259
Molecular and Cellular ~~ocr~nulo#, 82 it991 1259-263 0 1991 Elsevier Scientific Publishers Iretand, Ltd. 03W7207/91/$03.50 MOLCEL 02654
l?Iormonaf regulation of ~~nad~tr~~~u receptor mRNA in rat ovary during folIicular growth and luteinization
&Y WO&: Ovary; Follicle-stimulating Down-regulation
hormone receptor; Luteinizing hormone/human
chorionic gonadatrupjn
receptor; mRNA;
To investigate the regulation of the follicIe-stimulating hormone (FSH1 and luteinizing bo~one/human chorionic gonadotropin (LH/hCG) receptor genes by gonadotropins, we examined the effect of pregnant mare’s serum gonadotropin (PMSG) or PMSG-hCG on the expression of FSH and LH/hCG receptors in rat ovaries. After administration of PMSG, Northern blot analysis using the FSH receptor cDNA probe revealed that a major band of 2400 nudeotides was detected which reached the maximal level on day 3. On the other hand, the IeveI of LH/hCG receptor mRNA, a major mRNA of 5400 nucIeotides and minor species of 7500, 3600,230O and 1200 nucleotides, increased progressively during 4 days. Treatment with hCG resulted in a decrease of FSH and LH/hCG receptor mRNA levels, and the level of FSH receptor mRNA was completely suppressed. Although the Ievel of LH/hCG receptor mRNA was also suppressed from 3 h to an aImost undetectable level at 24 h after hCG injection, it recovered to the control level by 48 h and exceeded this level several fold by 72 h. The reappearance of LH/hCG receptors following desensitization was preceded by an increase in mRNA levels. These studies demonstrate that hormonal regulation of gonadotropin receptor mRNAs on rat ovary reflects the changes in go~ladotropin receptor levels.
Introduction
During follicular development, follicle-stimulating hormone (FSH) and iuteinizing hormone/ human chorionic gonadotropin iLHf hCG1 receptors play an important role in the response to
Address for correspondence: Takashi Minegishi. Department of Obstetrics and Gynecology, Gunma University, School of Medicine, Maebashi, Gunma 371, Japan.
circuIating gonadotropin. Previous in vivo and in vitro studies demonstrated that FSH induced FSH and LH/hCG receptors and that there are FSH receptors in granuiosa ceils from follicles of all sizes (Erickson et af., 1979; Richard, 19801, LHJhCG receptors are found only in granulosa ceils of large preovutatory follicles and LH surge or an ovulatory dose of hCG decreases both FSH and LH/hCG receptor numbers from the ovarian cellular surface (Richard et al., 1976, 1979, 1980), followed by a secondary increase in LH/hCG
260
receptor numbers coincident with corpora lutea formation (Richard and Williams, 1976). Recent cloning of rat ovarian and porcine testicular LH/hCG receptor cDNA (Loosefelt et al., 1989; McFarland et al., 1989) and rat testicular FSH receptor cDNA (Sprengel et al., 1990) has allowed us to investigate the regulation of gonadotropin receptor transcriptions. Recently it has been reported that the down-regulation of LH/hCG receptor following hCG injection is accompanied by a decrease in LH/hCG receptor mRNA (Hu et al., 1990; LaPolt et al., 1990). We have previously reported the effect of pregnant mare’s serum gonadotropin (PMSG)hCG treatment on LH/hCG receptor mRNA during luteinization (Nakamura et al., 1990). In this study, we describe changes in both FSH and LH/hCG receptor gene transcriptions in response to PMSG or PMSG-hCG exposure. Materials
and methods
25day-old immature female rats of the Wistar strain were primed with 30 IU PMSG alone or 30 IU PMSG and 20 IU hCG 60 h later. After each rat was sacrificed at selected intervals, ovaries were removed, immediately stored in liquid nitrogen until preparing the membrane fraction and extracting RNA for subsequent receptor binding and Northern blot analysis. The ovaries were minced and homogenized in ice-cold phosphatebuffered saline (PBS) using a glass homogenizer. The homogenate was centrifuged for 10 min at 600 xg twice, then supernatant was collected and centrifuged for 30 min at 10,000 X g. The precipitates were then suspended in PBS and used as receptor preparations (Kusuda and Dufau, 1986). Protein concentrations were determined by the method of Bradford (1976). Incubation of the receptor preparation with radioiodinated hormone (FSH or hCG) was carried out in the following system: 100 ~1 of receptor preparation, 100 ~1 of standard hormone solution, 100 ~1 of I’251]hormone (FSH or hCG). In order to estimate non-specific binding, 50 IU PMSG or hCG (Pregnyl) was used instead of standard hormone solution. The mixture was incubated at room temperature for 12 h. The reaction was stopped by adding 500 ~1 of ice-cold PBS containing 0.1%
