- Email: [email protected]
Effect of gonadectomy on pituitary levels of mRNA encoding gonadotropin subunits and secretion of luteinizing hormone
Molecular and Celltdar Endocrinology, 35 (1984) 83-87 Elsevier Scientific Publishers Ireland, Ltd.
83
MCE 01126
Effect of gonadectomy on pituitary levels of mRNA encoding gonadotropin subunits and secretion of luteinizing hormone Maithk Corbani, Raymond Counis, Anna Starzec and Marian Jutisz * Laboratoire des Hormones Polypeptidiques, (Received
Keywords:
pituitary
mRNA;
gonadotropin
15 September
synthesis;
CNRS, 91190
1983; accepted
cell-free
translation;
Gif -SW- Yvette (France)
16 December
gonadectomy
1983)
in rats; LH secretion.
Summary
Using cell-free translation of pituitary mRNAs we have investigated how, following gonadectomy in rats, the translational capacity of the specific messages encoding precursors to gonadotropin subunits LY, LH-fl and FSH-JI increases with time. In parallel, serum LH was assayed in order to compare release and synthesis patterns. We observed a rapid rise in the rate of synthesis of all three precursors, with a significant increase already detectable 4 days after gonadectomy, and a plateau reached after 21 days. The kinetics were similar in both males and females, but maximal translational values for a-subunit were slightly higher in males. During the same time period, serum LH rapidly increased in the males, while in the females the rise of circulating LH was somewhat delayed. Although no direct correlation seems to exist between synthesis and release processes of gonadotropins, it is evident from our previous findings and the present data that both phenomena are dependent on gonadal steroids. In this respect, estradiol has been shown to regulate negatively, via different routes, the synthesis as well as the secretion of pituitary gonadotropins.
A number of studies have now established that, in the rat, gonadotropin secretion increases in response to gonadectomy (McCann and Ramirez, 1964; Gay and Bogdanove, 1969; Gay and Midgley, 1969). Supplementation of operated animals with gonadal steroids has shown that the rise in serum gonadotropins is reversible, thus confirming the steroid-mediated negative feedback on the hypothalamus-anterior pituitary axis. While the effect of gonadal steroids on pituitary and serum levels of gonadotropins is fairly well documented, until recently nothing has been known about the influence of gonadectomy on either the biosynthesis of gonadotropin subunit precursors, or the * To whom correspondence 0303-7207/84/$03.~
should
be addressed.
0 1984 Elsevier Scientific
Publishers
Ireland,
regulation of their messenger RNAs. In recent work we observed that ovariectomy in rats results in the enhancement of the levels of precursors to LY,LH-@ and FSH-P encoded by specific mRNAs (Counis et al., 1982a, b, c). It has also been recently reported that orchidectomy of male sheep (Alexander and Miller, 1982) and ovariectomy of ewes (Landefeld et al., 1983) enhances the levels of pituitary precursors to FSH-/3- and -a-subunits, respectively. Treatment of gonadectomized animals with 17&estradiol (E2) reverses the effects of gonadectomy (Alexander and Miller, 1982; Landefeld et al., 1983; Counis et al., 1983a, b). As all these studies were performed using long-term castrated animals, a further step in our study of the regulation of mRNAs encoding rat LH and Ltd.
