined by addition of ovine LH (NIH-LHS16) or ovine FSH (NIH-FSH-S9) to a final concentration of 20 @g/ml. Reaction was started by adding 20 ~1 of granulosa cell homogenate containing 0.1-0.2 mg of protein to prewarmed incubation tubes; the final assay volume was 50 fl. The linearity of the reaction with regard to homogenate protein concentration was tested by measuring the fluoride-stimulated enzymic activity, and the linearity with regard to time was tested by measuring basal, LH- and FSH-stimulated activities. The incubation temperature was 30’. Unless otherwise stated, the reaction time was 2Cl min. The reaction was stopped by addition of 1 ml of 1 M HCI; most of the remaining ATP was destroyed by heating the samples at 90’ for 10 min, and the samples were then neutralized by adding 0.5 ml of 2 M KOH. Thereafter the samples were precipitated by adding 0.5 ml of 0.15 M Ba(OH)2 (Merck) and 0.5 ml of 5% ZnSO4 . 7 Hz0 (Merck), and were centrifuged. In this procedure, ATP, ADP, AMP and phosphate adsorb to the precipitate and cyclic AMP remains in the supernatant. The precipitation was repeated twice by taking 1 ml of the supernatarit. 1 ml of the final supernatant was taken for the liquid-scintillation counting. Measurement of unoccupied LH,hCG-binding sites For measurement of available LH/hCG-binding sites, portions of pooled granuloss cell suspensions were taken before homogenization. The binding assays were conducted in triplicate in polypropylene tubes precoated with bovine serum albumin. The assay tubes contained an aliquot of granulosa cell suspension equivalent to 0.1-0.2 mg of protein (20 d), [1251]hCG, 200 000 cpm (10 PI), an excess of unlabelled crude hCG (10 A) (30 IU) (Pregnyl, Organon) or an equivalent volume of buffer and 0.1% bovine serum albumin in PBS (pH 7.0) to give a final volume of 120 ~1. After incubation of the mixture for 4 h at 22” with continuous shaking, 3 ml of ice-cold PBS was added, and the granulosa cells were immediately sedimented by
148
Kalle J,i~skeEnen,
Tapani HyvBnen, Hannu Rajaniemi
centrifugation at 1200 X g for 15 min. The rinse of the cells with ice-cold PBS was repeated, and pellets were counted in an automatic gamma counter with 50% efiiciency. [ r2’I]hCG at about 100000-150000 cpm was sufficient to saturate all available binding sites in receptor assays; higher concentrations of [12’I]hCG did not increase the amount of bound hormone. Measurement of uptake of hCG in vivo After injection of the rats with the mixture of [‘251]hCG and unlabelled hCG 2 rats were decapitated at each time, blood was collected and an aliquot of serum counted in the gamma counter. Ovaries were removed and granulosa cells were isolated by rupturing follicles and expressing cells into PBS (pH 7.0); the cells were washed once with PBS and counted for 1251 radioactivity in the gamma counter. The amounts of hormone in serum and in granulosa cells were calculated from the known specific activities of the injection mixtures. Protein determination Protein was determined by the method serum albumin as a standard.
of Lowry et al. (1951) with bovine
RESULTS Linearity of the adenylate cyclase assay with protein concentration and time The time course of the adenylate cyclase reaction is shown in Fig. 1. The concentration of granulosa cell homogenate protein in each assay tube was 208 pg/50 ~1. The linearity of the basal adenylate cyclase activity was maintained for 25 min and the linearities of LH- and FSH-stimulated activities for 20 min. Fluoride-stimulated adenylate cyclase activity as a function of granulosa cell homogenate protein is shown in Fig. 2. The incubation time was 20 min. The enzymic activity was linearly proportional to homogenate protein concentration over a range up to 250 pg of protein in 50 /_d of assay volume. The ATP-regenerating system is essential for the linearity of the reaction (Nakai and Brooker, 1975). We found that, after an incubation of 20 min, 80% of [a-32P]ATP remained intact in the assay mixture. Adenylate cyclase and unoccupied LH/hCG receptors Rats pretreated with PMSG were injected intravenously with 1 or 100 pg of hCG l-2 h before the onset of endogenous gonadotropin surge. The adenylate cyclase activities of the granulosa cell homogenates after injection of 100 pg of hCG are shown in Fig. 3. The ability of LH to stimulate adenylate cyclase disappeared within 15 min. The loss of enzymic response to FSH was slower. After 1 h, FSH stimulation was still 2-fold. FSH receptors were evidently responsible for the stimulation of adenylate cyclase activity by. FSH. After luteinization the adenylate cyclase activity of rat luteal homogenates could be stimulated by hCG and by LH
ofaden~~~e cyclase and gonadotropin receptors
Regulation
5
10 15 INCUBATION TIME
20 irncni
25
149
,ug
HOMOGENATE
PROTEIN
Fig. 1. Adenylate cyclase activities of granulosa cell homogenates as a function of incubation time. The assays were performed as described in Materials and Methods. The drugs were present at the fo~ow~~ concelltrations: LH, 20 &g/ml: FSH, 20 fig/ml. The concentration of the homogenate protein in each assay tube was 208 @g/SOPI. Each point represents the mean (+SElM) value of triplicate determinations. Fig. 2. Fluoride-stimulated adenylate cyclase activity as a function of the quantity ofgranulosa celI homogenate protein in the assay volume of 50 pi. The assays were performed as described in Materials and Methods. The concentration of KF was IO mM. Each point represents the mean (+SEM) vaIue of triplicate determinations.