bovine serum albumin (BSA) to each tube, then the mixture was centrifuged for 20 min at 3000 x g. The supernatant was removed and the precipitate was counted for radioactivity by an automatic gamma counter. Scatchard analysis of FSH and LH/hCG receptors was performed with increasing amounts of non-labeled hormone (FSH or hCG) added to a constant quantity of membrane fraction. Purified FSH (AFP-9864B), hCG (CR119) was iodinated using the chloramine T method (Hunter and Greenwood, 1962), and the specific activity was about 30 pCi/pg. A size-selected (1 S-5 kb) randomly primed rat ovary cDNA was prepared by reverse transcription of poly(A)” RNA. PCR amplification was performed using this template with two oligonucleotide primers based on the published sequence of FSH receptor cDNA (Sprengel, 1990). A polymerase chain reaction (PCR) product (735 bp) was used to isolate full-length coding cDNA which was prepared from the same random-primed rat ovary cDNA packaged into AgtlO. We isolated and sequenced several clones with a sequence kit (Takara) using the dideoxy chain termination method (Hattori and Sakai, 1986) to obtain rat FSH receptor cDNA containing an almost full coding region ( - 61 to 1832). LH/hCG receptor cDNA probe was prepared as described previously (McFarland, 1989). 15 pg of total RNA which was extracted from the ovaries by the guanidinium thiocyanate method was fractionated by electrophoresis through a 1% agarose ge1 (Han et al., 1987). The RNA was transferred to nyion membrane (Hybond-N; Amersham) and hybridized to FSH receptor and LH/hCG receptor cDNA probes that were labeled by nick translation with [a-j2P]dCTP (Amersham). After hybridization, the membrane was washed and autoradiographed, as previously described (Nakamura et al.. 1990). Results and discussion
This study has revealed the relationship between gonadotropin receptor expressions and their mRNA transcriptions under the effects of gonadotropin, PMSG or PMSG-hCG in the ovary. FSH or PMSG injection induced FSH and LH/hCG receptor expressions in vivo and in
261
her than that of LH receptor and slightly decreased by day 4 of the experiment, while the mRNA level of LH/hCG receptor progressively increased. These data showed good agreement with previous data in which FSH stimulated the FSH binding and the follicular growth indicated by the appearance of LH/hCG receptor. We have previously reported that the changes of LH/hCG receptor message levels preceded to those of receptor binding sites in the membrane during luteinization (Nakamura et al., 19901. We tried to investigate the changes of FSH and LH/hCG receptor mRNAs following the administration of hCG to PMSG-primed immature rats. In Fig. 2, Northern blot analysis revealed that hCG injection decreased mRNA of both receptors. hCG induced the down-regulation of FSH receptor mRNA level after follicle maturation, associated with the down-regulation of LH/hCG receptor mRNA. Although the mRNA level of FSH receptor had been completely diminished by hCG injection, the mRNA level of LH/hCG receptor was suppressed by 12-24 h after injection, increased to control level by 48 h and this was followed by a progressive increase. These
vitro (Erickson et al., 1979; Richard, 1980). After PMSG priming, we attempted to investigate mRNA transcription of FSH and LH/hCG receptor. As shown in Fig. 1, Northern blot analysis of FSH receptor revealed a major RNA of 2400 nucleotides which reached a peak on day 3, while the level of LH/hCG receptor mRNA, which included a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300 and 1200 nucleotides, was markedly elevated from day 2 on and progressively increased throughout the experiment (Fig. 1). On the other hand, FSH and LH/hCG receptor binding assays indicated a maximum binding of FSH receptor at day 4, and a continuous increase for 5 days in LH,/hCG receptor (data not shown), identical to our previous results (Wakabayashi et al., 1980). Previous studies of our own and others indicated that FSH increased the FSH binding site and stimulated folli~ular growth. In this study, the PMSG treatment increased mRNA IeveI of both FSH and LH/hCG receptors followed by an increase of receptor numbers in the membrane. The mRNA level of FSH receptor increased ear-
A.