84
FSH subunits by E, was to determine the time dependence of the effects of gonadectomy. With this object, we have prepared RNAs from anterior pituitaries of male and female rats at different time periods following gonadectomy and translated them in a cell-free system. The precursors to each subunit were then isolated and quantified. In addition, radioimmunoassay (RIA) of serum LH was performed in parallel using the same series of experimental animals, allowing us to compare the patterns of serum LH with messenger translational capacity. Materials and methods Experiments were carried out with 3-month-old adult male and female Wistar rats (laboratory strain). Rats, maintained in common cages under artificial light (14 h light, 10 h dark), were fed ad libitum. Gonadectomies were performed under ether anaesthesia. Rats were sacrificed by decapitation after appropriate delays (0, 4, 7, 14, 21 and 28 days following gonadectomy), and always at the same time of the day (16.00 h). Blood was collected in polyethylene tubes and pituitaries were immediately excised; the anterior lobes were dissected and stored in liquid nitrogen. LH in the serum was assayed using the NIAMDDK rat RIA kit. The procedure was that of Kerdelhue et al. (1971), except that protein A (Pansorbin, Calbiochem Behring) was substituted for the second antibody. All tubes were duplicated and two concentrations of serum were assayed in each case. Results were evaluated statistically using Student’s t-test and expressed in terms of the NIAMDDK rat LH-RP2. RNA was extracted from pituitaries in each group in parallel, according to the microscaled method previously described (Counis et al., 1981b). In brief, deep-frozen rat pituitaries (7/group) were homogenized in a lysis buffer consisting of 4 M guanidinium chloride, 1% sarcosyl (Ciba-Geigy, Basel, Switzerland), 0.1 M mercaptoethanol in 50 mM sodium acetate, pH 5. The homogenate was layered over a 6 M caesium chloride cushion containing 0.1 M EDTA and centrifuged at 130000 X g, for 15 h at 25°C. The pelleted RNA was solubilized, twice reprecipitated in 70% ethanol and finally solubilized in 35 ~1 of 10 mM Tris-HCI, pH
7.4. This methodology gave highly reproducible results. Messenger RNAs were then translated under optimal conditions, in a wheat-germ cell-free system pretreated with nuclease (Counis et al., 1981a). Translation was performed in 50 ~1 of medium containing RNA extracted from 0.8 pituitary gland (non-saturating optimal dose), 1.5 mCi/ml [ 35S]methionine (1100 Ci/ mmole, CEA, France), 1.5 mCi/ml [35S]cysteine (1000 Ci/ mmole, NEN, U.S.A.) and 300 U/ml placental ribonuclease inhibitor (Bolton Biologicals, U.S.A.). Precursors to the gonadotropin subunits were isolated by immunoprecipitation with antisera directed against cy, LH-fi and FSH-/? previously denatured via reduction and carboxymethylation (RCXM subunits) (Counis et al., 1982b). Following SDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) and fluorography, bands corresponding to precursors were excised and counted after solubilization with H,O,. Results were expressed as cpm % of total radioactivity incorporated into total protein. Results The translational capacity of mRNAs extracted at different times following castration was investigated in three independent experiments performed under the same conditions. Fig. 1 shows a typical pattern of incorporation of radioactivity into the cy, LH-/3 and FSH-@ precursors as ‘% of total protein synthesized in response to mRNAs. In the male (Fig. 1A) as well as in the female (Fig. 1B) rat, the rate of synthesis of the LYprecursor can be divided into two phases: first, a phase of rapid increase until the 7th day after surgery, followed by a phase of slower increase which finally reaches a plateau after about 21 days. Although starting from the same basal values, (0.27-0.29% of the total protein), the maximal increase was greater in the male (2.9%) than in the female (2.3%). A comparison of the rates of synthesis of LH-/3 and FSH-J? precursors shows that the proportion of these precursors in the translation media was approximately 5-30-fold lower compared to the cy precursor. Therefore Fig. 1 does not accurately represent the real variations of LH-p and FSH-P precursors observed following castration. In the case of LH-/3, the values increased from 0.05%
85
Females
=1
1.5_ */
A
l.O_ 0.5.
o
/ A
I 0
I
Time
1
after
1
I
2
3
castration
I
4
0,*&O , 0
(weeks)
0
-D-D-D-0 I
Time
-
/c/o1
after
I
I
8
2
3
crstr8tion(weeks)
4
Fig. 1. Time-course of the gonadectomy-induce increase in gonadotropin subunit precursor levels in gonadectomized male (A) and female rats (B). Pituitary RNAs were prepared at appropriate times following gonadectomy, translated in cell-free conditions, and the precursors characterized as described above (see the text for further details). Precursor levels are expressed as cpm % of total protein: A, A, a precursor; l, 0, LH-P precursor; n, 0, FSH-fl precursor, in males and females, respectively.