but not by the FSH concentration (20 p&/ml) used in our adenylate cyclase assays (results not shown). Adenylate cyclase activity measured in the presence of fluoride ion fell by about 40% within 1 h. Basal activity reached a maximum at 6 h, but after 24 h it had returned to the initial level. Fig. 4 shows the adenylate cydase activities after injection of 1 ,ug of hCG. A decrease in both LH and FSH responses was seen after 1 h; after 6 h both responses had disappeared. Fluoride-stimulated activity fell by about 30%. Basal activity had a maximum at 6 h, but after 24 h it had declined to the initial level. To obtain an insight into the me~h~isms of the desensjt~~ation we measured the levels of free LH/hCG receptors of the same granulosa cells we used in measuring the adenylate cyclase activities. Receptor levels compared with initial values are shown in Fig. 5. Injection of 100 pp of hCG saturated all measurable LH/hCG receptor sites within a few minutes. After administration of 1 ,ug of hCG the level of free ~~/hCG receptors had declined at 1 h by 35% and at 6 h by 90%. With the Iatter dose, loss of LH sensitivity of adenylate cyclase seemed to be slightly faster than the loss of free LH/hCG receptors. To control the possible desensitizing effect of endogenous gonadotropins on adenylate cyclase, PMSG-treated rats were injected with PBS instead of hCG. The results from one injection group to another varied. Adenylate cyclase activities of one group of animals are shown in Fig. 6. Basal activity had a slight maximum after 6 h from PBS injections. After 24 h, gonadotropin responses had disappeared. The
150
K&e J&iskeli:inen, Tapani ffyvhen, Hannu Rajaniemi
t
lh
hCG~lK)uy
2h IV) TIME
3h
&h
AFTER
5h
6h
2Lh
INJECTION
Fig. 3. Adenylate cyclase activities of granulosa cell homogenates before and after an intravenous injection of 100 pg of hCG. Triplicate assays were performed as described in Materials and Metbods. The drugs were present at the following co~c~n~at~ons: LH, 20 &g/ml; FSH, 20 &g/ml; KF, 10 mM. Each point represents the mean (+SEM) value of a cell pool collected from the ovaries of 3 rats.
lh 2h hCG(l,ug iv) TIME
t
3h 4h 5h AFTER INJECTION
Gh
Uh
J
Fig. 4. Adenylate cyclase activities of granulosa cell homogenates before and after an intravenous injection of 1 pg of hCG. Triplicate assays were performed as described in Materials and Methods. The drugs were present at the following concentrations: LH, 20 pg/ml; FSH, 20 rrg/ ml; KF, 10 mM. Each point represents the mean (*SEM) value of a cell pool collected from the ovaries of 3 rats.
Regulation of adenylate cyclase and gonadotropin receptors
Lh
.3h hCGllv
I
TIME
AFTER
5h INJECTION
Gh
151
24h
Fig. 5. Unoccupied LM/hCG-binding sites of granulosa cells after intravenous injections of hCG or PBS. Triplicate binding assays were performed as described in Materials and Methods. The results are expressed as percentage of initial binding sites calculated on a protein basis. Each point represents the mean (iSEM) value of a cell pool collected from the ovaries of 3 rats.
level of free LH/hCG receptors after 24 h was still 35% of the initial level (Fig. 5). Stereomicroscopic examination of the ovaries from this injection group showed that a majority of the follicles had ovulated. Within another group of rats, 7 from the total of 9 had ovaries that showed no ovulation at 24 h. The basal, LH- and FSH-stimulated adenylate cyclase activities of the granulosa cell homogenates from
f4 PBS
6h iv
TIME
AFTER
24h
INJECTION
Fig. 6. Adenylate cyclase activities of granulosa cell homogenates before and after an intravenous injection of PBS. Triplicate assays were performed as described in Materials and Methods. The drugs were present at the following concentrations: LH, 20 pg/ml; FSH, 20 pg/ml; KF, 10 mM. Each point represents the mean (+SEM) value of a cell pool collected from the ovaries of 3 rats.