FSH
receptor
Kb
n
LH/hCG
receptor
Kb
7.5
Kb -
9.5
-
7.5
-
4.4
-
2.4
-
1.4
5.4 3.6 2.4
2.3
01234
0 1 2 3 4 DAY
Fig. 1. The effect of PMSG on FSH and LH/hCG receptor mRNA levels in immature rat ovary. From each time point 15 pg of total RNA was prepared and fractionated through a 1% agarose gel and blotted as described under Materials and methods. Blots were probed with 32P-labeled (A) FSH receptor cDNA or(B) LH/hCG receptor cDNA. The filters were exposed on Kodak film.
262
data show that the mRNA level of FSH was suppressed by hCG injection which stimulated luteinization of the ovary, and this phenomenon was well correlated to the decrease of FSH binding sites in the membrane after luteinization. In terms of LH/hCG receptor, our data confirmed previous data (Hu et al., 1990; LaPolt et al., 1990) in which hCG induced down-regulation of the mRNA level of LH/hCG receptor followed by down-regulation of membrane receptor level. The study by LaPolt et al. showed that the levels of LH/hCG receptor mRNA were maximal prior to administration of hCG, and LH/hCG receptor content increased a further 5 days after hCG treatment, without apparent rise in receptor message. However, in our experiment, the receptor mRNA level recovered by 48 h to the level that was observed prior to hCG treatment and exceeded this level several fold by 72 h after hCG treatment; these changes were well correlated to the following increase in LH/hCG receptor level. Thus, the observed changes in LH/hCG receptor mRNA levels and LH/hCG receptor number during luteinization were similar to the study by Hu et al. (1990). Gonadotropin stimulates adenylate cyclase activity (Richard et al., 1979; Knecht
A.
FSH
receptor
et al., 1983) and induces accumulation of CAMP, leading to FSH and LH/hCG receptor formation (Knecht et al., 1983; Sanders and Midgley, 1983). Thus, it is generally accepted that FSH and LH regulate gonadotropin receptors by CAMP-dependent events. Although hCG treatment following FSH in vivo increases CAMP content in granulosa cells (Jonassen and Richard, 1980), this second peak of CAMP presumably causes FSH receptor degradation and LH/hCG receptor down-regulation. Since the response of adenylate cyclase to gonadotropin was different in the various stages of follicular development (Jonassen et al., 1982), we speculate that some factor intracellularly works to modify the adenylate cyclase pathway according to follicular development. More recent studies (Hadcock and Malbon, 1988) have shown that CAMP is involved in the down-regulation of &-adrenergic receptor mRNA levels, but as yet no mechanism has been described to explain this observation. In addition, in the case of tyrosine aminotransferase gene, temporally distinct changes in the transcription rate and in the turnover of tyrosine aminotransferase mRNA have been observed, depending upon the duration of exposure to CAMP analogs
B.
LH/hCG
receptor Kb
Kb
Kb
9.5
7.5+ 5.4--,
2.4 -
7.5
3.6-
4.4
2.3-
2.4
1.2--,
-48 -24
0
3
6
12 24 48 72
-48
-24 0
3
6 12 24 48 72 HR
Fig. 2. Down-regulation of FSH and LH/hCG receptor mRNAs in preovulatory follicles of rats injected with hCG. administration of PMSG and hCG, the ovaries were removed at the indicated times and total RNA prepared. From each point 15 wg of total RNA was fractionated through a 1% agarose gel and blotted as described under Materials and methods. were probed with “P-labeled (A) FSH receptor cDNA or (B) LH/hCG receptor cDNA. The filters were exposed on Kodak film.