(660 cpm) and 0.04% (530 cpm) (day 0) of the total protein (1.32 x lo6 cpm) to 0.70% (9250 cpm) and 0.43% (5670 cpm) at day 28, in male and female rats, respectively. The increase in FSH-P was from 0.009% (120 cpm) and 0.012% (160 cpm) to 0.111% (1460 cpm) and 0.074% (975 cpm) in males and females, respectively. As Fig. 1 does not give a real account of variations in LH-j3 and FSH-fl synthesis, we show in Fig. 2 an example of a fluorograph of the 35S-labelled FSH-/3 precursor immunoprecipitated with antiserum to RCXM-FSH-j3 from the translation media of mRNAs derived from male rats at different times following orchidectomy and electrophoresed on SDS-PAGE. The band, already well perceptible 4 days following orchidectomy, increases to maximum intensity on day 21; the same pattern is observed for the iy and LH-/3 precursors. In normal rats, the band corresponding to the FSH-fi precursor is hardly detectable on the fluorograph (Counis et al., 1983b). The alteration in the Dattern of serum LH in response to gonadectomy (Fig. 3) differs somewhat between male and female rats. The level of LH in the male increased rapidly to 7 ng/ml after 4 days following gonadectomy and to more than 15 ng/ml A
days 0
FSHB-.
after 4
7
castration 14
21
28
‘
Fig. 2. Fluorograph of 3sS-labelled FSH-8 precursor synthesized in a wheat-germ system in response to mRNA derived from normal male rats (day 0) or from rats at different times after orchidectomy (4, 7, 14, 21 and 28 days). Labelled FSH+
precursor was isolated by specific immunoprecipitation and SDS-PAGE
as indicated
in the text.
OJ
t
0 Time
1
1
1
1
2
3
after
castration
1
4 (weeks)
Fig. 3. Serum LH levels (ng/mlfSEM) in male (+) and female (0) rats as a function of time after gonadectomy. LH was assayed using radioimmunoassay. Results are expressed in terms of the NIAMDDK rat LH-RP2.
after 28 days. In the female, the serum LH level decreased up to the 4th day (from 3.2 to 2 ng/ml) and then increased almost linearly to about 12 ng/ml after 28 days. The levels of LH were always somewhat lower in the female than in the male. On the other hand, while the rate of synthesis of gonadotropin subunits in both males and females usually plateaued 21 days after surgery, the levels of LH continued to increase for 28 days following gonadectomy and even afterwards (unpublished results). Discussion It has been repeatedly established that gonadectomy of rats results in a dramatic rise in plasma LH and FSH (McCann and Ramirez, 1964; Schally et al., 1972). A number of studies (Gay and Midgley, 1969; Yamamoto et al., 1970) have shown that the rise in serum LH in response to gonadectomy differs markedly between male and female rats. The initial response in adult females is less marked than in adult males (Yamamoto et al., 1970). Our results confirm these data and show, in addition, a small decrease in circulating LH 4 days after surgery in the female, while males exhibit a
dramatic increase in serum LH levels during the same time period. Although gonadectomy results in a rapid increase in the rate of synthesis of all three subunit precursors, examination of the time-course of their synthesis shows that no direct correlation seems to exist between the synthesis and the release pattern. Although some interconnections might exist between the two processes, it is evident that the synthesis and release of gonadotropins are relayed via different mechanisms, which implies that they are regulated in different manners. In connection with the increased synthesis of the gonadotropin subunits and the increased secretion of LH following gonadectomy, the role of GnRH has to be taken into account. First, it is well known that gonadectomy of male and female rats results in a significant elevation in GnRH in trunk plasma (Wheaton and McCann, 1976). Second, the number of pituitary GnRH receptors also increases in gonadectomized rats (Frager et al., 1981; Clayton and Catt, 1981; Aubert et al., 1983). These latter changes, together with the increase in gonadotropin synthesis, thus contribute to the elevation in levels of serum LH. Furthermore, additional data from our laboratory (Khar et al., 1978), suggesting