Kalle JciCskelCinen, Tapani HyvBnen, Hannu Rajaniemi
152
lh hCGllv1
2h 3h Lh 5h TIME AFTER INJECTION
Fh
24h
Fig. 7. Serum concentrations of hCG after intravenous injections of 0.7 &i of ‘2SI-labelled hCG together with 1 pg of unlabelled hCG or 3.3 nCi of 1 2 5 I-labelled hCG with 100 ng of unlabelled hCG. Each point represents the mean (+SEM) value for 2 rats.
these unovulated follicles at 24 h after PBS injections were 3.1, 10.3 and 10.5 pmoles CAMP/mm X mg homogenate protein, resp. The level of free LH/hCG receptors was 50% higher than the initial receptor level. The value of basal adenylate cyclase activity after 6 h from PBS injections was 2.7 pmoles cAMP/min X mg homogenate protein. Kostyk et al. (1978) reported that the responses of immature rats to exogenous gonadotropin treatment are dependent upon the type and quantity of injected gonadotropin. The percentage of animals ovulating at 72 h after a single S.C.injection of 15 IU of PMSG was about 35.
z B z 20 x
A k! 10
/‘%CG
PLUS
‘,,cJ OF
‘L&C
KC
3 3 2
2
:-_:t
hCGllv
lh
1
2h TIME
3h 4h 5h AFTER INJECTION
Gh
24h
Fig. 8. Occupancy of granulosa cells by hCG after intravenous injections of 0.7 nCi of 1251labelled hCG together with 1 gg of unlabelled hCG or 3.3 PCi of 1251-labelled hCG with 100 Mg of unlabelled hCG. The specific radioactivities of the gonadotropin fractions bound to granulosa cells were assumed to be the same as the specific radioactivities of the injection mixtures. Each point represents the mean (+SEM) value for 2 rats.
Regulation of adenylate cyclase and gonadotropin
receptors
153
Serum and granulosa cell hCG levels Serum and granulosa-cell hCG levels after intravenous injections of 1 or 100 pg hCG together with 1251-labelled hCG are shown in Figs. 7 and 8. By gel filtration on Sepharose 6B, 90-95% of the serum radioactivity was identified to be intact [‘251]hCG (results not shown). The radioactivity of the granulosa cells showed a maximum after 2 h from the injection of hCG (Fig. 8). By comparing the hCG concentrations bound to the granulosa cells (Fig. 8) and the concentrations of free receptors (Fig. 5) one can conclude that occupation of a small portion of LH/hCG receptors induces the loss of all high-affinity LH/hCG-receptor sites. After injection of 1 pug of hCG, the loss of free receptors seemed to be quantitatively more important than the loss of the occupied receptor sites. By gel filtration on Sepharose 6B, 70-90% of the radioactivity of the granulosa cells was identified as receptor-[1251]hCG complex.
DISCUSSION The results of the present study suggest that intravenous injections of microgram quantities of hCG, in 6 h, render adenylate cyclase of rat pre-ovulatory granulosa cells refractory to further stimulation by LH or FSH. Conti et al. (1976) found that, after 6 h from an S.C. injection of 20 IU of hCG (2 pg of hCG), the adenylate cyclase of luteinized rat ovaries can no longer be stimulated by LH. The loss of LH/ hCG receptors is complete after 24 h. Later, Conti et al. (1977a) reported that the lowest dose of hCG to cause a measurable loss in LH/hCG receptors of luteinized ovaries is about 20 ng/lOO g of body weight, which gives a serum concentration of hCG within the physiological serum concentration of LH. Conti et al. (1977b) also reported that, in desensitized rat luteal cells, the decrease in LH/hCG-receptor level is accompanied by a lesion in the progesterone biosynthetic pathway beyond the level of cyclic AMP production. The results of our control experiments (PBS instead of hCG injections) were not reproducible at 24 h from one injection group of rats to another. However, the desensitization of the adenylate cyclase to gonadotropins found at 24 h after injection correlated positively with the follicular ovulation. In addition, the control experiments showed that the desensitization of the adenylate cyclase to gonadotropins occurs without a total .loss of the receptors. Hunzicker-Dunn and Birnbaumer (1976b) report that, after 3 days from treating prepubertal rats with PMSG, a synchronous ovulation has occurred with a concomitant loss in the ability of LH and FSH to stimulate adenylate cyclase of ovarian homogenates. Adenylate cyclase activities measured in homogenates of ovarian follicles of cycling rats also become desensitized to LH and FSH by the morning of oestrus (Hunzicker-Dunn and Birnbaumer, 1976b). The present results are contradictory to the results of Nimrod et al. (1977) and to the results of Hamberger et al. (1979). Nimrod et al. (1977) used as a model sys-
154
Kalle Jriiiskehiinen, Tapani HyvSnen, Hannu Rajaniemi