After time Blots XRP
263
(Smith and Liu, 1988). This type of complex regulation may also exist for the gonadotropin receptors during down-regulation. This area requires further studies to establish a comprehensive understanding of the mechanisms of down-regulation of the FSH and LH/hCG receptor. Acknowledgements The authors wish to express their gratitude to Dr. A.F. Parlow, the NIAMDD Hormone Distribution Program, and the National Pituitary Agency, for supply of a radioimmunoassay kit for rat gonadotropins. This work was supported by a grant from the Ministry of Education for Science and Culture of Japan (02670732) and the Uehara Memorial Foundation. References Bradford, M.M. (1976) Anal. Biochem. 72, 248-251. Erickson, G.F., Wang, C. and Hsueh, A.J.W. (1979) Nature 279, 336-338. Hadcock, Jr., P. and Malbon, C.C. (1988) Proc. Nat]. Acad. Sci. U.S.A. 85, 5021-5025. Han, J.H., Stratowa, C. and Rutter, W.J. (1987) Biochemistry 26, 1617-162s. Hattori, M. and Sakai, Y. (1986) Anal. B&hem. lS2,232-238. Hu, Z-Z., Tsai-Morris, C.-H., Buzko, E. and Dufau, M.L. (1990) FEBS Lett. 274, 181-184. Hunter, W.M. and Greenwood, F.C. (1962) Nature 194, 49S497. Jonassen, J.A. and Richards, J.S. (1980) Endocrinology 106, 1786-1794.
Jonassen, J.A., Bose, K. and Richards, J.S. (1982) Endocrinology 111, 74-79. Knecht, M., Ranta, T. and Catt, K.J. (1983a) Endocrinology 113, 949-956. Knecht, M., Ranta, T., Katz, S.Z. and Catt, K.J. (1983b) Endocrinology 112, 1247-1255. Kusuda, S. and Dufau, M.L. (19861 J. Biol. Chem. 261, 16161-16168. LaPolt, P.S., Oikawa, M., Jia, X.-C., Damn, C. and Hsueh, A.J.W. (1990) Endocrinology 126, 3277-3279. Loosefelt, H., Misrahi, M., Atger, M., Salesse, R., Vu, H.-L., Thi, M., Johvet, A., Guiochon-Mantel, A., Sar, S., Jallel, B., Garnier, J. and Milgrom, E. (1989) Science 245, 525. 528. McFarland, KC., Sprengel, R., Philhps, H.S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (1989) Science 245,494-499. Nakamura, K., Minegishi, T., Takakura, Y., Miyamoto, K., Hasegawa, Y., Ibuki, Y. and Igarashi, M. (1990) Biochem. Biophys. Res. Commun. 172, 786-792. Richard, J.S. (1979) Recent Prog. Horm. Res. 35, 343-373. Richard, J.S. (1980) Physiol. Rev. 60, 51-89. Richard, J.S. and Williams, J.J. (1976) Endocrinology 99, 1571-1581. Richard, J.S., Ireland, J.J., Rao, M.C., Bernath, G.A., Midgley, Jr., A.R. and Reichert, Jr., LE. (1976) Endocrinology 99, 1562-1570. Richards, J.S., Jonassen, J.A., Rolfes, A.I., Kersey, K. and Reichert, Jr., L.E. (1979) Endocrinology 104, 765-773. Sanders, M.M. and Midgley, Jr., A.R. (1983) Endocrinology 112, 1382-1388. Smith, J.D. and Liu, A.Y.-C. (1988) EMBO J. 7, 3711-1728. Sprengel, R., Braun, T., Nikollics, K., Segabff, D.L. and Seeburg, P.H. 11990) Mol. Endocrinol. 4, 525-530. Wakabayashi, K., Minegishi, T., Yorozu, Y., Igarashi, M. and Ichnoe, K. (1980) Endocrinol. Jpn. 27, 87-93.