that GnRH stimulates (directly or indirectly) the biosynthesis of the polypeptide chains of LH and FSH, support the conclusion that GnRH plays a significant determinant role in the post-castration increase in the rate of synthesis of gonadotropin subunits and the elevation of circulating LH. We recently reported (Counis et al., 1983b) that supplementation of gonadectomized rats with estradiol rapidly reverses the stimulatory effect of gonadectomy on the synthesis of (Y, LH-P and FSH-/3 precursors almost to the levels observed in normal rats; progesterone has no such effect. Sirnilar data on the inhibition of the synthesis of FSH subunits (Alexander and Miller, 1982; Landefeld et al., 1983) with estradiol in castrated sheep have been reported. These results thus demonstrate that estradiol negatively regulates the synthesis of pituitary gonadotropins, and suggest that the regulation occurs via changes in specific mRNA levels. This hypothesis has recently been confirmed in our laboratory using synthetic oligodeoxynucleotides (15-16 bases) corresponding to partial se-
87
quences of a, LH-j3 and FSH-/I as probes (results not shown). Using these oligomers which hybridize to the corresponding mRNA sequences with high specificity, we demonstrated that the number of copies of specific mRNAs was lower in normal than in gonadectomized rats. This observation confirms that the inhibitory action of estradiol is on the expression of specific genes. Acknowledgements
We wish to thank Dr. A. Parlow and the National Hormone and Pituitary Program (NIAMDDK) for rat LH-RP2, Dr. J.G. Pierce for his gift of antisera to RCXM bovine LH subunits, Dr. A. Berault and Dr. M. Theoleyre for their help in performing radioimmunoassays and Mr. M. Poissonnier for his help in preparing antisera to RCXM ovine (Y,LH-j3 and FSH-/3. The valuable technical assistance of Mme. G. Ribot is acknowledged. This work was supported by a grant from the Fondation de Recherche en Hormonologie. M.C. is a recipient of a Fondation pour la Recherche M&iicale fellowship. References Alexander, D.C. and Miller, W.L. (1982) J. Biol. Chem. 2.57, 2282-2286. Aubert, M.L. Conne, B.S., Winniger, B.P., Lang, U. and Sizonenko, PC. (1983). In: Multihormonal Regulation in Neuroendocrine Cells, Eds.: A. Tixier-Vidal and P. Richard, Proceedings of the INSERM European Symposium, Colmar (France), pp. 319-346. Clayton, R.N. and Catt, K.J. (1981) Endocrinology 108, 887-895.
Counis, R., Ribot, G., Corbani, M., Poissonnier, M. and Jutisz, M. (1981a) FEBS Lett. 123, 151-155. Counis, R., Corbani, M., Berault, A., Theoleyre, M., Jansem, M. and Jutisz, M. (1981b) CR. Acad. Sci. (Paris) Ser. III 293, 115-118. Counis, R., Corbani, M. and Jutisz, M. (1982a) Program of the 64th Annual Meeting of the Endocrine Society, abstr. 195. Counis, R., Corbani, M., Poissonnier, M. and Jutisz, M. (1982b) B&hem. Biophys. Res. Commun. 107,998-1005. Counis, R., Corbani, M. and Jutisz, M. (1982~) In: Pituitary Hormones and Related Peptides, Eds.: M. Motta, M. Zanisi and F. Piva (Academic Press, New York) pp. 49-61. Counis, R., Corbani, M. and Jutisz, M. (1983a) In: Multihormonal Regulation in Neuroendocrine Cells, Eds.: A. Tixier-Vidal and P. Richard, Proceedings of the INSERM European Symposium, Colmar (France), pp. 509-523. Counis, R., Corbani, M. and Jutisz, M. (1983b) B&hem. Biophys. Res. Commun. 114, 65-72. Frager, M.S., Pieper, D.R., Tonetta, A., Duncan, J.A. and Marshall, J.C. (1981) J. Clin. Invest. 67, 615-623. Gay, V.L. and Bogdanove, E.M. (1969) End~~nology 84, 1132-1142. Gay, V.L. and Midgley Jr., A.R. (1969) Endocrinology 84, 1359-1364. Kerdelhut, B., Pitoulis, S. and Jutisz, M. (1971) C.R. Acad. Sci. (Paris) 273, 511-514. Khar, A., Debeljuk, L. and Jutisz, M. (1978) Mol. Cell. Endocrinol. 12, 53-65. Landefeld, T.D., Kepa, J. and Karsch, F.J. (1983) J. Biol. Chem. 258, 2390-2393. McCann S.M. and Ramirez, V.D. (1964) Recent Progr. Hormone Res. 20, 131-181. Schally, A.V., Kastin, A.J. and Arimura, A. (1972) Vitamins and Hormones 30.83-164. Wheaton, J.E. and McCann, SM. (1976) Neur~nd~~nology 20,296-310. Yamamoto, M., Diebel, N.D. and Bogdanove, E.M. (1970) Endocrinology 86, 1102-1111.