tern normal cycling rats and they injected the rats i.p. at 14.00 h on the day of prooestrus with 50 IU of hCG. They did not find a loss of LH sensitivity of adenylate cyclase in granulosa cells collected at 24.00 h either from follicles of hCG-treated rats or from follicles of untreated rats. Hamberger et al. (1979) used PMSG-treated rats and collected the granulosa cells either before or after the endogenous gonadotropin peak. Adenylate cyclase of granulosa cells collected a few hours after the endogenous LH-FSH peak did not show-a desensitization to LH stimulation. Intraperitoneal injections of LH or FSH (10 pg/rat) did not desensitize adenylate cyclase to LH or FSH when granulosa cells were collected after 2 h from the injections. We think it is important to follow the time course of adenylate cyclase activities for longer times than 10 h after endogenous surges or exogenous gonadotropin injection. In the control experiment of Fig. 6 the desensitization to gonadotropin stimulation was seen at 24 h but not yet at 6 h after PBS injections. It is also possible that intraperitoneally injected hormones are effectively taken up by the liver and catabolized (Markkanen et al., 1979). We found that, after injection of 1 pg of hCG, occupancy of a limited portion of LH/hCG receptors initiated desensitization of adenylate cyclase and loss of occupied as well as of free LH/hCG receptors. The,rapidity of the loss of free LH/hCG receptors suggests that aggregation or clustering of the receptors may form one step in their inactivation. Especially after endogenous gonadotropin surge, the desensitization of adenylate cyclase to LH seemed to be faster than the loss of free LH/hCG receptors, which suggests that, besides the disappearance of receptors, other mechanisms may be involved in the desensitization process. Su et al. (1979) incubated human astrocytoma cells with low concentrations of isoproterenol and compared the decrease in /3-adrenergic receptor density with a reduction in isoproterenolstimulated adenylate cyclase activity. 2 different reactions were found to’ be involved in the desensitization process: initially a loss of adenylate cyclase activity occurs with little loss of fl-adrenergic receptors, and a slower reaction results in a marked decrease of fl-adrenergic receptors. Su et al. (1979) suggest that the loss of fl-adrenergic receptors occurs as a result of the initial uncoupling of the P-receptorlinked adenylate cyclase. In pre-ovulatory follicles, theta interna cells and peripheral granulosa cells contam the highest level of LH/hCG receptors, and FSH receptors are distributed evenly among the granulosa cells (Zeleznik et al., 1974; Louvet and Vaitukaitis, 1976; Rajaniemi et al., 1977). The ability of FSH to stimulate the adenylate cyclase of the cell homogenates indicates that the cell pools obtained consist mainly of granulosa cells. Desensitization of adenylate cyclase to FSH was relatively slow and followed a similar pattern after both doses of hCG (Figs. 3 and 4). Perhaps intercellular communication through gap junctions (Lindner et al., 1977; Gilula et al., 1978) is a prerequisite for desensitization of innermost granulosa cells to FSH stimulation. Different results have been obtained for the hormone specificity of the desensiti-
Regulation of adenylate cyclase and gonadotropin receptors
155
zation of ovarian adenylate cyclase. Zor et al. (1976) found that culture of preovulatory rat follicles with LH, FSH or prostaglandin Ez for 24 h desensitized adenylate cyclase to homologous hormone but not to heterologous hormones. Richards et al. (1979) found that iv. injection of hCG to hypophysectomized, oestradiol-, and FSH-primed rats renders adenylate cyclase refractory to FSH stimulation in 2 h. In the reverse experiment, FSH rendered adenylate cyclase refractory to hCG stimulation in 2 h. In our experiments, adenylate cyclase of granulosa cells became refractory to FSH stimulation in 6 h after hCG injections and in 24 h after PBS injections (control experiment of Fig. 6). After hCG injection, and also in the control experiment of Fig. 6, basal adenylate cyclase activity reached a transient maximum at 6 h. Bauminger et al. (1978) found that the cyclic AMP level of rat pre-ovulatory follicles increases by about 40% after 3-5 h from the onset of endogenous gonadotropin surge and that the cyclic AMP level returns to the initial level at the time of ovulation. The results of the present study suggest that the activation of granulosa cell adenylate cyclase by endogenous LH-FSH surge and its further desensitization to gonadotropins cause the fluctuations in the cyclic AMP levels, and that these changes have a physiological role in the positive ovulatory response of rat Graatian follicles.
ACKNOWLEDGMENTS We thank Prof. A.R. Midgley Jr., University of Michigan, for a donation of highly purified hCG. The skilful technical and secretarial assistance of Miss Arja Venakiinen and Sirkka Pennanen is greatly appreciated. Financial support was given by the Academy of Finland and by the Cultural Foundation of Finland.
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Kalle Jb%kel&en,
Tapani Hyvdnen, Hannu Rajaniemi
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