Molecular and Cellular ~~ocr~nulo#, 82 it991 1259-263 0 1991 Elsevier Scientific Publishers Iretand, Ltd. 03W7207/91/$03.50 MOLCEL 02654
l?Iormonaf regulation of ~~nad~tr~~~u receptor mRNA in rat ovary during folIicular growth and luteinization
&Y WO&: Ovary; Follicle-stimulating Down-regulation
hormone receptor; Luteinizing hormone/human
chorionic gonadatrupjn
receptor; mRNA;
To investigate the regulation of the follicIe-stimulating hormone (FSH1 and luteinizing bo~one/human chorionic gonadotropin (LH/hCG) receptor genes by gonadotropins, we examined the effect of pregnant mare’s serum gonadotropin (PMSG) or PMSG-hCG on the expression of FSH and LH/hCG receptors in rat ovaries. After administration of PMSG, Northern blot analysis using the FSH receptor cDNA probe revealed that a major band of 2400 nudeotides was detected which reached the maximal level on day 3. On the other hand, the IeveI of LH/hCG receptor mRNA, a major mRNA of 5400 nucIeotides and minor species of 7500, 3600,230O and 1200 nucleotides, increased progressively during 4 days. Treatment with hCG resulted in a decrease of FSH and LH/hCG receptor mRNA levels, and the level of FSH receptor mRNA was completely suppressed. Although the Ievel of LH/hCG receptor mRNA was also suppressed from 3 h to an aImost undetectable level at 24 h after hCG injection, it recovered to the control level by 48 h and exceeded this level several fold by 72 h. The reappearance of LH/hCG receptors following desensitization was preceded by an increase in mRNA levels. These studies demonstrate that hormonal regulation of gonadotropin receptor mRNAs on rat ovary reflects the changes in go~ladotropin receptor levels.
Introduction
During follicular development, follicle-stimulating hormone (FSH) and iuteinizing hormone/ human chorionic gonadotropin iLHf hCG1 receptors play an important role in the response to
Address for correspondence: Takashi Minegishi. Department of Obstetrics and Gynecology, Gunma University, School of Medicine, Maebashi, Gunma 371, Japan.
circuIating gonadotropin. Previous in vivo and in vitro studies demonstrated that FSH induced FSH and LH/hCG receptors and that there are FSH receptors in granuiosa ceils from follicles of all sizes (Erickson et af., 1979; Richard, 19801, LHJhCG receptors are found only in granulosa ceils of large preovutatory follicles and LH surge or an ovulatory dose of hCG decreases both FSH and LH/hCG receptor numbers from the ovarian cellular surface (Richard et al., 1976, 1979, 1980), followed by a secondary increase in LH/hCG
260
receptor numbers coincident with corpora lutea formation (Richard and Williams, 1976). Recent cloning of rat ovarian and porcine testicular LH/hCG receptor cDNA (Loosefelt et al., 1989; McFarland et al., 1989) and rat testicular FSH receptor cDNA (Sprengel et al., 1990) has allowed us to investigate the regulation of gonadotropin receptor transcriptions. Recently it has been reported that the down-regulation of LH/hCG receptor following hCG injection is accompanied by a decrease in LH/hCG receptor mRNA (Hu et al., 1990; LaPolt et al., 1990). We have previously reported the effect of pregnant mare’s serum gonadotropin (PMSG)hCG treatment on LH/hCG receptor mRNA during luteinization (Nakamura et al., 1990). In this study, we describe changes in both FSH and LH/hCG receptor gene transcriptions in response to PMSG or PMSG-hCG exposure. Materials
and methods
25day-old immature female rats of the Wistar strain were primed with 30 IU PMSG alone or 30 IU PMSG and 20 IU hCG 60 h later. After each rat was sacrificed at selected intervals, ovaries were removed, immediately stored in liquid nitrogen until preparing the membrane fraction and extracting RNA for subsequent receptor binding and Northern blot analysis. The ovaries were minced and homogenized in ice-cold phosphatebuffered saline (PBS) using a glass homogenizer. The homogenate was centrifuged for 10 min at 600 xg twice, then supernatant was collected and centrifuged for 30 min at 10,000 X g. The precipitates were then suspended in PBS and used as receptor preparations (Kusuda and Dufau, 1986). Protein concentrations were determined by the method of Bradford (1976). Incubation of the receptor preparation with radioiodinated hormone (FSH or hCG) was carried out in the following system: 100 ~1 of receptor preparation, 100 ~1 of standard hormone solution, 100 ~1 of I’251]hormone (FSH or hCG). In order to estimate non-specific binding, 50 IU PMSG or hCG (Pregnyl) was used instead of standard hormone solution. The mixture was incubated at room temperature for 12 h. The reaction was stopped by adding 500 ~1 of ice-cold PBS containing 0.1%