83
MCE 01126
Effect of gonadectomy on pituitary levels of mRNA encoding gonadotropin subunits and secretion of luteinizing hormone Maithk Corbani, Raymond Counis, Anna Starzec and Marian Jutisz * Laboratoire des Hormones Polypeptidiques, (Received
Keywords:
pituitary
mRNA;
gonadotropin
15 September
synthesis;
CNRS, 91190
1983; accepted
cell-free
translation;
Gif -SW- Yvette (France)
16 December
gonadectomy
1983)
in rats; LH secretion.
Summary
Using cell-free translation of pituitary mRNAs we have investigated how, following gonadectomy in rats, the translational capacity of the specific messages encoding precursors to gonadotropin subunits LY, LH-fl and FSH-JI increases with time. In parallel, serum LH was assayed in order to compare release and synthesis patterns. We observed a rapid rise in the rate of synthesis of all three precursors, with a significant increase already detectable 4 days after gonadectomy, and a plateau reached after 21 days. The kinetics were similar in both males and females, but maximal translational values for a-subunit were slightly higher in males. During the same time period, serum LH rapidly increased in the males, while in the females the rise of circulating LH was somewhat delayed. Although no direct correlation seems to exist between synthesis and release processes of gonadotropins, it is evident from our previous findings and the present data that both phenomena are dependent on gonadal steroids. In this respect, estradiol has been shown to regulate negatively, via different routes, the synthesis as well as the secretion of pituitary gonadotropins.
A number of studies have now established that, in the rat, gonadotropin secretion increases in response to gonadectomy (McCann and Ramirez, 1964; Gay and Bogdanove, 1969; Gay and Midgley, 1969). Supplementation of operated animals with gonadal steroids has shown that the rise in serum gonadotropins is reversible, thus confirming the steroid-mediated negative feedback on the hypothalamus-anterior pituitary axis. While the effect of gonadal steroids on pituitary and serum levels of gonadotropins is fairly well documented, until recently nothing has been known about the influence of gonadectomy on either the biosynthesis of gonadotropin subunit precursors, or the * To whom correspondence 0303-7207/84/$03.~
should
be addressed.
0 1984 Elsevier Scientific
Publishers
Ireland,
regulation of their messenger RNAs. In recent work we observed that ovariectomy in rats results in the enhancement of the levels of precursors to LY,LH-@ and FSH-P encoded by specific mRNAs (Counis et al., 1982a, b, c). It has also been recently reported that orchidectomy of male sheep (Alexander and Miller, 1982) and ovariectomy of ewes (Landefeld et al., 1983) enhances the levels of pituitary precursors to FSH-/3- and -a-subunits, respectively. Treatment of gonadectomized animals with 17&estradiol (E2) reverses the effects of gonadectomy (Alexander and Miller, 1982; Landefeld et al., 1983; Counis et al., 1983a, b). As all these studies were performed using long-term castrated animals, a further step in our study of the regulation of mRNAs encoding rat LH and Ltd.