bovine serum albumin (BSA) to each tube, then the mixture was centrifuged for 20 min at 3000 x g. The supernatant was removed and the precipitate was counted for radioactivity by an automatic gamma counter. Scatchard analysis of FSH and LH/hCG receptors was performed with increasing amounts of non-labeled hormone (FSH or hCG) added to a constant quantity of membrane fraction. Purified FSH (AFP-9864B), hCG (CR119) was iodinated using the chloramine T method (Hunter and Greenwood, 1962), and the specific activity was about 30 pCi/pg. A size-selected (1 S-5 kb) randomly primed rat ovary cDNA was prepared by reverse transcription of poly(A)” RNA. PCR amplification was performed using this template with two oligonucleotide primers based on the published sequence of FSH receptor cDNA (Sprengel, 1990). A polymerase chain reaction (PCR) product (735 bp) was used to isolate full-length coding cDNA which was prepared from the same random-primed rat ovary cDNA packaged into AgtlO. We isolated and sequenced several clones with a sequence kit (Takara) using the dideoxy chain termination method (Hattori and Sakai, 1986) to obtain rat FSH receptor cDNA containing an almost full coding region ( - 61 to 1832). LH/hCG receptor cDNA probe was prepared as described previously (McFarland, 1989). 15 pg of total RNA which was extracted from the ovaries by the guanidinium thiocyanate method was fractionated by electrophoresis through a 1% agarose ge1 (Han et al., 1987). The RNA was transferred to nyion membrane (Hybond-N; Amersham) and hybridized to FSH receptor and LH/hCG receptor cDNA probes that were labeled by nick translation with [a-j2P]dCTP (Amersham). After hybridization, the membrane was washed and autoradiographed, as previously described (Nakamura et al.. 1990). Results and discussion
This study has revealed the relationship between gonadotropin receptor expressions and their mRNA transcriptions under the effects of gonadotropin, PMSG or PMSG-hCG in the ovary. FSH or PMSG injection induced FSH and LH/hCG receptor expressions in vivo and in
261
her than that of LH receptor and slightly decreased by day 4 of the experiment, while the mRNA level of LH/hCG receptor progressively increased. These data showed good agreement with previous data in which FSH stimulated the FSH binding and the follicular growth indicated by the appearance of LH/hCG receptor. We have previously reported that the changes of LH/hCG receptor message levels preceded to those of receptor binding sites in the membrane during luteinization (Nakamura et al., 19901. We tried to investigate the changes of FSH and LH/hCG receptor mRNAs following the administration of hCG to PMSG-primed immature rats. In Fig. 2, Northern blot analysis revealed that hCG injection decreased mRNA of both receptors. hCG induced the down-regulation of FSH receptor mRNA level after follicle maturation, associated with the down-regulation of LH/hCG receptor mRNA. Although the mRNA level of FSH receptor had been completely diminished by hCG injection, the mRNA level of LH/hCG receptor was suppressed by 12-24 h after injection, increased to control level by 48 h and this was followed by a progressive increase. These
vitro (Erickson et al., 1979; Richard, 1980). After PMSG priming, we attempted to investigate mRNA transcription of FSH and LH/hCG receptor. As shown in Fig. 1, Northern blot analysis of FSH receptor revealed a major RNA of 2400 nucleotides which reached a peak on day 3, while the level of LH/hCG receptor mRNA, which included a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300 and 1200 nucleotides, was markedly elevated from day 2 on and progressively increased throughout the experiment (Fig. 1). On the other hand, FSH and LH/hCG receptor binding assays indicated a maximum binding of FSH receptor at day 4, and a continuous increase for 5 days in LH,/hCG receptor (data not shown), identical to our previous results (Wakabayashi et al., 1980). Previous studies of our own and others indicated that FSH increased the FSH binding site and stimulated folli~ular growth. In this study, the PMSG treatment increased mRNA IeveI of both FSH and LH/hCG receptors followed by an increase of receptor numbers in the membrane. The mRNA level of FSH receptor increased ear-
A.
FSH
receptor
Kb
n
LH/hCG
receptor
Kb
7.5
Kb -
9.5
-
7.5
-
4.4
-
2.4
-
1.4
5.4 3.6 2.4
2.3
01234
0 1 2 3 4 DAY
Fig. 1. The effect of PMSG on FSH and LH/hCG receptor mRNA levels in immature rat ovary. From each time point 15 pg of total RNA was prepared and fractionated through a 1% agarose gel and blotted as described under Materials and methods. Blots were probed with 32P-labeled (A) FSH receptor cDNA or(B) LH/hCG receptor cDNA. The filters were exposed on Kodak film.
262
data show that the mRNA level of FSH was suppressed by hCG injection which stimulated luteinization of the ovary, and this phenomenon was well correlated to the decrease of FSH binding sites in the membrane after luteinization. In terms of LH/hCG receptor, our data confirmed previous data (Hu et al., 1990; LaPolt et al., 1990) in which hCG induced down-regulation of the mRNA level of LH/hCG receptor followed by down-regulation of membrane receptor level. The study by LaPolt et al. showed that the levels of LH/hCG receptor mRNA were maximal prior to administration of hCG, and LH/hCG receptor content increased a further 5 days after hCG treatment, without apparent rise in receptor message. However, in our experiment, the receptor mRNA level recovered by 48 h to the level that was observed prior to hCG treatment and exceeded this level several fold by 72 h after hCG treatment; these changes were well correlated to the following increase in LH/hCG receptor level. Thus, the observed changes in LH/hCG receptor mRNA levels and LH/hCG receptor number during luteinization were similar to the study by Hu et al. (1990). Gonadotropin stimulates adenylate cyclase activity (Richard et al., 1979; Knecht
A.
FSH
receptor
et al., 1983) and induces accumulation of CAMP, leading to FSH and LH/hCG receptor formation (Knecht et al., 1983; Sanders and Midgley, 1983). Thus, it is generally accepted that FSH and LH regulate gonadotropin receptors by CAMP-dependent events. Although hCG treatment following FSH in vivo increases CAMP content in granulosa cells (Jonassen and Richard, 1980), this second peak of CAMP presumably causes FSH receptor degradation and LH/hCG receptor down-regulation. Since the response of adenylate cyclase to gonadotropin was different in the various stages of follicular development (Jonassen et al., 1982), we speculate that some factor intracellularly works to modify the adenylate cyclase pathway according to follicular development. More recent studies (Hadcock and Malbon, 1988) have shown that CAMP is involved in the down-regulation of &-adrenergic receptor mRNA levels, but as yet no mechanism has been described to explain this observation. In addition, in the case of tyrosine aminotransferase gene, temporally distinct changes in the transcription rate and in the turnover of tyrosine aminotransferase mRNA have been observed, depending upon the duration of exposure to CAMP analogs
B.
LH/hCG
receptor Kb
Kb
Kb
9.5
7.5+ 5.4--,
2.4 -
7.5
3.6-
4.4
2.3-
2.4
1.2--,
-48 -24
0
3
6
12 24 48 72
-48
-24 0
3
6 12 24 48 72 HR
Fig. 2. Down-regulation of FSH and LH/hCG receptor mRNAs in preovulatory follicles of rats injected with hCG. administration of PMSG and hCG, the ovaries were removed at the indicated times and total RNA prepared. From each point 15 wg of total RNA was fractionated through a 1% agarose gel and blotted as described under Materials and methods. were probed with “P-labeled (A) FSH receptor cDNA or (B) LH/hCG receptor cDNA. The filters were exposed on Kodak film.