84
FSH subunits by E, was to determine the time dependence of the effects of gonadectomy. With this object, we have prepared RNAs from anterior pituitaries of male and female rats at different time periods following gonadectomy and translated them in a cell-free system. The precursors to each subunit were then isolated and quantified. In addition, radioimmunoassay (RIA) of serum LH was performed in parallel using the same series of experimental animals, allowing us to compare the patterns of serum LH with messenger translational capacity. Materials and methods Experiments were carried out with 3-month-old adult male and female Wistar rats (laboratory strain). Rats, maintained in common cages under artificial light (14 h light, 10 h dark), were fed ad libitum. Gonadectomies were performed under ether anaesthesia. Rats were sacrificed by decapitation after appropriate delays (0, 4, 7, 14, 21 and 28 days following gonadectomy), and always at the same time of the day (16.00 h). Blood was collected in polyethylene tubes and pituitaries were immediately excised; the anterior lobes were dissected and stored in liquid nitrogen. LH in the serum was assayed using the NIAMDDK rat RIA kit. The procedure was that of Kerdelhue et al. (1971), except that protein A (Pansorbin, Calbiochem Behring) was substituted for the second antibody. All tubes were duplicated and two concentrations of serum were assayed in each case. Results were evaluated statistically using Student’s t-test and expressed in terms of the NIAMDDK rat LH-RP2. RNA was extracted from pituitaries in each group in parallel, according to the microscaled method previously described (Counis et al., 1981b). In brief, deep-frozen rat pituitaries (7/group) were homogenized in a lysis buffer consisting of 4 M guanidinium chloride, 1% sarcosyl (Ciba-Geigy, Basel, Switzerland), 0.1 M mercaptoethanol in 50 mM sodium acetate, pH 5. The homogenate was layered over a 6 M caesium chloride cushion containing 0.1 M EDTA and centrifuged at 130000 X g, for 15 h at 25°C. The pelleted RNA was solubilized, twice reprecipitated in 70% ethanol and finally solubilized in 35 ~1 of 10 mM Tris-HCI, pH
7.4. This methodology gave highly reproducible results. Messenger RNAs were then translated under optimal conditions, in a wheat-germ cell-free system pretreated with nuclease (Counis et al., 1981a). Translation was performed in 50 ~1 of medium containing RNA extracted from 0.8 pituitary gland (non-saturating optimal dose), 1.5 mCi/ml [ 35S]methionine (1100 Ci/ mmole, CEA, France), 1.5 mCi/ml [35S]cysteine (1000 Ci/ mmole, NEN, U.S.A.) and 300 U/ml placental ribonuclease inhibitor (Bolton Biologicals, U.S.A.). Precursors to the gonadotropin subunits were isolated by immunoprecipitation with antisera directed against cy, LH-fi and FSH-/? previously denatured via reduction and carboxymethylation (RCXM subunits) (Counis et al., 1982b). Following SDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) and fluorography, bands corresponding to precursors were excised and counted after solubilization with H,O,. Results were expressed as cpm % of total radioactivity incorporated into total protein. Results The translational capacity of mRNAs extracted at different times following castration was investigated in three independent experiments performed under the same conditions. Fig. 1 shows a typical pattern of incorporation of radioactivity into the cy, LH-/3 and FSH-@ precursors as ‘% of total protein synthesized in response to mRNAs. In the male (Fig. 1A) as well as in the female (Fig. 1B) rat, the rate of synthesis of the LYprecursor can be divided into two phases: first, a phase of rapid increase until the 7th day after surgery, followed by a phase of slower increase which finally reaches a plateau after about 21 days. Although starting from the same basal values, (0.27-0.29% of the total protein), the maximal increase was greater in the male (2.9%) than in the female (2.3%). A comparison of the rates of synthesis of LH-/3 and FSH-J? precursors shows that the proportion of these precursors in the translation media was approximately 5-30-fold lower compared to the cy precursor. Therefore Fig. 1 does not accurately represent the real variations of LH-p and FSH-P precursors observed following castration. In the case of LH-/3, the values increased from 0.05%
85
Females
=1
1.5_ */
A
l.O_ 0.5.
o
/ A
I 0
I
Time
1
after
1
I
2
3
castration
I
4
0,*&O , 0
(weeks)
0
-D-D-D-0 I
Time
-
/c/o1
after
I
I
8
2
3
crstr8tion(weeks)
4
Fig. 1. Time-course of the gonadectomy-induce increase in gonadotropin subunit precursor levels in gonadectomized male (A) and female rats (B). Pituitary RNAs were prepared at appropriate times following gonadectomy, translated in cell-free conditions, and the precursors characterized as described above (see the text for further details). Precursor levels are expressed as cpm % of total protein: A, A, a precursor; l, 0, LH-P precursor; n, 0, FSH-fl precursor, in males and females, respectively.
(660 cpm) and 0.04% (530 cpm) (day 0) of the total protein (1.32 x lo6 cpm) to 0.70% (9250 cpm) and 0.43% (5670 cpm) at day 28, in male and female rats, respectively. The increase in FSH-P was from 0.009% (120 cpm) and 0.012% (160 cpm) to 0.111% (1460 cpm) and 0.074% (975 cpm) in males and females, respectively. As Fig. 1 does not give a real account of variations in LH-j3 and FSH-fl synthesis, we show in Fig. 2 an example of a fluorograph of the 35S-labelled FSH-/3 precursor immunoprecipitated with antiserum to RCXM-FSH-j3 from the translation media of mRNAs derived from male rats at different times following orchidectomy and electrophoresed on SDS-PAGE. The band, already well perceptible 4 days following orchidectomy, increases to maximum intensity on day 21; the same pattern is observed for the iy and LH-/3 precursors. In normal rats, the band corresponding to the FSH-fi precursor is hardly detectable on the fluorograph (Counis et al., 1983b). The alteration in the Dattern of serum LH in response to gonadectomy (Fig. 3) differs somewhat between male and female rats. The level of LH in the male increased rapidly to 7 ng/ml after 4 days following gonadectomy and to more than 15 ng/ml A
days 0
FSHB-.
after 4
7
castration 14
21
28
‘
Fig. 2. Fluorograph of 3sS-labelled FSH-8 precursor synthesized in a wheat-germ system in response to mRNA derived from normal male rats (day 0) or from rats at different times after orchidectomy (4, 7, 14, 21 and 28 days). Labelled FSH+
precursor was isolated by specific immunoprecipitation and SDS-PAGE
as indicated
in the text.
OJ
t
0 Time
1
1
1
1
2
3
after
castration
1
4 (weeks)
Fig. 3. Serum LH levels (ng/mlfSEM) in male (+) and female (0) rats as a function of time after gonadectomy. LH was assayed using radioimmunoassay. Results are expressed in terms of the NIAMDDK rat LH-RP2.
after 28 days. In the female, the serum LH level decreased up to the 4th day (from 3.2 to 2 ng/ml) and then increased almost linearly to about 12 ng/ml after 28 days. The levels of LH were always somewhat lower in the female than in the male. On the other hand, while the rate of synthesis of gonadotropin subunits in both males and females usually plateaued 21 days after surgery, the levels of LH continued to increase for 28 days following gonadectomy and even afterwards (unpublished results). Discussion It has been repeatedly established that gonadectomy of rats results in a dramatic rise in plasma LH and FSH (McCann and Ramirez, 1964; Schally et al., 1972). A number of studies (Gay and Midgley, 1969; Yamamoto et al., 1970) have shown that the rise in serum LH in response to gonadectomy differs markedly between male and female rats. The initial response in adult females is less marked than in adult males (Yamamoto et al., 1970). Our results confirm these data and show, in addition, a small decrease in circulating LH 4 days after surgery in the female, while males exhibit a
dramatic increase in serum LH levels during the same time period. Although gonadectomy results in a rapid increase in the rate of synthesis of all three subunit precursors, examination of the time-course of their synthesis shows that no direct correlation seems to exist between the synthesis and the release pattern. Although some interconnections might exist between the two processes, it is evident that the synthesis and release of gonadotropins are relayed via different mechanisms, which implies that they are regulated in different manners. In connection with the increased synthesis of the gonadotropin subunits and the increased secretion of LH following gonadectomy, the role of GnRH has to be taken into account. First, it is well known that gonadectomy of male and female rats results in a significant elevation in GnRH in trunk plasma (Wheaton and McCann, 1976). Second, the number of pituitary GnRH receptors also increases in gonadectomized rats (Frager et al., 1981; Clayton and Catt, 1981; Aubert et al., 1983). These latter changes, together with the increase in gonadotropin synthesis, thus contribute to the elevation in levels of serum LH. Furthermore, additional data from our laboratory (Khar et al., 1978), suggesting