After time Blots XRP
263
(Smith and Liu, 1988). This type of complex regulation may also exist for the gonadotropin receptors during down-regulation. This area requires further studies to establish a comprehensive understanding of the mechanisms of down-regulation of the FSH and LH/hCG receptor. Acknowledgements The authors wish to express their gratitude to Dr. A.F. Parlow, the NIAMDD Hormone Distribution Program, and the National Pituitary Agency, for supply of a radioimmunoassay kit for rat gonadotropins. This work was supported by a grant from the Ministry of Education for Science and Culture of Japan (02670732) and the Uehara Memorial Foundation. References Bradford, M.M. (1976) Anal. Biochem. 72, 248-251. Erickson, G.F., Wang, C. and Hsueh, A.J.W. (1979) Nature 279, 336-338. Hadcock, Jr., P. and Malbon, C.C. (1988) Proc. Nat]. Acad. Sci. U.S.A. 85, 5021-5025. Han, J.H., Stratowa, C. and Rutter, W.J. (1987) Biochemistry 26, 1617-162s. Hattori, M. and Sakai, Y. (1986) Anal. B&hem. lS2,232-238. Hu, Z-Z., Tsai-Morris, C.-H., Buzko, E. and Dufau, M.L. (1990) FEBS Lett. 274, 181-184. Hunter, W.M. and Greenwood, F.C. (1962) Nature 194, 49S497. Jonassen, J.A. and Richards, J.S. (1980) Endocrinology 106, 1786-1794.
Jonassen, J.A., Bose, K. and Richards, J.S. (1982) Endocrinology 111, 74-79. Knecht, M., Ranta, T. and Catt, K.J. (1983a) Endocrinology 113, 949-956. Knecht, M., Ranta, T., Katz, S.Z. and Catt, K.J. (1983b) Endocrinology 112, 1247-1255. Kusuda, S. and Dufau, M.L. (19861 J. Biol. Chem. 261, 16161-16168. LaPolt, P.S., Oikawa, M., Jia, X.-C., Damn, C. and Hsueh, A.J.W. (1990) Endocrinology 126, 3277-3279. Loosefelt, H., Misrahi, M., Atger, M., Salesse, R., Vu, H.-L., Thi, M., Johvet, A., Guiochon-Mantel, A., Sar, S., Jallel, B., Garnier, J. and Milgrom, E. (1989) Science 245, 525. 528. McFarland, KC., Sprengel, R., Philhps, H.S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (1989) Science 245,494-499. Nakamura, K., Minegishi, T., Takakura, Y., Miyamoto, K., Hasegawa, Y., Ibuki, Y. and Igarashi, M. (1990) Biochem. Biophys. Res. Commun. 172, 786-792. Richard, J.S. (1979) Recent Prog. Horm. Res. 35, 343-373. Richard, J.S. (1980) Physiol. Rev. 60, 51-89. Richard, J.S. and Williams, J.J. (1976) Endocrinology 99, 1571-1581. Richard, J.S., Ireland, J.J., Rao, M.C., Bernath, G.A., Midgley, Jr., A.R. and Reichert, Jr., LE. (1976) Endocrinology 99, 1562-1570. Richards, J.S., Jonassen, J.A., Rolfes, A.I., Kersey, K. and Reichert, Jr., L.E. (1979) Endocrinology 104, 765-773. Sanders, M.M. and Midgley, Jr., A.R. (1983) Endocrinology 112, 1382-1388. Smith, J.D. and Liu, A.Y.-C. (1988) EMBO J. 7, 3711-1728. Sprengel, R., Braun, T., Nikollics, K., Segabff, D.L. and Seeburg, P.H. 11990) Mol. Endocrinol. 4, 525-530. Wakabayashi, K., Minegishi, T., Yorozu, Y., Igarashi, M. and Ichnoe, K. (1980) Endocrinol. Jpn. 27, 87-93.