that GnRH stimulates (directly or indirectly) the biosynthesis of the polypeptide chains of LH and FSH, support the conclusion that GnRH plays a significant determinant role in the post-castration increase in the rate of synthesis of gonadotropin subunits and the elevation of circulating LH. We recently reported (Counis et al., 1983b) that supplementation of gonadectomized rats with estradiol rapidly reverses the stimulatory effect of gonadectomy on the synthesis of (Y, LH-P and FSH-/3 precursors almost to the levels observed in normal rats; progesterone has no such effect. Sirnilar data on the inhibition of the synthesis of FSH subunits (Alexander and Miller, 1982; Landefeld et al., 1983) with estradiol in castrated sheep have been reported. These results thus demonstrate that estradiol negatively regulates the synthesis of pituitary gonadotropins, and suggest that the regulation occurs via changes in specific mRNA levels. This hypothesis has recently been confirmed in our laboratory using synthetic oligodeoxynucleotides (15-16 bases) corresponding to partial se-
87
quences of a, LH-j3 and FSH-/I as probes (results not shown). Using these oligomers which hybridize to the corresponding mRNA sequences with high specificity, we demonstrated that the number of copies of specific mRNAs was lower in normal than in gonadectomized rats. This observation confirms that the inhibitory action of estradiol is on the expression of specific genes. Acknowledgements
We wish to thank Dr. A. Parlow and the National Hormone and Pituitary Program (NIAMDDK) for rat LH-RP2, Dr. J.G. Pierce for his gift of antisera to RCXM bovine LH subunits, Dr. A. Berault and Dr. M. Theoleyre for their help in performing radioimmunoassays and Mr. M. Poissonnier for his help in preparing antisera to RCXM ovine (Y,LH-j3 and FSH-/3. The valuable technical assistance of Mme. G. Ribot is acknowledged. This work was supported by a grant from the Fondation de Recherche en Hormonologie. M.C. is a recipient of a Fondation pour la Recherche M&iicale fellowship. References Alexander, D.C. and Miller, W.L. (1982) J. Biol. Chem. 2.57, 2282-2286. Aubert, M.L. Conne, B.S., Winniger, B.P., Lang, U. and Sizonenko, PC. (1983). In: Multihormonal Regulation in Neuroendocrine Cells, Eds.: A. Tixier-Vidal and P. Richard, Proceedings of the INSERM European Symposium, Colmar (France), pp. 319-346. Clayton, R.N. and Catt, K.J. (1981) Endocrinology 108, 887-895.
Counis, R., Ribot, G., Corbani, M., Poissonnier, M. and Jutisz, M. (1981a) FEBS Lett. 123, 151-155. Counis, R., Corbani, M., Berault, A., Theoleyre, M., Jansem, M. and Jutisz, M. (1981b) CR. Acad. Sci. (Paris) Ser. III 293, 115-118. Counis, R., Corbani, M. and Jutisz, M. (1982a) Program of the 64th Annual Meeting of the Endocrine Society, abstr. 195. Counis, R., Corbani, M., Poissonnier, M. and Jutisz, M. (1982b) B&hem. Biophys. Res. Commun. 107,998-1005. Counis, R., Corbani, M. and Jutisz, M. (1982~) In: Pituitary Hormones and Related Peptides, Eds.: M. Motta, M. Zanisi and F. Piva (Academic Press, New York) pp. 49-61. Counis, R., Corbani, M. and Jutisz, M. (1983a) In: Multihormonal Regulation in Neuroendocrine Cells, Eds.: A. Tixier-Vidal and P. Richard, Proceedings of the INSERM European Symposium, Colmar (France), pp. 509-523. Counis, R., Corbani, M. and Jutisz, M. (1983b) B&hem. Biophys. Res. Commun. 114, 65-72. Frager, M.S., Pieper, D.R., Tonetta, A., Duncan, J.A. and Marshall, J.C. (1981) J. Clin. Invest. 67, 615-623. Gay, V.L. and Bogdanove, E.M. (1969) End~~nology 84, 1132-1142. Gay, V.L. and Midgley Jr., A.R. (1969) Endocrinology 84, 1359-1364. Kerdelhut, B., Pitoulis, S. and Jutisz, M. (1971) C.R. Acad. Sci. (Paris) 273, 511-514. Khar, A., Debeljuk, L. and Jutisz, M. (1978) Mol. Cell. Endocrinol. 12, 53-65. Landefeld, T.D., Kepa, J. and Karsch, F.J. (1983) J. Biol. Chem. 258, 2390-2393. McCann S.M. and Ramirez, V.D. (1964) Recent Progr. Hormone Res. 20, 131-181. Schally, A.V., Kastin, A.J. and Arimura, A. (1972) Vitamins and Hormones 30.83-164. Wheaton, J.E. and McCann, SM. (1976) Neur~nd~~nology 20,296-310. Yamamoto, M., Diebel, N.D. and Bogdanove, E.M. (1970) Endocrinology 86, 1102-1111.